[Show abstract][Hide abstract] ABSTRACT: CD8(+) cytotoxic T lymphocytes (CTL) are strong mediators of human immunodeficiency virus type 1 (HIV-1) control, yet HIV-1 frequently mutates to escape CTL recognition. In an analysis of sequences in the Los Alamos HIV-1 database, we show that emerging CTL escape mutations were more often present at lower frequencies than the amino acid(s) that they replaced. Furthermore, epitopes that underwent escape contained amino acid sites of high variability, whereas epitopes persisting at high frequencies lacked highly variable sites. We therefore infer that escape mutations are likely to be associated with weak functional constraints on the viral protein. This was supported by an extensive analysis of one subject for whom all escape mutations within defined CTL epitopes were studied and by an analysis of all reported escape mutations of defined CTL epitopes in the HIV Immunology Database. In one of these defined epitopes, escape mutations involving the substitution of amino acids with lower database frequencies occurred, and the epitope soon reverted back to the sensitive form. We further show that this escape mutation substantially diminished viral fitness in in vitro competition assays. Coincident with the reversion in vivo, we observed the fixation of a mutation 3 amino acids C terminal to the epitope, coincident with the ablation of the corresponding CTL response. The C-terminal mutation did not restore replication fitness reduced by the escape mutation in the epitope and by itself had little effect on replication fitness. Therefore, this C-terminal mutation presumably impaired the processing and presentation of the epitope. Finally, for one persistent epitope, CTL cross-reactivity to a mutant form may have suppressed the mutant to undetected levels, whereas for two other persistent epitopes, each of two mutants showed poor cross-reactivity and appeared in the subject at later time points. Thus, a viral dynamic exists between the advantage of immune escape, peptide cross-reactivity, and the disadvantage of lost replication fitness, with the balance playing an important role in determining whether a CTL epitope will persist or decline during infection.
Journal of Virology 12/2007; 81(22):12179-88. DOI:10.1128/JVI.01277-07 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Typically during human immunodeficiency virus type 1 (HIV-1) infection, a nearly homogeneous viral population first emerges and then diversifies over time due to selective forces that are poorly understood. To identify these forces, we conducted an intensive longitudinal study of viral genetic changes and T-cell immunity in one subject at < or =17 time points during his first 3 years of infection, and in his infecting partner near the time of transmission. Autologous peptides covering amino acid sites inferred to be under positive selection were powerful for identifying HIV-1-specific cytotoxic-T-lymphocyte (CTL) epitopes. Positive selection and mutations resulting in escape from CTLs occurred across the viral proteome. We detected 25 CTL epitopes, including 14 previously unreported. Seven new epitopes mapped to the viral Env protein, emphasizing Env as a major target of CTLs. One-third of the selected sites were associated with epitopic mutational escapes from CTLs. Most of these resulted from replacement with amino acids found at low database frequency. Another one-third represented acquisition of amino acids found at high database frequency, suggesting potential reversions of CTL epitopic sites recognized by the immune system of the transmitting partner and mutation toward improved viral fitness in the absence of immune targeting within the recipient. A majority of the remaining selected sites occurred in the envelope protein and may have been subjected to humoral immune selection. Hence, a majority of the amino acids undergoing selection in this subject appeared to result from fitness-balanced CTL selection, confirming CTLs as a dominant selective force in HIV-1 infection.
Journal of Virology 11/2006; 80(19):9519-29. DOI:10.1128/JVI.00575-06 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: HIV-1 is one of the fastest evolving entities known. Given that census population sizes of HIV-1 within individuals are much greater than the inverse mutation rate, every possible single point mutation in the viral genome occurs each generation. This enormous capability to generate genetic variation allows for escape from immune surveillance and antiviral therapy. However, compared to this potential, populations of HIV-1 within individuals exhibit little genetic variation. This discrepancy between the known mutation rate of HIV-1 and the average level of genetic variation in the env gene observed in vivo is reflected in comparisons of the actual numbers of productively infected cells, estimated as 10(7), and the effective population size, estimated as 10(3). Using approximate Bayesian computation, we evaluated several hypotheses based on a variety of selective and demographic processes to explain the low effective population size of HIV-1. Of the models we examined, the metapopulation model, in which HIV-1 evolves within an individual as a large collection of small subpopulations subject to frequent migration, extinction, and recolonization, was most consistent with the observed levels of genetic variation and the average frequencies of those variants. The metapopulation model links previous studies of viral dynamics and population genetics.
[Show abstract][Hide abstract] ABSTRACT: The molecular clock hypothesis states that the rate of nucleotide substitution per generation is constant across lineages. If generation times were equal across lineages, samples obtained at the same calendar time would have experienced the same number of generations since their common ancestor. However, if sequences are not derived from contemporaneous samples, differences in the number of generations may be misinterpreted as variation in substitution rates and hence may lead to false rejection of the molecular clock hypothesis. A recent study has called into doubt the validity of clock-like evolution for HIV-1, using molecular sequences derived from noncontemporaneous samples. However, after separating their within-individual data according to sampling time, we found that what appeared to be nonclock-like behavior could be attributed, in most cases, to noncontemporaneous sampling, with contributions also likely to derive from recombination. Natural selection alone did not appear to obscure the clock-like evolution of HIV-1.
[Show abstract][Hide abstract] ABSTRACT: Recombinants of preexisting human immunodeficiency virus type 1 (HIV-1) strains are now circulating globally. To increase our understanding of the importance of these recombinants, we assessed recombination within an individual infected from a single source by studying the linkage patterns of the auxiliary genes of HIV-1 subtype B. Maximum-likelihood phylogenetic techniques revealed evidence for recombination from topological incongruence among adjacent genes. Coalescent methods were then used to estimate the in vivo recombination rate. The estimated mean rate of 1.38 x 10(-4) recombination events/adjacent sites/generation is approximately 5.5-fold greater than the reported point mutation rate of 2.5 x 10(-5)/site/generation. Recombination was found to be frequent enough to mask evidence for purifying selection by Tajima's D test. Thus, recombination is a major evolutionary force affecting genetic variation within an HIV-1-infected individual, of the same order of magnitude as point mutational change.
[Show abstract][Hide abstract] ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) has high replication and mutation rates that generate large census populations and high levels of genetic variation. We examined the roles of natural selection, population growth, random genetic drift, and recombination in shaping the variation in 1509 C2-V5 env sequences derived from nine men with chronic HIV-1 infection. These sequences were obtained from clinical visits that reflect the first 6-13.7 years of infection. Pairwise comparisons of nonsynonymous and synonymous distances, Tajima's D test, Fu and Li's D* test, and a test of recurrent mutation revealed evidence for episodes of nonneutral evolution in a total of 22 out of 145 blood samples, representing six of the nine individuals. Using three coalescent-based maximum-likelihood estimators, we found viral effective population sizes in all nine individuals to be approximately 10(3). We also show that a previous estimate of the effective population size of approximately 10(5) based on rare haplotype frequencies decreases to approximately 10(3) upon correcting a biased sampling procedure. We conclude that the genetic variation in these data sets can be explained by a predominance of random genetic drift of neutral mutations with brief episodes of natural selection that were frequently masked by recombination.
[Show abstract][Hide abstract] ABSTRACT: Early in infection, human immunodeficiency virus type 1 (HIV-1) generally uses the CCR5 chemokine receptor (along with CD4) for cellular entry. In many HIV-1-infected individuals, viral genotypic changes arise that allow the virus to use CXCR4 (either in addition to CCR5 or alone) as an entry coreceptor. This switch has been associated with an acceleration of both CD3(+) T-cell decline and progression to AIDS. While it is well known that the V3 loop of gp120 largely determines coreceptor usage and that positively charged residues in V3 play an important role, the process of genetic change in V3 leading to altered coreceptor usage is not well understood. Further, the methods for biological phenotyping of virus for research or clinical purposes are laborious, depend on sample availability, and present biosafety concerns, so reliable methods for sequence-based "virtual phenotyping" are desirable. We introduce a simple bioinformatic method of scoring V3 amino acid sequences that reliably predicts CXCR4 usage (sensitivity, 84%; specificity, 96%). This score (as determined on the basis of position-specific scoring matrices [PSSM]) can be interpreted as revealing a propensity to use CXCR4 as follows: known R5 viruses had low scores, R5X4 viruses had intermediate scores, and X4 viruses had high scores. Application of the PSSM scoring method to reconstructed virus phylogenies of 11 longitudinally sampled individuals revealed that the development of X4 viruses was generally gradual and involved the accumulation of multiple amino acid changes in V3. We found that X4 viruses were lost in two ways: by the dying off of an established X4 lineage or by mutation back to low-scoring V3 loops.
Journal of Virology 01/2004; 77(24):13376-88. DOI:10.1128/JVI.77.24.13376-13388.2003 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Current therapies for treating HIV-1 infection are capable of suppressing virus load in blood to undetectable levels, and result in marked clinical improvement. Despite this suppression, HIV-1 infection persists and virus load quickly rebounds when therapy is interrupted. The origin of the rebounding virus is unknown, but is thought to result from continuing viral replication in anatomic or cellular compartments, and the release of virus from latent infection in reservoirs.
Current Opinion in Microbiology 09/2003; 6(4):410-6. DOI:10.1016/S1369-5274(03)00096-1 · 5.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In vivo virologic compartments are cell types or tissues between which there is a restriction of virus flow, while virologic reservoirs are cell types or tissues in which there is a relative restriction of replication. The distinction between reservoirs and compartments is important because therapies that would be effective against a reservoir may not be effective against viruses produced by a given compartment, and vice versa. For example, the use of cytokines to "flush out" long-lived infected cells in patients on highly active antiretroviral therapy (T. W. Chun, D. Engel, M. M. Berrey, T. Shea, L. Corey, and A. S. Fauci, Proc. Natl. Acad. Sci. USA 95:8869-8873, 1998) may be successful for a latent reservoir but may not impact a compartment in which virus continues to replicate because of poor drug penetration. Here, we suggest phylogenetic criteria to illustrate, define, and differentiate between reservoirs and compartments. We then apply these criteria to the analysis of simulated and actual human immunodeficiency virus type 1 sequence data sets. We report that existing statistical methods work quite well at detecting viral compartments, and we learn from simulations that viral divergence from a calculated most recent common ancestor is a strong predictor of viral reservoirs.
Journal of Virology 06/2003; 77(9):5540-6. DOI:10.1128/JVI.77.9.5540-5546.2003 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Current sitewise methods for detecting positive selection on gene sequences (the de facto standard being the CODEML method (Yang et al., 2000)) assume no recombination. This paper presents simulation results indicating that violation of this assumption can lead to false positive detection of sites undergoing positive selection. Through the use of population-scaled mutation and recombination rates, simulations can be performed that permit the generation of appropriate null distributions corresponding to neutral expectations in the presence of recombination, thereby allowing for a more accurate estimation of positive selection.
Genetics Research 05/2003; 81(2):115-21. DOI:10.1017/S0016672303006128 · 1.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although human immunodeficiency virus type 1 (HIV-1) recombinants have been found with high frequency, little is known about the forces that select for these viruses or their importance to pathogenesis. Here we document the emergence and dynamics of 11 distinct HIV-1 recombinants in a man who was infected with two subtype B HIV-1 strains and progressed rapidly to AIDS without developing substantial cellular or humoral immune responses. Although numerous frequency oscillations were observed, a single recombinant lineage eventually came to dominate the population. Numerical simulations indicate that the successive recombinant forms displaced each other too rapidly to be explained by any simple model of random genetic drift or sampling variation. All of the recombinants, including several resulting from independent recombination events, possessed the same sequence motif in the V3 loop, suggesting intense selection on this segment of the viral envelope protein. The outgrowth of the predominant V3 loop recombinants was not, however, associated with changes in coreceptor utilization. The final variant was instead notable for having lost 3 of 14 potential glycosylation sites. We also observed high ratios of synonymous-to-nonsynonymous nucleotide changes-suggestive of purifying selection-in all viral populations, with particularly high ratios in newly arising recombinants. Our study, therefore, illustrates the unusual and important patterns of viral adaptation that can occur in a patient with weak immune responses. Although it is hard to tease apart cause and effect in a single patient, the correlation with disease progression in this patient suggests that recombination between divergent viruses, with its ability to create chimeras with increased fitness, can accelerate progression to AIDS.
Journal of Virology 12/2002; 76(21):10674-84. DOI:10.1128/JVI.76.21.10674-10684.2002 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transfer of nascent DNA from an RNA template (donor) to the homologous region of a second RNA template (acceptor) was studied.
The templates were designed to assess the roles of the sequences proximal (3′ relative to the transferring DNA) to the point
of transfer. The donor template was primed with a specific 18 nucleotide DNA such that extension by reverse transcriptase
to the end of the template produced a 79 nucleotide product. Homologous strand transfer and subsequent extension on the acceptors
produced longer products allowing distinction between strand transfer and donor-directed synthesis. The donor and one particular
acceptor shared a region of homology which included 8 tandem 5′-CAGU-3′ repeats followed at the 3′ end by a 17 nucleotide
region of random homologous sequence. Derivatives of the acceptor either completely lacked the 17 nucleotide region or were
progressively truncated resulting in a shorter region. With the acceptor lacking this region, prominent transfer products
differing in length by 4 nucleotides were observed. Presumably this occurs because the transferring DNA can base-pair with
several copies of the repeat elements of the acceptor. Addition of 5 or more of the 17 random nucleotides to the 3′ end of
the acceptor resulted in transfer products of essentially one length, and consistent with the transferring DNA correctly base-pairing
with the 3′ nucleotides. Results suggest that the transferring DNA interacts with the acceptor over several bases to form
the most energetically stable hybrid duplex prior to extension on the acceptor. Hybrids formed from shorter interactions between
the 3′ end of the DNA and acceptor are either realinged prior to extension or precluded due to the mechanism of transfer.
Archives of Virology 08/1997; 142(9):1797-1812. DOI:10.1007/s007050050198 · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The rate of progression to AIDS in human immunodeficiency virus type 1 (HIV-1)-infected individuals is determined by a complex series of interactions between the host and virus. Here we evaluate virologic properties and host responses in two men near-simultaneously infected with HIV-1 from the same sexual partner--one individual progressed to AIDS in less than 2 years, and the other remains asymptomatic 3 years postinfection. Distinct neutralizing antibody and cellular immune responses were evident, with the slower progressor exhibiting generally stronger and broader responses, except for cytotoxic T-lymphocyte responses early in infection. Virtually identical, homogeneous virus populations were found in both patients in the first sample obtained; however, a second unrelated HIV-1 virus population was also found in the fast progressor. Whether the second population emanated from an additional source of infection or the two were transmitted from the original source could not be determined. The virus population in the slower progressor turned over and diversified rapidly, whereas both virus populations in the rapid progressor evolved at a much slower rate. In addition, the character of mutational changes underlying these diversities appeared to be distinct, with a bias for diversifying selection developing in the slower progressor and a reciprocal bias towards purifying selection maintained in both populations in the fast progressor. Thus, the rapid evolution that is a hallmark of HIV replication may be a reflection of strong host resistance against emerging virus variants and a longer period of asymptomatic infection. Furthermore, rapid progression was not linked to a collapse of any appreciable immune response following attainment of some threshold of antigenic diversity but rather to a failure to drive this diversification and a condition of relatively unimpeded expansion of variants with optimized replicative capacity within the host.
Journal of Virology 07/1997; 71(6):4284-95. · 4.44 Impact Factor