ABSTRACT
Chromatin remodeling proteins are frequently dysregulated in human cancer, yet little is known about how they control tumorigenesis. Here, we uncover an epigenetic program mediated by the NAD(+)-dependent histone deacetylase Sirtuin 6 (SIRT6) that is critical for suppression of pancreatic ductal adenocarcinoma (PDAC), one of the most lethal malignancies. SIRT6 inactivation accelerates PDAC progression and metastasis via upregulation of Lin28b, a negative regulator of the let-7 microRNA. SIRT6 loss results in histone hyperacetylation at the Lin28b promoter, Myc recruitment, and pronounced induction of Lin28b and downstream let-7 target genes, HMGA2, IGF2BP1, and IGF2BP3. This epigenetic program defines a distinct subset with a poor prognosis, representing 30%-40% of human PDAC, characterized by reduced SIRT6 expression and an exquisite dependence on Lin28b for tumor growth. Thus, we identify SIRT6 as an important PDAC tumor suppressor and uncover the Lin28b pathway as a potential therapeutic target in a molecularly defined PDAC subset. PAPERCLIP.
Subject(s)
Adenocarcinoma/genetics , Gene Expression Regulation, Neoplastic , Pancreatic Neoplasms/genetics , RNA-Binding Proteins/genetics , Sirtuins/genetics , Acetylation , Animals , Cell Line, Tumor , Chromatin Assembly and Disassembly , Epigenesis, Genetic , Female , Genes, ras , Histones/metabolism , Humans , Male , Mice , Mice, Knockout , RNA-Binding Proteins/metabolism , Tumor Suppressor Proteins/metabolismABSTRACT
The 2013-2015 West African epidemic of Ebola virus disease (EVD) reminds us of how little is known about biosafety level 4 viruses. Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever with high case fatality rates. We generated a genomic catalog of almost 200 LASV sequences from clinical and rodent reservoir samples. We show that whereas the 2013-2015 EVD epidemic is fueled by human-to-human transmissions, LASV infections mainly result from reservoir-to-human infections. We elucidated the spread of LASV across West Africa and show that this migration was accompanied by changes in LASV genome abundance, fatality rates, codon adaptation, and translational efficiency. By investigating intrahost evolution, we found that mutations accumulate in epitopes of viral surface proteins, suggesting selection for immune escape. This catalog will serve as a foundation for the development of vaccines and diagnostics. VIDEO ABSTRACT.
Subject(s)
Genome, Viral , Lassa Fever/virology , Lassa virus/genetics , RNA, Viral/genetics , Africa, Western/epidemiology , Animals , Biological Evolution , Disease Reservoirs , Ebolavirus/genetics , Genetic Variation , Glycoproteins/genetics , Hemorrhagic Fever, Ebola/virology , Humans , Lassa Fever/epidemiology , Lassa Fever/transmission , Lassa virus/classification , Lassa virus/physiology , Murinae/genetics , Mutation , Nigeria/epidemiology , Viral Proteins/genetics , Zoonoses/epidemiology , Zoonoses/virologyABSTRACT
Due to the stringent population bottleneck that occurs during sexual HIV-1 transmission, systemic infection is typically established by a limited number of founder viruses. Elucidation of the precise forces influencing the selection of founder viruses may reveal key vulnerabilities that could aid in the development of a vaccine or other clinical interventions. Here, we utilize deep sequencing data and apply a genetic distance-based method to investigate whether the mode of sexual transmission shapes the nascent founder viral genome. Analysis of 74 acute and early HIV-1 infected subjects revealed that 83% of men who have sex with men (MSM) exhibit a single founder virus, levels similar to those previously observed in heterosexual (HSX) transmission. In a metadata analysis of a total of 354 subjects, including HSX, MSM and injecting drug users (IDU), we also observed no significant differences in the frequency of single founder virus infections between HSX and MSM transmissions. However, comparison of HIV-1 envelope sequences revealed that HSX founder viruses exhibited a greater number of codon sites under positive selection, as well as stronger transmission indices possibly reflective of higher fitness variants. Moreover, specific genetic "signatures" within MSM and HSX founder viruses were identified, with single polymorphisms within gp41 enriched among HSX viruses while more complex patterns, including clustered polymorphisms surrounding the CD4 binding site, were enriched in MSM viruses. While our findings do not support an influence of the mode of sexual transmission on the number of founder viruses, they do demonstrate that there are marked differences in the selection bottleneck that can significantly shape their genetic composition. This study illustrates the complex dynamics of the transmission bottleneck and reveals that distinct genetic bottleneck processes exist dependent upon the mode of HIV-1 transmission.
Subject(s)
HIV Infections/transmission , HIV-1/genetics , Evolution, Molecular , Genetic Variation , Genome, Viral , HIV Envelope Protein gp120/genetics , Humans , Male , Models, Theoretical , Polymerase Chain Reaction , Selection, Genetic/geneticsABSTRACT
Developing a vaccine for human immunodeficiency virus (HIV) may be aided by a complete understanding of those rare cases in which some HIV-infected individuals control replication of the virus. Most of these elite controllers express the histocompatibility alleles HLA-B*57 or HLA-B*27 (ref. 3). These alleles remain by far the most robust associations with low concentrations of plasma virus, yet the mechanism of control in these individuals is not entirely clear. Here we vaccinate Indian rhesus macaques that express Mamu-B*08, an animal model for HLA-B*27-mediated elite control, with three Mamu-B*08-restricted CD8(+) T-cell epitopes, and demonstrate that these vaccinated animals control replication of the highly pathogenic clonal simian immunodeficiency virus (SIV) mac239 virus. High frequencies of CD8(+) T cells against these Vif and Nef epitopes in the blood, lymph nodes and colon were associated with viral control. Moreover, the frequency of the CD8(+) T-cell response against the Nef RL10 epitope (Nef amino acids 137-146) correlated significantly with reduced acute phase viraemia. Finally, two of the eight vaccinees lost control of viral replication in the chronic phase, concomitant with escape in all three targeted epitopes, further implicating these three CD8(+) T-cell responses in the control of viral replication. Our findings indicate that narrowly targeted vaccine-induced virus-specific CD8(+) T-cell responses can control replication of the AIDS virus.
Subject(s)
AIDS Vaccines/immunology , Acquired Immunodeficiency Syndrome/virology , CD8-Positive T-Lymphocytes/immunology , SAIDS Vaccines/immunology , Simian Acquired Immunodeficiency Syndrome/prevention & control , Simian Immunodeficiency Virus/immunology , Virus Replication/immunology , Animals , Disease Models, Animal , Epitopes, T-Lymphocyte/immunology , Female , HIV-1/immunology , HLA-B27 Antigen/immunology , Humans , Immunodominant Epitopes/immunology , Macaca mulatta/immunology , Macaca mulatta/virology , Male , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/growth & development , Simian Immunodeficiency Virus/pathogenicity , Viral Load , Viremia/immunology , Viremia/prevention & controlABSTRACT
Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia.
Subject(s)
Genome, Viral/genetics , Genome-Wide Association Study , HIV Infections/virology , HIV-1/genetics , Immune Evasion/immunology , CD8-Positive T-Lymphocytes/immunology , Genetic Variation , Genomic Structural Variation , HIV Infections/immunology , HIV Infections/prevention & control , HIV-1/immunology , HIV-1/pathogenicity , Humans , Immune Evasion/genetics , Oligonucleotide Array Sequence Analysis , RNA, Viral/analysis , Sequence Analysis, RNA , Viral Vaccines/immunologyABSTRACT
While human leukocyte antigen B57 (HLA-B57) is associated with the spontaneous clearance of hepatitis C virus (HCV), the mechanisms behind this control remain unclear. Immunodominant CD8(+) T cell responses against the B57-restricted epitopes comprised of residues 2629 to 2637 of nonstructural protein 5B (NS5B(2629-2637)) (KSKKTPMGF) and E2(541-549) (NTRPPLGNW) were recently shown to be crucial in the control of HCV infection. Here, we investigated whether the selection of deleterious cytotoxic T lymphocyte (CTL) escape mutations in the NS5B KSKKTPMGF epitope might impair viral replication and contribute to the B57-mediated control of HCV. Common CTL escape mutations in this epitope were identified from a cohort of 374 HCV genotype 1a-infected subjects, and their impact on HCV replication assessed using a transient HCV replicon system. We demonstrate that while escape mutations at residue 2633 (position 5) of the epitope had little or no impact on HCV replication in vitro, mutations at residue 2629 (position 1) substantially impaired replication. Notably, the deleterious mutations at position 2629 were tightly linked in vivo to upstream mutations at residue 2626, which functioned to restore the replicative defects imparted by the deleterious escape mutations. These data suggest that the selection of costly escape mutations within the immunodominant NS5B KSKKTPMGF epitope may contribute in part to the control of HCV replication in B57-positive individuals and that persistence of HCV in B57-positive individuals may involve the development of specific secondary compensatory mutations. These findings are reminiscent of the selection of deleterious CTL escape and compensatory mutations by HLA-B57 in HIV-1 infection and, thus, may suggest a common mechanism by which alleles like HLA-B57 mediate protection against these highly variable pathogens.
Subject(s)
HLA-B Antigens/immunology , Hepacivirus/immunology , Mutation, Missense , Suppression, Genetic , T-Lymphocytes, Cytotoxic/immunology , Viral Nonstructural Proteins/metabolism , Virus Replication , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , Hepacivirus/genetics , Hepacivirus/physiology , Humans , T-Lymphocytes, Cytotoxic/virology , Viral Nonstructural Proteins/geneticsABSTRACT
The control of human immunodeficiency virus type 1 (HIV-1) associated with particular HLA class I alleles suggests that some CD8(+) T-cell responses may be more effective than others at containing HIV-1. Unfortunately, substantial diversities in the breadth, magnitude, and function of these responses have impaired our ability to identify responses most critical to this control. It has been proposed that CD8 responses targeting conserved regions of the virus may be particularly effective, since the development of cytotoxic T-lymphocyte (CTL) escape mutations in these regions may significantly impair viral replication. To address this hypothesis at the population level, we derived near-full-length viral genomes from 98 chronically infected individuals and identified a total of 76 HLA class I-associated mutations across the genome, reflective of CD8 responses capable of selecting for sequence evolution. The majority of HLA-associated mutations were found in p24 Gag, Pol, and Nef. Reversion of HLA-associated mutations in the absence of the selecting HLA allele was also commonly observed, suggesting an impact of most CTL escape mutations on viral replication. Although no correlations were observed between the number or location of HLA-associated mutations and protective HLA alleles, limiting the analysis to mutations selected by acute-phase immunodominant responses revealed a strong positive correlation between mutations at conserved residues and protective HLA alleles. These data suggest that control of HIV-1 may be associated with acute-phase CD8 responses capable of selecting for viral escape mutations in highly conserved regions of the virus, supporting the inclusion of these regions in the design of an effective vaccine.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , HIV-1/genetics , HIV-1/immunology , Mutation, Missense/immunology , HIV Core Protein p24/genetics , HIV Core Protein p24/immunology , Humans , Molecular Sequence Data , Sequence Analysis, DNA , nef Gene Products, Human Immunodeficiency Virus/genetics , nef Gene Products, Human Immunodeficiency Virus/immunology , pol Gene Products, Human Immunodeficiency Virus/genetics , pol Gene Products, Human Immunodeficiency Virus/immunologyABSTRACT
Human immunodeficiency virus effectively evades CD8(+) T-cell responses through the development of CD8 escape mutations. Recent reports documenting reversion of transmitted mutations and the impact of specific escape mutations upon viral replication suggest that complex forces limit the accumulation of CD8 escape mutations at the population level. However, the presence of compensatory mutations capable of alleviating the impact of CD8 escape mutations on replication capacity may enable their persistence in an HLA-mismatched host. Herein, we illustrate the long-term stability of stereotypic escape mutations in the immunodominant HLA-B27-restricted epitope KK10 in p24/Gag following transmission when accompanied by a specific compensatory mutation.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , HIV/growth & development , HIV/immunology , Mutation, Missense/immunology , Amino Acid Sequence , Animals , HIV/genetics , HIV Core Protein p24/genetics , Humans , Molecular Sequence Data , Virus ReplicationABSTRACT
BACKGROUND: The robustness of ChIP-seq datasets is highly dependent upon the antibodies used. Currently, polyclonal antibodies are the standard despite several limitations: They are non-renewable, vary in performance between lots and need to be validated with each new lot. In contrast, monoclonal antibody lots are renewable and provide consistent performance. To increase ChIP-seq standardization, we investigated whether monoclonal antibodies could replace polyclonal antibodies. We compared monoclonal antibodies that target five key histone modifications (H3K4me1, H3K4me3, H3K9me3, H3K27ac and H3K27me3) to their polyclonal counterparts in both human and mouse cells. RESULTS: Overall performance was highly similar for four monoclonal/polyclonal pairs, including when we used two distinct lots of the same monoclonal antibody. In contrast, the binding patterns for H3K27ac differed substantially between polyclonal and monoclonal antibodies. However, this was most likely due to the distinct immunogen used rather than the clonality of the antibody. CONCLUSIONS: Altogether, we found that monoclonal antibodies as a class perform equivalently to polyclonal antibodies for the detection of histone post-translational modifications in both human and mouse. Accordingly, we recommend the use of monoclonal antibodies in ChIP-seq experiments.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies/immunology , Histones/metabolism , Animals , Chromatin Immunoprecipitation , DNA/chemistry , DNA/isolation & purification , DNA/metabolism , Histones/chemistry , Histones/immunology , Humans , K562 Cells , Mice , Mouse Embryonic Stem Cells , Peptide Mapping , Protein Processing, Post-Translational , Sequence Analysis, DNAABSTRACT
How embryonic stem cells (ESCs) commit to specific cell lineages and yield all cell types of a fully formed organism remains a major question. ESC differentiation is accompanied by large-scale histone and DNA modifications, but the relations between these epigenetic categories are not understood. Here we demonstrate the interplay between the histone deacetylase sirtuin 6 (SIRT6) and the ten-eleven translocation enzymes (TETs). SIRT6 targets acetylated histone H3 at Lys 9 and 56 (H3K9ac and H3K56ac), while TETs convert 5-methylcytosine into 5-hydroxymethylcytosine (5hmC). ESCs derived from Sirt6 knockout (S6KO) mice are skewed towards neuroectoderm development. This phenotype involves derepression of OCT4, SOX2 and NANOG, which causes an upregulation of TET-dependent production of 5hmC. Genome-wide analysis revealed neural genes marked with 5hmC in S6KO ESCs, thereby implicating TET enzymes in the neuroectoderm-skewed differentiation phenotype. We demonstrate that SIRT6 functions as a chromatin regulator safeguarding the balance between pluripotency and differentiation through Tet-mediated production of 5hmC.
Subject(s)
Cell Differentiation , Cell Lineage , Cytosine/analogs & derivatives , DNA-Binding Proteins/metabolism , Embryonic Stem Cells/enzymology , Proto-Oncogene Proteins/metabolism , Sirtuins/metabolism , 5-Methylcytosine/analogs & derivatives , Acetylation , Animals , Cells, Cultured , Chromatin Assembly and Disassembly , Cytosine/metabolism , DNA-Binding Proteins/genetics , Dioxygenases , Embryonic Stem Cells/pathology , Embryonic Stem Cells/transplantation , Gene Expression Regulation, Developmental , Genotype , Histones/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Induced Pluripotent Stem Cells/enzymology , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, SCID , Nanog Homeobox Protein , Neurogenesis , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Phenotype , Proto-Oncogene Proteins/genetics , RNA Interference , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Signal Transduction , Sirtuins/deficiency , Sirtuins/genetics , Teratoma/enzymology , Teratoma/pathology , TransfectionABSTRACT
We have developed a robust RNA sequencing method for generating complete de novo assemblies with intra-host variant calls of Lassa and Ebola virus genomes in clinical and biological samples. Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA. This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries. We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries. These protocols have enabled rapid deep sequencing of both Lassa and Ebola virus and are broadly applicable to other viral genomics studies.
Subject(s)
Ebolavirus/genetics , High-Throughput Nucleotide Sequencing/methods , Lassa virus/genetics , Hemorrhagic Fever, Ebola/genetics , Hemorrhagic Fever, Ebola/virology , Humans , Lassa Fever/genetics , Lassa Fever/virology , RNA, ViralABSTRACT
In its largest outbreak, Ebola virus disease is spreading through Guinea, Liberia, Sierra Leone, and Nigeria. We sequenced 99 Ebola virus genomes from 78 patients in Sierra Leone to ~2000× coverage. We observed a rapid accumulation of interhost and intrahost genetic variation, allowing us to characterize patterns of viral transmission over the initial weeks of the epidemic. This West African variant likely diverged from central African lineages around 2004, crossed from Guinea to Sierra Leone in May 2014, and has exhibited sustained human-to-human transmission subsequently, with no evidence of additional zoonotic sources. Because many of the mutations alter protein sequences and other biologically meaningful targets, they should be monitored for impact on diagnostics, vaccines, and therapies critical to outbreak response.
Subject(s)
Disease Outbreaks , Ebolavirus/genetics , Epidemiological Monitoring , Hemorrhagic Fever, Ebola/transmission , Hemorrhagic Fever, Ebola/virology , Base Sequence , Ebolavirus/isolation & purification , Genetic Variation , Genome, Viral/genetics , Genomics/methods , Hemorrhagic Fever, Ebola/epidemiology , Humans , Mutation , Sequence Analysis, DNA , Sierra Leone/epidemiologyABSTRACT
The development of mouse/human chimeras through the engraftment of human immune cells and tissues into immunodeficient mice, including the recently described humanized BLT (bone marrow, liver, thymus) mouse model, holds great promise to facilitate the in vivo study of human immune responses. However, little data exist regarding the extent to which cellular immune responses in humanized mice accurately reflect those seen in humans. We infected humanized BLT mice with HIV-1 as a model pathogen and characterized HIV-1-specific immune responses and viral evolution during the acute phase of infection. HIV-1-specific CD8(+) T cell responses in these mice were found to closely resemble those in humans in terms of their specificity, kinetics, and immunodominance. Viral sequence evolution also revealed rapid and highly reproducible escape from these responses, mirroring the adaptations to host immune pressures observed during natural HIV-1 infection. Moreover, mice expressing the protective HLA-B*57 allele exhibited enhanced control of viral replication and restricted the same CD8(+) T cell responses to conserved regions of HIV-1 Gag that are critical to its control of HIV-1 in humans. These data reveal that the humanized BLT mouse model appears to accurately recapitulate human pathogen-specific cellular immunity and the fundamental immunological mechanisms required to control a model human pathogen, aspects critical to the use of a small-animal model for human pathogens.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , HIV-1/immunology , Animals , HIV Infections/immunology , HLA-B Antigens/metabolism , Humans , Immunity, Cellular/immunology , MiceABSTRACT
After acute HIV infection, CD8(+) T cells are able to control viral replication to a set point. This control is often lost after superinfection, although the mechanism behind this remains unclear. In this study, we illustrate in an HLA-B27(+) subject that loss of viral control after HIV superinfection coincides with rapid recombination events within two narrow regions of Gag and Env. Screening for CD8(+) T cell responses revealed that each of these recombination sites (approximately 50 aa) encompassed distinct regions containing two immunodominant CD8 epitopes (B27-KK10 in Gag and Cw1-CL9 in Env). Viral escape and the subsequent development of variant-specific de novo CD8(+) T cell responses against both epitopes were illustrative of the significant immune selection pressures exerted by both responses. Comprehensive analysis of the kinetics of CD8 responses and viral evolution indicated that the recombination events quickly facilitated viral escape from both dominant WT- and variant-specific responses. These data suggest that the ability of a superinfecting strain of HIV to overcome preexisting immune control may be related to its ability to rapidly recombine in critical regions under immune selection pressure. These data also support a role for cellular immune pressures in driving the selection of new recombinant forms of HIV.
Subject(s)
HIV Infections/immunology , HIV Infections/virology , HIV/genetics , HIV/immunology , Superinfection/immunology , Superinfection/virology , Base Sequence , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , Case-Control Studies , DNA Primers/genetics , DNA, Viral/genetics , Genes, env , Genes, gag , Genetic Variation , HIV Infections/genetics , HLA-B27 Antigen/genetics , HLA-B27 Antigen/immunology , Humans , Immunodominant Epitopes/genetics , Recombination, Genetic , Selection, Genetic , Superinfection/genetics , Viremia/genetics , Viremia/immunology , Viremia/virology , Virus Replication/genetics , Virus Replication/immunologyABSTRACT
The error-prone replication of human immunodeficiency virus type 1 (HIV-1) enables it to continuously evade host CD8+ T-cell responses. The observed transmission, and potential accumulation, of CD8+ T-cell escape mutations in the population may suggest a gradual adaptation of HIV-1 to immune pressures. Recent reports, however, have highlighted the propensity of some escape mutations to revert upon transmission to a new host in order to restore efficient replication capacity. To more specifically address the role of reversions in early HIV-1 evolution, we examined sequence polymorphisms arising across the HIV-1 genome in seven subjects followed longitudinally 1 year from primary infection. As expected, numerous nonsynonymous mutations were associated with described CD8+ T-cell epitopes, supporting a prominent role for cellular immune responses in driving early HIV-1 evolution. Strikingly, however, a substantial proportion of substitutions (42%) reverted toward the clade B consensus sequence, with nearly one-quarter of them located within defined CD8 epitopes not restricted by the contemporary host's HLA. More importantly, these reversions arose significantly faster than forward mutations, with the most rapidly reverting mutations preferentially arising within structurally conserved residues. These data suggest that many transmitted mutations likely incur a fitness cost that is recovered through retrieval of an optimal, or ancestral, form of the virus. The propensity of mutations to revert may limit the accumulation of immune pressure-driven mutations in the population, thus preserving critical CD8+ T-cell epitopes as vaccine targets, and argue against an unremitting adaptation of HIV-1 to host immune pressures.