Can proteins from different viruses be gathered in one virus?

Can proteins from different viruses be gathered in one virus?

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There is a biology project I must do with some of my classmates and we're facing a problem. We would like to choose proteins from different viruses that seem interesting to us (for example one protein from a negative-strand ARN virus and the other one from a DNA virus) and gather them (theory) together through this process called "directed mutagenesis". Is it possible ? If no, isn't there another mechanism that allows the gathering ?

Thank you ! (PS: Sorry for the english)

If I understand your question, you're asking if you can take a protein from Virus A, and make Virus B produce that protein. The answer is yes, you certainly can do that, although depending on the proteins and viruses there may be some constraints. There are many examples of this being done, and even more examples of making a virus produce proteins from humans or other species.

The actual process of inserting a new gene can be pretty trivial; you can pick up handy kits of viruses specifically designed to have new genes inserted into them. If you're putting the gene into a virus of your interest, the design process can be a little trickier; you need to be sure that the inserted protein doesn't disrupt something essential for the recipient, isn't too large for its genome, and so on.

But in principle, it's pretty simple.

Some Weird Truths About Viruses, And The Covid-19 Virus

Images combined from a 3D medical animation, depicting the shape of coronavirus as well as the . [+] cross-sectional view. Image shows the major elements including the Spike S protein, HE protein, viral envelope, and helical RNA.

Credit: This work is free and may be used by anyone for any purpose.

The virus that has devastated the world this year, SARS-CoV-2, is not a living organism. Viruses are not alive. Think of them instead as biological machines, incredibly small ones.

What, exactly, is a virus? Many people outside the world of science and medicine don’t really know, so today I’m going to describe just a few of their essential features.

Viruses have, in general, just two functions: they invade your cells, and then they borrow your own cells’ machinery to copy themselves. (Note: for simplicity I’m describing viruses that infect humans, but in reality they infect pretty much every living thing, from bacteria to plants to animals.) After making many copies, they break out, usually destroying the cell they’ve invaded, and do it again.

Here’s a weird thing about viruses. All living things on this planet are made from instructions encoded in DNA. Some viruses are also made of DNA, but many are made of RNA instead. RNA is a lot like DNA, but it doesn’t have that famous double-stranded helix structure instead, it’s just a single strand.

Now consider how small they are. The Covid-19 virus, SARS-CoV-2, has just 29 genes that are encoded in just under 30,000 letters of RNA. Other viruses can be even smaller: the influenza virus has just 10 genes, encoded in 13,588 letters of RNA. In contrast, the human genome has about 3 billion letters of DNA, and over 20,000 genes. In other words, our genome has 100,000 times more information encoded in it than the Covid-19 virus.

And yet these simple machines with a handful of genes can destroy us. Think of it like throwing a wrench into a running engine: the wrench is simple, but that doesn’t mean it can’t gum up the works of a far more complicated device. So too with viruses and their hosts.

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It’s not just that they have a small genetic code: viruses are also physically small. So small, in fact that they cannot be seen under a normal microscope. Bacteria are huge compared to viruses in fact, bacteria suffer viral infections just like humans do.

(Aside: the exciting new technology known as CRISPR is actually a mechanism created by bacteria to fight off viral infections!)

One consequence of virus’s tiny size is that when the 1918 flu pandemic swept the world, no one knew it was caused by a virus. Scientists didn’t have the technology to see a virus at that time. The influenza virus–the true cause of flu–wasn’t discovered until 15 years later, in 1933.

(Another aside: a bacterium called Haemophilus influenzae was given its name because scientists thought it caused the flu. It doesn’t. It does cause ear infections and sometimes-deadly meningitis, though, and for that reason the Hib vaccine, which prevents infection from this bacterium, is a critical part of the childhood vaccine schedule.)

Another odd fact about viruses: they’re not cells. They don’t have a proper cell wall, as such, just a shell made out of a few proteins. The shells encapsulate the tiny genetic code of the virus. We call them “particles” for lack of a better word.

Viruses are everywhere, and they are far more numerous than bacteria. Bacteria, in turn, are far more numerous than plants and animals. Viruses are also devastatingly effective at what they do (infecting living cells and hijacking those cells to make more viruses), which is why we will never rid ourselves of them.

While we can’t get rid of them, we can fight the viruses that cause human diseases like Covid-19. The best way to do that is to prevent viruses from invading our cells. How? There’s only one good way that we know of so far, and that’s to use the human immune system to fight them off at the molecular level. (While viruses are simple, the immune system is really complicated. I can’t possibly explain it here, but check out Ed Yong’s recent story at The Atlantic for an excellent attempt to de-mystify the immune system.)

This is where vaccination comes in. When a virus invades us, our immune system creates custom-designed cells (see that Ed Yong article) that recognize and destroy the virus. Then it becomes a race: if the immune system wins, it destroys all of the viral particles. If the virus overwhelms the host, the result can be fatal.

For Covid-19, most people mount an immune response quickly enough to avoid getting seriously ill. However, for those that don’t, the results are extremely serious. A vaccine works by “showing” the immune system part of the virus, but doing this in a way that isn’t actually an infection. One strategy used by several of the Covid-19 vaccines under development is to just package up one of the SARS-CoV-2 proteins, without the rest of the virus. The vaccine itself will prime the immune system to recognize the Covid-19 virus without actually causing an infection. Then, if that person is actually infected, the immune system swings into action quickly, and fights off Covid-19 before it ever gets established.

So that’s it. Covid-19 is caused by a tiny, sub-microscopic biological machine, a virus with just 29 genes. The virus can be ruthlessly effective, but our immune system can wipe it out if we give it the right clues. Let’s hope we’ll have a vaccine soon.

Disclaimer: the content on this site is my personal opinion and is independent of my affiliation with Johns Hopkins University.

Steps of Virus Infections

A virus must use its host-cell processes to replicate. The viral replication cycle can produce dramatic biochemical and structural changes in the host cell, which may cause cell damage. These changes, called cytopathic effects , can change cell functions or even destroy the cell. Some infected cells, such as those infected by the common cold virus known as rhinovirus, die through lysis (bursting) or apoptosis (programmed cell death or “cell suicide”), releasing all progeny virions at once. The symptoms of viral diseases result both from such cell damage caused by the virus and from the immune response to the virus, which attempts to control and eliminate the virus from the body.

Many animal viruses, such as HIV (human immunodeficiency virus) , leave the infected cells of the immune system by a process known as budding , where virions leave the cell individually. During the budding process, the cell does not undergo lysis and is not immediately killed. However, the damage to the cells that the virus infects may make it impossible for the cells to function normally, even though the cells remain alive for a period of time. Most productive viral infections follow similar steps in the virus replication cycle: attachment, penetration, uncoating, replication, assembly, and release (Figure 1).


A virus attaches to a specific receptor site on the host cell membrane through attachment proteins in the capsid or via glycoproteins embedded in the viral envelope. The specificity of this interaction determines the host—and the cells within the host—that can be infected by a particular virus. This can be illustrated by thinking of several keys and several locks, where each key will fit only one specific lock.

Surprise: A virus-like protein is important for cognition and memory

A protein involved in cognition and storing long-term memories looks and acts like a protein from viruses. The protein, called Arc, has properties similar to those that viruses use for infecting host cells, and originated from a chance evolutionary event that occurred hundreds of millions of years ago.

The prospect that virus-like proteins could be the basis for a novel form of cell-to-cell communication in the brain could change our understanding of how memories are made, according to Jason Shepherd, a neuroscientist at University of Utah Health and senior author of the study publishing in the journal Cell on Jan. 11.

PHOTO CREDIT: Charlie Ehlert

Jason Shepherd, Rachel Kearns, and Elissa Pastuzyn, from the Department of Neurobiology and Anatomy, University of Utah Health

Shepherd first suspected that something was different about Arc when his colleagues captured an image of the protein showing that Arc was assembling into large structures. With a shape that resembles a capsule from a lunar lander, these structures looked a lot like the retrovirus, HIV.

“At the time, we didn’t know much about the molecular function or evolutionary history of Arc,” says Shepherd who has researched the protein for 15 years. “I had almost lost interest in the protein, to be honest. After seeing the capsids, we knew we were onto something interesting.”

The gap in research was not for want of an interesting subject. Prior work had shown that mice lacking Arc forgot things they had learned a mere 24 hours earlier. Further, their brains lacked plasticity. There is a window of time early in life when the brain is like a sponge, easily soaking up new knowledge and skills. Without Arc, the window never opens.

Scientists had never considered that mechanisms responsible for acquiring knowledge could stem from foreign origins. Now, the work by Shepherd and his team has raised this intriguing possibility.

Everything Old is New Again

Seeing Arc’s unusual propensity to form virus-like structures prompted Shepherd to scrutinize the protein sequence with a new set of eyes. He found that regions of the code were similar to that from viral capsids. An essential tool for viral infection, capsids carry virus’ genetic information and deliver it from cell to cell in its victim.

Given that Arc looks like a viral protein, Shepherd and his colleagues designed a set of experiments to test whether it also acts like one. They first determined that several copies of Arc self-assemble into hollow virus-like capsids and stash its own genetic material, in this case mRNA, inside them. When the scientists added the capsids to mouse brain cells, or neurons, growing in a dish, Arc transferred its genetic cargo into the cells.

After viruses invade host cells, they emerge ready to infect once again. It appears that Arc works in a similar way. The scientists gathered Arc that had been released from mouse neurons and determined that the proteins and their cargo could be taken up by another set of neurons. Unlike for viruses, activating neurons mobilizes Arc, triggering the release of capsids.

PHOTO CREDIT: Chris Manfre

A protein important for cognition and memory named Arc can encapsulate genetic material (polyhedron enveloping the ribbon-like strands) and deliver it to brain cells in a manner similar to the way in which viruses infect host cells.

“We went into this line of research knowing that Arc was special in many ways, but when we discovered that Arc was able to mediate cell-to-cell transport of RNA, we were floored,” says the study’s lead author, postdoctoral fellow Elissa Pastuzyn. “No other non-viral protein that we know of acts in this way.”

When Lightning Strikes Twice

The story of Arc’s origin is relayed through the genomes of animals throughout evolutionary time. 350-400 million years ago, a chance occurrence struck four-limbed creatures that roamed the earth. An ancestor to retroviruses, called retrotransposons, inserted its genetic material into the animals’ DNA. The event led to the mammalian Arc that we know today.

The significance of such an event is hinted at by the fact that it happened more than once. An accompanying paper in the same issue of Cell shows that a version of Arc found in flies also looks and acts like a viral capsid. Vivian Budnik’s lab at the University of Massachusetts shows that fly Arc transports RNA from neurons to muscles to control movement. Even though mammalian and fly Arc evolved from the same class of retrotransposons, the event in flies occurred about 150 million years later.

“As an evolutionary biologist this is what is the most exciting to me,” says co-author Cédric Feschotte, a professor at Cornell University. “The fact that it happened at least twice makes us think that it happened even more.”

Shepherd believes this could mean that it is advantageous to have this viral-inspired system in place, and it may represent a novel form of intercellular communication. This hypothesis remains to be tested in mammals. “Knowing what cargo Arc vesicles transport in living animals will be critical to understanding the function of this pathway,” he says.

Remember the unusual viral-like protein that you just learned about? It could be controlling your memory. Watch this video to learn more:

The research was supported by the National Institutes of Health and will be published as “The Neuronal Gene Arc Encodes a Repurposed Retrotransposon Gag Protein that Mediates Intercellular mRNA Transfer” online in Cell.

In addition to Shepherd, Pastuzyn, and Feschotte, co-authors are Cameron Day, Rachel Kearns, Madeleine Kyrke-Smith, Andrew Taibi, John McCormick, Nathan Yoder, and David Belnap from the University of Utah, and Simon Erlendsson, Dustin Morado, and John Briggs from the MRC Lab of Molecular Biology at the University of Cambridge.


Viruses have evolved to enter cells from all three domains of life — Bacteria, Archaea and Eukaryotes. Of more than 3,600 known viruses, hundreds can infect human cells and most of those are associated with disease. To gain access to the cell interior, animal viruses attach to host-cell receptors. Advances in our understanding of how viral entry proteins interact with their host-cell receptors and undergo conformational changes that lead to entry offer unprecedented opportunities for the development of novel therapeutics and vaccines.

Probably the first observation of specific attachment of a virus to a cell was made at the start of the twentieth century by d'Herelle 1 . He cultured Shigella and observed occasional clear spots — lysed bacteria — in a lawn of bacterial growth on a solid agar medium which he called plaques. The viruses that had lysed Shigella were named bacteriophages. Using co-sedimentation experiments, he showed that the attachment of the virus to the host cell is the first step in infection, and that attachment only occurred when the virus was mixed with bacteria that were susceptible to the virus. This early study showed that the host range of a virus was determined by the attachment step. A century later, we are beginning to understand the details of an increasing number of virus–receptor interactions at the atomic level. All viruses contain nucleic-acid genomes (either RNA or DNA), which are packaged with proteins that are encoded by the viral genome. Viruses can be divided into two main categories enveloped viruses, which have a lipid membrane (envelope) that is derived from the host cell and non-enveloped viruses, which lack a membrane. Viruses from 24 different families can cause, or are associated with, diseases in humans (Table 1), so it is crucial to understand how different viruses solve the problem of entry into cells, and how this process can be inhibited. This review summarizes recent advances in our understanding of virus entry mechanisms at the molecular level and options for therapeutic intervention of these processes.

Both non-enveloped and enveloped viruses share the same main steps and routes of virus entry — which begin with attachment to cell-surface receptors and end with the delivery of the viral genome to the cell cytoplasm (Fig. 1). After binding to receptors — which can be proteins, carbohydrates or lipids — viruses use two main routes to enter the cell — the endocytic and non-endocytic routes. The endocytic route is usually by transport in clathrin-coated vesicles or pits, but non-clathrin-coated pits, macropinocytosis or caveolae are also used 2 . Some viruses can induce internalization by endocytosis — for example, simian virus 40 (SV40), which induces local actin polymerization and dynamin recruitment at the site of entry 3 (Fig. 1). The non-endocytic route of entry involves directly crossing the plasma membrane at neutral pH (Fig. 1). Viruses that use the non-endocytic route can also enter cells by the endocytic pathway — for example, human immunodeficiency virus type 1 (HIV-1). Membrane fusion — a basic cellular process that is essential for phagocytosis, pinocytosis and vesicular trafficking — is a basic mode of entry by enveloped viruses that use the endocytic or non-endocytic routes. The process is regulated and is mediated by membrane proteins once the membranes are in close proximity to each other. For both enveloped and non-enveloped viruses, entry into cells involves important conformational changes of the viral ENTRY PROTEINS or the host-cell receptors, which are induced by low endosomal pH. This can occur either by penetration (for non-enveloped viruses) or fusion (for enveloped viruses). After entry into the host cell, many viruses, including HIV-1 and SV40, are transported through the cytoplasm as nucleoprotein complexes. Surface-exposed nuclear localization signals on the nucleoprotein complex allow targeting to and entry into the nucleus, and infection of non-dividing cells.

a | Clathrin-mediated endocytosis, for example, adenovirus. Endocytosis by caveolae can also occur, for example, SV40. b | Fusion at the cell membrane, for example, HIV.Fusion can also occur from inside an endosome, for example, influenza.

Entry of viruses such as SV40, echovirus 1 (EV1), HIV-1, measles virus, Ebola virus and Epstein–Barr virus (EBV), can be enhanced by lipid microdomains, known as LIPID RAFTS 4,5 . Other viruses, including influenza, can use lipid rafts as a platform on which to concentrate a sufficient number of viral molecules into virions for efficient exit from one cell and entry into another cell 6 . However, fusion of Semliki Forest virus (SFV) or Sindbis virus (SIN) with LIPOSOMES does not require rafts, and the presence of rafts can be inhibitory to membrane fusion 7 . It remains to be established whether rafts are involved in the infectious cell entry of SFV and SIN 8 .

Some viruses enter cells through direct cell-to-cell contacts, using structures that are formed by the polarized cytoskeleton, adhesion molecules and viral proteins at the infected cell junction, which is known as the 'virological synapse' 9 . Direct cell-to-cell transmission of viruses by this process — for example, retroviruses (such as human T-cell lymphoma-leukaemia virus type 1 (HTLV-1) and HIV-1), herpesviruses (such as herpes simplex virus (HSV) and varicella-zoster virus) and poxviruses (such as vaccinia virus) — is poorly understood. In many cases it is not clear whether cell-to-cell transmission by this route involves membrane fusion or penetration, or direct transfer of the virus through cell junctions. Efficient and rapid cell-to-cell transmission of some viruses, such as HIV-1, could alternatively be mediated by virions that are budding or have just been released into the space between the closely apposed interacting cells or by cell-to-cell fusion. Cell-to-cell transmission might protect viruses from the actions of the immune system and could be an important route of transmission in vivo.

Viruses such as HIV-1 and poliovirus can enter and exit cells without crossing membranes by a process known as transcytosis 10 . Transcytosis — vesicular transport from one side of a cell to the other — is used by multicellular organisms to selectively move material (usually macromolecules) through cells between two environments without modifying it. Viruses have usurped this mechanism to cross the epithelial cell barrier and infect the underlying cells.

The kinetics and efficiency of entry vary greatly between viruses from different families, between viruses within a family, between viruses within a genus and even between isolates of the same species. Some viruses, such as adeno-associated virus serotype 2 (AAV-2), SFV and influenza can cross the endosomal membranes very rapidly (within seconds), and the efficiency of entry can be as much, or more than, 50% (which means that 50% of attached viruses enter cells). Single AAV-2 virions can cross membranes in less than a second 11 and individual influenza virions cross membranes in as little as one or more seconds 12,13 . Other viruses, such as HIV-1, take one or more minutes to enter cells, and the efficiency of entry is poor compared with AAV-2 — often as low as 0.1% (Refs 14,15). The kinetics and efficiency of virus entry might be related to the virus structure and it seems that the best kinetics and efficiency of entry are observed for viruses that use low pH as an entry trigger and have flattened structures — such as SFV. Cell-bound SFV can fuse in seconds with an efficiency of 80% (Ref. 16). Membrane lipid composition and structure also affect the kinetics and efficiency of virus entry.

Both non-enveloped and enveloped viruses can use the energy of METASTABLE STATES in viral entry proteins to expose hydrophobic sequences 17,18 that can destabilize host-cell membranes. However, after this, the formation of different intermediates leads to the formation of membrane pores (in the case of non-enveloped viruses) or membrane fusion pores (in the case of enveloped viruses) (Fig. 1). Often, conformational changes in a single virus protein can mediate membrane fusion. Enveloped viruses can fuse with the plasma membrane or from inside an endosome. The penetration and entry of non-enveloped viruses might resemble the entry of toxins, such as anthrax toxin 19 . Entry of enveloped viruses has similarities with intracellular fusion processes, such as exocytosis 20 .

Virus structure and receptor recognition

Virus evolution has resulted in several receptor-recognizing surface structures, which frequently have protrusions (spikes) about 10 nm or longer that are formed by the entry proteins — for example, coronaviruses (Fig. 2d) and AAVs (Fig. 2b) — or canyons — for example, the picornaviruses human rhinovirus 14 (HRV14) 21 and poliovirus 22 (Fig. 2a). Non-enveloped viruses are often small and stable, and can form crystals that diffract to good resolution, so the structures of a relatively large number of representatives from different virus families in this group have been solved by X-ray crystallography to high resolution — typically about 2 Å. By contrast, only a few structures of whole enveloped viruses — for example, SFV 23 (Fig. 2c) and dengue virus 24 — have been published, which have been solved to no more than 9-Å resolution by cryoelectron microscopy.

a | Structure of the 160S poliovirus particle. Reproduced with permission from Ref. 106 © (2000) American Society for Microbiology. b | Surface topology of adeno-associated virus 2. The protruding spikes are coloured white. Reproduced with permission from Ref. 105 © (2002) National Academies of Sciences, USA. c | Structure of Semliki Forest virus. The colour scheme reflects the radial distance from the centre of the virion, increasing from blue to red. Reproduced with permission from Ref. 23 © (2000) Elsevier Science. d | Structure of the severe acute respiratory syndrome (SARS) coronavirus. The coronavirus particle has club-shaped surface projections — known as spikes. Adeno-associated virus 2 and Semliki Forest virus particles (not to scale) are comparable in size to Poliovirus. Reproduced with permission from Ref. 107 © (2003) Massachusetts Medical Society.

Structure of viral entry proteins. Virion-associated entry proteins are typically glycosylated oligomers — for example, homodimeric tick-borne encephalitis virus (TBE) E protein, homotrimeric adenovirus fibre and influenza haemagglutinin (HA) (Fig. 3). The ATTACHMENT PROTEINS of some viruses — for example, adenoviruses and retroviruses — are unlikely to significantly interact with each other, whereas the entry proteins of other viruses — including alphaviruses, such as SFV — form heterodimers with other proteins and interact with each other extensively to form a lattice of interacting proteins (Fig. 2c). The entire CAPSID of many non-enveloped viruses — for example the picornaviruses human rhinovirus 14 (HRV14) 21 and poliovirus 22 (Fig. 2a) — is formed by a network of interacting proteins that are involved in entry.

a | Ribbon tracing of reovirus attachment protein σ1. Reproduced with permission from Ref. 108 © (2003) Wiley. b | Ribbon tracing of adenovirus fibre. Reproduced with permission from Ref. 22 © (1985) American Association of Sciences. Both reovirus attachment protein σ1 (a) and adenovirus fibre (b) are homotrimers. The three monomers in each trimer are shown in red, orange and blue. Both proteins have head-and-tail morphology, with a triple β–spiral domain forming the tail and an eight-stranded-sandwich domain forming the head. c | Cleaved influenza haemagglutinin trimer 26 . Reproduced with permission from Ref. 38 © (2000) Annual Reviews. d | Model of the respiratory syncitial virus F protein structure (RSV-F), which is based on amino-acid sequence homology with the structure of the Newcastle disease virus F protein 27 . Reproduced with permission from Ref. 110 © (2003) Elsevier Science.

The topologies of viral entry proteins vary from those that form spikes and which comprise a stem and a globular head (Fig. 3) — for example reoviruses, adenoviruses, orthomyxoviruses and paramyxoviruses — to those that form relatively 'flat' structures, for example, the alphaviruses (family togaviridae) (Fig. 2c). The similarity between the topologies of the non-enveloped reovirus and adenovirus attachment proteins extends to their three-dimensional structures even though the amino-acid sequences of these two proteins share no sequence similarity 25 (Fig. 3a,b). The similarity between the 3D structures, together with the conservation of function, indicates a common ancestor for these proteins, but does not preclude convergent evolution. The overall similarities between the topologies of the non-enveloped reovirus and adenovirus attachment proteins, and between the topologies of the ENVELOPE GLYCOPROTEINS (Envs) from enveloped viruses, such as influenza HA 26 and the respiratory syncitial virus (RSV) fusion protein (which has been predicted based on sequence similarity with the homologous fusion protein of the Newcastle disease virus (NDV) 27 ) is notable, particularly owing to the lack of amino-acid sequence identity between these proteins (Fig. 3). However, the stems of the reovirus and adenovirus attachment proteins are stabilized by triple β-spirals, whereas the stems of influenza HA and respiratory syncitial virus F protein are stabilised by α-helical coiled-coils, which indicates that these viruses have different ancestors. The structures of the enveloped flavivirus E proteins from TBE 28 and dengue virus 29 and the enveloped alphavirus SFV E1 protein 30 are similar to each other, which, in combination with similar functions, might indicate the existence of a common ancestor for the entry proteins of flaviviruses and alphaviruses 30 . Virus entry proteins have been divided into two classes that are dependent on several criteria, including mechanism of action, whether the entry protein is cleaved and whether the entry protein is complexed with other viral proteins. Envs that contain coiled-coils, such as influenza HA, paramyxovirus fusion protein F and HIV glycoprotein 160 (gp160), have been designated class I FUSION PROTEINS and the Envs of alphaviruses and flaviviruses have been designated class II fusion proteins 30,31 .

Viral entry proteins have diverse amino-acid sequences, but many of those for which the structure has been solved contain similar 3D structural motifs — although the overall topology can be different. This is especially notable for non-enveloped viruses the viral proteins that are exposed to the environment and recognize receptor molecules from several plant, insect and mammalian viruses contain the same basic core structure, which consists of an 8-stranded β-barrel with a jelly-roll motif 18 . Although only a few Env structures have been solved, it seems that they might use similar types of structural motifs as non-enveloped viruses for the recognition of host cells. The globular head of the best-characterized viral Env that mediates entry — the HA of the orthomyxovirus influenza — contains a β-barrel-type structure 26 (Fig. 3). The head of the NDV F protein, comprises an immunoglobulin-type β-sandwich domain and a highly twisted β-sheet domain 32 . Fragments of two other Envs — the HSV glycoprotein D 33 and the receptor-binding domain of Friend murine leukaemia virus (Fr-MLV) 34 — also contain β-barrel structures with similarity to the immunoglobulin-like fold. Analysis of this limited number of structures indicates that viruses use conserved frameworks of β-sheets joined by variable loops that can allow rapid adaptation to new receptors. The other three X-ray crystal structures of Envs with receptor-binding domains in an unbound state are soluble fragments of the main Envs of the flaviviruses TBE 28 and dengue virus 29 , and a fragment of E1 from SFV 30 , which contain predominantly β-strands. Most of these β-strands are packed in sheets, including a six-stranded β-barrel in the second domain, and an immunoglobulin-like β-barrel fold in the third domain that could be important for binding to cellular receptors.

Recognition of virus receptors on host cells. Although VIRUS RECEPTORS have diverse sequences, structures and cellular functions 35,36 , there is a preference for molecules that are involved in cell adhesion and recognition by reversible, multivalent AVIDITY-determined interactions. Viruses might have evolved to bind to abundant cellular receptors, or to bind to cellular receptors that have relatively low affinity for their natural ligands with high affinity 37 or to bind to receptors that have both of these characteristics (Table 2).

The receptor-recognition interactions of different viruses — and even different isolates of the same virus — can vary significantly. Typically, low-affinity (μM–mM) binding interactions between viral attachment proteins and their cognate receptors involve a small area of interaction between the viral and cellular receptors and do not lead to conformational changes in the entry proteins. For example, binding of influenza HA and SV40 to sialic acid 38 . High-affinity (nM–pM) interactions with virus receptors involve a large area of interaction (about 10 nm 2 ) between the viral and cellular receptors, and often involve large conformational changes. For example, the binding of HIV-1 gp120 to CD4 and one of the CO-RECEPTORS CCR5 or CXCR4, and poliovirus to CD155, both result in conformational changes 18,39 . One exception is the high-affinity binding of the adenovirus fibre to the coxsackievirus-adenovirus receptor (CAR), which does not involve significant conformational changes (Fig. 4).

a | A molecular-surface representation of the interface between the adenovirus 12 (Ad12) knob and the coxsackievirus-adenovirus receptor D1 (CAR D1). The figure shows two adjacent Ad12 knob monomers, viewed at the interface between the Ad12 and CAR D1 molecules and coloured on a scale from yellow (contact with CAR D1) to red (no contact with CAR D1). Atoms in contact with CAR D1 are shared between the two Ad12 monomers. From Ref. 111 © (1999) American Association of Sciences. b | The HIV-1 gp120 and CD4 receptor contact surface. The gp120 surface is shown in red, with the surface that is 3.5 Å distant from the CD4 receptor (surface-to-atom-centre distance) shown in yellow. From Ref. 39 © (1998) Nature, Macmillan Magazines. c | Interaction of rhinovirus and poliovirus with their receptors. Comparison of poliovirus binding to its receptor CD155 and rhinovirus binding to its receptor ICAM-1. The figure shows the electron density of a poliovirus-bound CD155 molecule (green), the poliovirus molecular surface (yellow), including the canyon, and a rhinovirus-bound IC1 molecule (red). Structures of poliovirus and rhinovirus were superimposed to generate the figure. Reproduced with permission from Ref. 112 © (2000) Oxford University Press. d | The conformational change of soluble glycoprotein D (gD285) fragment. The left panel shows the gD285 fragment, in complex with herpesvirus entry mediator A (HveA). The amino-terminal portion of the HveA binding hairpin (red) can be seen interacting with residues 224–240 of an α-helix of gD285 (white). The right panel shows unliganded gD285. From Ref. 33 © (2003) American Society for Microbiology.

The high-affinity receptor-binding site can be located in a deep crevice (or canyon) on the viral protein, such as in the picornaviruses, or can contain loops, cavities and channels, such as the adenovirus knob and HIV gp120 (Fig. 4). Rather unusually, the HveA receptor-binding site on HSV-1 glycoprotein D is situated on an amino-terminal extension at one edge of the glycoprotein D molecule rather than being assembled from many parts of the glycoprotein D sequence, as for a typical binding surface or binding pocket. It undergoes conformational changes on binding to the receptor 33 . There is no correlation between the structure of the viral entry protein structure and the structure of the cellular receptor. Viruses from the same family, such as retroviruses, can bind to different cellular receptors, and the same cellular molecule, for example, sialic acid, can serve as a receptor for several different viruses.

The conformational changes that are induced by interactions with one receptor can be required to expose the binding site for another receptor, for example the interaction between CD4 and gp120 induces the exposure of a high-affinity binding site for a co-receptor (typically CCR5 or CXCR4) on HIV-1 gp120. In this case, CD4 serves as an 'attachment' receptor that ensures specific binding to CD4-expressing cells and the co-receptor serves as a 'fusion' receptor that induces conformational changes that lead to exposure of fusogenic sequences. In some strains of HIV-1, co-receptors can mediate both attachment and fusion in the absence of CD4. Entry can also be initiated by binding to a low-affinity receptor, such as heparin sulphate, followed by higher affinity interactions. The role of many receptors in entry remains unresolved or controversial. Entry into cells through interactions with more than one receptor seems to be widely used by viruses, especially for the infection of specific types of cells in vivo. In the case that a specific cell receptor is absent, ALTERNATIVE VIRUS RECEPTORS have also been identified for some viruses. For example, galactosyl ceramide 40 and its sulphated derivative (sulphatide) can support low level HIV-1 infections in some CD4-negative cell lines, although the roles of such alternative receptors in vivo remain unknown.

Although the number of identified receptors for human viruses has increased rapidly during the past two decades 41,42,43,44,45,46 , most virus receptors remain uncharacterized, and there are only a few X-ray crystal or cryoelectron microscopy structures of entry proteins in complex with receptors (Fig. 4). Identification of new receptors is important for understanding virus tropism, pathogenicity and the mechanisms of entry. Recently, the receptor for the severe acute respiratory syndrome coronavirus (SARS-CoV) — the angiotensin-converting enzyme 2 (ACE2) — was identified, only months after the virus was discovered 47 , and the receptor-binding domain has been localized to amino-acid residues 303–537 of the SARS-CoV entry protein 48 .

Virus-receptor function is affected by membrane organization. Lipid rafts have been intensively studied to determine any possible role in virus entry 5 . Although rafts are well characterized, their role in virus entry is controversial. Studies on HIV-1 entry illustrate the controversies that still exist with regard to the role of rafts in virus entry. Together, depletion of cholesterol and inhibition of glycosphingolipid synthesis decrease the efficiency of HIV-1 Env-mediated membrane fusion — typically by about two-fold — which could indicate effects on membrane fusion owing to the disruption of raft integrity 5 . The raft component glycosphingolipids Gb3 and GM3 seem to interact with gp120 in the presence of CD4, which could also indicate that rafts are involved in HIV-1 entry 49 . However, recent data indicate that HIV-1 infection does not depend on the presence of CD4 and CCR5 in rafts and it has been proposed that cholesterol modulates HIV-1 entry by an independent mechanism, perhaps related to membrane merging or modulation of co-receptor binding 50 . Although the exact role of rafts in receptor-expressing host cells is controversial, recent experiments have clearly shown the importance of rafts for membrane fusion by clustering sufficient numbers of influenza HA molecules in rafts 200–280 nm in diameter 6 . So, it appears that in both receptor-expressing and Env-expressing cells the function of rafts is mainly to increase the local concentrations of molecules that are involved in entry. Glycosphingolipids might not only provide the structural basis for raft formation, but could also interact directly with viral entry proteins and cellular receptor molecules.

Conformational changes of entry proteins

After receptor recognition, viral entry proteins undergo marked conformational changes that drive the entry process to completion. One hypothesis is that entry proteins from many viruses, including poliovirus, influenza, HIV, TBE and SFV, are in a metastable high-energy state 17,18 . However, recent differential scanning-calorimetric measurements showed that the unfolding of influenza HA at neutral pH is an endothermic process, which might indicate that it is not in a metastable high-energy state 51,52 . According to the metastable-state hypothesis, receptor binding or a pH change (or possibly both events 53,54 , but this possibility is debated 55 ) can provide the activation energy that is required to overcome energy barriers for the viral entry protein to reach a stable, energetically favourable state. The energy of the transition to this state is used to externalize sequences from internal parts of entry proteins that can destabilize membranes. The structural transition of the virus entry protein and subsequent action of the externalized membrane-destabilizing sequence induces the formation of membrane pores and membrane fusion pores.

Enveloped viruses: class I, class II and unclassified fusion proteins. The prototype class I fusion protein is influenza HA. Perhaps the best-characterized fusion intermediates are the helical coiled-coils that are formed by fragments from fusion proteins of representative orthomyxo-, paramyxo-, retro-, filo-, and coronaviruses 38,56,57 . These structures are thought to represent the lowest energy state, which is reached after a series of conformational changes that are induced by receptor binding or low pH. In addition to the formation of coiled-coils during fusion, the Envs from many of these viruses mature by proteolytic cleavage of precursor proteins to yield membrane-anchored subunits, which contain N-terminal fusion peptides, but some coronaviruses, including SARS-CoV, are not cleaved and remain trimeric throughout the fusion process 30,31 . A similar, but four-stranded, coiled-coil is involved in a number of intracellular fusion processes, which include synaptic vesicle fusion 20 . Although the formation of the coiled-coils is irreversible, it does involve some reversible steps — for example, in the case of influenza HA 58 . The molecular details of the pathway that leads to the formation of these structures are not fully understood, but include several intermediates that can be distinguished by kinetic measurements and structural analysis 56,59 (Fig. 5). Molecular dynamics simulations of the conformational transitions of the influenza HA that are induced by a change from a neutral to a low pH might indicate that a complete dissociation of the globular domains of the HA proteins could expose the fusion peptides and reorientate the peptides towards the target membrane, which is consistent with a spring-loaded conformational-change hypothesis 60 .

a | Schematic representation of a working model for viral membrane fusion mediated by class I fusion proteins. Influenza virus, which is internalized into an endosome, is shown as an example. In the native state of the fusion protein — which is a trimer — most of the surface subunit (green) is exposed. Part of the transmembrane subunit, including the fusion peptide, is not exposed. Following fusion-activating conditions, conformational changes occur to 'free' the fusion peptide (red) from its previously unexposed location. In the case of influenza HA, this occurs by a 'spring-loaded' mechanism. The 'pre-hairpin' intermediate spans two membranes — with the transmembrane domain positioned in the viral membrane and the fusion peptide inserted into the host-cell membrane. The pre-hairpin intermediate forms a trimer of hairpins, and membrane fusion occurs, which leads to pore formation and release of the viral genome into the cytoplasm. Modified with permission from Ref. 56 © (2001) Annual Reviews. b | Conformational changes of class II viral fusion proteins and entry. Structure of fragments of the class II fusion (E) glycoprotein from dengue virus 29 . The polypeptide chain begins in the central domain (domain I, red), which is an eight-stranded β-barrel with up-and-down topology. Two long insertions between strands in the central domain form the dimerization domain (domain II, yellow). The carboxy-terminal domain (domain III, blue) is an antiparallel β-barrel with an immunoglobulin-like topology, which is stabilized by three disulphide bridges. The domain definition is also highlighted on the peptide sequence (top). During entry, the oligomeric structure is reorganized. The configuration of dengue virus glycoproteins on the virion surface at neutral pH and the proposed configuration at low pH are shown. The E glycoproteins are shown as yellow cylinders and the fusion peptide is green. Reproduced from Refs 24,29 © (2002) Elsevier (2003) National Academies of Sciences.

Class II fusion proteins are not proteolytically cleaved and have internal, rather than N-terminal fusion peptides 30,31 . They are synthesized as a complex with a second membrane glycoprotein, and the activation of the fusogenic potential of the class II fusion proteins involves the cleavage of this accessory protein. X-ray crystallography of three class II fusion proteins — the E proteins of TBE and dengue virus, and the E1 protein of SFV — has revealed a common fold for these proteins, which is structurally unrelated to the class I viral fusion proteins. Class II fusion proteins fold as heterodimers with the companion (chaperone) glycoprotein — for example, pE2 with pE1 in alphaviruses and prM with pE in flaviviruses — and form a protein network at the viral surface. The fusogenic conformational changes lead to a reversible dissociation of the dimers to release monomers, followed by irreversible reassociation into stable homotrimers in the presence of membranes (Fig. 5). Although the alphaviruses SFV and SIN require the membrane components cholesterol and sphingolipids for the binding and initiation of conformational changes, the flavivirus TBE does not require these membrane components — but binding to target membranes and trimerization of the TBE E protein might involve interactions with cholesterol 61 .

Unlike class I and class II fusion proteins, the conformational changes of the G proteins of rabies virus and the vesicular stomatitis virus (VSV) (family rhabdoviridae) that are induced by a low pH are reversible, which indicates that the low pH does not trigger a transition through a high-energy intermediate state 62,63 . However, interactions with membranes could induce irreversible conformational changes. VSV and rabies virus can both fuse with pure lipid vesicles, although neither virus requires any specific lipids for fusion. The transition between equilibrium states of the entry proteins could provide free energy to overcome the membrane fusion barrier, but formation of the fusion site might require many more trimers acting cooperatively than the 5–6 that have typically been estimated to be involved 64 . The G proteins of rhabdoviruses have several characteristics in common with class I fusion proteins (for example, they have an internal fusion peptide and are not complexed with other proteins at the virion surface) and class II fusion proteins (for example, they are not cleaved and do not have heptad sequences that are predictive of coiled-coils). The X-ray crystal structure of the rhabdovirus G protein has not been solved, and it remains to be seen whether it will become the founding member of a new class III fusion protein family.

Enveloped viruses: membrane fusion. An important role of the conformational changes that all classes of fusion proteins undergo is to overcome energy barriers to enable membrane destabilization and the formation of fusion pores. The energy barrier for the fusion of VSV with a cell is estimated to be about 42 kcal mol −1 (Ref. 65). For protein-free model lipid bilayers, the activation energies are comparable — estimated values of 37, 27 and 22 kcal mol −1 were reported for the formation of a reversible first intermediate, its conversion to a second, semi-stable intermediate and irreversible fusion-pore formation, respectively 66 , which indicates that the basic molecular mechanisms of viral fusion might involve similar lipid molecular rearrangements to those observed in the fusion of model lipid membranes.

How the energy that is released by conformational changes of the viral protein is used to overcome the membrane fusion energy barriers is unclear. For class I fusion proteins it was recently found that the formation of six-helix bundles stabilizes fusion-pore formation 67 . A spring-loaded conformational change 68 (Fig. 5) is required, but might not be sufficient, for HA-mediated fusion, and the transition to the membrane fusion pore could depend, in part, on the subsequent action of the HA fusion peptide and transmembrane domain 69 . For class II fusion proteins it was proposed that the pH-triggered conformational changes result in insertion of class II protein β-barrels into the host membrane, which, in turn, leads to fusion-pore formation 24 . It seems that once the viral protein conformational changes have provided sufficient energy to enable close membrane apposition (less than 1 nm) and destabilization, formation of the fusion pore can proceed through intermediates, stalks and hemifusion diaphragms, which might be common to all known viral membrane fusion processes 70,71 . Data obtained for baculovirus gp64 are consistent with this hypothesis 72 .

A number of models have been suggested for fusion-pore formation by lipid–protein complexes 52,70 . To ensure the transfer of the viral nucleoprotein complex, which is typically stable in solution, it seems that the internal lining of the fusion pore must be hydrophilic. Fusion-pore formation requires a minimal number (5–6) of oligomeric viral entry proteins to form a supramolecular lipid–protein complex. It is a dynamic process and small pores can open and close reversibly. Models of fusion-pore formation are mainly based on fusion mediated by influenza HA, but might prove generally valid. The enlargement of the fusion pore that allows transfer of the genome into the cell occurs by an unknown mechanism.

Non-enveloped viruses: membrane penetration. The mechanism of penetration of non-enveloped viruses remains poorly understood, although they are relatively small and simple in structure 18 . Fusion of the more complex enveloped viruses is better understood owing to the similarities between enveloped viruses and cells — both are surrounded by membranes and their fusion does not necessarily require a significant reorganization of the viral nucleoprotein complex. By contrast, either the whole non-enveloped virus must cross the membrane, or it must undergo important conformational changes and transfer the genome through the membrane. In the latter case, the energy of the metastable state must be used not only to destabilize the cell membrane, but also to reorganize the nucleoprotein complex so that the genome can be released through the destabilized membrane either through pores or other structures. Although the structures of several key intermediates in the conformational changes of viral entry proteins have now been solved to atomic resolution, it is clear that there is a great deal left to learn owing to the difficulties that are associated with studying the rapid conformational changes of only a few molecules of each virion against a background of large numbers of surface molecules on the virion that undergo no structural changes.

Entry proteins and pathogenesis

Viral proteins can destabilize not only plasma and endosomal membranes during entry, but can also destabilize other membranes inside cells to which the protein is in close proximity if there is a suitable trigger, such as a receptor, low pH or low calcium concentration. For example, such conditions arise during transportation of viral proteins after synthesis in the host cell in acidic Golgi vesicles. It seems that viruses have developed strategies to cope with this problem. For example, the G protein of rhabdoviruses, such as rabies and VSV, can exist in native, activated and inactive conformations. It was proposed that the inactive state helps to avoid membrane fusion during the transport of the G protein 73 . However, the HIV-1 Env can induce cell lysis after interaction with receptors — probably through disruption of important intracellular membranes — so methods to cope with destabilization of cell functions are not universal 74 . The significance of membrane fusion effects in human pathogenesis is unclear. Perhaps viruses that are well adapted to their hosts do not induce significant pathogenic effects that could lead to a reduction of virus production. However, viruses that infect a new host, such as HIV and SARS, might not be well adapted to this host and their entry proteins could cause CYTOPATHIC EFFECTS. Cell fusion — perhaps mediated by Envs — by endogenous retroviruses could contribute to cancer 75 .

Entry inhibitors, antibodies and vaccines

Entry is an attractive target for inhibition because the entry machinery is extracellular and it is therefore easier for drug molecules to reach than intracellular targets. Any step(s) of the entry process can be targeted by an entry inhibitor. Various types of molecules, such as proteins, peptides, carbohydrates, small organic molecules, nucleic acids and supramolecular structures, including liposomes and phage, have been found to inhibit entry. Yet, out of more than 30 antiviral drugs 76 , there are only two entry inhibitors — Synagis 77 and T-20 (Ref. 78) — that have been approved by the US Food and Drug Administration (FDA) for clinical use (excluding human immune globulin for use against hepatitis A and measles, and virus-specific polyclonal human immune globulins for use against cytomegalovirus, hepatitis B, rabies, RSV, vaccinia and varicella-zoster 79 ). Only T-20 (which is marketed as enfuvirtide) is used for the treatment of ongoing viral (HIV-1) infection. The humanized monoclonal antibody Synagis (which is also known as palivizumab) is used for the prevention of RSV infections in neonates and immunocompromised individuals. T-20 is not a small molecule — regarded as the 'gold standard' for a drug — but is a peptide that cannot be taken orally. A small organic molecule entry inhibitor (pleconaril) showed promising results for the treatment of infections caused by the picornaviruses that cause the common cold 80 , but was not approved by the FDA owing to concerns about potential interactions with other drugs — although different formulations are presently being evaluated for use in life-threatening disease. At present, a number of compounds are in clinical trials, including small organic molecules that bind to the HIV-1 co-receptor CCR5, and entry inhibitors are also being tested for efficacy as microbicides.

Challenges in the development of virus entry inhibitors. Major problems in the development of effective entry inhibitors include the generation of virus mutants that are resistant to such drugs, a low potency in vivo and toxic side effects. Although these problems are common to the development of other antiviral (and cancer) drugs and limit their efficacy, the development of entry inhibitors might also face specific challenges. Viruses have evolved to recognize cellular receptors and enter cells despite the presence of the host immune system. Antibodies that exhibit inhibitory effects on virus entry must bind viruses that have already developed a number of strategies for immune evasion, including the use of immunodominant variable loops and incomplete complementary binding surfaces, oligomeric occlusion, glycosylation 81 , conformational masking 82 and multivalent interactions. Some problems that are inherent to the design of any inhibitor are especially challenging for those targeted to virus entry, such as the inhibition of high-affinity protein–protein interactions by small molecules. Another strategy used by viruses to counteract the action of antibodies and other entry inhibitors is the direct cell-to-cell transmission of viruses, which avoids or reduces the exposure of the virus to inhibitors that are not designed to penetrate through membranes or other barriers. This could be one important reason for the low efficacy that is observed in vivo of otherwise potent in vitro inhibitors. Low inhibitor potency combined with a high virus mutation rate is perhaps the most challenging problem in the development of entry inhibitors.

Although the rapid progress in our understanding of the structural mechanisms of virus entry promises new approaches that could 'outsmart' the virus, no entry inhibitors or treatment protocols in clinical use have so far been developed on the basis of predictions made by structural models, and the main source of new inhibitors is still from screening large libraries of small organic molecules, natural products, peptides and antibodies. However, structures of entry proteins have been invaluable for the development of our understanding of the mechanisms of inhibition and should allow further improvement of the inhibitors. It seems likely that sooner or later structure-based design will yield entry inhibitors that will be in clinical use.

Conserved entry intermediates as targets for inhibitors multivalent inhibitors and other approaches. One direction of research that could hasten the arrival of entry inhibitors to the clinics is the development of compounds that interact with conserved intermediates of the entry process or with the protein structures that, on binding to receptors, trigger the conformational changes that lead to the formation of these entry intermediates. Typically, such intermediates are only transiently exposed, so viruses might not have evolved strategies to avoid inhibitors targeted to these structures. In addition, conserved intermediates are usually important for virus entry and presumably cannot easily be substituted by other structures after mutation.

An example of the successful design of an entry inhibitor that shows proof of the concept is the 5-Helix protein, which interferes with a conserved intermediate in the entry of HIV-1 (Ref. 83). A related example is a class of peptides that could have broad applications to several viruses containing class I fusion proteins. The peptides are derived from regions of fusion proteins (heptad repeats) that have a propensity to form coiled-coils and which serve as fusion intermediates and enable oligomerization of proteins. T-20 (also known as DP178) is derived from the carboxy-terminal heptad region of the HIV-1 gp41 and showed potent inhibitory activity in vivo 78 . Similar peptides from other viruses, including HTLV-1, RSV, measles virus, Nipah virus, Hendra virus, Ebola virus and SARS-CoV, are also promising entry inhibitor candidates. However, Ebola virus is inhibited only at very high concentrations, influenza HA-mediated fusion is not inhibited and SARS-CoV infection might not be inhibited either (K. Bossart and C. Broder, personal communication), perhaps owing to the endocytic entry route of these viruses. Although peptides have certain promising features, including a relatively small size that might ensure good penetration combined with high binding affinity, the lack of oral formulations, short half-life, possible toxicity and immunogenicity might limit their application.

Recently, a small molecule, BMS-378806, was identified that inhibits the entry of a broad range of HIV-1 isolates by a mechanism which was attributed to competition with the CD4 receptor for binding to gp120 (Ref. 84). However, this compound inhibited the entry of one isolate of HIV-1 (HIV-1JRFL) with an IC50 of 1.5 nM, and the binding of CD4 to gp120 from the same isolate with an IC50 of 100 nM. So, at inhibitory concentrations, BMS-378806 does not interfere with CD4 binding, indicating a different inhibitory mechanism. By analogy with inhibitors of picornavirus entry, such as pleconaril, BMS-378806 might inhibit the entry of HIV-1 by binding to conserved structures that are important for the conformational changes which gp120 must undergo for viral entry 80 . Potent virus-specific inhibitors of the viral-membrane-merging step have not been identified yet.

Another promising direction is the development of multivalent inhibitors that can overcome problems caused by mutation of viral proteins to escape inhibition because multivalent inhibitors bind to several regions of the same (or different) protein(s) on the viral surface. One example of a multivalent inhibitor is the multimeric soluble receptors of influenza and HIV-1 (Ref. 82), which are potent inhibitors of entry in vitro and, to some extent, in vivo. The use of entry inhibitors in combination or as fusion proteins could also result in increased efficiency. Finally, improvement of current methods for structure-based design by accounting for protein flexibility and dynamics in binding to ligands 85 , and screening methods for inhibitors 86,87 would certainly expand the range of possible inhibitors that can be tested.

Neutralizing antibodies and vaccine immunogen design. Neutralizing antibodies usually inhibit virus entry by preventing attachment of the virus to the cell or by binding to entry intermediates 88,89,90 . Human immunoglobulin composed of concentrated antibodies collected from pooled human plasma has been successfully used as a preventative treatment for virus infections, including rabies, hepatitis A and B, measles, mumps, varicella, cytomegalovirus and arenaviruses. Antibodies can completely prevent infection, but once infection is established they are a much less efficient treatment. The only monoclonal antibody in clinical use today to treat a viral disease — Synagis (MEDI-493) — is more potent than the polyclonal immunoglobulin that is presently in use, and is broadly active against numerous RSV type A and B clinical isolates 91 . It binds to the F protein of RSV with high affinity (3 nM) and inhibits virus entry and cell fusion in vitro with an IC50 of approximately 0.1 μg ml −1 . It seems that the efficacy of Synagis in vivo is correlated with the high affinity of binding and potency of this antibody in vitro 94 . However, Synagis had no measurable clinical efficacy after administration as a single 15 mg kg −1 intravenous dose to infants that were hospitalized with established RSV infection, although it did significantly reduce RSV concentrations in tracheal aspirates 92 .

The X-ray crystal structures of rhinovirus 21 and poliovirus 22 indicate a possible mechanism by which picornaviruses can avoid neutralization by antibodies through the mutation of non-conserved amino acid residues surrounding the receptor-binding site — a 2 nm deep and 2 nm wide canyon (Fig. 4c). It was initially hypothesized that the conserved amino acid residues of the canyon are not accessible by antibodies however, it was later shown that a strongly neutralizing antibody, Fab17, can penetrate deep within the receptor-binding canyon by undergoing a large conformational change without inducing conformational changes in the virus 90,93 . Unusually, not only the hypervariable residues but also residues from the framework region of Fab17 contact the canyon. Yet another remarkable mechanism of immune recognition of viruses is the recently discovered receptor mimicry by post-translational modification (tyrosine sulphation) of antibodies (Ref. 94 Huang, C. et al., manuscript in preparation). It seems that any accessible viral surface can be recognized by antibodies. Rapidly mutating viruses can escape neutralizing antibodies even if they bind to structures that are essential for virus replication, such as receptor-binding sites, unless they bind with energetically identical profiles 95 . Whether a virus will escape neutralization by antibodies depends on the interplay between the antibody affinity (avidity) and kinetics of binding, generation rate, concentration and the viral mutation rate and fitness. Mutations of immunodominant structural loops that form antibody-binding sites and mutations leading to changes in oligosaccharide attachment to viral entry proteins are common mechanisms by which viruses avoid neutralization 90,96 .

Mutations of conserved residues that have a role in the entry mechanism typically result in reduction or loss of infectivity. Antibodies or their derivatives that bind to epitopes where residues contribute most of the binding energy could have potential as entry inhibitors. Epitopes that are exposed after virus binding to receptors are typically well conserved — for example, the 17b 39 and X5 (Ref. 97) epitopes on HIV-1 gp120. One potential problem with using antibodies as entry inhibitors in this case could be limited access to the post-receptor-binding state of the viral entry protein due to the relatively large size of the whole antibody. Solving this, and other problems, could lead to the development of potent broadly neutralizing antibodies which could limit the generation of resistant viruses, especially if these inhibitors are used in combination with other antibodies or inhibitory molecules.

Many viruses, especially RNA viruses such as HIV-1, exist as swarms of virions inside an infected individual, and might significantly differ in sequence between isolates. So, elicitation of potent, broadly neutralizing antibodies is an important goal for vaccine development. Potent, broadly neutralizing antibodies for HIV-1 Env do exist — for example, b12, 2G12, 447-52D, X5, 2F5 and 4E10/Z13. However, elicitation of these antibodies in vivo has not been successful. Identification of broadly neutralizing antibodies and the characterization of their epitopes could help to design vaccine immunogens that would be able to elicit these neutralizing antibodies in vivo — so-called retrovaccinology 89 . At present, all vaccines that elicit antibodies against entry proteins have been developed empirically using an antigen, rather than by designing an immunogen on the basis of the antibodies produced.

The important advantages of human antibodies as therapeutics are low or negligible toxicity combined with high potency and a long half-life. However, drawbacks include the generation of neutralization-resistant virus mutants, limited access of the large antibody molecules to the site of virus replication, lack of oral formulations and the high cost of production and storage.

Virus entry physiology and retargeting

Viruses are usually associated with disease. However, some viruses can be beneficial. Human endogenous retrovirus W (HERV-W) might be essential in human physiology — for the formation of the placenta. The HERV-W Env, known as syncytin, is fusogenic and has a role in human trophoblast cell fusion and differentiation 98 . Retroviral particles have been observed in the placenta, along with fused placental cells, which are morphologically reminiscent of virally induced syncytia. These studies led to the proposal that an ancient retroviral infection might have been a pivotal event in mammalian evolution 99 .

Viruses have long been used to transfer genes into cells. During the last decade, another important application has been the viral delivery of genes and drugs to treat cancer. A major challenge has been to develop virus entry proteins to deliver molecules to specific cells with high efficiency. To achieve this goal it is often desirable to engineer viruses that do not infect cells expressing the native receptor, but instead target a cell of choice. Engineering of entry proteins in this way is known as transductional retargeting 100 .

A conceptually simple approach to transductional retargeting is to incorporate the protein that determines cell tropism into the infecting virion of choice — known as virus 'pseudotyping'. This has been used in both retroviruses and adenoviruses, and does not require prior knowledge of specific virus–receptor interactions. In a related approach, viral entry proteins are used to produce drug and gene delivery vehicles, for example, the F protein of Sendai virus has been incorporated into liposomes to form virosomes 101 and the L protein of hepatitis B has been incorporated into yeast-derived lipid vesicles 102 . Retargeting of retroviruses, adenoviruses and AAVs has been achieved by conjugation of entry proteins with molecular adaptors, such as bi-specific antibodies that have particular receptor-binding properties. Modification of the entry proteins so that the normal receptor-binding property is abolished, or a ligand for alternative receptor binding is incorporated has also been successful at redirecting adenovirus tropism in cell culture, but is unlikely to work for the entry of viruses that require receptor-induced conformational changes, such as retroviruses, unless detailed molecular mechanisms of those conformational changes are better understood. A related approach is based on screening libraries of chimaeric Envs from different strains of MLV 103 , or randomized peptides inserted at tolerant sites in viral proteins, such as VP3 of AAV 104 . This approach seems promising for the selection of specific retargeting vectors. Understanding the structure of AAV 105 and other viruses could help to further improve the specificity and efficiency of retargeting. Retargeting viruses with complex entry mechanisms that involve several proteins, such as those of herpes viruses and poxviruses, remains challenging.

Challenges and perspectives

Elucidation of the molecular mechanisms and the dynamics of the conformational changes driving virus entry remains a significant challenge. It requires the development of new approaches to study the rapid conformational changes of a small number of membrane-interacting protein molecules that are surrounded by many more non-interacting molecules. A more realistic goal is the determination of the structures of proteins that mediate the entry of all human viruses and the identification of the cognate cellular receptors. If research continues at the present pace, this goal could be accomplished within the next decade. Identification of all the cellular receptors for human viruses would be an important contribution to our understanding of virus tropism and pathogenesis. The various, and in many cases unexpected, ways that entry proteins can affect pathogenesis could offer new opportunities for intervention. Rational structure/mechanism-based design of entry inhibitors and vaccine immunogens that are capable of eliciting potent, broadly neutralizing antibodies of known epitopes is expected to contribute towards the development of clinically useful therapeutics and vaccines. The development of panels of human monoclonal antibodies against every entry-related protein of all pathogenic human viruses could accelerate our understanding of entry mechanisms and help to fight viral diseases. Recent progress in virus retargeting also raises hopes for the possibility of designing entry machines that can deliver genes and other molecules to any cell of choice.

Vibrations of coronavirus proteins may play a role in infection

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When someone struggles to open a lock with a key that doesn’t quite seem to work, sometimes jiggling the key a bit will help. Now, new research from MIT suggests that coronaviruses, including the one that causes Covid-19, may use a similar method to trick cells into letting the viruses inside. The findings could be useful for determining how dangerous different strains or mutations of coronaviruses may be, and might point to a new approach for developing treatments.

Studies of how spike proteins, which give coronaviruses their distinct crown-like appearance, interact with human cells typically involve biochemical mechanisms, but for this study the researchers took a different approach. Using atomistic simulations, they looked at the mechanical aspects of how the spike proteins move, change shape, and vibrate. The results indicate that these vibrational motions could account for a strategy that coronaviruses use, which can trick a locking mechanism on the cell’s surface into letting the virus through the cell wall so it can hijack the cell’s reproductive mechanisms.

The team found a strong direct relationship between the rate and intensity of the spikes’ vibrations and how readily the virus could penetrate the cell. They also found an opposite relationship with the fatality rate of a given coronavirus. Because this method is based on understanding the detailed molecular structure of these proteins, the researchers say it could be used to screen emerging coronaviruses or new mutations of Covid-19, to quickly assess their potential risk.

The findings, by MIT professor of civil and environmental engineering Markus Buehler and graduate student Yiwen Hu, are being published today in the journal Matter.

All the images we see of the SARS-CoV-2 virus are a bit misleading, according to Buehler. “The virus doesn’t look like that,” he says, because in reality all matter down at the nanometer scale of atoms, molecules, and viruses “is continuously moving and vibrating. They don’t really look like those images in a chemistry book or a website.”

Buehler’s lab specializes in atom-by-atom simulation of biological molecules and their behavior. As soon as Covid-19 appeared and information about the virus’ protein composition became available, Buehler and Hu, a doctoral student in mechanical engineering, swung into action to see if the mechanical properties of the proteins played a role in their interaction with the human body.

The tiny nanoscale vibrations and shape changes of these protein molecules are extremely difficult to observe experimentally, so atomistic simulations are useful in understanding what is taking place. The researchers applied this technique to look at a crucial step in infection, when a virus particle with its protein spikes attaches to a human cell receptor called the ACE2 receptor. Once these spikes bind with the receptor, that unlocks a channel that allows the virus to penetrate the cell.

That binding mechanism between the proteins and the receptors works something like a lock and key, and that’s why the vibrations matter, according to Buehler. “If it's static, it just either fits or it doesn't fit,” he says. But the protein spikes are not static “they’re vibrating and continuously changing their shape slightly, and that's important. Keys are static, they don't change shape, but what if you had a key that's continuously changing its shape — it's vibrating, it's moving, it's morphing slightly? They're going to fit differently depending on how they look at the moment when we put the key in the lock.”

The more the “key” can change, the researchers reason, the likelier it is to find a fit.

Buehler and Hu modeled the vibrational characteristics of these protein molecules and their interactions, using analytical tools such as “normal mode analysis.” This method is used to study the way vibrations develop and propagate, by modeling the atoms as point masses connected to each other by springs that represent the various forces acting between them.

They found that differences in vibrational characteristics correlate strongly with the different rates of infectivity and lethality of different kinds of coronaviruses, taken from a global database of confirmed case numbers and case fatality rates. The viruses studied included SARS-CoV, MERS-CoV, SATS-CoV-2, and of one known mutation of the SARS-CoV-2 virus that is becoming increasingly prevalent around the world. This makes this method a promising tool for predicting the potential risks from new coronaviruses that emerge, as they likely will, Buehler says.

In all the cases they have studied, Hu says, a crucial part of the process is fluctuations in an upward swing of one branch of the protein molecule, which helps make it accessible to bind to the receptor. “That movement is of significant functional importance,” she says. Another key indicator has to do with the ratio between two different vibrational motions in the molecule. “We find that these two factors show a direct relationship to the epidemiological data, the virus infectivity and also the virus lethality,” she says.

The correlations they found mean that when new viruses or new mutations of existing ones appear, “you could screen them from a purely mechanical side,” Hu says. “You can just look at the fluctuations of these spike proteins and find out how they may act on the epidemiological side, like how infectious and how serious would the disease be.”

Potentially, these findings could also provide a new avenue for research on possible treatments for Covid-19 and other coronavirus diseases, Buehler says, speculating that it might be possible to find a molecule that would bind to the spike proteins in a way that would stiffen them and limit their vibrations. Another approach might be to induce opposite vibrations to cancel out the natural ones in the spikes, similarly to the way noise-canceling headphones suppress unwanted sounds.

As biologists learn more about the various kinds of mutations taking place in coronaviruses, and identify which areas of the genomes are most subject to change, this methodology could also be used predictively, Buehler says. The most likely kinds of mutations to emerge could all be simulated, and those that have the most dangerous potential could be flagged so that the world could be alerted to watch for any signs of the actual emergence of those particular strains. Buehler adds, “The G614 mutation, for instance, that is currently dominating the Covid-19 spread around the world, is predicted to be slightly more infectious, according to our findings, and slightly less lethal.”

Mihri Ozkan, a professor of electrical and computer engineering at the University of California at Riverside, who was not connected to this research, says this analysis “points out the direct correlation between nanomechanical features and the lethality and infection rate of coronavirus. I believe his work leads the field forward significantly to find insights on the mechanics of diseases and infections.”

Ozkan adds that “If under the natural environmental conditions, overall flexibility and mobility ratios predicted in this work do happen, identifying an effective inhibitor that can lock the spike protein to prevent binding could be a holy grail of preventing SARS-CoV-2 infections, which we all need now desperately.”

The research was supported by the MIT-IBM Watson AI Lab, the Office of Naval Research, and the National Institutes of Health.

Influenza Virus Genome Sequencing and Genetic Characterization

Influenza viruses are constantly changing, in fact all influenza viruses undergo genetic changes over time (for more information, see How the Flu Virus Can Change: &ldquoDrift&rdquo and &ldquoShift&rdquo). An influenza virus&rsquo genome consists of all genes that make up the virus. CDC conducts year-round surveillance of circulating influenza viruses to monitor changes to the genome (or parts of the genome) of these viruses. This work is performed as part of routine U.S. influenza surveillance and as part of CDC&rsquos role as a World Health Organization (WHO) Collaborating Center for Reference and Research on Influenza. The information CDC collects from studying genetic changes (also known as &ldquosubstitutions,&rdquo &ldquovariants&rdquo or &ldquomutations&rdquo) in influenza viruses plays an important public health role by helping to determine whether vaccines and antiviral drugs will work against currently-circulating influenza viruses, as well as helping to determine the potential for influenza viruses in animals to infect humans.

Genome sequencing reveals the sequence of the nucleotides in a gene, like alphabet letters in words. Nucleotides are organic molecules that form the structural unit building block of nucleic acids, such as RNA or DNA. All influenza viruses consist of single-stranded RNA as opposed to dual-stranded DNA. The RNA genes of influenza viruses are made up of chains of nucleotides that are bonded together and coded by the letters A, C, G and U, which stand for adenine, cytosine, guanine, and uracil, respectively. Comparing the composition of nucleotides in one virus gene with the order of nucleotides in a different virus gene can reveal variations between the two viruses.

Genetic variations are important because they can affect the structure of an influenza virus&rsquo surface proteins. Proteins are made of sequences of amino acids.

The substitution of one amino acid for another can affect properties of a virus, such as how well a virus transmits between people, and how susceptible the virus is to antiviral drugs or current vaccines.

Genome sequencing reveals the sequence of the nucleotides in a gene, like alphabet letters in words. Comparing the composition of nucleotides in one virus gene with the order of nucleotides in a different virus gene can reveal variations between the two viruses.

Genetic variations are important because they affect the structure of an influenza virus&rsquo surface proteins. Proteins are made of sequences of amino acids.

The substitution of one amino acid for another can affect properties of a virus, such as how well a virus transmits between people, and how susceptible the virus is to antiviral drugs or current vaccines.

Influenza A and B viruses &ndash the primary influenza viruses that infect people &ndash are RNA viruses that have eight gene segments. These genes contain &lsquoinstructions&rsquo for making new viruses, and it&rsquos these instructions that an influenza virus uses once it infects a human cell to trick the cell into producing more influenza viruses, thereby spreading infection.

Influenza genes consist of a sequence of molecules called nucleotides that bond together in a chain-like shape. Nucleotides are designated by the letters A, C, G and U.

Genome sequencing is a process that determines the order, or sequence, of the nucleotides (i.e., A, C, G and U) in each of the genes present in the virus&rsquos genome. Full genome sequencing can reveal the approximately 13,500-letter sequence of all the genes of the virus&rsquo genome.

Each year CDC performs whole genome sequencing on about 7,000 influenza viruses from original clinical samples collected through virologic surveillance. An influenza A or B virus&rsquo genome contains eight gene segments that encode (i.e., determine the structure and features of) the virus&rsquo 12 proteins, including its two primary surface proteins: hemagglutinin (HA) and neuraminidase (NA). An influenza virus&rsquo surface proteins determine important properties of the virus, including how the virus responds to certain antiviral drugs, the virus&rsquo genetic similarity to current influenza vaccine viruses, and the potential for zoonotic (animal origin) influenza viruses to infect human hosts.

Genetic Characterization

CDC and other public health laboratories around the world have been sequencing the genes of influenza viruses since the 1980s. CDC contributes gene sequences to public databases, such as GenBank external icon and the Global Initiative on Sharing Avian Influenza Data (GISAID) external icon , for use by public health researchers. The resulting libraries of gene sequences allow CDC and other laboratories to compare the genes of currently circulating influenza viruses with the genes of older influenza viruses and viruses used in vaccines. This process of comparing genetic sequences is called genetic characterization. CDC uses genetic characterization for the following reasons:

  • To determine how closely &ldquorelated&rdquo or similar flu viruses are to one another genetically
  • To monitor how flu viruses are evolving
  • To identify genetic changes that affect the virus&rsquo properties. For example, to identify the specific changes that are associated with influenza viruses spreading more easily, causing more-severe disease, or developing resistance to antiviral drugs
  • To assess how well an influenza flu vaccine might protect against a particular influenza virus based on its genetic similarity to the virus
  • To monitor for genetic changes in influenza viruses circulating in animal populations that could enable them to infect humans.

The relative differences among a group of influenza viruses are shown by organizing them into a graphic called a &lsquophylogenetic tree.&rsquo Phylogenetic trees for influenza viruses are like family (genealogy) trees for people. These trees show how closely &lsquorelated&rsquo individual viruses are to one another. Viruses are grouped together based on whether their genes&rsquo nucleotides are identical or not. Phylogenetic trees of influenza viruses will usually display how similar the viruses&rsquo hemagglutinin (HA) or neuraminidase (NA) genes are to one another. Each sequence from a specific influenza virus has its own branch on the tree. The degree of genetic difference (number of nucleotide differences) between viruses is represented by the length of the horizontal lines (branches) in the phylogenetic tree. The further apart viruses are on the horizontal axis of a phylogenetic tree, the more genetically different the viruses are to one another.

Figure. A phylogenetic tree.

For example, after CDC sequences an influenza A(H3N2) virus collected through surveillance, the virus sequence is cataloged with other virus sequences that have a similar HA gene (H3), and a similar NA gene (N2). As part of this process, CDC compares the new virus sequence with the other virus sequences, and looks for differences among them. CDC then uses a phylogenetic tree to visually represent how genetically different the A(H3N2) viruses are from each other.

CDC performs genetic characterization of influenza viruses year round. This genetic data is used in conjunction with virus antigenic characterization data to help determine which vaccine viruses should be chosen for the upcoming Northern Hemisphere or Southern Hemisphere influenza vaccines. In the months leading up to the WHO vaccine consultation meetings in February and September, CDC collects influenza viruses through surveillance and compares the HA and NA gene sequences of current vaccine viruses against those of circulating flu viruses. This is one way to assess how closely related the circulating influenza viruses are to the viruses the seasonal flu vaccine was formulated to protect against. As viruses are collected and genetically characterized, differences can be revealed.

For example, sometimes over the course of a season, circulating viruses will change genetically, which causes them to become different from the corresponding vaccine virus. This is one indication that a different vaccine virus may need to be selected for the next flu season&rsquos vaccine, although other factors, including antigenic characterization findings, heavily influence vaccine decisions. The HA and NA surface proteins of influenza viruses are antigens, which means they are recognized by the immune system and are capable of triggering an immune response, including production of antibodies that can block infection. Antigenic characterization refers to the analysis of a virus&rsquos reaction with antibodies to help assess how it relates to another virus.

Methods of Flu Genome Sequencing

One influenza sample contains many influenza virus particles that were grown in a test tube and that often have small genetic differences in comparison to one another among the whole population of sibling viruses.

Traditionally, scientists have used a sequencing technique called &ldquothe Sanger reaction&rdquo to monitor influenza evolution as part of virologic surveillance. Sanger sequencing identifies the predominant genetic sequence among the many influenza viruses found in an isolate. This means small variations in the population of viruses present in a sample are not reflected in the final result. Scientists often use the Sanger method to conduct partial genome sequencing of influenza viruses, while newer technologies (see next paragraph) are better suited for whole genome sequencing.

Over the past five years, CDC has been using &ldquoNext Generation Sequencing (NGS)&rdquo methodologies, which have greatly expanded the amount of information and detail that sequencing analysis can provide. NGS uses advanced molecular detection (AMD) to identify gene sequences from each virus in a sample. Therefore, NGS reveals the genetic variations among many different influenza virus particles in a single sample, and these methods also reveal the entire coding region of the genomes. This level of detail can directly benefit public health decision-making in important ways, but data must be carefully interpreted by highly-trained experts in the context of other available information. See AMD Projects: Improving Influenza Vaccines for more information about how NGS and AMD are revolutionizing flu genome mapping at CDC.

The deadly spike protein, take two

About three weeks ago, antivaxxers started pointing to a study from the Salk Institute as yet more “proof” that the spike protein used in COVID-19 vaccines is toxic and deadly. For instance, behold Alex Berenson, the “pandemic’s wrongest man“, crowing about the study:

As smoking guns go, this study is high-caliber. @UCSanDiego and Chinese researchers showed that the #SARSCoV2 spike protein – the one the vaccines make you produce – can all by itself cause major damage to the walls of blood vessels.

&mdash Alex Berenson (@AlexBerenson) May 2, 2021

I was amused when I saw these Tweets to see Berenson use a term like “off-target effects” as if he actually knows what it means.

It turns out that this study on a preprint server has been published in Circulation Research . It also turns out—surprise! surprise!—to definitely not to be “smoking gun” evidence for Berenson’s claims. Unlike the case of many papers cherry picked by antivaxxers to support their claims, it’s not that the paper is horrible, either. It’s not. It’s pretty decent, actually, at least as a preliminary, primarily observational study. Even more amusing, in it the authors expressly describe how their work actually demonstrates why vaccines that use spike protein as the antigen are so effective, and the Salk Institute press release even includes a disclaimer that the spike proteins made in cells by SARS-CoV-2 “behave very differently than those safely encoded by vaccines”.

Let’s look at the paper itself. The first thing that those of you with access to the paper will notice is how short it is: Three pages, one figure. That’s because it’s not a full research paper, but rather a research letter. As a result, there’s no detailed Methods section, and the results are very briefly described (much too briefly, for my liking). To be honest, for some of the experiments, due to the brevity of the paper, I had a bit of a hard time making heads or tails of what, exactly, the investigators did. I’ll do my best trying to explain, however.

In brief, the researchers used a “pseudovirus” that was surrounded by a “crown” of spike protein, like the coronavirus, but did not contain actual virus, dubbed Pseu-Spike by the authors. What is a pseudovirus? A reasonable question. In brief, a pseudovirus is a construct that has the external proteins of the virus of interest. There are a variety of pseudoviruses now, as described in this article in The Scientist :

Among these, researchers turned to models of the pathogen such as pseudoviruses and chimeric viruses that can be studied safely in labs with lower biosafety level (BSL) clearance than required for studying the wildtype version, in an effort to expand the study of the novel coronavirus. Pseudoviruses don’t replicate, rendering them harmless, but by replacing their surface envelope proteins with those of SARS-CoV-2, researchers can glean insights into the ways the pathogen infects cells.

Pseudoviruses were first developed in the 1960s, after scientists began studying a vesicular stomatitis virus (VSV) isolated from cattle. In addition to replicating well in culture, they later learned that its surface protein, VSV-G, facilitates entry into all eukaryotic cells, making the virus a useful vector not only as a pseudovirus but as a ferry to deliver DNA into cells for therapeutic purposes. The first Ebola vaccine was developed using a VSV platform, and more recently, the virus has been engineered to seek out and destroy cancer cells.

HIV-based platforms, which came about in the 1980s, have since replaced VSV as the most common model for developing both pseudo- and chimeric viruses. Unlike VSV’s negative-strand RNA genome that must be transcribed once inside the cell, HIV’s positive-strand RNA genome can instantly begin translation, making pseudoviruses based on HIV faster to produce. HIV-based model viruses have now been used in many of the same applications as VSV, with scientists applying them to the study of diseases such as AIDS, SARS, MERS, and influenza.

Also, compared with natural virus, a pseudovirus can only infect cells in a single round, has broad host range, high titer, and is not easily inactivated by serum complement.

Unfortunately, it is not clear from the paper which of these platforms was used to produce the pseudovirus in the experiments or how that pseudovirus was developed and produced. This is the sort of information that a full-length research paper would describe in the Methods section and it’s important information for determining whether the pseudovirus used was likely to be a good model. In another issue with this paper, the authors also do not describe the “mock virus” that they used as a control or how it was constructed. As a result, I find it very difficult to interpret their results. In fairness, some of this confusion might be because I am not highly knowledgeable about this particular system and don’t have the background knowledge about methodology that the authors clearly assume that the reader possesses. On the other hand, in a paper this in a journal like Circulation Research , which is not a virology journal, and particularly given that this is a paper that was likely to make the news and be misused by antivaxxers after its release, explanatory details that allow scientists from other fields with knowledge of molecular biology (but who are not experts in this field) to understand what was done are critical. A Research Letter does not accomplish this.

My concerns aside, let’s look at the experiments. The authors took pseudovirus or mock virus and instilled it into the tracheas of Syrian hamsters, three animals per experimental group. Another aspect of this study caught my eye, namely the amount of virus used, 5 x 10 8 pfu. For those of you not knowing what “pfu” stands for, it stands for “plaque-forming units.” Basically it’s a measure of the number of viable virus particles, virus particles that can infect cells and cause a plaque on a confluent layer of cells. That’s half a billion particles, far, far more of a viral challenge than the amount of virus launching any “natural” infection by SARs-CoV-2.

Using what is a highly artificial system, the authors compared the levels of a whole slate of protein markers related to cell signaling and oxidative stress in the mock- and Pseu-Spike-treated hamsters, as well as the histology of the lungs. I won’t go into detail about all of the markers examined, but rather will step back to take a longer view because it is not important for a lay person to understand all the phosphorylation of this protein or ubiquitination of that protein measured. (It’s also easy to get lost in the weeds of a study like this.) As stated, the authors found signs of inflammation in the alveoli (air sacs) of the Pseu-Spike-treated lungs, including thickened walls and inflammatory cells. They measured the levels of various proteins they deemed relevant:

AMPK (AMP-activated protein kinase) phosphorylates ACE2 Ser-680, MDM2 (murine double minute 2) ubiquitinates ACE2 Lys-788, and crosstalk between AMPK and MDM2 determines the ACE2 level.4 In the damaged lungs, levels of pAMPK (phospho-AMPK), pACE2 (phospho-ACE2), and ACE2 decreased but those of MDM2 increased (Figure [B], i). Furthermore, complementary increased and decreased phosphorylation of eNOS (endothelial NO synthase) Thr-494 and Ser-1176 indicated impaired eNOS activity. These changes of pACE2, ACE2, MDM2 expression, and AMPK activity in endothelium were recapitulated by in vitro experiments using pulmonary arterial ECs infected with Pseu-Spike which was rescued by treatment with N-acetyl-L-cysteine, a reactive oxygen species inhibitor (Figure [B], ii).

Translation: Compared to mock virus, Pseu-Spike altered signaling due to the ACE2 receptor, which is not surprising given that it’s been known for a year now that spike protein latches onto the ACE2 receptor in order to get SARS-CoV-2 into the cell. As a result, there was a lower level of ACE2 in the hamster lung tissue treated with Pseu-Spike, although looking at the Western blots in Figure 1B I am not particularly impressed by the magnitude of the decrease in protein level.

Also observed in the Pseu-Spike-treated hamster lung was decreased activity of eNOS, an enzyme that generates nitric oxide, as well as damage to the mitochondria, the “power plants” of the cell. The authors also did the same experiments in cell culture alone using pulmonary vascular endothelial cells (the cells the line the inside of the arteries in the lung), reporting that they recapitulated their findings, although they used spike protein at a rather high concentration (4 μg/ml). They also tested whether similar changes occurred in vascular endothelial cells genetically engineered to make a more stable and less stable version of ACE2. They did, although this is only suggestive, not slam dunk evidence, that it is the spike protein-induced degradation of ACE2 that results in these intracellular changes. The authors also reported that in pulmonary arteries isolated from the hamsters vasodilation induced by a drug called nitroprusside was not affected by Pseu-Spike, but the vasodilation caused by acetylcholine was impaired. Nitroprusside works by breaking down in the presence of oxyhemoglobin to produce, among other things, nitric oxide, while acetylcholine binds to specific protein receptors on the cell surface.

To be honest, I’ve never been a fan of papers this short (e.g., some Nature or Science papers, which can be even shorter than this) because I can never quite figure out what the authors did this is one of the rare cases of a paper that to me screams out for an online Supplemental Data and Supplemental Figures section, and I say this as someone who generally detests the trend in scientific publications to dump all sorts of data into supplemental sections.

Let’s, for the sake of argument, take the results at face value and assume that this study shows what the authors say it shows, namely that spike protein damages endothelium, “manifested by impaired mitochondrial function and eNOS activity”. and can cause oxidative stress that destabilizes the ACE2 receptor, leading to lower levels of the receptor. The authors themselves note that by decreasing the level of ACE2, spike protein could actually decrease the infectivity of SARs-CoV-2, given that the coronavirus needs to bind to ACE2 to get into cells, while speculating that the dysfunction of endothelial cells could result in endotheliitis, or inflammation of the endothelium that more than makes up for the decreased infectivity.

But here’s the kicker, taken right from the final paragraph of the paper:

Collectively, our results suggest that the S protein-exerted EC damage overrides the decreased virus infectivity. This conclusion suggests that vaccination-generated antibody and/or exogenous antibody against S protein not only protects the host from SARS-CoV-2 infectivity but also inhibits S protein imposed endothelial injury.

In other words, the vaccine could be protective not just against infection by SARS-CoV-2 but also against endothelial injury from the spike protein.

I just want to reiterate again that this is a contrived system. It’s far from a worthless system, as pseudovirus systems have value in studying the role of spike protein in the pathogenesis of COVID-19. However, given the crapton of pseudovirus used in this hamster model, I really question any relevance of this system to vaccine safety issues with respect to mRNA- or adenovirus-based vaccines that produce the spike protein as an antigen. Why? The mRNA or adenovirus from the vaccines does not distribute extensively given that it’s an intramuscular injection, and the spike protein is highly unlikely to attain concentrations in the circulation anywhere near the high levels produced by the model used in these experiments. Moreover, the spike protein from the vaccine is not attached in a crown-like array on a virus particle (or pseudovirus particle), but rather exists as naked single protein molecules, and, as has been described before, it’s unclear that in this form spike protein, compared to the “crown of spikes” that gives coronaviruses their name, is anywhere near as effective at causing these downstream effects in cells. Add to that the fact that mRNA, even the modified mRNA in the vaccine, doesn’t hang around very long and therefore doesn’t generate spike protein for very long. (Doubters should consider this: Why do the mRNA vaccines both require a second dose 3-4 weeks after the first dose if, as many antivaxxers claim, the vaccines crank out spike protein indefinitely?)

Indeed, one of the authors pointed out this very issue and took antivaxxers to task for misusing their study:

i’m going to give a full response asap. but quickly for the record:
1) the (relatively) small amount of spike protein produced by the mRNA vaccine would not be nearly enough to do any damage
2) i happily got the mRNA vaccine, FWIW
3) i encourage everyone to get it

&mdash Uri Manor (@manorlaboratory) May 2, 2021

very important: while the mRNA codes for spike protein, the transfected cells degrade it and only present small chunks via MHC-I on their surfaces. the amount of full length spike protein entering circulation must be *infinitesimal*. this video explains:

&mdash Uri Manor (@manorlaboratory) May 2, 2021

a couple prelim responses to anti-vaxxers misrepresenting these findings (here: tldr: mRNA vaccine is waaaaay safer than COVID19 and everyone should get it – I did and everyone in my family did as well! Our paper just shows this disease really sucks.

&mdash Uri Manor (@manorlaboratory) May 2, 2021

Since I first discovered this study, it’s just amused me how obvious it is that the antivaxxers citing this study have obviously not actually read the study itself, nor have they considered the background science and knowledge behind the study. They’ve just read the press release. What do you expect, though? They’re antivaxxers. This study by Uri Manor’s laboratory is interesting and potentially important because it begins to elucidate the role of the spike protein itself in the pathophysiology of SARS-CoV-2 infection and how the spike protein alone can cause damage, but it does not in any way suggest that spike protein made by a COVID-19 vaccine is in any way toxic at the concentrations it’s produced, much less that it’s in any way “shed” or that the “shed” spike protein can cause disease or miscarriages in the unvaccinated who encounter the vaccinated.

Which brings me to the last of the three studies, which was published late last week.

Ancient Viruses as Gene Therapy Vectors

Ashley P. Taylor
Jul 31, 2015

Retinal targeting by Anc80 LIVIA CARVALHO The immune system is designed to protect the body, but it sometimes gets in the way&mdashby rejecting potentially life-saving blood transfusions or organ transplants, for example. Because one of the most commonly used methods for delivering gene therapies involves viruses as vectors, scientists developing such treatments are working to circumnavigate the host immune response.

Adeno-associated viruses (AAVs) have shown promise as gene-therapy delivery vehicles in clinical trials evaluating treatments for hemophilia and a genetic form of blindness. Problem is, anywhere from 30 percent to 90 percent of people have already been exposed to AAVs&mdashwhich are not pathogenic&mdashand have developed immunity to them, said Luk Vandenberghe of the Schepens Eye Research Institute and Massachusetts Eye and Ear Infirmary in Boston. As a result, they are ineligible for AAV-based therapies. &ldquoAnd it could, for some of these diseases, actually be a life-or-death differentiation&mdashenrolling in a.

In an effort to generate gene therapy vectors that could evade the immune system, Vandenberghe and his colleagues deduced the evolutionary history of today’s AAVs. They then synthesized the predicted ancestral viruses and tested them as gene therapy vectors in mammalian tissues. Their results were published today (July 30) in Cell Reports.

“This is a very thorough, creative, and carefully done study,” Jean Bennett of the University of Pennsylvania, who has collaborated with Vandenberghe but was not involved in the work, told The Scientist in an e-mail. “The ancestral AAVs have promise with respect to use, although, ultimately, human testing would reveal their utility.”

“This is on par with . . . other approaches” to designing immune detection-evading viral vectors, said R. Jude Samulski, director of the University of North Carolina Gene Therapy Center in Chapel Hill, North Carolina, who also was not involved in the work.

Researchers have used a variety of approaches to modify AAVs most involve rearranging coat proteins called capsids, rendering the viruses unrecognizable to hosts. No matter the method, “everybody’s looking for capsids that change the surface enough so that the pre-existing neutralizing antibodies don’t recognize it,” said Samulski.

Vandenberghe and his colleagues gathered the amino-acid sequences of capsid proteins from 75 viruses circulating today in primates. They then created a putative evolutionary tree for the viral family, which included nine ancestral viruses leading back to the oldest common ancestor, “Anc80.” In several places, the amino-acid sequence of Anc80 was ambiguous, with two amino acids possible at a given position, so the researchers created a library of all 2,048 possible sequences. They selected one, “Anc80L65,” based on its ability to assemble into viral particles, package the therapeutic transgene DNA, and infect mammalian cells in culture.

Anc80L65 successfully expressed a transgene in the mouse retina, skeletal muscle, and liver, and the expression was as good or better than that of AAV8—a commonly used gene therapy vector tested as a control.

“In some animals, not in all, we were able to achieve gene transfer even though these animals have pre-existing immunity against AAV8,” said Vandenberghe. “So this is one way of testing this initial hypothesis that we generated an immunologically distinct virus that can circumvent this pre-existing immunity problem.”

Beyond Anc80L65, the researchers recreated eight additional ancient viruses that represented different branching points on AAV evolutionary tree. The hope is that these viruses will further help researchers understand viral architecture and evolutionary history. “The structure-function relationships themselves will be useful for further improving gene therapy vectors,” said Bennett.

When it comes to designing AAV-vectors for gene therapy, “no one has any idea which one or if any of them are going to work,” said Samulski. “It may be a combination of ancestral mixed with library mixed with rational design [approaches]. . . . At this point in time, anything else you can add to the arsenal to attack the question in hand is valuable to the research community.”

Viruses and Cancer

There are many different causes of cancer, or unregulated cell growth and reproduction. Some known causes include exposure to certain chemicals or UV light. There are also certain viruses that have a known associated with the development of cancer. Such viruses are referred to as oncoviruses. Oncoviruses can cause cancer by producing proteins that bind to host proteins known as tumor suppressor proteins, which function to regulate cell growth and to initiate programmed cell death, if needed. If the tumor suppressor proteins are inactivated by viral proteins then cells grow out of control, leading to the development of tumors and metastasis, where the cells spread throughout the body.

Key Words

virus, obligate intracellular parasite, capsid, bacteriophage, capsomere, nucleocapsid, envelope, peplomer, virion, dsDNA, ssDNA, dsRNA, +ssRNA, -ssRNA, helical viruses, icosahedral viruses, complex viruses, attachment, penetration, viral entry, synthesis, assembly, release, naked virus, endocytosis, budding, bacteriophage, phage, virulent phage, lytic cycle, temperate phage, lysogeny, lysogenic cycle, prophage, lysogen, lysogenic bacterium, induction, lysogenic conversion, virulent infection, latent infection, persistent infection, transformation, oncovirus, tumor suppressor proteins.