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How does HIV survive natural selection? And how has it managed to kill far more than any non-airborne virus in recorded history?
Human Immunodeficiency Virus is a mutated form of Simian Immunodeficiency Virus. In simians (Apes and Monkeys, not including humans), SIV is not pathogenic, in most cases, however, when the mutated form made the jump to humans, it became highly contagious and virulent. You can find a basic description in the wikipedia article here: Simian Immunodeficiency virus.
As for your second question, I would likely say that influenza has killed more humans in total than HIV, so you would have to provide a reference for your claim.
However, because HIV is a retrovirus that incorporates into the genome of the immune cells of its host, it can survive in the body for long periods of time in a dormant state. A person may not know they are infected for months or years, and they can pass the virus on to other people. Slow killing viruses such as HIV are very dangerous, as they can be transmitted to large swaths of the population before people are aware they are sick.
Based on comments, there may be a misunderstanding of the term virulence. From The Karolinska Instetutet medical subject heading (MeSH) definition:
The degree of pathogenicity within a group or species of microorganisms or viruses as indicated by case fatality rates and/or the ability of the organism to invade the tissues of the host. The pathogenic capacity of an organism is determined by its VIRULENCE FACTORS.
-The Karolinska Institutet; Virulence
The emphasis is mine, but virulence also refers to the ability of the virus to infect its host, and does not necessary lead to the hosts death. The virus that causes the common cold is highly virulent, but for almost the entire human population it causes nothing more than discomfort.
A virus like Ebola on the other hand is highly virulent in both senses of the term. The reason that there are far fewer cases of Ebola is that it kills the human host so quickly, that an afflicted person will on average only go on to infect about two other individuals, mainly because victims 1) die quickly, 2) are only contagious after symptoms manifest themselves, and 3) requires direct contact with the infected person's bodily fluids.
Contrast that with Influenza which a single person can infect tens to hundreds of people and is contagious before they are displaying symptoms and spreads it with casual contact, or as stated above, with HIV, where a person can be contagious for weeks, months, or even years before they are aware that they are infected, and you see why HIV is very sustainable in the Human population.
HIV also has the added weapon in its arsenal that it can lay dormant in cells for long periods of time so as to escape the detection of the host immune system, and as the disease attacks and kills immune cells, when the dormant virus activates again there are far fewer specialized cells to address the active infection.
Another thing is it is the comorbidity with other diseases that often results in the death of a patient with an HIV infection. AIDS results from other diseases having the opportunity to go unchecked after HIV has decimated the host immune system.
History of HIV/AIDS
AIDS is caused by a human immunodeficiency virus (HIV), which originated in non-human primates in Central and West Africa. While various sub-groups of the virus acquired human infectivity at different times, the global pandemic had its origins in the emergence of one specific strain – HIV-1 subgroup M – in Léopoldville in the Belgian Congo (now Kinshasa in the Democratic Republic of the Congo) in the 1920s. 
There are two types of HIV: HIV-1 and HIV-2. HIV-1 is more virulent, easily transmitted and is the cause of the vast majority of HIV infections globally.  The pandemic strain of HIV-1 is closely related to a virus found in chimpanzees of the subspecies Pan troglodytes troglodytes, which live in the forests of the Central African nations of Cameroon, Equatorial Guinea, Gabon, the Republic of the Congo (or Congo-Brazzaville), and the Central African Republic. HIV-2 is less transmittable and is largely confined to West Africa, along with its closest relative, a virus of the sooty mangabey (Cercocebus atys atys), an Old World monkey inhabiting southern Senegal, Guinea-Bissau, Guinea, Sierra Leone, Liberia, and western Ivory Coast.  
The existence of long-lived reservoirs of latently infected CD4+ T cells is the major barrier to curing HIV, and has been extensively studied in this light. However, the effect of these reservoirs on the evolutionary dynamics of the virus has received little attention. Here, we present a within-host quasispecies model that incorporates a long-lived reservoir, which we then nest into an epidemiological model of HIV dynamics. For biologically plausible parameter values, we find that the presence of a latent reservoir can severely delay evolutionary dynamics within a single host, with longer delays associated with larger relative reservoir sizes and/or homeostatic proliferation of cells within the reservoir. These delays can fundamentally change the dynamics of the virus at the epidemiological scale. In particular, the delay in within-host evolutionary dynamics can be sufficient for the virus to evolve intermediate viral loads consistent with maximising transmission, as is observed, and not the very high viral loads that previous models have predicted, an effect that can be further enhanced if viruses similar to those that initiate infection are preferentially transmitted. These results depend strongly on within-host characteristics such as the relative reservoir size, with the evolution of intermediate viral loads observed only when the within-host dynamics are sufficiently delayed. In conclusion, we argue that the latent reservoir has important, and hitherto under-appreciated, roles in both within- and between-host viral evolution.
Horton, R.E., McLaren, P.J., Fowke, K., Kimani, J. & Ball, T.B. Cohorts for the study of HIV-1-exposed but uninfected individuals: benefits and limitations. J. Infect. Dis. 202 (suppl. 3), S377–S381 (2010).
An, P. & Winkler, C.A. Host genes associated with HIV/AIDS: advances in gene discovery. Trends Genet. 26, 119–131 (2010).
Lane, J. et al. A genome-wide association study of resistance to HIV infection in highly exposed uninfected individuals with hemophilia A. Hum. Mol. Genet. 22, 1903–1910 (2013).
McLaren, P.J. et al. Association study of common genetic variants and HIV-1 acquisition in 6,300 infected cases and 7,200 controls. PLoS Pathog. 9, e1003515 (2013).
Gurdasani, D. et al. A systematic review of definitions of extreme phenotypes of HIV control and progression. AIDS 28, 149–162 (2014).
Kaslow, R.A. et al. Influence of combinations of human major histocompatibility complex genes on the course of HIV-1 infection. Nat. Med. 2, 405–411 (1996).
Carrington, M. et al. HLA and HIV-1: heterozygote advantage and B*35-Cw*04 disadvantage. Science 283, 1748–1752 (1999).
Gao, X. et al. Effect of a single amino acid change in MHC class I molecules on the rate of progression to AIDS. N. Engl. J. Med. 344, 1668–1675 (2001).
Keet, I.P. et al. Consistent associations of HLA class I and II and transporter gene products with progression of human immunodeficiency virus type 1 infection in homosexual men. J. Infect. Dis. 180, 299–309 (1999).
Martin, M.P. & Carrington, M. Immunogenetics of HIV disease. Immunol. Rev. 254, 245–264 (2013).
Fellay, J. et al. Common genetic variation and the control of HIV-1 in humans. PLoS Genet. 5, e1000791 (2009).
Pereyra, F. et al. The major genetic determinants of HIV-1 control affect HLA class I peptide presentation. Science 330, 1551–1557 (2010).
Carrington, M., Bashirova, A.A. & McLaren, P.J. On stand by: host genetics of HIV control. AIDS 27, 2831–2839 (2013).
Pyo, C.W. et al. Recombinant structures expand and contract inter and intragenic diversification at the KIR locus. BMC Genomics 14, 89 (2013).
Fellay, J. et al. A whole-genome association study of major determinants for host control of HIV-1. Science 317, 944–947 (2007). This is the first genome-wide association study on an infectious disease trait. This study demonstrates that variation in the MHC region is the primary host genetic influence on HIV outcome in an unbiased, genome-wide screen.
Migueles, S.A. et al. HLA B*5701 is highly associated with restriction of virus replication in a subgroup of HIV-infected long-term nonprogressors. Proc. Natl. Acad. Sci. USA 97, 2709–2714 (2000).
Jia, X. et al. Imputing amino acid polymorphisms in human leukocyte antigens. PLoS ONE 8, e64683 (2013).
Dunstan, S.J. et al. Variation at HLA-DRB1 is associated with resistance to enteric fever. Nat. Genet. 46, 1333–1336 (2014).
Patsopoulos, N.A. et al. Fine-mapping the genetic association of the major histocompatibility complex in multiple sclerosis: HLA and non-HLA effects. PLoS Genet. 9, e1003926 (2013).
Okada, Y. et al. Fine mapping major histocompatibility complex associations in psoriasis and its clinical subtypes. Am. J. Hum. Genet. 95, 162–172 (2014).
Raychaudhuri, S. et al. Five amino acids in three HLA proteins explain most of the association between MHC and seropositive rheumatoid arthritis. Nat. Genet. 44, 291–296 (2012).
Stranger, B.E. et al. Population genomics of human gene expression. Nat. Genet. 39, 1217–1224 (2007).
Thomas, R. et al. HLA-C cell surface expression and control of HIV/AIDS correlate with a variant upstream of HLA-C. Nat. Genet. 41, 1290–1294 (2009).
Kaufman, J. & Salomonsen, J. The “minimal essential MHC” revisited: both peptide-binding and cell surface expression level of MHC molecules are polymorphisms selected by pathogens in chickens. Hereditas 127, 67–73 (1997).
Koch, M. et al. Structures of an MHC class I molecule from B21 chickens illustrate promiscuous peptide binding. Immunity 27, 885–899 (2007).
Corrah, T.W. et al. Reappraisal of the relationship between the HIV-1-protective single-nucleotide polymorphism 35 kilobases upstream of the HLA-C gene and surface HLA-C expression. J. Virol. 85, 3367–3374 (2011).
Pulit, S.L., Voight, B.F. & de Bakker, P.I. Multiethnic genetic association studies improve power for locus discovery. PLoS ONE 5, e12600 (2010).
Apps, R. et al. Influence of HLA-C expression level on HIV control. Science 340, 87–91 (2013). The authors determine the cell surface expression dynamics of all common classical HLA-C allotypes . Accounting for linkage disequilibrium with known allelic effects at HLA-A and HLA-B, they demonstrate an independent impact of HLA-C expression level on HIV control.
Kulkarni, S. et al. Differential microRNA regulation of HLA-C expression and its association with HIV control. Nature 472, 495–498 (2011).
Kulkarni, S. et al. Genetic interplay between HLA-C and MIR148A in HIV control and Crohn disease. Proc. Natl. Acad. Sci. USA 110, 20705–20710 (2013).
Blais, M.E. et al. High frequency of HIV mutations associated with HLA-C suggests enhanced HLA-C-restricted CTL selective pressure associated with an AIDS-protective polymorphism. J. Immunol. 188, 4663–4670 (2012).
Kiepiela, P. et al. Dominant influence of HLA-B in mediating the potential co-evolution of HIV and HLA. Nature 432, 769–775 (2004).
Kiepiela, P. et al. CD8 + T-cell responses to different HIV proteins have discordant associations with viral load. Nat. Med. 13, 46–53 (2007).
Petersdorf, E.W. et al. HLA-C expression levels define permissible mismatches in hematopoietic cell transplantation. Blood 124, 3996–4003 (2014).
Thomas, R. et al. A novel variant marking HLA-DP expression levels predicts recovery from hepatitis B virus infection. J. Virol. 86, 6979–6985 (2012).
Duggal, P. et al. Genome-wide association study of spontaneous resolution of hepatitis C virus infection: data from multiple cohorts. Ann. Intern. Med. 158, 235–245 (2013).
Wissemann, W.T. et al. Association of Parkinson disease with structural and regulatory variants in the HLA region. Am. J. Hum. Genet. 93, 984–993 (2013).
Reits, E.A. et al. Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy. J. Exp. Med. 203, 1259–1271 (2006).
Faroudi, M. et al. Lytic versus stimulatory synapse in cytotoxic T lymphocyte/target cell interaction: manifestation of a dual activation threshold. Proc. Natl. Acad. Sci. USA 100, 14145–14150 (2003).
Stahl, E.A. et al. Bayesian inference analyses of the polygenic architecture of rheumatoid arthritis. Nat. Genet. 44, 483–489 (2012).
Yang, J. et al. Common SNPs explain a large proportion of the heritability for human height. Nat. Genet. 42, 565–569 (2010). Missing heritabililty is the proportion of trait variability known to be attributable to genetic effects that has not been explained by GWAS. Using human height as a model, the authors describe a method for quantifying the heritability explained by all common SNPs and show that this accounts for a large fraction of the missing heritability.
Gusev, A. et al. Partitioning heritability of regulatory and cell-type-specific variants across 11 common diseases. Am. J. Hum. Genet. 95, 535–552 (2014).
Carlson, J.M. et al. HIV transmission. Selection bias at the heterosexual HIV-1 transmission bottleneck. Science 345, 1254031 doi:10.1126/science.1254031 (2014).
Fraser, C. et al. Virulence and pathogenesis of HIV-1 infection: an evolutionary perspective. Science 343, 1243727 doi:10.1126/science.1243727 (2014).
Fraser, C., Hollingsworth, T.D., Chapman, R., de Wolf, F. & Hanage, W.P. Variation in HIV-1 set-point viral load: epidemiological analysis and an evolutionary hypothesis. Proc. Natl. Acad. Sci. USA 104, 17441–17446 (2007).
van Dorp, C.H., van Boven, M. & de Boer, R.J. Immuno-epidemiological modeling of HIV-1 predicts high heritability of the set-point virus load, while selection for CTL escape dominates virulence evolution. PLoS Comput. Biol. 10, e1003899 (2014).
Bartha, I. et al. A genome-to-genome analysis of associations between human genetic variation, HIV-1 sequence diversity, and viral control. Elife 2, e01123 (2013). In a combined analysis of host and pathogen genetics, the authors use HIV sequence diversity as phenotype in multiple GWAS. The authors demonstrated that using sites of viral escape as an intermediate phenotype was more powerful than using clinical markers of disease progression to detect HLA associations.
Mackelprang, R.D. et al. Host genetic and viral determinants of HIV-1 RNA set-point among HIV-1 seroconverters from sub-Saharan Africa. J. Virol. 89, 2104–2111 (2015).
Frater, A.J. et al. Effective T-cell responses select human immunodeficiency virus mutants and slow disease progression. J. Virol. 81, 6742–6751 (2007).
Leslie, A.J. et al. HIV evolution: CTL escape mutation and reversion after transmission. Nat. Med. 10, 282–289 (2004).
Martinez-Picado, J. et al. Fitness cost of escape mutations in p24 Gag in association with control of human immunodeficiency virus type 1. J. Virol. 80, 3617–3623 (2006).
Schneidewind, A. et al. Escape from the dominant HLA-B27-restricted cytotoxic T-lymphocyte response in Gag is associated with a dramatic reduction in human immunodeficiency virus type 1 replication. J. Virol. 81, 12382–12393 (2007).
Kawashima, Y. et al. Adaptation of HIV-1 to human leukocyte antigen class I. Nature 458, 641–645 (2009). In a study of HIV diversity in multiple geographic regions, the authors show a correlation between frequency of viral escape mutations and the restricting HLA alleles. This adaptation resulted in the loss of protective effect of HLA-B*51 in a Japanese sample over time.
Cotton, L.A. et al. Genotypic and functional impact of HIV-1 adaptation to its host population during the North American epidemic. PLoS Genet. 10, e1004295 (2014).
Payne, R. et al. Impact of HLA-driven HIV adaptation on virulence in populations of high HIV seroprevalence. Proc. Natl. Acad. Sci. USA 111, E5393–E5400 (2014).
Herbeck, J.T. et al. Is the virulence of HIV changing? A meta-analysis of trends in prognostic markers of HIV disease progression and transmission. AIDS 26, 193–205 (2012).
Regoes, R.R. et al. Disentangling human tolerance and resistance against HIV. PLoS Biol. 12, e1001951 (2014).
Abecasis, G.R. et al. A map of human genome variation from population-scale sequencing. Nature 467, 1061–1073 (2010).
Jägger, S. et al. Global landscape of HIV–human protein complexes. Nature 481, 365–370 (2012).
Zhu, J. et al. Comprehensive identification of host modulators of HIV-1 replication using multiple orthologous RNAi reagents. Cell Reports 9, 752–766 (2014).
Akashi, H., Osada, N. & Ohta, T. Weak selection and protein evolution. Genetics 192, 15–31 (2012).
Farzadegan, H. et al. Sex differences in HIV-1 viral load and progression to AIDS. Lancet 352, 1510–1514 (1998).
Sterling, T.R. et al. Sex differences in longitudinal human immunodeficiency virus type 1 RNA levels among seroconverters. J. Infect. Dis. 180, 666–672 (1999).
Konttinen, Y.T., Hanninen, A. & Fuellen, G. Plasmacytoid dendritic cells, Janus-faced sentinels: progesterone, guilty or innocent? Immunotherapy 1, 929–931 (2009).
Kiezun, A. et al. Exome sequencing and the genetic basis of complex traits. Nat. Genet. 44, 623–630 (2012).
MacArthur, D.G. et al. Guidelines for investigating causality of sequence variants in human disease. Nature 508, 469–476 (2014).
Chang, J. et al. Polymorphisms in interferon regulatory factor 7 reduce interferon-alpha responses of plasmacytoid dendritic cells to HIV-1. AIDS 25, 715–717 (2011).
Lodi, S. et al. Immunovirologic control 24 months after interruption of antiretroviral therapy initiated close to HIV seroconversion. Arch. Intern. Med. 172, 1252–1255 (2012).
Sáez-Cirión, A. et al. Post-treatment HIV-1 controllers with a long-term virological remission after the interruption of early initiated antiretroviral therapy ANRS VISCONTI Study. PLoS Pathog. 9, e1003211 (2013).
Ge, D. et al. Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 461, 399–401 (2009).
Prentice, H.A. et al. HLA class I, KIR, and genome-wide SNP diversity in the RV144 Thai phase 3 HIV vaccine clinical trial. Immunogenetics 66, 299–310 (2014).
Deeks, S.G. & Walker, B.D. Human immunodeficiency virus controllers: mechanisms of durable virus control in the absence of antiretroviral therapy. Immunity 27, 406–416 (2007).
Bartha, I., McLaren, P.J., Ciuffi, A., Fellay, J. & Telenti, A. GuavaH: a compendium of host genomic data in HIV biology and disease. Retrovirology 11, 6 (2014).
HIV transmission via arthropods was a serious concern upon the discovery of this virus. Experiments and epidemiological data have unequivocally demonstrated, however, that such vector transmission does not occur at any significant level, and various aspects of HIV biology have been implicated as proximate reasons ( Bockarie and Paru 1996 ). These reasons do not offer an explanation for why vector transmission has not evolved, however, and as Weiss (2001) points out, we ought to seriously consider whether such evolution might occur in the future (for a summary, see Table 1).
|Evidence of capability of HIV for vector transmission||Evidence of vector transmission in related viruses||Evidence of genetic constraint||Why a lack of vector transmission?|
|Mechanism of vector transmission||Mechanical vector acts solely as a means of physical transport of viral particles||HIV remains viable for considerable time in ticks ( Humphrey-Smith and Chastel 1988 Humphrey-Smith et al. 1993 ) and C. hemipterus ( Webb et al. 1989 ).||In Bovine leukemia virus, Friend murine leukemia virus, equine infectious anemia virus ( Foil and Issel 1991 Humphrey-Smith et al. 1993 ).||Data not consistent with a genetic constraint.||Selectively disadvantageous since it requires higher levels of viremia, resulting in faster onset of AIDS.|
|Biological virus replicates within the vector||Little evidence of replication within potential vectors ( Srinivasan et al. 1987 ).||No evidence ( Foil and Issel 1991 Kuno 2004 Kuno and Chang 2005 Webb et al. 1989)||Data is consistent with a genetic constraint.||Genetic trade-off between replication in human host and insect vector.|
Existing data suggest that the lack of mechanical vector transmission in HIV is not due to genetic constraints. While ecological constraints, such as number of vectors and biting rates, may limit vector transmission in certain areas, these constraints would likely not explain why HIV has not evolved this form of transmission in areas where vector-borne diseases (e.g. malaria) are endemic. Rather, there must presumably be a reason why such transmission is selectively disadvantageous in HIV. The calculations presented above offer one possibility. Effective mechanical vector transmission can be brought about only through the evolution of higher levels of viremia, and this also results in a more rapid onset of AIDS. This reduces the duration over which such strains can be transmitted from an infected human, more than is made up for by the occurrence of vector transmission. It also remains possible that insufficient time has elapsed for the evolution of vector transmission to occur, but our calculations suggest that this is not a very compelling possibility.
On the other hand, existing data is largely consistent with the hypothesis that biological vector transmission has not evolved in HIV because of genetic constraints. At the same time, it is not possible to rule out a selective explanation instead. In particular, if there is a genetic trade-off between efficient replication in humans and replication in arthropod vectors, then a conflict between selection favoring effective replication within humans, and selection favoring arthropod transmission between humans can readily prevent biological transmission from evolving. This is particularly likely when the mutation rate of the virus is high, and thus might provide an explanation for the lack of biological vector transmission in all retroviruses.
Our analysis might also be extended to include other forms of transmission, for instance needle transmission (see Bruneau et al. 1997 for the efficiency of needle exchange programs). Several evolutionary consequences of this are possible depending both on the level of viremia required for such transmission to occur and the resulting transmission rate. For instance, if needle transmission can be achieved with a lower viremia than sexual transmission, and if this leads to a sufficiently high transmission rate, less virulent strains could be favored. Conversely, if needle transmission requires a high viremia and leads to a sufficiently high transmission rate, more virulent strains would be favored. The only situation in which enhanced needle use could lead to the evolution of vector-borne transmission would be if effective needle transmission requires a viremia close to that of vector-borne transmission, while leading to a much higher transmission rate than vector-borne transmission. This way, strains with high viremia could be maintained in the population through needle transmission, and vector-borne transmission would then occur largely as a byproduct.
Our conclusions in this article are necessarily speculative, but such speculation is a necessary part of the initial stages of any research. One of our aims is to stimulate future research into the evolutionary biology of HIV transmission. From the results presented here, a number of different directions might be taken to ground these evolutionary ideas more firmly in empirical data. One possibility would be to examine more closely mechanical vector transmission in immunodeficiency viruses of other species. For example, more data on the epidemiological patterns of SIV and its potential for alternative routes of transmission would be enormously useful. Since SIV is believed to be at the evolutionary ancestor of HIV, it would be very interesting to know if the longer evolutionary history it has had with its host has resulted in different transmission patterns. To the best of our knowledge, there are no empirical studies testing the potential for vector transmission of SIV. Another fruitful approach might be to conduct artificial selection experiments with HIV in arthropod tissue culture. Experiments have demonstrated that HIV cannot currently replicate significantly in arthropod cells, but no study to our knowledge has attempted to select for the evolution of HIV replication in such cells. One could even imagine doing such experiments with both mammalian and arthropod cell cultures to determine of the evolutionary trade-off postulated here actually occurs.
Ultimately, it will require innovative experiments and empirical studies to push the boundaries of our knowledge of HIV, and the use of evolutionary biology as a powerful tool for designing sensible intervention strategies. These kinds of studies are beginning to appear for other aspects of HIV biology (e.g., see Müller et al. 2006 for an interesting evolutionary analysis of HIV virulence) but more work on transmission biology would be useful. For example, if further empirical research validated the hypothesis presented here, that mechanical vector transmission has not evolved because of its associated mortality costs, this would then have important implications for how we attempt to stop the spread of HIV. Strategies such as condom use, while beneficial for reducing the extent of sexual transmission, could thereby enhance the relative benefit of vector transmission, potentially resulting in the evolution of this new route of transmission. The use of antibiotics against bacterial pathogens has clearly brought home the fact that pathogens can readily evolve the means to circumvent our control measures, and there is no reason to expect things to be any different for other control measures. The use of antiviral medication, on the other hand, not only reduces sexual transmission but also the level of viremia, and therefore would presumably not move the selective balance more towards vector transmission. It is only by asking these kinds of questions, however, that we will have a chance at preventing adverse future outcomes.
Finally, the question of biological vector transmission addressed here is really a special case of the more general question of the evolution of a pathogen’s host range. Why do some pathogens have a relatively broad taxonomic host range while others are much more conservative? This continues to be an interesting and important question in the evolutionary ecology of parasites ( Poulin 2007 ) and there are some theoretical results predicting when we might expect different outcomes ( Gandon 2004 ). From the standpoint of human diseases this is also clearly an important question since emerging diseases, such as pandemic influenza, are precisely instances in which a pathogen evolves a different host range. A better understanding of the evolutionary biology of parasite host ranges is an important goal for future research.
Marx, A. & Alian, A. The Road Less Traveled: HIV’s Use of Alternative Routes through Cellular Pathways. J Virol 89, 5204–5212, doi: 10.1128/JVI.03684-14 (2015).
Socolich, M. et al. Evolutionary information for specifying a protein fold. Nature 437, 512–518, doi: 10.1038/nature03991 (2005).
Kowarsch, A., Fuchs, A., Frishman, D. & Pagel, P. Correlated mutations: a hallmark of phenotypic amino acid substitutions. Plos Comput Biol 6, doi: 10.1371/journal.pcbi.1000923 (2010).
Marx, A. & Alian, A. Rerouting Resistance: Escaping Restriction Using Alternative Cellular Pathways. Trends Microbiol 23, 595–597, doi: 10.1016/j.tim.2015.08.005 (2015).
Ovchinnikov, S. et al. Large-scale determination of previously unsolved protein structures using evolutionary information. Elife 4, doi: 10.7554/eLife.09248 (2015).
Jacob, E., Unger, R. & Horovitz, A. Codon-level information improves predictions of inter-residue contacts in proteins by correlated mutation analysis. Elife 4, doi: 10.7554/eLife.08932 (2015).
Li, G. et al. Functional conservation of HIV-1 Gag: implications for rational drug design. Retrovirology 10, 126, doi: 10.1186/1742-4690-10-126 (2013).
Flynn, W. F. et al. Deep sequencing of protease inhibitor resistant HIV patient isolates reveals patterns of correlated mutations in Gag and protease. Plos Comput Biol 11, e1004249, doi: 10.1371/journal.pcbi.1004249 (2015).
Le Sage, V., Mouland, A. J. & Valiente-Echeverria, F. Roles of HIV-1 capsid in viral replication and immune evasion. Virus Res 193, 116–129, doi: 10.1016/j.virusres.2014.07.010 (2014).
Campbell, E. M. & Hope, T. J. HIV-1 capsid: the multifaceted key player in HIV-1 infection. Nat Rev Microbiol 13, 471–483, doi: 10.1038/nrmicro3503 (2015).
Gallay, P. A. et al. Correlation of naturally occurring HIV-1 resistance to DEB025 with capsid amino acid polymorphisms. Viruses 5, 981–997, doi: 10.3390/v5030981 (2013).
Dahirel, V. et al. Coordinate linkage of HIV evolution reveals regions of immunological vulnerability. Proc Natl Acad Sci USA 108, 11530–11535, doi: 10.1073/pnas.1105315108 (2011).
Kalinina, O. V. et al. Computational identification of novel amino-acid interactions in HIV Gag via correlated evolution. Plos One 7, e42468, doi: 10.1371/journal.pone.0042468 (2012).
Zhao, Y., Wang, Y., Gao, Y., Li, G. & Huang, J. Integrated analysis of residue coevolution and protein structures capture key protein sectors in HIV-1 proteins. Plos One 10, e0117506, doi: 10.1371/journal.pone.0117506 (2015).
Shi, J. et al. Compensatory substitutions in the HIV-1 capsid reduce the fitness cost associated with resistance to a capsid-targeting small-molecule inhibitor. J Virol 89, 208–219, doi: 10.1128/JVI.01411-14 (2015).
Zhou, J., Price, A. J., Halambage, U. D., James, L. C. & Aiken, C. HIV-1 Resistance to the Capsid-Targeting Inhibitor PF74 Results in Altered Dependence on Host Factors Required for Virus Nuclear Entry. J Virol 89, 9068–9079, doi: 10.1128/JVI.00340-15 (2015).
Yang, R. et al. Second-site suppressors of HIV-1 capsid mutations: restoration of intracellular activities without correction of intrinsic capsid stability defects. Retrovirology 9, 30, doi: 10.1186/1742-4690-9-30 (2012).
Rihn, S. J. et al. Extreme genetic fragility of the HIV-1 capsid. Plos Pathog 9, e1003461, doi: 10.1371/journal.ppat.1003461 (2013).
Rolland, M. et al. HIV-1 conserved-element vaccines: relationship between sequence conservation and replicative capacity. J Virol 87, 5461–5467, doi: 10.1128/JVI.03033-12 (2013).
Bienzle, D. FIV in cats–a useful model of HIV in people? Vet Immunol Immunopathol 159, 171–179, doi: 10.1016/j.vetimm.2014.02.014 (2014).
Poeschla, E. M. Primate and feline lentiviruses in current intrinsic immunity research: the cat is back. Vet Immunol Immunopathol 143, 215–220, doi: 10.1016/j.vetimm.2011.06.014 (2011).
Galilee, M. & Alian, A. Identification of Phe187 as a crucial dimerization determinant facilitates crystallization of a monomeric retroviral integrase core domain. Structure 22, 1512–1519, doi: 10.1016/j.str.2014.08.001 (2014).
Esteva, M. J., Affranchino, J. L. & Gonzalez, S. A. Lentiviral Gag assembly analyzed through the functional characterization of chimeric simian immunodeficiency viruses expressing different domains of the feline immunodeficiency virus capsid protein. Plos One 9, e114299, doi: 10.1371/journal.pone.0114299 (2014).
Ivanov, D. et al. Domain-swapped dimerization of the HIV-1 capsid C-terminal domain. Proc Natl Acad Sci USA 104, 4353–4358, doi: 10.1073/pnas.0609477104 (2007).
von Schwedler, U. K., Stray, K. M., Garrus, J. E. & Sundquist, W. I. Functional surfaces of the human immunodeficiency virus type 1 capsid protein. J Virol 77, 5439–5450, doi: 10.1128/JVI.77.9.5439-5450.2003 (2003).
Obal, G. et al. Conformational plasticity of a native retroviral capsid revealed by x-ray crystallography. Science 349, 95–98, doi: 10.1126/science.aaa5182 (2015).
Bayro, M. J., Chen, B., Yau, W. M. & Tycko, R. Site-specific structural variations accompanying tubular assembly of the HIV-1 capsid protein. J Mol Biol 426, 1109–1127, doi: 10.1016/j.jmb.2013.12.021 (2014).
Pornillos, O., Ganser-Pornillos, B. K., Banumathi, S., Hua, Y. & Yeager, M. Disulfide bond stabilization of the hexameric capsomer of human immunodeficiency virus. J Mol Biol 401, 985–995, doi: 10.1016/j.jmb.2010.06.042 (2010).
Ternois, F., Sticht, J., Duquerroy, S., Krausslich, H. G. & Rey, F. A. The HIV-1 capsid protein C-terminal domain in complex with a virus assembly inhibitor. Nat Struct Mol Biol 12, 678–682, doi: 10.1038/nsmb967 (2005).
Gres, A. T. et al. X-ray crystal structures of native HIV-1 capsid protein reveal conformational variability. Science 349, 99–103, doi: 10.1126/science.aaa5936 (2015).
Freed, E. O. HIV-1 assembly, release and maturation. Nat Rev Microbiol 13, 484–496, doi: 10.1038/nrmicro3490 (2015).
Meng, X. et al. Protease cleavage leads to formation of mature trimer interface in HIV-1 capsid. Plos Pathog 8, e1002886, doi: 10.1371/journal.ppat.1002886 (2012).
Bartonova, V. et al. Residues in the HIV-1 capsid assembly inhibitor binding site are essential for maintaining the assembly-competent quaternary structure of the capsid protein. J Biol Chem 283, 32024–32033, doi: 10.1074/jbc.M804230200 (2008).
Zhao, G. et al. Mature HIV-1 capsid structure by cryo-electron microscopy and all-atom molecular dynamics. Nature 497, 643–646, doi: 10.1038/nature12162 (2013).
Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. HKL-3000: the integration of data reduction and structure solution--from diffraction images to an initial model in minutes. Acta crystallographica. Section D, Biological crystallography 62, 859–866, doi: 10.1107/S0907444906019949 (2006).
McCoy, A. J. et al. Phaser crystallographic software. J Appl Crystallogr 40, 658–674, doi: 10.1107/S0021889807021206 (2007).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta crystallographica. Section D, Biological crystallography 66, 486–501, doi: 10.1107/S0907444910007493 (2010).
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta crystallographica. Section D, Biological crystallography 67, 235–242, doi: 10.1107/S0907444910045749 (2011).
Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta crystallographica. Section D, Biological crystallography 67, 355–367, doi: 10.1107/S0907444911001314 (2011).
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Pathogenesis-Informed Approaches to Vaccines
Nonviable Brucella vaccines historically have a poor record of success that started with heat-killed Brucella, crude extracts, and then subunit and DNA vaccines. This poor track record is primarily explained by a failure to induce an effective Th1 response comparable with live, attenuated vaccines (LAVs). 91 In contrast, LAVs have a long history of successful use against brucellosis and other intracellular pathogens in laboratory animal models and in target species, such as ruminants. However, given the safety concerns associated with human use, including the threat of persistent infection or reversion to virulence extreme, caution must be used in LAV development.
Prevention of animal disease was found to provide substantial protection against human disease. Historically, the focus of immune protection against Brucella infection in animals has been the use of spontaneously attenuated strains. 92,93 Their stable and effective use in animals over decades was used to justify support for human trials. 94 Three vaccines are used extensively in animals to provide immune protection: B. abortus S19 and RB51 and B. melitensis Rev.1. 95 S19 and Rev.1 are fortuitously attenuated isolates. One strain was obtained accidentally, and the second was obtained after a stepwise process to identify streptomycin-dependent and then streptomycin-independent isolates. The two strains are considered to be smooth (expressing intact LPS with O-polysaccharide) which distinguishes them from RB51, a rough strain lacking the O-polysaccharide. S19 and Rev.1 provide superior protection but exhibit substantial human virulence and cannot be used in gravid female animals. RB51 is considered to be safe for use in gravid females and does not induce O-polysaccharide antibodies that can be used to distinguish field strain-infected from vaccinated animals. Despite well-defined differences in immune potential among these vaccines, no marker or correlate has been identified that can be used to predict immune protection.
Attenuated virulence may be derived from simple point mutations or from genetic rearrangements, including gene deletion. Although the potential for reversion of a mutant that bears a complete gene deletion is already small, the potential for reversion to virulence can be made infinitesimal by the introduction of a secondary mutation. Optimally, the additional mutation would affect the same pathway as the primary mutation, serving only as a backup to prevent reversion, but inducing no additional reduction in virulence or negative effect on protective immunity.
Obviously, care must be taken so that a balance is struck that supports survival sufficiently to enhance immune protection without posing a risk of inducing disease. One approach to this task is encapsulation of the attenuated vaccine strain to release the organism over time and to provide the added advantage of providing a natural booster response. 68,98 This approach uses a vaccine depot from which the attenuated Brucella is gradually released over a 30-day period and significantly enhances immune protection by using highly attenuated LAVs to improve both efficacy and safety. 99 In addition, recent cell biology findings have revealed the dependence of Brucella infection on the UPR, specifically IRE1α. 20,42 This dependence may be exploited in an effort to provide LAVs that provide enhanced immune protection. ER stress and TLR signaling provide a synergistic stimulation of the proinflammatory response. 100 The key to an optimal LAV development strategy is to identify vaccine candidates that fail to restrict the innate immune response and as a result induce an effective adaptive immune response without safety or reversion concerns.
Vaccine-driven virulence evolution: consequences of unbalanced reductions in mortality and transmission and implications for pertussis vaccines
Many vaccines have heterogeneous effects across individuals. Additionally, some vaccines do not prevent infection, but reduce disease-associated mortality and transmission. Both of these factors will alter selection pressures on pathogens and thus shape the evolution of pathogen virulence. We use a mathematical modelling framework to show that (i) the balance of how vaccines reduce transmission versus mortality and (ii) individual variability in protection conferred both shape the evolution of pathogen virulence. Epidemiological (burden of disease) and evolutionary (pathogen virulence) outcomes are both worse when vaccines confer smaller reductions in transmission than in mortality. Furthermore, outcomes are modulated by variability in vaccine effects, with increased variability limiting the extent of virulence evolution but in some cases preventing eradication. These findings are pertinent to current concerns about the global resurgence of pertussis and the efficacy of pertussis vaccines, as the two classes of these vaccines may reduce disease symptoms without preventing infection and differ in their ability to reduce transmission. Furthermore, these findings point to the importance of generating precise predictions for virulence evolution in Bordetella pertussis (and other similar pathogens) by incorporating empirical characterizations of vaccine effects into models capturing the epidemiological details of this system.
Vaccination is one of the most powerful public health tools available . When vaccines block within and/or between host propagation of pathogens, they provide both individual benefits through their direct protective effects and population benefits ranging from herd immunity to eradication. However, vaccination can fundamentally change the ecology of infectious disease systems and introduce novel selective pressures that drive pathogen evolution, potentially reducing or negating these benefits. Pathogen life-history traits can evolve in response to vaccination when pathogens retain the ability to infect some or all of the host population. One such pathogen life-history trait is virulence, or the rate of disease-associated host death. Virulence evolution has been a general focus in the fields of epidemiology and evolutionary biology [2–4] and has received particular attention in the context of vaccination.
Virulence determines a pathogen's effects on morbidity and mortality, as it describes the aggressiveness of the pathogen's host manipulation strategy . When host death truncates transmission, virulence is inversely proportional to transmission time. Thus, virulence in itself is never adaptive, but it is often maintained as an unavoidable pleiotropic byproduct of transmission. Theoretical and empirical evidence points towards a saturating relationship between virulence and transmission, creating a trade-off that shapes pathogens' life-history strategies [5–8]. This transmission–virulence trade-off has been shown to emerge from within-host processes  and is often reflected phenomenologically in population-level evolutionary epidemiological models . In most scenarios, intermediate virulence strategies confer the highest fitness because increased transmission rate comes at the price of increased host mortality and decreased transmission time (, equation (2.1)). Here, we define virulence as the rate of disease-associated mortality in hosts with no mortality-blocking immunity, noting that the operational definition of virulence varies between studies and that the term ‘virulence’ itself is sometimes used to describe infectivity rather than aggressiveness in plant systems .
Vaccination can drive virulence evolution by inducing forms of immunity that alter the relationship between transmission rate and duration. Some vaccines are thought to have an ‘anti-growth’ effect (‘r2’ parameter in the models developed by Gandon et al. [11,12]) by slowing the within-host replication rate of a pathogen, leading to coupled reductions in both transmission and mortality. In a population-level model, Gandon et al.  (see also ) found that vaccines that moderately reduce pathogen replication rate select for increased pathogen virulence, but lead to reductions in prevalence. Analogous within-host models that consider transmission and mortality to be functions of within-host pathogen density also predict that pathogens should evolve increased virulence when vaccine-induced immunity impedes the replication of a pathogen [13,14], and that virulence should increase with increasing immune efficacy .
Other vaccines are thought to only block mortality effects without conferring any reduction in transmission. Gandon et al.  showed theoretically that the use of such mortality-blocking (‘r4’ in their model specification) vaccines that do not reduce transmission can drive the evolution of very high pathogen virulence. Normally, highly virulent strategies confer low fitness because rapid host mortality truncates the transmission period. But when vaccines remove or reduce this cost of mortality, pathogens are able to increase their total fitness by increasing their transmission rate. Non-immunized individuals are disproportionately affected by the evolution of increased virulence—they experience drastically increased mortality while vaccinated individuals are at least partially protected. Several empirical examples support the predictions of Gandon et al. . Read et al.  showed convincingly that when vaccines blocked the mortality effects of Marek's disease virus in commercial poultry the virus evolved higher virulence to the point where vaccinated hosts lost all benefits of vaccine-induced immunity and non-vaccinated individuals experienced increased mortality. Recently, Fleming-Davies et al.  showed that incomplete acquired immunity (analogous to vaccination) to the bacterial pathogen Mycoplasma gallisepticum in finches selects for increased virulence, as low-virulence strains are unable to infect previously infected hosts while high-virulence strains retain this ability.
Despite the development of a rich theoretical framework for investigating vaccine-driven virulence evolution, this approach has not yet been applied to many important vaccine types. In particular, there is a clear knowledge gap about vaccines with unbalanced effects on transmission and mortality, whose effects are not fully or accurately captured by either the anti-growth or mortality-blocking models of vaccine action. These unbalanced effects may be characteristic of vaccines targeted towards pathogen proteins like toxins that cause damage to the host, since toxins generally have a dual effect, reducing host survival (as considered in Gandon et al. ) and also increasing pathogen growth, often via immunosuppressive effects. As a result, toxin-targeting vaccines can slightly reduce transmission and also have significant mortality-reducing effects. Investigating how such vaccines might drive virulence evolution requires a model that can consider both effects independently in the same framework. Previous model formulations are unsuitable for investigating this range of vaccine effects, because they either consider reductions in transmission and mortality to be necessarily coupled via a reduction in pathogen replication rate (e.g. ) or assume that transmission is necessarily reduced to a greater degree than mortality by layering anti-growth and transmission-blocking effects. Here, we present a modification to previous modelling frameworks that allows us to investigate and compare between the effects of various modes of vaccine action on the evolution of pathogen virulence (figure 1).
Figure 1. A model schematic encompassing multiple modes of vaccine action. In the model framework that we present, vaccine-induced immunity can act in two non-exclusive ways. First, immunity can have a mortality-reducing effect (x-axis). Second, immunity can have a transmission-reducing effect (y-axis) by decreasing the within-host replication rate of the pathogen, which has a nonlinear effect on transmission. Thus, our model allows us to consider not only ‘anti-growth’ and ‘anti-toxin’ effects but also scenarios in which vaccines have unequal effects on transmission and mortality. An important aspect of this model is that immunity does not reduce the rate at which susceptible individuals become infected when challenged—consistent with the action of Bordetella pertussis vaccines. The shaded regions show the parameter spaces that correspond to a greater percentage decrease in a disease-associated mortality rate or an onward transmission rate for an individual treated with a vaccine. The boundaries of the shaded areas were generated from the equations for transmission and disease-associated mortality given below, with c1 = 1.0, c2 = 0.33 and i= 1.0. (Online version in colour.)
This model framework might provide particularly important insights into the B. pertussis system. Both whole-cell pertussis (wP) vaccines, which contain killed B. pertussis bacteria, and acellular pertussis (aP) vaccines, which contain a small subset of the repertoire of proteins B. pertussis would normally present to the human immune system , are known to significantly reduce disease-associated damage. However, evidence from a primate model system suggests that, unlike naturally acquired immunity, neither form of vaccine-induced immunity is able to prevent infection [20–22]. Epidemiological patterns suggest that both do moderately reduce onward transmission [23,24], but that aP vaccines reduce transmission to a lesser extent [25,26]. Thus, vaccinated individuals challenged by B. pertussis can become asymptomatically infected and can carry out some amount of transmission. The model framework presented in figure 1 is able to represent these effects, which are intermediate between anti-growth and anti-toxin.
Another aspect of vaccine-driven virulence evolution that we seek to investigate relates to the structure of vaccine-induced immunity in host populations. While the importance of characterizing ‘landscapes of immunity’ and predicting their evolutionary and epidemiological effects is increasingly recognized , complexity has been conspicuously lacking in current modelling efforts, despite empirical evidence for its existence. To date, only the simplest distributions of immunity have been considered almost all models (with the exception of [14,28–30]) consider only ‘binary’ distributions of immunity under the assumption that vaccination has a ‘fixed effect’ in all individuals. Yet, it has long been known that vaccines can have a ‘variable effect’, creating continuous distributions of immunity within populations [31,32], and recent efforts have sought to characterize these patterns . Variability in age, sex or environmental factors could create population-level immunological heterogeneity in the absence of vaccination. These factors could also contribute to heterogeneity in combination with vaccination or through interactions with vaccine efficacy.
Incorporating immunological heterogeneity into models could potentially reveal safe implementation strategies for mortality-blocking vaccines, which have previously been found to have dangerous long-term effects as they drive the evolution of hypervirulent pathogens at any level of coverage. Gandon et al.  found that mortality-blocking vaccines drove the evolution of hypervirulent pathogens while assuming that vaccination had a fixed effect, creating a binary distribution of immunity in the host population (figure 2). The experimental work of Read et al.  that produced similar results used host populations that exhibited such homogeneity all vaccinated individuals were age matched and kept in identical conditions to reduce the variance in vaccine effect. In both cases, the optimum pathogen virulence strategies in the two host categories were extremely different, especially when vaccination completely eliminated mortality effects. Highly virulent strategies emerged as pathogens gained most of their fitness through the vaccinated host group. Because there were no hosts with intermediate immunity, the costs of a hypervirulent strategy were only incurred in unvaccinated individuals. The few inquiries into the consequences of immunological heterogeneity for virulence evolution have indeed produced intriguing results. For example, Ganusov et al.  found that, in a within-host model, heterogeneity in the lethal pathogen density slightly decreased the evolutionarily stable degree of virulence.
Figure 2. Immunity distributions. Vaccines that have a variable effect create continuous distributions of immunity (modelled here as pseudo-continuous distributions), while vaccines that have a fixed effect create a binary distribution of immunity. In all plots, θ = 0.5.
Here, we apply adaptive dynamics to a non-system-specific epidemiological model to quantify the extent to which patterns of vaccine-driven virulence evolution and their associated epidemiological outcomes are dependent upon (i) the relative strength of mortality and transmission-reducing vaccine effects and (ii) variation in the effects of vaccination between individuals. We find that drastically different evolutionary and epidemiological outcomes can result from the use of vaccines that differ only slightly in their effects on mortality and transmission and/or in their variability in effect among individuals, and that immunological variability can buffer against virulence evolution.
To investigate the evolutionary and epidemiological consequences of the use of vaccines that block mortality, transmission or a combination of the two, we constructed an evolutionary epidemiological model that follows a susceptible–infected–recovered (SIR) framework in which susceptible hosts become infected in a density-dependent fashion and then permanently recover with no chance of reinfection. We include two classes of infected hosts in order to explore competition between two pathogen strains of differing virulence and assume no co-infection or superinfection. The susceptible and infected host populations are structured by immunity. The relative frequencies of individuals born into each immunity class in the susceptible population are fixed, consistent with a constant vaccination rate and outcome and no host evolution. We vary how immunity affects transmission and mortality, and characterize the dynamics of virulence evolution as pathogens adapt to immunity distributions of various shapes using an adaptive dynamics approach. We then explore the epidemiological outcomes associated with evolutionarily stable pathogen virulence, as well as the potential for pathogen eradication. We note that these methods are not intended to make specific predictions about virulence evolution in B. pertussis or any other disease system. Rather, they are designed to explore how certain mechanistic details of vaccine action (that do differ between the two classes of pertussis vaccines) might generally shape pathogen evolution. Definitions of the parameters and variables used throughout the paper are given in table 1.
The HIV-1 polypurine tracts are highly conserved genetic elements required for initiation of plus-strand DNA synthesis and intriguing candidates for siRNA targeting. The data presented here provide insight into (i) whether HIV replication can be effectively suppressed in this manner, (ii) which siRNAs most effectively target the PPT, (iii) the degree to which HIV-1 PPTs are genetically flexible and (iv) what nucleotide changes are observed in HIV-1 escape mutants.
Our study demonstrated that siRNA targeting HIV-1 3’PPT region could effectively inhibit virus replication. However, it is not immediately clear why, of the three siRNAs tested, only siRNA-PPT1 was consistently effective in suppressing HIV-1 replication. The explanation for this may lie in the idiosyncrasies of RISC target recognition or perhaps in the unique structure of duplexes containing 3′PPT RNA. Standard synthetic siRNAs are comprised of 19 nt RNA duplexes with 2 nt 5′ overhangs on either side . The complementary 21 nt oligoribonucleotides that constitute these siRNAs are functionally asymmetrical, as it is hybridization of the guide strand to its target that is essential for ultimate degradation of the mRNA. In general, the RNA strand with greater base-pairing thermostability toward the 5′ terminus is preferentially selected as a guide strand in the RISC [44, 45]. However, of the siRNAs utilized in the present study, siRNA-PPT1 does not seem to have an advantage in this regard. Instead, it is possible that the full length PPT sequence (which is only included in siRNA-PPT1) possesses unusual structural features that promote formation of RISC and increase RISC cleavage efficiency just as they direct RT-associated RNase H to generate the viral plus-strand primers. The fact that RNases H and the RISC protein argonaute are in the same gene family makes this possibility even more intriguing. On the other hand, as showed in Fig 4 , interestingly, the inhibition efficiency of siRNA inhibiting HIV-1 replication was not significantly decreased when siRNA-PPT1 was mutated with one nucleotide mismatching to its target sequence, which is possibly due to the mutation not sitting in the critical position, but may again suggest the above mentioned possibility.
The notion that duplexes containing the intact PPT are in some way exceptional is supported by predictions that hybrid duplexes containing an intact Mo-MLV PPT are more stable than those containing other Mo-MLV genomic RNA sequences of the same length . Alternatively, perhaps hybridization of oligonucleotides comprised of mixed PPT/non-PPT sequence (like siRNA-PPT3) is inherently disfavored. The HIV-1 genomic sequence itself may lend support to this hypothesis. Specifically, there are numerous purine rich sequences throughout the genome that share partial homology with the central and 3′PPTs (e.g., 5′-AAACACAGTGGGGGGACA, 5′-AAAAAACATCAGAAAGAA, etc.) yet do not effectively serve as plus-strand primers. It is possible that the structural and thermodynamic properties that make these sequences unsuitable for plus-strand priming also render PPT targeting with siRNA-PPT2 and/or-PPT3 unfavorable.
The lone escape mutant identified by siRNA targeting of the 3′PPT contained two nucleotide substitutions, G(-1)A and T(-16)A, located at opposite ends of the conserved purine rich element. Together, these mutations conferred both limited resistance to siRNA-PPT1 and reduced fitness in the context of the NL4-3 strain of HIV-1. Interestingly, nucleotide substitution between positions -1 and -16 of the 3′PPT were not observed, suggesting that (i) such mutations would not confer resistance to siRNA or (ii) more significant changes to the PPT could not be tolerated by the virus. We suspect the latter conclusion is more likely, as HIV-1 RT and the HIV-1 PPTs are likely to have co-evolved into a tight functional symbiosis. A comparison of PPT sequences and RT structures among retroviruses tends to support this notion i.e., differences in PPT sequence between two retroviruses are usually matched by corresponding differences in RT structure suggesting the existence of co-evolution between RT and PPT sequence .
As discussed previously, placing the T(-16)A mutation in the context of the cPPT completely abolished virus replication, most likely because this nucleotide substitution changes the integrase coding sequence to produce a defective mutant enzyme. Viruses containing any of the other PPT mutations were viable, despite the fact that introducing T(-16)A or G(-1)A mutations into the 3′PPT either truncates or introduces a point mutation into the nef protein, respectively. Even though nef is important for disease progression and is considered a pathogenic factor in primate lentiviridae , it is not essential for viral replication . Because the effects of disrupting nef function under these conditions appear to be negligible, we conclude that the reduced fitness observed for virus containing T(-16)A, G(-1)A or both mutations in the 3′PPT region is due mainly to aberrant 3′PPT processing.
Precise generation and removal of plus-strand primers from the 3′ and central PPTs has previously been shown to be crucial for HIV-1 replication [2, 14, 50]. While most RNase H-mediated hydrolysis can be imprecise without impairing synthesis of pre-integrative viral DNA, the cleavage events that generate and remove the plus-strand primers must be specific in order to produce linear DNAs that are appropriate substrates for integration. All RNase H cleavage is catalyzed by HIV-1 RT, a p66/p51 heterodimer housing DNA polymerase and RNase H active sites spaced
18 bp apart relative to duplex nucleic acid substrates. Plus-strand primer processing presents a unique problem for RT in that the enzyme must first bind the PPT/DNA hybrid in an orientation that positions the RNase H domain for cleavage at the PPT 3′ terminus, then re-bind in the opposite orientation to initiate DNA synthesis from the nascent plus-strand primer. This 𠇏lipping” on the substrate has been observed in single molecule FRET studies of HIV-1 RT-PPT/DNA complexes, where it appears to occur more frequently than on generic RNA/DNA hybrids [51, 52]. PPTs must also be refractory to internal cleavage, since a smaller RNA fragment would likely not remain hybridized to minus-strand DNA long enough to promote priming, nor could RT efficiently bind to or initiate DNA synthesis from a truncated PPT primer.
The structural determinants that dictate RT binding orientation, direct proper RNase H cleavage, prevent internal PPT cleavage and promote plus-strand priming have been extensively studied yet remain poorly understood [4, 25, 53]. Numerous contact points exist between primer grip/RNase H primer grip residues in HIV-1 RT and the DNA strand of an RNA/DNA hybrid, some of which have been suggested to play a role in specific recognition of the PPT/DNA hybrid . On the nucleic acid side, previous work has demonstrated that introducing G-to-A substitutions at positions -2 and/or -4 in the HIV-1 PPT resulted in enhanced internal cleavage, while a contiguous stretch of 3′ terminal Gs has been shown to promote priming of DNA synthesis in both RNA/DNA and DNA/DNA contexts [25, 57]. The role of the 5′-terminal A-tracts in HIV-1 PPT function is less apparent, as only modest effects on PPT processing are observed upon site-directed mutagenesis of individual A-tract nucleotides [25, 53]. However, complete removal of one or both A-tracts greatly reduces the efficiency of plus-strand primer generation and utilization . Some evidence also suggests that the U-A junction at the PPT 5′ terminus is important for preventing slippage during reverse transcription of HIV-1 RNA, as demonstrated in SIV mutants in which the U-tract immediately upstream from the 3′PPT had been deleted .
Introducing the G(-1)A mutation into a synthetic PPT/DNA hybrid increases the rate of internal PPT cleavage, particularly at the -2/-1 position. This effect is not negated in the G(-1)A/T(-16)A tandem mutation, suggesting that, in the context of an in vitro RNase H assay, the second mutation is relatively inconsequential. Internal cleavage of the PPT at any position is likely to reduce the efficiency of plus-strand priming both because a shorter RNA primer will not remain stably associated with the minus-strand DNA complement and because fewer contact sites will be available for RT binding (and initiation events will therefore be less frequent). In addition, HIV-1 pre-integrative DNA would be expected to be one-or-more nucleotides longer, depending upon whether the truncated 3′PPT is successfully removed, which is likely to affect integration.
Pre-integrative DNA 3′-processing exposes the 3′ hydroxyl in the integrase/dsDNA complex for nucleophilic attack on the host genomic DNA𠅊n event known as strand transfer. Retroviral integrase attachment sites (att) are found at the extreme U3 and U5 ends of the linear proviral genome, as long as the pre-integrative DNA is correctly processed . The att is comprised of at least 7 and as many as 20 bp starting from the highly conserved 3′-ACTG-5′ at the terminus of U3. Extensive mutational analyses of this relatively well-conserved U3 terminus revealed that nucleotide substitutions in numerous positions are important for IN recognition [59, 61]. However, to our knowledge, there have been no reports on the impact of U3 terminal extensions on integrase activity and specificity. It is quite possible that a single base pair extension on the end of U3, or a more significant extension in the event the plus-strand primer is not properly removed, could reduce IN binding and dinucleotide cleavage. Dicker et al.  compared the relative binding affinity of inhibitors and various mutant att U3 sequences for integrase. Because these ligands/inhibitors bind integrase cooperatively, mutations within the terminal four base pairs of att U3 (or att U5) not only reduce the affinity of integrase for its nucleic acid substrates but also, indirectly, for the inhibitors. Interestingly, our NL4-3 virus with G(-1)A or the G(-1)A/T(-16)A substitutions in the 3′PPT were slightly less sensitive to the integrase inhibitor Raltegravir than the wild-type virus (data not shown). These findings suggest that a 1 nt U3 extension may exist in viral DNA and that this extension may reduce integrase activity.