A spatio-temporal assessment of simian/human immunodeficiency virus (SHIV) evolution reveals a highly dynamic process within the host.

The process by which drug-resistant HIV-1 arises and spreads spatially within an infected individual is poorly understood. Studies have found variable results relating how HIV-1 in the blood differs from virus sampled in tissues, offering conflicting findings about whether HIV-1 throughout the body is homogeneously distributed. However, most of these studies sample only two compartments and few have data from multiple time points. To directly measure how drug resistance spreads within a host and to assess how spatial structure impacts its emergence, we examined serial sequences from four macaques infected with RT-SHIVmne027, a simian immunodeficiency virus encoding HIV-1 reverse transcriptase (RT), and treated with RT inhibitors. Both viral DNA and RNA (vDNA and vRNA) were isolated from the blood (including plasma and peripheral blood mononuclear cells), lymph nodes, gut, and vagina at a median of four time points and RT was characterized via single-genome sequencing. The resulting sequences reveal a dynamic system in which vRNA rapidly acquires drug resistance concomitantly across compartments through multiple independent mutations. Fast migration results in the same viral genotypes present across compartments, but not so fast as to equilibrate their frequencies immediately. The blood and lymph nodes were found to be compartmentalized rarely, while both the blood and lymph node were more frequently different from mucosal tissues. This study suggests that even oft-sampled blood does not fully capture the viral dynamics in other parts of the body, especially the gut where vRNA turnover was faster than the plasma and vDNA retained fewer wild-type viruses than other sampled compartments. Our findings of transient compartmentalization across multiple tissues may help explain the varied results of previous compartmentalization studies in HIV-1.

Dating phylogenies with sequentially sampled tips.

We develop a Bayesian Markov chain Monte Carlo (MCMC) algorithm for estimating divergence times using sequentially sampled molecular sequences. This type of data is commonly collected during viral epidemics and is sometimes available from different species in ancient DNA studies. We derive the distribution of ages of nodes in the tree under a birth-death-sequential-sampling (BDSS) model and use it as the prior for divergence times in the dating analysis. We implement the prior in the MCMCtree program in the PAML package for divergence dating. The BDSS prior is very flexible and, with different parameters, can generate trees of very different shapes, suitable for examining the sensitivity of posterior time estimates. We apply the method to a data set of SIV/HIV-2 genes in comparison with a likelihood-based dating method, and to a data set of influenza H1 genes from different hosts in comparison with the Bayesian program BEAST. We examined the impact of tree topology on time estimates and suggest that multifurcating consensus trees should be avoided in dating analysis. We found posterior time estimates for old nodes to be sensitive to the priors on times and rates and suggest that previous Bayesian dating studies may have produced overconfident estimates.

HandAlign: Bayesian multiple sequence alignment, phylogeny and ancestral reconstruction.

UNLABELLED: We describe handalign, a software package for Bayesian reconstruction of phylogenetic history. The underlying model of sequence evolution describes indels and substitutions. Alignments, trees and model parameters are all treated as jointly dependent random variables and sampled via Metropolis-Hastings Markov chain Monte Carlo (MCMC), enabling systematic statistical parameter inference and hypothesis testing. handalign implements several different MCMC proposal kernels, allows sampling from arbitrary target distributions via Hastings ratios, and uses standard file formats for trees, alignments and models. AVAILABILITY AND IMPLEMENTATION: Installation and usage instructions are at http://biowiki.org/HandAlign.

Nef gene evolution from a single transmitted strain in acute SIV infection.

BACKGROUND: The acute phase of immunodeficiency virus infection plays a crucial role in determining steady-state virus load and subsequent progression of disease in both humans and nonhuman primates. The acute period is also the time when vaccine-mediated effects on host immunity are likely to exert their major effects on virus infection. Recently we developed a Monte-Carlo (MC) simulation with mathematical analysis of viral evolution during primary HIV-1 infection that enables classification of new HIV-1 infections originating from multiple versus single transmitted viral strains and the estimation of time elapsed following infection. RESULTS: A total of 322 SIV nef SIV sequences, collected during the first 3 weeks following experimental infection of two rhesus macaques with the SIVmac239 clone, were analyzed and found to display a comparable level of genetic diversity, 0.015% to 0.052%, with that of env sequences from acute HIV-1 infection, 0.005% to 0.127%. We confirmed that the acute HIV-1 infection model correctly identified the experimental SIV infections in rhesus macaques as "homogenous" infections, initiated by a single founder strain. The consensus sequence of the sampled strains corresponded to the transmitted sequence as the model predicted. However, measured sequential decrease in diversity at day 7, 11, and 18 post infection violated the model assumption, neutral evolution without any selection. CONCLUSION: While nef gene evolution over the first 3 weeks of SIV infection originating from a single transmitted strain showed a comparable rate of sequence evolution to that observed during acute HIV-1 infection, a purifying selection for the founder nef gene was observed during the early phase of experimental infection of a nonhuman primate.

Standardized assessment of NAb responses elicited in rhesus monkeys immunized with single- or multi-clade HIV-1 envelope immunogens.

The genetic diversity of HIV-1 envelope glycoproteins (Env) remains a major obstacle to the development of an antibody-based AIDS vaccine. The present studies examine the breadth and magnitude of neutralizing antibody (NAb) responses in rhesus monkeys after immunization with DNA prime-recombinant adenovirus (rAd) boost vaccines encoding either single or multiple genetically distant Env immunogens, and subsequently challenged with a pathogenic simian-human immunodeficiency virus (SHIV-89.6P). Using a standardized multi-tier panel of reference Env pseudoviruses for NAb assessment, we show that monkeys immunized with a mixture of Env immunogens (clades A, B, and C) exhibited a greater breadth of NAb activity against neutralization-sensitive Tier 1 viruses following both vaccination and challenge compared to monkeys immunized with a single Env immunogen (clade B or C). However, all groups of Env-vaccinated monkeys demonstrated only limited neutralizing activity against Tier 2 pseudoviruses, which are more characteristic of the neutralization sensitivity of circulating HIV-1. Notably, the development of a post-challenge NAb response against SHIV-89.6P was similar in monkeys receiving either clade B, clade C, or clade A+B+C Env immunogens, suggesting cross-clade priming of NAb responses. In addition, vaccines encoding Env immunogens heterologous to SHIV-89.6P primed for a rapid anamnestic NAb response following infection compared to vaccines lacking an Env component. These results show that DNA/rAd immunization with multiple diverse Env immunogens is a viable approach for enhancing the breadth of NAb responses against HIV-1, and suggest that Env immunogens can prime for anamnestic NAb responses against a heterologous challenge virus.

In vivo fitness costs of different Gag CD8 T-cell escape mutant simian-human immunodeficiency viruses for macaques.

The kinetics of immune escape and reversion depend upon the efficiency of CD8 cytotoxic T lymphocytes (CTL) and the fitness cost of escape mutations. Escape kinetics of three simian immunodeficiency virus Gag CTL epitopes in pigtail macaques were variable; those of KP9 and AF9 were faster than those of KW9. Kinetics of reversion of escape mutant virus to wild type upon passage to naïve major histocompatibility complex-mismatched macaques also varied. Rapid reversion occurred at KP9, gradual biphasic reversion occurred at AF9, and escape mutant KW9 virus failed to revert. The fitness impact of these mutations is KP9 > AF9 > KW9. These data provide insights into the differential utility of CTL in controlling viremia.

Incorporating indel information into phylogeny estimation for rapidly emerging pathogens.

BACKGROUND: Phylogenies of rapidly evolving pathogens can be difficult to resolve because of the small number of substitutions that accumulate in the short times since divergence. To improve resolution of such phylogenies we propose using insertion and deletion (indel) information in addition to substitution information. We accomplish this through joint estimation of alignment and phylogeny in a Bayesian framework, drawing inference using Markov chain Monte Carlo. Joint estimation of alignment and phylogeny sidesteps biases that stem from conditioning on a single alignment by taking into account the ensemble of near-optimal alignments. RESULTS: We introduce a novel Markov chain transition kernel that improves computational efficiency by proposing non-local topology rearrangements and by block sampling alignment and topology parameters. In addition, we extend our previous indel model to increase biological realism by placing indels preferentially on longer branches. We demonstrate the ability of indel information to increase phylogenetic resolution in examples drawn from within-host viral sequence samples. We also demonstrate the importance of taking alignment uncertainty into account when using such information. Finally, we show that codon-based substitution models can significantly affect alignment quality and phylogenetic inference by unrealistically forcing indels to begin and end between codons. CONCLUSION: These results indicate that indel information can improve phylogenetic resolution of recently diverged pathogens and that alignment uncertainty should be considered in such analyses.

Estimating Costs and Benefits of CTL Escape Mutations in SIV/HIV Infection.

Mutations that allow SIV/HIV to avoid the cytotoxic T lymphocyte (CTL) response are well documented. Recently, there have been a few attempts at estimating the costs of CTL escape mutations in terms of the reduction in viral fitness and the killing rate at which the CTL response specific to one viral epitope clears virus-infected cells. Using a mathematical model we show that estimation of both parameters depends critically on the underlying changes in the replication rate of the virus and the changes in the killing rate over time (which in previous studies were assumed to be constant). We provide a theoretical basis for estimation of these parameters using in vivo data. In particular, we show that 1) by assuming unlimited virus growth one can obtain a minimal estimate of the fitness cost of the escape mutation, and 2) by assuming no virus growth during the escape, one can obtain a minimal estimate of the average killing rate. We also discuss the conditions under which better estimates of the average killing rate can be obtained.

Setting the stage for bench-to-bedside movement of anti-HIV RNA inhibitors-gene therapy for AIDS in macaques.

Despite significant progress over the last two decades, treatment of HIV infection remains a tremendous challenge. Although antiretroviral therapy has proved quite effective in most HIV-infected patients, increasing recognition of toxicity and the emergence of multidrug resistant HIV strains has fueled the development of alternative therapeutic approaches. Introduction of genes to inhibit HIV replication into CD4+ T lymphocytes or hematopoietic stem cells represents a potentially attractive but still unproven strategy. Despite the availability of a diverse range of molecular strategies that are able to provide potent inhibition of HIV replication in the laboratory, translation of these in vitro successes to in vivo therapies has been difficult. Fundamental challenges facing AIDS gene therapy at the present time includes the need to increase the efficiency of gene transfer in vivo, to confer upon genetically-modified T cells the ability to have a selective growth advantage in vivo, and the development of additional techniques to decrease the probability of emergence of resistant viruses. As one of the leading animal models for AIDS and for hematopoietic stem cell gene therapy, nonhuman primates are ideally suited to help address many of these basic questions. This review will provide a general overview of RNA-based genetic strategies for inhibition of HIV and SIV replication, criteria to be considered in the selection of promising inhibitory strategies for in vivo use, and key questions that can be addressed in the macaque model.

Fitness costs limit viral escape from cytotoxic T lymphocytes at a structurally constrained epitope.

The intense selection pressure exerted by virus-specific cytotoxic T lymphocytes (CTL) on replicating human immunodeficiency virus and simian immunodeficiency virus results in the accumulation of CTL epitope mutations. It has been assumed that fitness costs can limit the evolution of CTL epitope mutations. However, only a limited number of studies have carefully examined this possibility. To explore the fitness costs associated with viral escape from p11C, C-M-specific CTL, we constructed a panel of viruses encoding point mutations at each position of the entire p11C, C-M epitope. Amino acid substitutions at positions 3, 4, 5, 6, 7, and 9 of the epitope significantly impaired virus replication by altering virus production and Gag protein expression as well as by destabilizing mature cores. Amino acid substitutions at position 2 of the epitope were tolerated but required reversion or additional compensatory mutations to generate replication-competent viruses. Finally, while amino acid substitutions at positions 1 and 8 of the p11C, C-M epitope were functionally tolerated, these substitutions were recognized by p11C, C-M-specific CTL and therefore provided no selection advantage for the virus. Together, these data suggest that limited sequence variation is tolerated by the region of the capsid encoding the p11C, C-M epitope and therefore that only a very limited number of mutations can allow successful viral escape from the p11C, C-M-specific CTL response.

HIV-1 and HIV-2 LTR nucleotide sequences: assessment of the alignment by N-block presentation, "retroviral signatures" of overrepeated oligonucleotides, and a probable important role of scrambled stepwise duplications/deletions in molecular evolution.

Previous analyses of retroviral nucleotide sequences, suggest a so-called "scrambled duplicative stepwise molecular evolution" (many sectors with successive duplications/deletions of short and longer motifs) that could have stemmed from one or several starter tandemly repeated short sequence(s). In the present report, we tested this hypothesis by focusing on the long terminal repeats (LTRs) (and flanking sequences) of 24 human and 3 simian immunodeficiency viruses. By using a calculation strategy applicable to short sequences, we found consensus overrepresented motifs (often containing CTG or CAG) that were congruent with the previously defined "retroviral signature." We also show many local repetition patterns that are significant when compared with simply shuffled sequences. First- and second-order Markov chain analyses demonstrate that a major portion of the overrepresented oligonucleotides can be predicted from the dinucleotide compositions of the sequences, but by no means can biological mechanisms be deduced from these results: some of the listed local repetitions remain significant against dinucleotide-conserving shuffled sequences; together with previous results, this suggests that interspersed and/or local mononucleotide and oligonucleotide repetitions could have biased the dinucleotide compositions of the sequences. We searched for suggestive evolutionary patterns by scrutinizing a reliable multiple alignment of the 27 sequences. A manually constructed alignment based on homology blocks was in good agreement with the polypeptide alignment in the coding sectors and has been exhaustively assessed by using a multiplied alphabet obtained by the promising mathematical strategy called the N-block presentation (taking into account the environment of each nucleotide in a sequence). Sector by sector, we hypothesize many successive duplication/deletion scenarios that fit our previous evolutionary hypotheses. This suggests an important duplication/deletion role for the reverse transcriptase, particularly in inducing stuttering cryptic simplicity patterns.

The assessment of nucleotide sequence diversity by the polymerase chain reaction is highly reproducible.

The polymerase chain reaction (PCR) is often used to assess the diversity of viral nucleotide sequences present in various biological samples (e.g., blood and tissues). However, it is not clear how reproducible this approach may be. DNA was extracted from the peripheral blood lymphocytes of a macaque that had been infected, experimentally, with SIVmac251-32H 6 months previously. The nef gene was then amplified by the PCR on three separate occasions from this same template preparation. A panel of clones was prepared from the product of each PCR, the entire nef gene was sequenced and the sequences obtained compared with each other. Phenogram analysis revealed that within each panel the same degree of sequence diversity was observed between clones. Furthermore, when the sequences obtained from all three panels were compared, the overall sequence diversity observed was no greater than that observed for each panel individually. These data indicate that the analysis of sequence diversity in SIV 'quasi-species' populations by the PCR is reliable and, more important, reproducible.

Detection of unusual RNA folding regions in HIV and SIV sequences.

We have developed a method for detecting more stable and significant folding regions relative to others in the sequence. The algorithm is based on the calculation of the lowest free energy of RNA secondary structures and Monte Carlo simulation. For any given RNA segment, the stability and statistical significance of RNA folding are assessed by two measures: the stability score and the significance score. The stability score measures the degree of thermodynamic stability of the segment between all possible biological segments in the RNA sequence. The significance score characterizes the specific arrangement of the nucleotides in the segment that could imply a structural role for the sequence information. Using these two measures, we are able to detect a series of distinct folding regions where highly stable and statistically significant secondary structures occur in human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) sequences.