Information

The effect of low pH on HIV virus

The effect of low pH on HIV virus


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

I've read a couple of articles that state that HIV is an enveloped Retrovirus that is so sensitive to the acidity of the environment and it cannot survive or will be inactivated at pH below 7. What exactly happens to the virus when exposed to an acidic environment. Does it denature the GP120 AND GP41 proteins on the virus envelope and make them ineffective? If not, how do hydrogen ions in the acid react with the virus?

Thanks for your attention.


Breakthrough discovery in HIV research opens path to new, better therapies

New research on the structure of the human immunodeficiency virus (HIV) has revealed a promising novel drug target for treating HIV infection, which affects more than 1 million Americans and 40 million people worldwide. The findings, published today in Science, show that the virus's genetic code can be read in two different ways by cells the virus has infected. The result is that infected cells make two different forms of the virus's RNA.

"This functional diversity is essential for the virus to replicate in the body. The virus has to have a proper balance between the two forms of RNA," says Joshua Brown, the lead author on the study. "For decades, the scientific community has known that two different structural forms of HIV RNA exist -- they just didn't know what controls that balance. We've discovered that a single nucleotide is having a huge effect, which is a paradigm shift in understanding how HIV works."

Crucially, "You can imagine that if you could come up with a drug that would target the genetic code at that one specific spot, and shift it to one form only, then it could prevent further infection, theoretically," says Brown, who earned his Ph.D. from UMBC in 2018 and continues to conduct research there while completing his M.D.

A new trajectory

"One of the things we're working on now is testing different molecules that could shift the equilibrium between the two forms, so that it could potentially be used as a treatment for HIV," says Issac Chaudry, a junior at UMBC and an author on the paper.

This exciting work comes from a research group led by Michael Summers, Robert E. Meyerhoff Chair for Excellence in Research and Mentoring and Distinguished University Professor at UMBC. Summers has been conducting groundbreaking research on HIV for decades. Typically, the group's focus is on basic science.

"Drug discovery isn't the direction that the Summers lab usually goes, but this was such an impactful finding in a very attractive area, we took the initiative to start looking into it," Brown says. "But we're still in the very early stages."

More effective treatments for more patients

Thanks to significant research on HIV over the last few decades, today AIDS is a manageable disease. Still, therapies can come with side effects, medication regimens can be complex, and treatment options can be limited for patients with other conditions, such as liver or kidney problems.

Many therapies, even if they come in the form of a single pill, contain several drugs targeting different parts of the virus's replication cycle. That's necessary because the HIV genetic code, which is made of RNA, mutates rapidly. This allows the virus to adapt and become resistant to current HIV therapies. If a drug targets an area that has mutated in a given patient, the drug may no longer work for them. By using several drugs at once, it's more likely that the regimen will continue to work for longer.

But the area of the HIV RNA genome that this new research focuses on is "highly conserved." This means the rate of mutation is less than other places in the genome, explains Ghazal Becker, a 2019 UMBC alumna and an author on the paper. The result is "there's more chance of a drug that targets that region being effective for longer," she says.

It might also mean that one drug would be enough, rather than patients needing several drugs to get the job done. "If you're targeting a conserved region, you can potentially come up with a treatment plan that uses only one drug," says Aishwarya Iyer, a 2018 UMBC alumna, current M.D./Ph.D. at the University of Maryland School of Medicine, and an author on the paper. "It might have fewer side effects and could offer more treatment options to people with different health conditions."

Expanding the research horizon

This new research opens up a range of opportunities for Brown's research group and others. "We're very interested to see how other labs will interpret our results, expand upon them, and possibly find other applications for this type of RNA function," Brown says.

Those future results and any new therapies they enable could have a major impact. "Every time we get a new drug in HIV, we exponentially improve the chances of individuals finding a drug that works for them, where resistance is a little less likely," says Hannah Carter, a 2017 UMBC alumna, current M.D./Ph.D. student at University of Michigan, and an author on the paper. "Every time a new drug can get on the scene, that's a significant improvement for the lives of HIV patients."

The research could have effects beyond HIV, too. "Some HIV research has laid the groundwork in how we understand coronaviruses," Carter adds. "All basic science in HIV ends up having a ripple effect throughout all of virology."

The ripple effect might go even farther. "The idea that a single nucleotide difference is changing the structure and function of RNA that is thousands of nucleotides long could open up a whole new aspect of cell biology," Chaudry says. "It could be possible that there are mammalian genes that operate in a similar manner, and the entire mechanism might be something that's applicable to other human genes as well. I think that whole paradigm could provide a new perspective for RNA biology."


CoronaVirus Bundle

Learn Dr Sircus protocol including dosages, methods, side effects and contra-indications. This bundle includes the special edition of Transdermal Magnesium Therapy, Iodine and Sodium Bicarbonate eBooks.

Dr. Mark Sircus AC., OMD, DM (P)

Professor of Natural Oncology, Da Vinci Institute of Holistic Medicine
Doctor of Oriental and Pastoral Medicine
Founder of Natural Allopathic Medicine

Never miss Dr. Sircus updates. Join 90,000 others in my newsletter and get a free ebook!


The effect of low pH on HIV virus - Biology

Evolutionary biologists can help uncover clues to new ways to treat or vaccinate against HIV. These clues emerge from the evolutionary origins of the virus, how human populations have evolved under pressure from other deadly pathogens, and how the virus evolves resistance to the drugs we've designed. Controlling the disease may be a matter of controlling the evolution of this constantly adapting virus.

The human immunodeficiency virus (HIV, shown here budding from a white blood cell) is one of the fastest evolving entities known. It reproduces sloppily, accumulating lots of mutations when it copies its genetic material. It also reproduces at a lightning-fast rate — a single virus can spawn billions of copies in just one day. To fight HIV, we must understand its evolution within the human body and then ultimately find a way to control its evolution.

Taking an evolutionary perspective on HIV has led scientists to look in three new directions in their search for treatments and vaccines:

  • What are the evolutionary origins of HIV?
  • Why are some people resistant to HIV?
  • How can we control HIV's evolution of resistance to our drugs?

However, studies of these related viral lineages showed something surprising: primates with SIV and wild cats with FIV don't seem to be harmed by the viruses they carry. If scientists can figure out how non-human primates and wild cats are able to live with these viruses, they may learn how to better treat HIV infections or prevent them altogether.

2. Why are some people resistant to HIV?
HIV is by no means the first plague that human populations have weathered. Many pathogens have deeply affected our evolutionary history. In fact, the human genome is littered with the remnants of our past battles with pathogens — and one of these remnants, a mutation to a gene called CCR5, may lead researchers to a new treatment for HIV.

The mutant CCR5 allele probably began to spread in northern Europe during the past 700 years when the population was ravaged by a plague. (It may have been bubonic plague or some other pathogen research on this topic continues.) The mutant CCR5 probably made its bearers resistant to the disease, and so its frequency increased.

In some parts of Europe today, up to 20% of the population carry at least one copy of the protective allele. However, the populations of Asia and Africa were not exposed to the same epidemics very few Asians and Africans now carry the allele (see map above). Thus, CCR5 is fairly common in northern Europe but its frequency diminishes as one moves south, and the mutation is rare in the rest of the world.

We now know that the mutant CCR5 allele has an unexpected side effect: it confers resistance to HIV. Scientists hope that studying this by-product of past selection will help them develop new treatments for the HIV epidemic ravaging human populations today.

3. How can we control HIV's evolution of resistance to our drugs?
HIV evolves so quickly that it evolves right out from under our treatments. When a patient begins taking an HIV drug, the drug keeps many of the viruses from reproducing, but some survive because they happen to have a certain level of resistance. Because of HIV's speedy evolution, it responds to selection pressures quickly: viruses that happen to survive the drug are favored, and resistant virus strains evolve within the patient, sometimes in just a few weeks. However, basic evolutionary theory points out a way that this evolution of resistant viral strains can be delayed. Patients are prescribed "drug cocktails" — several different HIV drugs taken together.

When taking any single drug, it is fairly likely that some mutant virus in the patient might happen to be resistant, survive the onslaught, and spawn a resistant lineage.

But the probability that the patient hosts a mutant virus that happens to be resistant to several different drugs at the same time is much lower. Although multiple-drug-resistant HIV strains do eventually evolve, drug cocktails delay their evolution.

An evolutionary trade-off
If a patient is already infected with a drug-resistant HIV strain, basic evolutionary theory has also pointed out a way to make the drug useful again. Studies of the evolution of resistance often show that you don't get something for nothing. Specifically, it "costs" a pest or pathogen to be resistant to a pesticide or drug. If you place resistant and non-resistant organisms in head-to-head competition in the absence of the pesticide or drug, the non-resistant organisms generally win.

Consider a patient who takes a particular drug and winds up with viruses resistant to the drug. If the patient stops taking the drug for a while, evolutionary theory predicts that her viral load will evolve back towards a non-resistant strain. If she then takes very strong doses of the drug, it may be able to halt the replication of those non-resistant viruses and reduce her viral load to very low levels.

This therapy has shown early, promising results — it may not eliminate HIV, but it could keep patients' virus loads low for a long time, slowing progression of the disease.

Ultimately, understanding the evolutionary history of HIV and its pattern of evolutionary change may help us control this disease.


Comprehensive, up-to-date information on HIV/AIDS treatment and prevention from the University of California San Francisco

Bringing the global HIV epidemic under control will require more effective approaches to prevent the spread of the retrovirus, as well as broader use of existing and future antiretroviral drugs. These interventions must be applicable in the developing world, where HIV has the most severe impact. Understanding the dynamic interplay of HIV with its cellular host provides the biological basis for controlling the epidemic. This chapter reviews current understanding of the HIV life cycle, with particular attention to the interactions between viral proteins and cellular machinery, and highlights promising future points of attack.

The genetic material of HIV, an RNA molecule 9 kilobases in length, contains 9 different genes encoding 15 proteins. Considerable insights have been gained into the function of these different gene products.(Figure 1) To productively infect a target cell, HIV must introduce its genetic material into the cytoplasm of this cell. The process of viral entry involves fusion of the viral envelope with the host cell membrane and requires the specific interaction of the envelope with specific cell surface receptors. The two viral envelope proteins, gp120 and gp41, are conformationally associated to form a trimeric functional unit consisting of three molecules of gp120 exposed on the virion surface and associated with three molecules of gp41 inserted into the viral lipid membrane. Trimeric gp120 on the surface of the virion binds CD4 on the surface of the target cell, inducing a conformational change in the envelope proteins that in turn allows binding of the virion to a specific subset of chemokine receptors on the cell surface.(1)(Figure 2) These receptors normally play a role in chemoattraction, in which hematopoietic cells move along chemokine gradients to specific sites. Although these receptors, which contain seven membrane-spanning domains, normally transduce signals through G proteins,(2) signaling is not required for HIV infection.

Twelve chemokine receptors can function as HIV coreceptors in cultured cells, but only two are known to play a role in vivo.(2) One of these, CCR5, binds macrophage-tropic, non-syncytium-inducing (R5) viruses, which are associated with mucosal and intravenous transmission of HIV infection. The other, CXCR4, binds T-cell-tropic, syncytium-inducing (X4) viruses, which are frequently found during the later stages of disease.(3) In up to 13% of individuals of northern European descent, a naturally occurring deletion of 32 base pairs in the CCR5 gene results in a mutant CCR5 receptor that never reaches the cell surface.(4,5) Individuals homozygous for this mutation (1-2% of the Caucasian population) are almost completely resistant to HIV infection.(4,5) These observations emphasize the pivotal role of CCR5 in the spread of HIV and suggest that small molecules that prevent HIV interaction with CCR5 might form a promising new class of antiretroviral drugs.

Both CD4 and chemokine coreceptors for HIV are found disproportionately in lipid rafts in the cell membrane.(6) These cholesterol- and sphingolipid-enriched microdomains likely provide a better environment for membrane fusion, perhaps by mirroring the optimal lipid bilayer of the virus.(7) Removing cholesterol from virions, producer cells, or target cells greatly decreases the infectivity of HIV.(8) Studies currently under way are exploring whether cholesterol-depleting compounds might be efficacious as topically applied microbicides to inhibit HIV transmission at mucosal surfaces. The development of effective microbicides represents an important component of future HIV prevention strategies.

The binding of surface gp120, CD4, and the chemokine coreceptors produces an additional radical conformational change in gp41.(9) Assembled as a trimer on the virion membrane, this coiled-coil protein springs open, projecting three peptide fusion domains that "harpoon" the lipid bilayer of the target cell. The fusion domains then form hairpin-like structures that draw the virion and cell membranes together to promote fusion, leading to the release of the viral core into the cell interior.(9) The fusion inhibitors T-20 and T-1249 act to prevent fusion by blocking the formation of these hairpin structures.

HIV virions can also enter cells by endocytosis. Usually, productive infection does not result, presumably reflecting inactivation of these virions within endosomes. However, a special form of endocytosis has been demonstrated in submucosal dendritic cells. These cells, which normally process and present antigens to immune cells, express a specialized attachment structure termed DC-SIGN.(10) This C-type lectin binds HIV gp120 with high affinity but does not trigger the conformational changes required for fusion. Instead, virions bound to DC-SIGN are internalized into an acidic compartment and subsequently displayed on the cell surface after the dendritic cell has matured and migrated to regional lymph nodes, where it engages T cells.(11) Thus, dendritic cells expressing DC-SIGN appear to act as "Trojan horses" facilitating the spread of HIV from mucosal surfaces to T cells in lymphatic organs.

Once inside the cell, the virion undergoes uncoating, likely while still associated with the plasma membrane.(Figure 2) This poorly understood process may involve phosphorylation of viral matrix proteins by a mitogen-activated protein (MAP) kinase(12) and additional actions of cyclophilin A(13) and the viral proteins Nef(14) and Vif.(15) Nef associates with a universal proton pump, V-ATPase,(16) which could promote uncoating by inducing local changes in pH in a manner similar to that of the M2 protein of influenza.(17) After the virion is uncoated, the viral reverse transcription complex is released from the plasma membrane.(18) This complex includes the diploid viral RNA genome, lysine transfer RNA (tRNA Lys ) which acts as a primer for reverse transcription, viral reverse transcriptase, integrase, matrix and nucleocapsid proteins, viral protein R (Vpr), and various host proteins. The reverse transcription complex docks with actin microfilaments.(19) This interaction, mediated by the phosphorylated matrix, is required for efficient viral DNA synthesis. By overcoming destabilizing effects of a recently identified protein termed CEM15/APOBEC3G, Vif stabilizes the reverse transcription complex in most human cells.(15-20)

Reverse transcription yields the HIV preintegration complex (PIC), composed of double-stranded viral cDNA, integrase, matrix, Vpr, reverse transcriptase, and the high mobility group DNA-binding cellular protein HMGI(Y).(21) The PIC may move toward the nucleus by using microtubules as a conduit.(22) Adenovirus and herpes simplex virus 1 also dock with microtubules and use the microtubule-associated dynein molecular motor for cytoplasmic transport. This finding suggests that many viruses use these cytoskeletal structures for directional movement. How the switch from actin microfilaments to microtubules is orchestrated remains unknown.

Recent studies have revealed a mechanism by which the target cell defends against the HIV intruder.(23,24) Within 30 minutes of infection, select host proteins including the integrase interactor 1 (also known as INI-1, SNF5, or BAF47), a component of the SWI/SNF chromatin remodeling complex, and PML, a protein present in promyelocytic oncogenic domains, translocate from the nucleus into the cytoplasm.(24)(Figure 2) Addition of arsenic trioxide sharply blocks PML movement and enhances the susceptibility of cells to HIV infection raising the possibility that the normal function of PML is to oppose viral infection.(24) The binding of integrase to integrase interactor 1 may be a viral adaptation that recruits additional chromatin remodeling factors. Whether these complexes influence the site of viral integration or improve subsequent proviral gene expression is not known.

Unlike most animal retroviruses, HIV can infect nondividing cells, such as terminally differentiated macrophages.(25) This requires an ability to cross the intact nuclear membrane. With a Stokes radius of approximately 28 nm or roughly the size of a ribosome, the PIC is roughly twice as large as the maximal diameter of the central aqueous channel in the nuclear pore.(26) The 3 µm contour length of viral DNA must undergo significant compaction, and the import process must involve considerable molecular gymnastics.

One of the most contentious areas of HIV research involves the identification of key viral proteins that mediate the nuclear import of the PIC. Integrase,(27) matrix,(28) and Vpr(29) have been implicated.(Figure 2) Because plus-strand synthesis is discontinuous in reverse transcription, a triple helical DNA domain or "DNA flap" results that may bind a host protein containing a nuclear targeting signal.(30) Matrix contains a canonical nuclear localization signal that is recognized by the importins alpha and beta, which are components of the classical nuclear import pathway. However, a recent publication calls into question the contributions both of the nuclear import signal in integrase and of the DNA flap to the nuclear uptake of the PIC.(31) The HIV Vpr gene product contains at least three noncanonical nuclear targeting signals.(32) Vpr may bypass the importin system altogether, perhaps mediating the direct docking of the PIC with one or more components of the nuclear pore complex. The multiple nuclear targeting signals within the PIC may function in a cooperative manner or play larger roles individually in different target cells. For example, while Vpr is not needed for infection of nondividing, resting T cells,(33) it enhances viral infection in nondividing macrophages.(34) The finding that both matrix(35) and Vpr(32) shuttle between the nucleus and cytoplasm explains their availability for incorporation into new virions.

Once inside the nucleus, the viral PIC can establish a functional provirus.(Figure 2) Integration of double-stranded viral DNA into the host chromosome is mediated by integrase, which binds the ends of the viral DNA.(21) The host proteins HMGI(Y) and barrier to autointegration (BAF) are required for efficient integration, although their precise functions remain unknown.(36) Integrase removes terminal nucleotides from the viral DNA, producing a two-base recess and thereby correcting the ragged ends generated by the terminal transferase activity of reverse transcriptase.(21) Integrase also catalyzes the subsequent joining reaction that establishes the HIV provirus within the chromosome.

Not all PICs that enter the nucleus result in a functional provirus. The ends of the viral DNA may be joined to form a 2-LTR circle containing long terminal repeat sequences from both ends of the viral genome, or the viral genome may undergo homologous recombination yielding a single-LTR circle. Finally, the viral DNA may auto-integrate into itself, producing a rearranged circular structure. Although some circular forms may direct the synthesis of the transcriptional transactivator Tat or the accessory protein Nef, none produces infectious virus.(37) In a normal cellular response to DNA fragments, the nonhomologous end-joining (NHEJ) system may form 2-LTR circles to protect the cell.(38) This system is responsible for rapid repair of double-strand breaks, thereby preventing an apoptotic response. A single double-strand break within the cell can induce G1 cell-cycle arrest. The ability of the free ends of the viral DNA to mimic such double-strand chromosomal breaks may contribute to the direct cytopathic effects observed with HIV.

Integration can lead to latent or transcriptionally active forms of infection.(39) HIV's transcriptional latency explains the inability of potent antiviral therapies to eradicate the virus from the body. Moreover, despite a vigorous immune response early in infection, these silent proviruses are a reservoir that allows reemergence of HIV when the body's defenses grow weaker. Understanding latency and developing approaches to target latent virus are essential goals if eradication of HIV infection is ever to be achieved.

The chromosomal environment likely shapes the transcriptional activity of the provirus.(40) For example, proviral integration into repressed heterochromatin might result in latency.(Figure 3) Other causes of latency may include cell type differences in the availability of activators that bind to the transcriptional enhancer in the HIV LTR or the lack of Tat. However, of the multiple copies of provirus that are usually integrated in a given infected cell, at least one is likely to be transcriptionally active. This fact may explain why the number of latently infected cells (10 5 -10 6 ) in infected patients is small.

In the host genome, the 5´ LTR functions like other eukaryotic transcriptional units. It contains downstream and upstream promoter elements, which include the initiator (Inr), TATA-box (T), and three Sp1 sites.(41) These regions help position the RNA polymerase II (RNAPII) at the site of initiation of transcription and to assemble the preinitiation complex. Slightly upstream of the promoter is the transcriptional enhancer, which in HIV-1 binds nuclear factor [kappa]B (NF-[kappa]B), nuclear factor of activated T cells (NFAT), and Ets family members.(42) NF-[kappa]B and NFAT relocalize to the nucleus after cellular activation. NF-[kappa]B is liberated from its cytoplasmic inhibitor, I[kappa]B, by stimulus-coupled phosphorylation, ubiquitination, and proteosomal degradation of the inhibitor.(43) NFAT is dephosphorylated by calcineurin (a reaction inhibited by cyclosporin A) and, after its nuclear import, assembles with AP1 to form the fully active transcriptional complex.(44) NF-[kappa]B, which is composed of p50 and p65 (RelA) subunits, increases the rates of initiation and elongation of viral transcription.(45) Since NF-[kappa]B is activated after several antigen-specific and cytokine-mediated events, it may play a key role in rousing transcriptionally silent proviruses

When these factors engage the LTR, transcription begins, but in the absence of Tat described below the polymerase fails to elongate efficiently along the viral genome.(Figure 3) In the process, short nonpolyadenylated transcripts are synthesized, which are stable and persist in cells due to the formation of an RNA stem loop called the transactivation response (TAR) element.(46)

Tat significantly increases the rate of viral gene expression. With cyclin T1 (CycT1), Tat binds to the TAR RNA stem-loop structure and recruits the cellular cyclin-dependent kinase 9 (Cdk9) to the HIV LTR.(47)(Figure 3) Within the positive transcription elongation factor b (P-TEFb) complex, Cdk9 phosphorylates the C-terminal domain of RNAPII, marking the transition from initiation to elongation of eukaryotic transcription.(48) Other targets of P-TEFb include negative transcription elongation factors (N-TEF), such as the DRB-sensitivity inducing (DSIF) and negative elongation (NELF) factors.(48) The high efficiency with which the HIV LTR attracts these negative transcription factors in vivo may explain why the LTR is a poor promoter in the absence of Tat. The arginine-rich motif (ARM) within Tat binds the 5´ bulge region in TAR. A shorter ARM in cyclin T1, which is also called the Tat-TAR recognition motif (TRM), binds the central loop of TAR.(47)

Binding of the Tat cyclin T1 complex to both the bulge and loop regions of TAR strengthens the affinity of this interaction. All of these components are required for Tat transactivation. In the presence of the complex between Tat and P-TEFb, the RNAPII elongates efficiently. Because murine CycT1 contains a cysteine at position 261, the complex between Tat and murine P-TEFb binds TAR weakly.(49) Thus, Tat transactivation is severely compromised in murine cells. Cdk9 also must undergo autophosphorylation of several serine and threonine residues near its C-terminus to allow productive interactions between Tat, P-TEFb, and TAR.(50) Additionally, basal levels of P-TEFb may be low in resting cells or only weakly active due to the interaction between P-TEFb and 7SK RNA.(51) All of these events may contribute to postintegration latency.

Transcription of the viral genome results in more than a dozen different HIV-specific transcripts.(52) Some are processed cotranscriptionally and, in the absence of inhibitory RNA sequences (IRS), transported rapidly into the cytoplasm.(53) These multiply spliced transcripts encode Nef, Tat, and Rev. Other singly spliced or unspliced viral transcripts remain in the nucleus and are relatively stable. These viral transcripts encode the structural, enzymatic, and accessory proteins and represent viral genomic RNAs that are needed for the assembly of fully infectious virions.

Incomplete splicing likely results from suboptimal splice donor and acceptor sites in viral transcripts. In addition, the regulator of virion gene expression, Rev, may inhibit splicing by its interaction with alternate splicing factor/splicing factor 2 (ASF/SF2)(54) and its associated p32 protein.(55)

Transport of the incompletely spliced viral transcripts to the cytoplasm depends on an adequate supply of Rev.(53) Rev is a small shuttling protein that binds a complex RNA stem-loop termed the Rev response element (RRE), which is located in the env gene. Rev binds first with high affinity to a small region of the RRE termed the stem-loop IIB.(56)(Figure 4) This binding leads to the multimerization of Rev on the remainder of the RRE. In addition to a nuclear localization signal, Rev contains a leucine-rich nuclear export sequence (NES).(53) Of note, the study of Rev was the catalyst for the discovery of such NES in many cellular proteins and led to identification of the complex formed between CRM1/exportin-1 and this sequence.(53)

The nuclear export of this assembly (viral RNA transcript, Rev, and CRM1/exportin 1) depends critically on yet another host factor, RanGTP. Ran is a small guanine nucleotide-binding protein that switches between GTP- and GDP-bound states. RanGDP is found predominantly in the cytoplasm because the GTPase activating protein specific for Ran (RanGAP) is expressed in this cellular compartment. Conversely, the Ran nucleotide exchange factor, RCC1, which charges Ran with GTP, is expressed predominantly in the nucleus. The inverse nucleocytoplasmic gradients of RanGTP and RanGDP produced by the subcellular localization of these enzymes likely plays a major role in determining the directional transport of proteins into and out of the nucleus. Outbound cargo is only effectively loaded onto CRM1/exportin-1 in the presence of RanGTP. However, when the complex reaches the cytoplasm, GTP is hydrolyzed to GDP, resulting in release of the bound cargo. The opposite relationship regulates the nuclear import by importins alpha and beta, where nuclear RanGTP stimulates cargo release.(53)

For HIV infection to spread, a balance between splicing and transport of viral mRNA species must be achieved. If splicing is too efficient, then only the multiply spliced transcripts appear in the cytoplasm. Although required, the regulatory proteins encoded by multiply spliced transcripts are insufficient to support full viral replication. However, if splicing is impaired, adequate synthesis of Tat, Rev, and Nef will not occur. In many non-primate cells, HIV transcripts may be overly spliced, effectively preventing viral replication in these hosts.(57)

In contrast to Tat and Rev, which act directly on viral RNA structures, Nef modifies the environment of the infected cell to optimize viral replication.(2)(Figure 4) The absence of Nef in infected monkeys and humans is associated with much slower clinical progression to AIDS.(58,59) This virulence caused by Nef appears to be associated with its ability to affect signaling cascades, including the activation of T-cell antigen receptor,(60) and to decrease the expression of CD4 on the cell surface.(61,62) Nef also promotes the production and release of virions that are more infectious.(63,64) Effects of Nef on the PI3-K signaling cascade--which involves the guanine nucleotide exchange factor Vav, the small GTPases Cdc42 and Rac1, and p21-activated kinase PAK--cause marked changes in the intracellular actin network, promoting lipid raft movement and the formation of larger raft structures that have been implicated in T-cell receptor signaling.(65) Indeed, Nef and viral structural proteins colocalize in lipid rafts.(64,66) Two other HIV proteins assist Nef in downregulating expression of CD4.(67) The envelope protein gp120 binds CD4 in the endoplasmic reticulum, slowing its export to the plasma membrane,(68) and Vpu binds the cytoplasmic tail of CD4, promoting recruitment of TrCP and Skp1p.(Figure 5) These events target CD4 for ubiquitination and proteasomal degradation before it reaches the cell surface.(69)

Nef acts by several mechanisms to impair immunological responses to HIV. In T cells, Nef activates the expression of FasL, which induces apoptosis in bystander cells that express Fas,(70) thereby killing cytotoxic T cells that might otherwise eliminate HIV-1 infected cells. Nef also reduces the expression of MHC I determinants on the surface of the infected cell(71)(Figure 4) and so decreases the recognition and killing of infected cells by CD8 cytotoxic T cells. However, Nef does not decrease the expression of HLA-C,(72) which prevents recognition and killing of these infected cells by natural killer cells.

Nef also inhibits apoptosis. It binds and inhibits the intermediate apoptosis signal regulating kinase-1 (ASK-1)(73) that functions in the Fas and TNFR death signaling pathways and stimulates the phosphorylation of Bad leading to its sequestration by 14-3-3 proteins.(74)(Figure 4) Nef also binds the tumor suppressor protein p53, inhibiting another potiential initator of apoptosis.(75) Via these different mechanisms, Nef prolongs the life of the infected host cell, thereby optimizing viral replication.

Other viral proteins also participate in the modification of the environment in infected cells. Rev-dependent expression of Vpr induces the arrest of proliferating infected cells at the G2/M phase of the cell cycle.(76) Since the viral LTR is more active during G2, this arrest likely enhances viral gene expression.(77) These cell-cycle arresting properties involve localized defects in the structure of the nuclear lamina that lead to dynamic, DNA-filled herniations that project from the nuclear envelope into the cytoplasm.(78)(Figure 4) Intermittently, these herniations rupture, causing the mixing of soluble nuclear and cytoplasmic proteins. Either alterations in the lamina structure or the inappropriate mixing of cell cycle regulators that are normally sequestered in specific cellular compartments could explain the G2 arresting properties of Vpr.

New viral particles are assembled at the plasma membrane.(Figure 5) Each virion consists of roughly 1500 molecules of Gag and 100 Gag-Pol polyproteins,(79) two copies of the viral RNA genome, and Vpr.(80) Several proteins participate in the assembly process, including Gag polyproteins and Gag-Pol, as well as Nef and Env. A human ATP-binding protein, HP68 (previously identified as an RNase L inhibitor), likely acts as a molecular chaperone, facilitating conformational changes in Gag needed for the assembly of viral capsids.(81) In primary CD4 T lymphocytes, Vif plays a key but poorly understood role in the assembly of infectious virions. In the absence of Vif, normal levels of virus are produced, but these virions are noninfectious, displaying arrest at the level of reverse transcription in the subsequent target cell. Heterokaryon analyses of cells formed by the fusion of nonpermissive (requiring Vif for viral growth) and permissive (supporting growth of Vif-deficient viruses) cells have revealed that Vif overcomes the effects of a natural inhibitor of HIV replication.(20,82) Recently this factor, initially termed CEM15/APOBEC3G, was identified(83) and shown to share homology with APOBEC1, an enzyme involved in RNA editing. Whether the intrinsic antiviral activity of CEM15 involves such an RNA editing function remains unknown. CEM15 is expressed in non-permissive but not in permissive cells and when introduced alone is sufficient to render permissive cells nonpermissive.

The Gag polyproteins are subject to myristylation,(84) and thus associate preferentially with cholesterol- and glycolipid-enriched membrane microdomains.(85) Virion budding occurs through these specialized regions in the lipid bilayer, yielding virions with cholesterol-rich membranes. This lipid composition likely favors release, stability, and fusion of virions with the subsequent target cell.(7)

The budding reaction involves the action of several proteins, including the "late domain"(86) sequence (PTAP) present in the p6 portion of Gag.(87)(Figure 5) The p6 protein also appears to be modified by ubiquitination. The product of the tumor suppressor gene 101 (TSG101) binds the PTAP motif of p6 Gag and also recognizes ubiquitin through its ubiquitin enzyme 2 (UEV) domain.(88,89) The TSG101 protein normally associates with other cellular proteins in the vacuolar protein sorting pathway to form the ESCRT-1 complex that selects cargo for incorporation into the multivesicular body (MVB).(90) The MVB is produced when surface patches on late endosomes bud away from the cytoplasm and fuse with lysosomes, releasing their contents for degradation within this organelle. In the case of HIV, TSG101 appears to be "hijacked" to participate in the budding of virions into the extracellular space away from the cytoplasm.

As the AIDS pandemic continues, advances in antiretroviral therapies have slowed its advance in the industrialized world, but have had little effect in developing countries. Because of its high rate of mutation, HIV is able to refine and optimize its interactions with various host proteins and pathways, thereby promoting its growth and spread. The virus ensures that the host cell survives until the viral replicative cycle is completed. Possibly even more damaging, HIV establishes stable latent forms that support the chronic nature of infection. Eradication of the virus appears unlikely until effective methods are developed to purge these latent viral reservoirs.

Basic science will clearly play a leading role in future attempts to solve the mysteries of viral latency and replication. A small-animal model that recapitulates the pathogenic mechanisms of HIV is sorely needed to study the mechanisms underlying viral cytopathogenesis. Virally induced cell death is not limited to infected targets but also involves uninfected bystander cells.(91) Murine cells support neither efficient virion assembly nor release of virions from the cell surface.(92) Currently, this defect represents a major impediment to the successful development of a rodent model of AIDS.

Proposed mechanisms for HIV killing of T cells include the formation of giant cell syncytia through the interactions of gp120 with CD4 and chemokine receptors,(93) the accumulation of unintegrated linear forms of viral DNA, the proapoptotic effects of the Tat,(94) Nef,(95) and Vpr(96) proteins, and the adverse effects conferred by the metabolic burden that HIV replication places on the infected cell.(97) Of note, expression of Nef alone as a transgene in mice recapitulates many of the clinical features of AIDS, including immunodeficiency and loss of CD4-positive cells.(98) All of these mechanisms suggest potential points of therapeutic intervention. Finally, future therapies will likely target viral proteins other than the reverse transcriptase, protease, and integrase enzymes. Clinical trials are already underway to study small molecules or short peptides that block the binding of HIV to cell-surface chemokine receptors or interfere with the machinery of viral-host cell fusion. Although not as advanced in development, small molecules have been found that block Tat transactivation(99) and Rev-dependent export of viral transcripts from the nucleus to the cytoplasm.(100) As a proof of principle, dominant-negative mutants of Tat, Rev, and Gag proteins have been shown to block viral replication. By increasing the number of antiviral compounds available to target different steps in the viral replicative cycle, in particular drugs that can be deployed in developing countries, research at the cellular level can serve to extend survival and to improve the quality of life for infected individuals, and to inhibit the spread of AIDS.

Warner C. Greene thanks Gary Howard and Stephen Ordway for editorial support, Robin Givens for administrative support, and the National Institutes of Health (R01 AI45234-02, R01 CA86814-02, P01 HD40543), the UCSF California AIDS Research Center (C99-SF-002), the James B. Pendleton Charitable Trust, and the J. David Gladstone Institutes for funding support.

B. Matija Peterlin thanks the National Institutes of Health (R01-AI38532, R01-AI46967, RO1-AI49104, and R01-AI51165-01) and the Universitywide AIDS Research Program (R00-SF-006) for funding support.


Results

Bioinformatics analysis of LLP12 domain

The mutations were designed in the LLP1 and LLP2 domains, respectively, or in combination to evaluate the significance of conserved arginine residues for the structural and functional properties of LLP12 domain (residues 768–855), with a similar experimental design of previously reported studies ( 9 ). Based on clustalx ( 15 ) analysis among over one thousand different HIV gp41 sequences from National Center for Biotechnology Information (NCBI), some positively charged arginine residues are highly conserved (>74% conservation in LLP2 and >97% in LLP1 Figure 1A). Also, cytoplasmic domains of 13 different viruses selected randomly from NCBI were compared with LLP12. This comparison indicated that the highest frequency of arginine residues was found in LLP12 (13.5%) of HIV-1 (Figure 1B). Based on these analyses, three mutants were constructed with substitutions of arginine to alanine in either the LLP1 (LLPM1) or the LLP2 (LLPM2) or in both domains (LLPM3) to study the effects of certain conserved arginine residues for the structure and function of LLP12 domain.

Secondary structure changes of LLP12 mutants

The prepared and purified LLP12 and mutants exhibited bands with a molecular weight of about 10 kDa and a few polymers with different size on SDS-PAGE gel, corresponding to the predicted molecular weight and characterization of LLP12 with its mutants (Figure 2). This series of peptides were difficult to purify using conventional methods relative to their hydrophobic properties, therefore we modified the protocol and solution formulation after repeated experiments (see Materials and methods, Figure 2A). Even in the reduced SDS-PAGE, some LLP12 peptide oligomeric forms could also be observed (Figure 2B), with similar results to previous studies ( 14 ).

Sodium dodecyl sulfate polyacrylamide gel analysis (reduced) of elution with various buffers to obtain GST–LLP12 fusion proteins from the affinity column, and purified LLP12 protein together with each mutant. (A) 1, The low molecular weight (LMW) markers (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA), consisting of phosphorylase b (97 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.4 kDa) were included for estimation of the polypeptide molecular weight. 2, After incubated with lysis supernatants, 5 μL resin in Tris-buffered saline (TBS) was taken out as the sample, showing the highly expressed GST–LLP12 protein. 3, Elution product with 50 m m reduced glutathione in 50 m m Tris (pH 8.0). 4, Elution product with 3 m NaCl in TBS (pH 8.0). 5, Elution product with 6 m urea in ddH2O (pH 8.0). 6, Elution product with 0.1% Triton-X-100 in 50 m m Tris (pH 8.0). 7, Elution product with 50 m m reduced glutathione and 0.1% Triton-X-100 in 50 m m Tris (pH 8.0). (B) 1, The LMW markers. 2, GST protein was taken as a second marker. 3–6, The purified proteins of LLP12, LLPM1, LLPM2, and LLPM3 in 1% Triton-X-100, showing the monomer and tetramer form. All of them were eluted from resin with 0.1% Triton-X-100 in 50 m m Tris (pH 8.0).

To investigate the changes in their secondary structures, the wild type and mutant LLP12 proteins were detected by CD. Surprisingly, substitutions of only a few arginines lead to distinct structural alterations. As illustrated in Figure 3A, a substantial change in the CD spectrum was observed for LLP12 mutants in comparison to the wild type. A more significant change in the spectrum was observed for LLPM3 mutants. The large positive increase in ellipticity at 222 nm is consistent with a structure change from α-helix to random coil.

(A) Secondary structural changes detected by CD spectroscopy. Circular dichroism spectra for wild type and LLP12 mutants of 300 μ m at 25 °C were showed by molar ellipticity versus wavelength. (B) Fast protein liquid chromatographic analysis (gel filtration chromatography) of LLP12 and mutants. Ba, LLP12 (solid line, 0) and LLPM1 (dotted line, I) Bb, LLP12 (solid line, 0) and LLPM2 (dotted line, II) Bc, LLP12 (solid line, 0) and LLPM3 (dotted line, III). Purified proteins was injected into a Superdex 200HR 10/30 size exclusion column and eluted with Tris-buffered saline containing 0.05% Triton-X-100 (pH 8.0). The protein elution pattern was measured by UV absorption at 280 nm. The elution peak of IgG (150 kDa), which was taken as a molecular mass standard, is marked at the top of the image with arrows. Bd, all the elution samples (the concentration of Triton-X-100 was adjusted to 1% before sodium dodecyl sulfate polyacrylamide gel) were analyzed by Western blotting using antisera.

Identification of multimerization potential of LLP12 and mutants

The multimerization potential of LLP12 and mutants was investigated by FPLC using a Superdex 200HR 10/30 column (gel filtration). During gel filtration chromatography, larger molecules cannot enter the gel’s pores consequently, larger molecules could be eluted faster. When the purified LLP12 protein (wild type) was eluted at 12 min, it was shown that LLP12 was prone to self-assemble into a complex over 150 kDa (Figure 3B). In comparison to LLP12, the fractionation peak of LLPM1 was observed about 1 min later. Therefore, the mutations in LLP1 domain demonstrably decreased the multimerization potential of LLP12 protein. However, the LLPM2 mutant was eluted almost at the same time as the wild type, suggesting that the mutants in LLP2 domain did not affect the multimerization formation of the whole protein (LLP12). Then LLPM3 mutant was eluted almost at the same time as LLPM1, which could be explained based on the previous results. The eluted proteins at each peak were collected and tested by Western blotting (Figure 3B), showing that these were indeed LLP12 peptides (about 10 kDa).

Identification of cellular membrane-binding ability of LLP12 and mutants

Another crucial reported function of LLP12 domain is the membrane-binding ability ( 12 ), which was also significantly affected by arginine substitutions in our experiments. In comparison with the LLP12 wild type, all LLP12 mutants showed an impaired ability in binding to the outer membrane of HeLa cells relative to flow cytometry analysis (Figure 4A–D). The binding affinities of the mouse antisera and all four involved proteins, together with GST as the proper control, were proved in ELISA (Figure 4E). To further test the changes in intermembrane-binding ability in vivo, LLP12 fused with EGFP was inserted into pCDNA3 plasmid, which was then transfected into 293T cells. These cells were examined under fluorescence microscope 24 h later (Figure 4F). There were intense fluorescence signals in the perinuclear area of EGFP-LLP12 in contrast to EGFP control, and this confirmed previous research studies on LLP12 intermembrane-binding ability ( 12 ). A similar phenomenon was observed in cells transfected with EGFP-LLPM1, EGFP-LLPM2, and EGFP-LLPM3, indicating that none of these mutants notably affected their intermembrane targeting signal on LLP12. A possibility exists that other membrane or scaffold proteins were involved in this membrane-binding process, as exemplified by the calmodulin system ( 7, 16-18 ).

(A–D) Flow cytometry analysis of LLP12 wild type and mutants binding to 293T cell membrane. I (black solid line), Negative control (293T cells alone), same in all four figures II (dotted line), 293T cells incubated with different samples. III (gray solid line), Positive control (293T cells incubated with 50 μg/mL LLP12), same in all four figures. AII, cells incubated with 25 μg/mL LLP12. BII, 25 μg/mL LLPM1. CII, 25 μg/mL LLPM2. DII, 25 μg/mL LLPM3. E, Identification of the binding affinity of the mouse antisera and all four involved proteins with GST in enzyme-linked immunosorbent assay. All five proteins were diluted (1:10, 2:5, 4:2.5, and 4:1.25 μg/mL) and coated on a microtiter plate. The binding of mouse antisera was detected by peroxidase-conjugated rabbit immunoglobulins to mouse IgG (Sigma, St Louis, MO, USA). F, Examination of subcellular localization of EGFP-LLP12 recombinant proteins by cell transfection under a fluorescence microscope (Leica Microsystems, Heidelberg GmbH, Germany). HeLa cells were transfected by pCDNA3/EGFP (A), pDNA3/EGFP-LLP12 (B), pCDNA3/EGFP-LLPM1 (C), pCDNA3/EGFP-LLPM2 (D), and pCDNA3/EGFP-LLPM3 (E). Cells were examined under the fluorescence microscope 24 h after transfection.


Conclusions and perspectives

HIV-1 has been analyzed by structural biology techniques more so than any other virus, with partial or complete structures known for all 16 of its protein components and additional structures determined for substrate- and host factor-bound complexes. Structural biology will continue to have a significant impact on HIV/AIDS research moving forward by providing high-resolution glimpses of target protein𠄽rug complexes and viral–host interactions, such as CA–TRIM5α, Vif𠄺POBEC3G or Vpu–tetherin, which will reveal novel druggable sites. Despite decades of research, the interactions between HIV-1 and host proteins that underlie some steps in the viral life cycle, for example the import of the preintegration complex into the nucleus ( Fig. 1 , step 5), are only now being illuminated. The simian immunodeficiency virus Vpx protein was moreover recently shown to counteract the SAMHD1 restriction factor that inhibits HIV-1 reverse transcription and infection of monocytic cells 160,161 , indicating that these protein complexes could define new pathways for antiviral drug developmentas well.

Notwithstanding the ongoing work with PIs, it will be interesting to see if structure-based substrate/inhibitor envelope hypotheses will apply to the development of other HIV-1 inhibitors. Because NNRTIs form induced fit binding pockets, they would appear to be poor candidates for this technique. The relatively tight overlay of multiple bound drugs at the IN active site and similarities in drug positions with the ejected terminal adenosine base 88 hints that INSTIs could be another drug class to benefit from such approaches. 3D structures of new drug targets as well as inhibitor or antibody-bound targets will predictably increase the pace of antiviral development and help guide vaccine development efforts 162,163 . The development of new technologies and improvements in existing methods will also significantly influence structural virology moving forward. Single-particle electron cryo-microscopy has recently yielded near-atomic resolution structures of a number of so-called naked viruses that, unlike HIV-1, lack an exterior envelope lipid bilayer 164 . Although the icosahedral symmetry underlying these structures greatly facilitated their determination, ongoing developments in instrumentation and computational science may very well yield similar resolution structures for particles that possess less inherent symmetry.

The development of HAART has dramatically changed the face of the HIV/AIDS epidemic since the disease was first recognized 30 years ago. Considered virtually a death sentence prior to the advent of anti-retroviral drugs, HIV-1 infection is now a manageable chronic disease. Yet, despite these remarkable advances, there remains significant room for improvement. Some of the drugs, in particular the PIs, exert toxic side-effects. More tolerable antiviral regimens could strengthen patient compliance and consequently reduce the emergence of resistant strains. Although the recently approved INSTI raltegravir is apparently non-toxic, the relative ease by which it selects for drug resistant strains highlights the need for second-generation INSTIs with more favorable genetic barriers to the resistance. The development of compounds that inhibit functions of less explored drug targets, in particular of the accessory HIV-1 proteins and host factors, would be of obvious benefit as well. The availability and efficacy of the current arsenal of anti-retroviral drugs should not be taken for granted. It is important to bear in mind, that the majority of the HIV-infected population do not have access to the advanced treatment options. Short of an effective vaccination strategy, the ongoing race against drug resistance can best be won by sustained effort to develop novel ever more potent and tolerable antiviral approaches.

At a glance

HIV-1 replication relies on the proper functioning of specific viral proteins and three of these, protease, integrase, and reverse transcriptase with associated RNase H activity, are enzymes. Antiviral drugs that inhibit protease, integrase and reverse transcriptase DNA polymerase activities are approved for treating AIDS patients. The highly active anti-retroviral therapy or HAART regimens utilize cocktails of three inhibitors to suppress HIV-1 replication and the outgrowth of drug resistant viral strains.

HIV-1 replication depends on a plethora of functional interactions between its proteins and those of the host. Other cellular proteins, which are referred to as restriction factors, work to counteract virus growth. TRIM5α, APOBEC3G, tetherin and SAMHD1 are examples of such restriction factors.

In addition to enzyme active sites, critical viral-host protein interactions define targets for therapeutic intervention. Drugs might block interactions between the virus and host proteins needed for replication, as is the case for the approved entry inhibitor maraviroc, or enhance the effects of cell restriction factors.

Neutralization of the viral envelope glycoprotein gp120 by the adaptive immune system underscores AIDS vaccine development strategies.

Structural biology studies yield three-dimensional glimpses of protein function at near atomic resolution. Such results form the cornerstones of modern antiviral drug and vaccine development efforts.


The HIV lifecycle

HIV infects a type of white blood cell in the body’s immune system called a T-helper cell (also called a CD4 cell). These vital cells keep us healthy by fighting off infections and diseases.

HIV cannot reproduce on its own. Instead, the virus attaches itself to a T-helper cell and fuses with it (joins together). It then takes control of the cell’s DNA, makes copies of itself inside the cell, and finally releases more HIV into the blood. HIV will continue to multiply and spread throughout the body – a process called the HIV lifecycle.

In this way, HIV weakens the body’s natural defences and over time severely damages the immune system. How quickly the virus develops depends on a person’s general health, how quickly they are diagnosed and start antiretroviral treatment, and how consistently they take their treatment.

Antiretroviral treatment and the HIV lifecycle

Antiretroviral treatment for HIV combines several different types of drugs, each of which targets a different stage in the HIV lifecycle. This means that the replication of HIV is stopped on multiple fronts, making it very effective.

If taken correctly, it keeps the immune system healthy, prevents the symptoms and illnesses associated with AIDS from developing, and means that people can enjoy long and healthy lives.

If someone doesn’t take their treatment correctly or consistently (at the right time every day), the level of HIV in their blood may increase and the drugs may no longer work. This is known as developing drug resistance.

Stages of the HIV lifecycle

Binding and fusion (attachment)

HIV attaches to a T-helper cell. It then fuses to it and releases its genetic information into the cell.

The types of drugs that stop this stage of the lifecycle are called fusion or entry inhibitor drugs – because they stop HIV from entering the cell.

Reverse transcription (conversion) and integration

Once inside the T-helper cell, HIV converts its genetic material into HIV DNA, a process called reverse transcription. The new HIV DNA then enters the nucleus of the host cell and takes control of it.

The types of drugs that stop this stage of the lifecycle are called NRTIs (nucleoside reverse transcriptase inhibitors), NNRTIs (non-nucleoside reverse transcriptase inhibitors) and integrase inhibitor drugs.

Transcription and translation (replication)

The infected T-helper cell then produces HIV proteins that are used to produce more HIV particles inside the cell.

Assembly, budding and maturation

The new HIV is put together and then released from the T-helper cell into the bloodstream to infect other cells and so the process begins again.

The type of drugs that stop this stage of the lifecycle are called protease inhibitor (PI) drugs.


How Viruses Replicate

Viruses were first discovered after the development of a porcelain filter, called the Chamberland-Pasteur filter, which could remove all bacteria visible under the microscope from any liquid sample. In 1886, Adolph Meyer demonstrated that a disease of tobacco plants, tobacco mosaic disease, could be transferred from a diseased plant to a healthy one through liquid plant extracts. In 1892, Dmitri Ivanowski showed that this disease could be transmitted in this way even after the Chamberland-Pasteur filter had removed all viable bacteria from the extract. Still, it was many years before it was proven that these &ldquofilterable&rdquo infectious agents were not simply very small bacteria but were a new type of tiny, disease-causing particle.

Virions, single virus particles, are very small, about 20&ndash250 nanometers (1 nanometer = 1/1,000,000 mm). These individual virus particles are the infectious form of a virus outside the host cell. Unlike bacteria (which are about 100 times larger), we cannot see viruses with a light microscope, with the exception of some large virions of the poxvirus family (Figure 17.1.2).

Figure 17.1.2: The size of a virus is very small relative to the size of cells and organelles.

It was not until the development of the electron microscope in the 1940s that scientists got their first good view of the structure of the tobacco mosaic virus (Figure) and others. The surface structure of virions can be observed by both scanning and transmission electron microscopy, whereas the internal structures of the virus can only be observed in images from a transmission electron microscope (Figure 17.1.3).

Figure 17.1.3: The ebola virus is shown here as visualized through (a) a scanning electron micrograph and (b) a transmission electron micrograph. (credit a: modification of work by Cynthia Goldsmith, CDC credit b: modification of work by Thomas W. Geisbert, Boston University School of Medicine scale-bar data from Matt Russell)

The use of this technology has allowed for the discovery of many viruses of all types of living organisms. They were initially grouped by shared morphology, meaning their size, shape, and distinguishing structures. Later, groups of viruses were classified by the type of nucleic acid they contained, DNA or RNA, and whether their nucleic acid was single- or double-stranded. More recently, molecular analysis of viral replication cycles has further refined their classification.

A virion consists of a nucleic-acid core, an outer protein coating, and sometimes an outer envelope made of protein and phospholipid membranes derived from the host cell. The most visible difference between members of viral families is their morphology, which is quite diverse. An interesting feature of viral complexity is that the complexity of the host does not correlate to the complexity of the virion. Some of the most complex virion structures are observed in bacteriophages, viruses that infect the simplest living organisms, bacteria.

Viruses come in many shapes and sizes, but these are consistent and distinct for each viral family (Figure 17.1.4). All virions have a nucleic-acid genome covered by a protective layer of protein, called a capsid. The capsid is made of protein subunits called capsomeres. Some viral capsids are simple polyhedral &ldquospheres,&rdquo whereas others are quite complex in structure. The outer structure surrounding the capsid of some viruses is called the viral envelope. All viruses use some sort of glycoprotein to attach to their host cells at molecules on the cell called viral receptors. The virus exploits these cell-surface molecules, which the cell uses for some other purpose, as a way to recognize and infect specific cell types. For example, the measles virus uses a cell-surface glycoprotein in humans that normally functions in immune reactions and possibly in the sperm-egg interaction at fertilization. Attachment is a requirement for viruses to later penetrate the cell membrane, inject the viral genome, and complete their replication inside the cell.

The T4 bacteriophage, which infects the E. coli bacterium, is among the most complex virion known T4 has a protein tail structure that the virus uses to attach to the host cell and a head structure that houses its DNA.

Adenovirus, a nonenveloped animal virus that causes respiratory illnesses in humans, uses protein spikes protruding from its capsomeres to attach to the host cell. Nonenveloped viruses also include those that cause polio (poliovirus), plantar warts (papillomavirus), and hepatitis A (hepatitis A virus). Nonenveloped viruses tend to be more robust and more likely to survive under harsh conditions, such as the gut.

Enveloped virions like HIV (human immunodeficiency virus), the causative agent in AIDS (acquired immune deficiency syndrome), consist of nucleic acid (RNA in the case of HIV) and capsid proteins surrounded by a phospholipid bilayer envelope and its associated proteins (Figure 17.1.4). Chicken pox, influenza, and mumps are examples of diseases caused by viruses with envelopes. Because of the fragility of the envelope, nonenveloped viruses are more resistant to changes in temperature, pH, and some disinfectants than enveloped viruses.

Overall, the shape of the virion and the presence or absence of an envelope tells us little about what diseases the viruses may cause or what species they might infect, but is still a useful means to begin viral classification.

Figure 17.1.4: Viruses can be complex in shape or relatively simple. This figure shows three relatively complex virions: the bacteriophage T4, with its DNA-containing head group and tail fibers that attach to host cells adenovirus, which uses spikes from its capsid to bind to the host cells and HIV, which uses glycoproteins embedded in its envelope to do so. Notice that HIV has proteins called matrix proteins, internal to the envelope, which help stabilize virion shape. HIV is a retrovirus, which means it reverse transcribes its RNA genome into DNA, which is then spliced into the host&rsquos DNA. (credit &ldquobacteriophage, adenovirus&rdquo: modification of work by NCBI, NIH credit &ldquoHIV retrovirus&rdquo: modification of work by NIAID, NIH)

Which of the following statements about virus structure is true?

  1. All viruses are encased in a viral membrane.
  2. The capsomere is made up of small protein subunits called capsids.
  3. DNA is the genetic material in all viruses.
  4. Glycoproteins help the virus attach to the host cell.

DNA viruses have a DNA core. The viral DNA directs the host cell&rsquos replication proteins to synthesize new copies of the viral genome and to transcribe and translate that genome into viral proteins. DNA viruses cause human diseases such as chickenpox, hepatitis B, and some venereal diseases like herpes and genital warts.

RNA viruses contain only RNA in their cores. To replicate their genomes in the host cell, the genomes of RNA viruses encode enzymes not found in host cells. RNA polymerase enzymes are not as stable as DNA polymerases and often make mistakes during transcription. For this reason, mutations, changes in the nucleotide sequence, in RNA viruses occur more frequently than in DNA viruses. This leads to more rapid evolution and change in RNA viruses. For example, the fact that influenza is an RNA virus is one reason a new flu vaccine is needed every year. Human diseases caused by RNA viruses include hepatitis C, measles, and rabies.

Viruses can be seen as obligate intracellular parasites. The virus must attach to a living cell, be taken inside, manufacture its proteins and copy its genome, and find a way to escape the cell so the virus can infect other cells and ultimately other individuals. Viruses can infect only certain species of hosts and only certain cells within that host. The molecular basis for this specificity is that a particular surface molecule, known as the viral receptor, must be found on the host cell surface for the virus to attach. Also, metabolic differences seen in different cell types based on differential gene expression are a likely factor in which cells a virus may use to replicate. The cell must be making the substances the virus needs, such as enzymes the virus genome itself does not have genes for, or the virus will not be able to replicate using that cell.

Steps of Virus Infections

A virus must &ldquotake over&rdquo a cell 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 (rhinovirus), die through lysis (bursting) or apoptosis (programmed cell death or &ldquocell suicide&rdquo), releasing all the progeny virions at once. The symptoms of viral diseases result from the immune response to the virus, which attempts to control and eliminate the virus from the body, and from cell damage caused by the virus. 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 HIV infects may make it impossible for the cells to function as mediators of immunity, 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.

A virus attaches to a specific receptor site on the host-cell membrane through attachment proteins in the capsid or proteins embedded in its envelope. The attachment is specific, and typically a virus will only attach to cells of one or a few species and only certain cell types within those species with the appropriate receptors.

View this video for a visual explanation of how influenza attacks the body.

Unlike animal viruses, the nucleic acid of bacteriophages is injected into the host cell naked, leaving the capsid outside the cell. Plant and animal viruses can enter their cells through endocytosis , in which the cell membrane surrounds and engulfs the entire virus. Some enveloped viruses enter the cell when the viral envelope fuses directly with the cell membrane. Once inside the cell, the viral capsid is degraded and the viral nucleic acid is released, which then becomes available for replication and transcription.

The replication mechanism depends on the viral genome. DNA viruses usually use host cell proteins and enzymes to make additional DNA that is used to copy the genome or be transcribed to messenger RNA (mRNA), which is then used in protein synthesis. RNA viruses, such as the influenza virus, usually use the RNA core as a template for synthesis of viral genomic RNA and mRNA. The viral mRNA is translated into viral enzymes and capsid proteins to assemble new virions (Figure). Of course, there are exceptions to this pattern. If a host cell does not provide the enzymes necessary for viral replication, viral genes supply the information to direct synthesis of the missing proteins. Retroviruses, such as HIV, have an RNA genome that must be reverse transcribed to make DNA, which then is inserted into the host&rsquos DNA. To convert RNA into DNA, retroviruses contain genes that encode the virus-specific enzyme reverse transcriptase that transcribes an RNA template to DNA. The fact that HIV produces some of its own enzymes, which are not found in the host, has allowed researchers to develop drugs that inhibit these enzymes. These drugs, including the reverse transcriptase inhibitor AZT, inhibit HIV replication by reducing the activity of the enzyme without affecting the host&rsquos metabolism.

The last stage of viral replication is the release of the new virions into the host organism, where they are able to infect adjacent cells and repeat the replication cycle. Some viruses are released when the host cell dies and other viruses can leave infected cells by budding through the membrane without directly killing the cell.

Figure 17.1.5: In influenza virus infection, glycoproteins attach to a host epithelial cell. As a result, the virus is engulfed. RNA and proteins are made and assembled into new virions.

Influenza virus is packaged in a viral envelope, which fuses with the plasma membrane. This way, the virus can exit the host cell without killing it. What advantage does the virus gain by keeping the host cell alive?

Click through this tutorial on viruses to identify structures, modes of transmission, replication, and more.

Viruses cause a variety of diseases in animals, including humans, ranging from the common cold to potentially fatal illnesses like meningitis (Figure 17.1.6). These diseases can be treated by antiviral drugs or by vaccines, but some viruses, such as HIV, are capable of avoiding the immune response and mutating so as to become resistant to antiviral drugs.

Figure 17.1.6: Viruses are the cause of dozens of ailments in humans, ranging from mild illnesses to serious diseases. (credit: modification of work by Mikael Häggström)

Vaccines for Prevention

While we do have limited numbers of effective antiviral drugs, such as those used to treat HIV and influenza, the primary method of controlling viral disease is by vaccination, which is intended to prevent outbreaks by building immunity to a virus or virus family. A vaccine may be prepared using weakened live viruses, killed viruses, or molecular subunits of the virus. In general, live viruses lead to better immunity, but have the possibility of causing disease at some low frequency. Killed viral vaccine and the subunit viruses are both incapable of causing disease, but in general lead to less effective or long-lasting immunity.

Weakened live viral vaccines are designed in the laboratory to cause few symptoms in recipients while giving them immunity against future infections. Polio was one disease that represented a milestone in the use of vaccines. Mass immunization campaigns in the U.S. in the 1950s (killed vaccine) and 1960s (live vaccine) essentially eradicated the disease, which caused muscle paralysis in children and generated fear in the general population when regional epidemics occurred. The success of the polio vaccine paved the way for the routine dispensation of childhood vaccines against measles, mumps, rubella, chickenpox, and other diseases.

Live vaccines are usually made by attenuation (weakening) of the &ldquowild-type&rdquo (disease-causing) virus by growing it in the laboratory in tissues or at temperatures different from what the virus is accustomed to in the host. For example, the virus may be grown in cells in a test tube, in bird embryos, or in live animals. The adaptation to these new cells or temperature induces mutations in the virus&rsquo genomes, allowing them to grow better in the laboratory while inhibiting their ability to cause disease when reintroduced into the conditions found in the host. These attenuated viruses thus still cause an infection, but they do not grow very well, allowing the immune response to develop in time to prevent major disease. The danger of using live vaccines, which are usually more effective than killed vaccines, is the low but significant risk that these viruses will revert back to their disease-causing form by back mutations. Back mutations occur when the vaccine undergoes mutations in the host such that it readapts to the host and can again cause disease, which can then be spread to other humans in an epidemic. This happened as recently as 2007 in Nigeria where mutations in a polio vaccine led to an epidemic of polio in that country.

Some vaccines are in continuous development because certain viruses, such as influenza and HIV, have a high mutation rate compared to other viruses or host cells. With influenza, mutation in genes for the surface molecules helps the virus evade the protective immunity that may have been obtained in a previous influenza season, making it necessary for individuals to get vaccinated every year. Other viruses, such as those that cause the childhood diseases measles, mumps, and rubella, mutate so little that the same vaccine is used year after year.

Vaccines and Antiviral Drugs for Treatment

In some cases, vaccines can be used to treat an active viral infection. In the case of rabies, a fatal neurological disease transmitted in the saliva of rabies virus-infected animals, the progression of the disease from the time of the animal bite to the time it enters the central nervous system may be two weeks or longer. This is enough time to vaccinate an individual who suspects being bitten by a rabid animal, and the boosted immune response from the vaccination is enough to prevent the virus from entering nervous tissue. Thus, the fatal neurological consequences of the disease are averted and the individual only has to recover from the infected bite. This approach is also being used for the treatment of Ebola, one of the fastest and most deadly viruses affecting humans, though usually infecting limited populations. Ebola is also a leading cause of death in gorillas. Transmitted by bats and great apes, this virus can cause death in 70&ndash90 percent of the infected within two weeks. Using newly developed vaccines that boost the immune response, there is hope that immune systems of affected individuals will be better able to control the virus, potentially reducing mortality rates.

Another way of treating viral infections is the use of antiviral drugs. These drugs often have limited ability to cure viral disease but have been used to control and reduce symptoms for a wide variety of viral diseases. For most viruses, these drugs inhibit the virus by blocking the actions of one or more of its proteins. It is important that the targeted proteins be encoded for by viral genes and that these molecules are not present in a healthy host cell. In this way, viral growth is inhibited without damaging the host. There are large numbers of antiviral drugs available to treat infections, some specific for a particular virus and others that can affect multiple viruses.

Antivirals have been developed to treat genital herpes (herpes simplex II) and influenza. For genital herpes, drugs such as acyclovir can reduce the number and duration of the episodes of active viral disease during which patients develop viral lesions in their skins cells. As the virus remains latent in nervous tissue of the body for life, this drug is not a cure but can make the symptoms of the disease more manageable. For influenza, drugs like Tamiflu can reduce the duration of &ldquoflu&rdquo symptoms by one or two days, but the drug does not prevent symptoms entirely. Other antiviral drugs, such as Ribavirin, have been used to treat a variety of viral infections.

By far the most successful use of antivirals has been in the treatment of the retrovirus HIV, which causes a disease that, if untreated, is usually fatal within 10&ndash12 years after being infected. Anti-HIV drugs have been able to control viral replication to the point that individuals receiving these drugs survive for a significantly longer time than the untreated.

Anti-HIV drugs inhibit viral replication at many different phases of the HIV replicative cycle. Drugs have been developed that inhibit the fusion of the HIV viral envelope with the plasma membrane of the host cell (fusion inhibitors), the conversion of its RNA genome to double-stranded DNA (reverse transcriptase inhibitors), the integration of the viral DNA into the host genome (integrase inhibitors), and the processing of viral proteins (protease inhibitors).

When any of these drugs are used individually, the virus&rsquo high mutation rate allows the virus to rapidly evolve resistance to the drug. The breakthrough in the treatment of HIV was the development of highly active anti-retroviral therapy (HAART), which involves a mixture of different drugs, sometimes called a drug &ldquococktail.&rdquo By attacking the virus at different stages of its replication cycle, it is difficult for the virus to develop resistance to multiple drugs at the same time. Still, even with the use of combination HAART therapy, there is concern that, over time, the virus will evolve resistance to this therapy. Thus, new anti-HIV drugs are constantly being developed with the hope of continuing the battle against this highly fatal virus.


HIV and AIDS: Tracking a Sexually Transmitted Virus

AIDS was a mystery disease when first formally recognized in patients in the U.S. in 1981. Transmission through sexual contact was rapidly recognized, but the source was initially unknown. Just three years later, inspired work by disease detectives (epidemiologists) identified the cause, later officially dubbed Human Immunodeficiency Virus (HIV). No vaccine or effective cure exists, and with untreated infections, the average survival is only 11 years.

Transmission of HIV

HIV belongs to the retroviruses, remarkably simple organisms with two tiny RNA molecules inside a spherical envelope that somehow escapes destruction by the host’s immune system. Each RNA strand contains just nine genes. Any retrovirus—hence the name—has the special feature that, after invading a cell, a DNA replica of its genetic material is spliced into the host’s genome. Once inserted, the viral genes exploit the cell’s machinery to produce hordes of new viral particles.

Among adults, HIV is transmitted mainly by unprotected sex but also by unsterile hypodermic needles and transfusions of contaminated blood. Mother-child transmission can occur during pregnancy, birth, or breastfeeding. Rather belatedly, it was recognized that HIV could pass from infected infants to breastfeeding mothers or female caretakers. According to a 2012 review by Kristen Little and colleagues, approximately half of women breastfeeding infected infants contract the disease.

Few symptoms are apparent at first infection, although a brief flu-like illness may ensue. A lengthy symptom-free stage typically follows, but the developing disease increasingly disrupts the immune system, opening the way for familiar infections (e.g., tuberculosis) and certain cancers (notably Kaposi’s sarcoma) that rarely occur with normally functioning immunity. During this final stage, weight loss is also common. The name Acquired Immunodeficiency Syndrome (AIDS) refers to this suite of symptoms. As a rule, in the absence of antiviral therapies, HIV infection takes about a decade to become full-blown AIDS.

Tracing HIV origins

It is rarely appreciated that investigation of HIV directly benefitted from already available computer programs for generating pedigrees from DNA sequences. Beginning in the 1960s, evolutionary biologists had developed and fine-tuned these crucial tools to explore relationships in the Tree of Life. Tried and tested procedures were thus at hand for swift redeployment to reveal HIV’s origins. As the virus proliferates within and between patients, it rapidly accumulates mutations, especially in regions of the envelope gene targetted by the host’s immune system. Analyses of those changes unveil its history.

HIV falls among the lentiviruses, which typically cause diseases with an extended incubation in many mammal species. All lentiviruses provoke long-term infections, but the outcome ranges between the complete absence of symptoms and the development of fatal immunodeficiency. From the outset, it seemed likely that HIV arose through cross-infection from another mammal.

However, in one of its first important findings, tree-building soon revealed two different types of HIV, now regarded as separate species. The first (particularly infective and virulent) type discovered, labeled HIV-1, is the predominant cause of AIDS worldwide. But further research identified a less virulent and less easily transmitted second type, dubbed HIV-2, confined to West Africa.

In 1994, Andrew Leigh Brown and Edward Holmes published a landmark review of the evolution of HIV. As it happens, Eddie Holmes began his university training as one of my Anthropology students at University College London (1983-1986). Years later, he told me that my lectures on primate evolution had inspired his research interest. His stellar career in the scientific study of viruses causing major human diseases—from hepatitis through HIV to COVID-19—corroborated my long-standing conviction that a clear understanding of human health demands familiarity with evolutionary biology.

Leigh Brown and Holmes analyzed several HIV-like viruses found in various African monkeys and apes—Simian Immunodeficiency Viruses (SIVs)—and examined their relationships using tree-building. It emerged that HIV-1 originated from a virus passed from common chimpanzees to human populations in equatorial West Africa, whereas HIV-2 groups with SIVs in African monkeys, being closest to one in sooty mangabeys.

Subsequently, in a 2010 review, Paul Sharp and Beatrice Hahn discussed updated evidence relating to the evolution of HIV-1. They confirmed that the closest relatives of HIV-1 are SIVs infecting the wild populations of chimpanzees and gorillas in West Africa. Analyses confirmed that the initial hosts were chimpanzees.

In fact, four lineages of HIV-1 evidently arose in chimpanzees. At least two were transmitted directly to humans, while one or two may have been transmitted indirectly via gorillas. Sharp and Hahn found that, as with human HIV, SIV infection in wild chimpanzees increases mortality by depleting particular helper cells (CD4+ T-cells) that trigger responses to infections.

The history of human AIDS

Although it is now well established that the AIDS pandemic originally began in Africa, the date long remained uncertain. To gain insight into underlying factors, inferring a reliable date for the source is very important. Because AIDS was first diagnosed in the U.S., the location and timing of the initial infection there has also been much discussed. An airline steward who died in 1984 ("Patient Zero") was named and unfairly decried as the likely source, notably by Randy Shilts in his 1987 book And the Band Played On.

A 2016 paper by Michael Worobey and colleagues eventually reconstructed the early history of HIV/AIDS in North America. They noted that identification of a particular strain of HIV-1—group M subtype B—was a crucial advance. Previous research had indicated that subtype B circulated undetected in the U.S. during the 1970s and even earlier in the Caribbean.

Using a method developed to recover viral RNA from degraded samples, Worobey and colleagues screened over 2,000 archived serum specimens from the 1970s. They managed to obtain eight complete HIV-1 sequences from 1978-1979. Analyses revealed that a pre-existing Caribbean epidemic was the likely original source, with the U.S. epidemic most probably beginning with an ancestral HIV in New York. Worobey and colleagues also sequenced the HIV-1 genome of "Patient Zero," finding that he was neither the source of subtype B nor even the primary case in the U.S.

In a very recent paper published in 2020, Sophie Gryseels and colleagues (including Worobey) reported findings from an almost complete HIV-1 genome dating back to 1966, the oldest so far recovered. They conducted an extensive search of 1,645 tissue samples collected in Central Africa in 1958-1966 for pathological study. Their needle-in-a-haystack search paid off. One sample, from Kinshasa in the Democratic Republic of Congo (DRC), was HIV-1 positive and could be allocated to a sister lineage to subtype C in group M. Overall analyses indicated that the pandemic lineage of HIV-1 originated at some time around 1900.

Tracing the origins of HIV from non-human primates in Africa has also clarified another issue. Edward Hooper, in his much-publicized 1999 book The River, claimed that the AIDS epidemic was incidentally triggered by an oral polio vaccine administered to hundreds of thousands of Africans in 1957-1960. He stated that the region concerned—DRC, Burundi, and Rwanda—was the source of the group M strain of HIV-1 virus. He further claimed that the polio vaccine was developed in the DRC using chimpanzee cells that contained an SIV and allegedly contaminated the vaccine. A special meeting convened at the Royal Society (London) in 2000 invalidated these claims. In fact, production took place in the U.S. at the Wistar Institute in Philadelphia, and no chimpanzee cells were used.

Applying the tree-building tools developed by evolutionary biologists to ever-larger large datasets eventually permitted reliable dating. It became increasingly clear that the AIDS pandemic began when HIV descended from a chimpanzee SIV around 1900, 50 years earlier than Hooper’s imagined and now rejected scenario.

An alternative, far less controversial, proposal mentioned but not really pursued by Hopper was that the re-use of unsterilized needles and transfusion of unscreened blood drove the proliferation of HIV in Africa. These factors undoubtedly contributed to the HIV pandemic.

From HIV to COVID-19

Procedures developed and lessons learned from the investigation of the AIDS pandemic greatly facilitated scientific responses to the current coronavirus pandemic. The same tree-building methods derived from evolutionary biology permitted prompt tracking of the galloping progress of COVID-19 around the world.

As with HIV, rapidly accumulating mutations complicate monitoring efforts but also yield valuable data for tree-building. One particularly significant application was testing a proposal that this coronavirus had been artificially created in a laboratory in China. Earlier this year, using well-established methods for tracking origins, an international team of authors, including Eddie Holmes, effectively ruled out purposeful manipulation as the source.

Andersen, K.G., Rambaut, A., Lipkin, W.I., Holmes, E.C. & Garry, R.F. (2020) The proximal origin of SARS-CoV-2. Nature Medicine 26:450-452.

Faria, N.R., Rambaut, A., Suchard, M.A., Baele, G., Bedford, T., Ward, M., Tatem, A.J., Sousa, J.D., Arinaminpathy, N., Pépin, J., Posada, D., Peeters, M., Pybus, O.G. & Lemey, P. (2014) The early spread and epidemic ignition of HIV-1 in human populations. Science 346:56-61.

GBD 2017 HIV Collaborators (2019) Global, regional, and national incidence, prevalence, and mortality of HIV, 1980–2017, and forecasts to 2030, for 195 countries and territories: a systematic analysis for the Global Burden of Diseases, Injuries, and Risk Factors Study 2017. Lancet HIV 6:e831-859.

Gryseels, S., Watts, T.D., Mpolesha, J.-M.K., Larsen, B.B., Lemey, P., Muyembe-Tamfum, J.-J., Teuwen, D.E. & Worobey, M. (2019) A near-full-length HIV-1 genome from 1966 recovered from formalin-fixed paraffin-embedded tissue. bioRxiv 1-15. [subsequently published in Proceedings of the National Academy of Sciences U.S.A. 117:12222-12229]

Holmes, E.C. (2001) On the origin and evolution of the human immunodeficiency virus (HIV). Biological Reviews 76:239-254.

Hooper, E. (1999) The River: A Journey to the Source of HIV and AIDS. New York: Little Brown & Co.



Comments:

  1. Kamau

    There is something in it, too, it seems to me an excellent idea. I agree with you.

  2. James

    You are making a mistake. I propose to discuss it. Email me at PM, we'll talk.



Write a message