Sergei L. Kosakovsky Pond

Persistent HIV-1 replication maintains the tissue reservoir during therapy

Lymphoid tissue is a key reservoir established by HIV-1 during acute infection. It is a site associated with viral production, storage of viral particles in immune complexes, and viral persistence. Although combinations of antiretroviral drugs usually suppress viral replication and reduce viral RNA to undetectable levels in blood, it is unclear whether treatment fully suppresses viral replication in lymphoid tissue reservoirs. Here we show that virus evolution and trafficking between tissue compartments continues in patients with undetectable levels of virus in their bloodstream. We present a spatial and dynamic model of persistent viral replication and spread that indicates why the development of drug resistance is not a foregone conclusion under conditions in which drug concentrations are insufficient to completely block virus replication. These data provide new insights into the evolutionary and infection dynamics of the virus population within the host, revealing that HIV-1 can continue to replicate and replenish the viral reservoir despite potent antiretroviral therapy.

Mode and rate of evolution of haemosporidian mitochondrial genomes: Timing the radiation of avian parasites

&NA; Haemosporidians are a diverse group of vector‐borne parasitic protozoa that includes the agents of human malaria; however, most of the described species are found in birds and reptiles. Although our understanding of these parasites’ diversity has expanded by analyses of their mitochondrial genes, there is limited information on these genes’ evolutionary rates. Here, 114 mitochondrial genomes (mtDNA) were studied from species belonging to four genera: Leucocytozoon, Haemoproteus, Hepatocystis, and Plasmodium. Contrary to previous assertions, the mtDNA is phylogenetically informative. The inferred phylogeny showed that, like the genus Plasmodium, the Leucocytozoon and Haemoproteus genera are not monophyletic groups. Although sensitive to the assumptions of the molecular dating method used, the estimated times indicate that the diversification of the avian haemosporidian subgenera/genera took place after the Cretaceous‐Paleogene boundary following the radiation of modern birds. Furthermore, parasite clade differences in mtDNA substitution rates and strength of negative selection were detected. These differences may affect the biological interpretation of mtDNA gene lineages used as a proxy to species in ecological and parasitological investigations. Given that the mitochondria are critically important in the parasite life cycle stages that take place in the vector and that the transmission of parasites belonging to particular clades has been linked to specific insect families/subfamilies, this study suggests that differences in vectors have affected the mode of evolution of haemosporidian mtDNA genes. The observed patterns also suggest that the radiation of haemosporidian parasites may be the result of community‐level evolutionary processes between their vertebrate and invertebrate hosts.

Compartmentalized HIV rebound in the central nervous system after interruption of antiretroviral therapy

To design effective eradication strategies, it may be necessary to target HIV reservoirs in anatomic compartments other than blood. This study examined HIV RNA rebound following interruption of antiretroviral therapy (ART) in blood and cerebrospinal fluid (CSF) to determine whether the central nervous system (CNS) might serve as an independent source of resurgent viral replication. Paired blood and CSF samples were collected longitudinally from 14 chronically HIV-infected individuals undergoing ART interruption. HIV env (C2-V3), gag (p24) and pol (reverse transcriptase) were sequenced from cell-free HIV RNA and cell-associated HIV DNA in blood and CSF using the Roche 454 FLX Titanium platform. Comprehensive sequence and phylogenetic analyses were performed to search for evidence of unique or differentially represented viral subpopulations emerging in CSF supernatant as compared with blood plasma. Using a conservative definition of compartmentalization based on four distinct statistical tests, nine participants presented a compartmentalized HIV RNA rebound within the CSF after interruption of ART, even when sampled within 2 weeks from viral rebound. The degree and duration of viral compartmentalization varied considerably between subjects and between time-points within a subject. In 10 cases, we identified viral populations within the CSF supernatant at the first sampled time-point after ART interruption, which were phylogenetically distinct from those present in the paired blood plasma and mostly persisted over time (when longitudinal time-points were available). Our data suggest that an independent source of HIV RNA contributes to viral rebound within the CSF after treatment interruption. The most likely source of compartmentalized HIV RNA is a CNS reservoir that would need to be targeted to achieve complete HIV eradication.

In Vivo VL-Targeted Microbial Superantigen Induced Global Shifts in the B Cell Repertoire

To subvert host defenses, some microbial pathogens produce proteins that interact with conserved motifs in V regions of B cell Ag receptor shared by large sets of lymphocytes, which define the properties of a superantigen. Because the clonal composition of the lymphocyte pool is a major determinant of immune responsiveness, this study was undertaken to examine the in vivo effect on the host immune system of exposure to a B cell superantigen, protein L (PpL), a product of the common commensal bacterial species, Finegoldia magna, which is one of the most common pathogenic species among Gram-positive anaerobic cocci. Libraries of Vκ L chain transcripts were generated from the spleens of control and PpL-exposed mice, and the expressed Vκ rearrangements were characterized by high-throughput sequencing. A total of 120,855 sequencing reads could be assigned to a germline Vκ gene, with all 20 known Vκ subgroups represented. In control mice, we found a recurrent and consistent hierarchy of Vκ gene usage, as well as patterns of preferential Vκ–Jκ pairing. PpL exposure induced significant targeted global shifts in repertoire with reduction of Vκ that contain the superantigen binding motif in all exposed mice. We found significant targeted reductions in the expression of clonotypes encoded by 14 specific Vκ genes with the predicted PpL binding motif. These rigorous surveys document the capacity of a microbial protein to modulate the composition of the expressed lymphocyte repertoire, which also has broad potential implications for host–microbiome and host–pathogen relationships.

Datamonkey 2.0: A Modern Web Application for Characterizing Selective and Other Evolutionary Processes

Inference of how evolutionary forces have shaped extant genetic diversity is a cornerstone of modern comparative sequence analysis. Advances in sequence generation and increased statistical sophistication of relevant methods now allow researchers to extract ever more evolutionary signal from the data, albeit at an increased computational cost. Here, we announce the release of Datamonkey 2.0, a completely re-engineered version of the Datamonkey web-server for analyzing evolutionary signatures in sequence data. For this endeavor, we leveraged recent developments in open-source libraries that facilitate interactive, robust, and scalable web application development. Datamonkey 2.0 provides a carefully curated collection of methods for interrogating coding-sequence alignments for imprints of natural selection, packaged as a responsive (i.e. can be viewed on tablet and mobile devices), fully interactive, and API-enabled web application. To complement Datamonkey 2.0, we additionally release HyPhy Vision, an accompanying JavaScript application for visualizing analysis results. HyPhy Vision can also be used separately from Datamonkey 2.0 to visualize locally-executed HyPhy analyses. Together, Datamonkey 2.0 and HyPhy Vision showcase how scientific software development can benefit from general-purpose open-source frameworks. Datamonkey 2.0 is freely and publicly available at http://www.datamonkey. org, and the underlying codebase is available from https://github.com/veg/datamonkey-js.

Relative evolutionary rate inference in HyPhy with LEISR

We introduce LEISR (Likehood Estimation of Individual Site Rates, pronounced “laser”), a tool to infer relative evolutionary rates from protein and nucleotide data, implemented in HyPhy. LEISR is based on the popular Rate4Site (Pupko et al., 2002) approach for inferring relative site-wise evolutionary rates, primarily from protein data. We extend the original method for more general use in several key ways: (i) we increase the support for nucleotide data with additional models, (ii) we allow for datasets of arbitrary size, (iii) we support analysis of site-partitioned datasets to correct for the presence of recombination breakpoints, (iv) we produce rate estimates at all sites rather than at just a subset of sites, and (v) we implemented LEISR as MPI-enabled to support rapid, high-throughput analysis. LEISR is available in HyPhy starting with version 2.3.8, and it is accessible as an option in the HyPhy analysis menu (“Relative evolutionary rate inference”), which calls the HyPhy batchfile LEISR.bf.

Evolution of gag and gp41 in Patients Receiving Ritonavir-Boosted Protease Inhibitors

Several groups have proposed that genotypic determinants in gag and the gp41 cytoplasmic domain (gp41-CD) reduce protease inhibitor (PI) susceptibility without PI-resistance mutations in protease. However, no gag and gp41-CD mutations definitively responsible for reduced PI susceptibility have been identified in individuals with virological failure (VF) while receiving a boosted PI (PI/r)-containing regimen. To identify gag and gp41 mutations under selective PI pressure, we sequenced gag and/or gp41 in 61 individuals with VF on a PI/r (n = 40) or NNRTI (n = 20) containing regimen. We quantified nonsynonymous and synonymous changes in both genes and identified sites exhibiting signal for directional or diversifying selection. We also used published gag and gp41 polymorphism data to highlight mutations displaying a high selection index, defined as changing from a conserved to an uncommon amino acid. Many amino acid mutations developed in gag and in gp41-CD in both the PI- and NNRTI-treated groups. However, in neither gene, were there discernable differences between the two groups in overall numbers of mutations, mutations displaying evidence of diversifying or directional selection, or mutations with a high selection index. If gag and/or gp41 encode PI-resistance mutations, they may not be confined to consistent mutations at a few sites.

Lorenzo-Redondo et al. reply

Despite antiretroviral therapy (ART), a latent reservoir of replicationcompetent HIV-1 persists in resting memory CD4+ T cells and precludes a cure1–6. Lorenzo-Redondo et al.7 analysed HIV-1 sequences collected from three individuals during the first six months of ART, discovered specific patterns of sequence evolution, and concluded that viral replication persists during therapy. We believe that these evolutionary patterns are artefacts of rapidly decaying viral subpopulations present during the first months of therapy and are not characteristic of the long-lived reservoir. We therefore argue that ref. 7 does not provide evidence that ongoing replication is an additional barrier to a cure for treated individuals who consistently maintain low viral loads. There is a Reply to this Comment by Lorenzo-Redondo, R. et al. Nature 551, http://doi.org/10.1038/nature24635 (2017). Lorenzo-Redondo et al.7 collected samples before and three and six months after treatment initiation, when labile viral populations dominate and change rapidly. Before treatment, most HIV-1 DNA in resting CD4+ T cells exists in an unintegrated state, decaying with a half-life of days8,9. Another major population of infected resting cells decays with a half-life of weeks10. The latent reservoir of integrated proviruses, observed in blood and lymphoid tissue1, is smaller and decays with half-life of approximately four years5,6. Lifelong persistence of this reservoir is determined by the longevity and proliferation of the infected cells11. Initiation of ART blocks new infection from replenishing these populations, revealing their different lifespans. Differential decay causes marked shifts in infected cell populations in the first six months of ART, bringing into question any conclusions about viral evolution gleaned from this period. Below, we support our claim by simulating differential decay and replicating the analysis of ref. 7 on the simulated data. We find that false signals of viral evolution—and ongoing viral replication—often appear. To estimate the size and decay of labile compartments, we examined a cohort of seven early-treated individuals, which we consider comparable to the two early-treated participants in ref. 7. The quantitative viral outgrowth assay (qVOA) has been used on blood samples10 to detect resting CD4+ T cells harbouring replication-competent HIV-1. At the onset of ART, infected cell frequencies greatly exceeded those of individuals on long-term ART. A multi-log, multi-phasic decay over the first year of therapy reduced frequencies to levels typically observed during long-term ART. Fitting to the most extensively sampled individual, we inferred a large, fast-decaying population, a smaller population with slower decay, and a very small, persistent reservoir, which was approximated as constant (Fig. 1). At zero, three, and six months after ART initiation, labile populations comprised 99.99%, 96.2%, and 76% of infected resting cells, respectively, masking the persistent reservoir. The RNA-based assays performed by Lorenzo-Redondo et al.7 on lymphoid tissue paint a similar picture: the infection decays rapidly over the first three to six months, eventually dwindling to a more stable state that is more than three orders of magnitude smaller than the pretreatment population (extended data fig. 1 of ref. 7). Regardless of sequencing depth, the limited number of infected cells in a blood draw or tissue biopsy is likely to prevent the persistent reservoir from being sequenced at early time points. The genetic diversity of this reservoir emerges only later. Latency studies are therefore generally restricted to participants who have received suppressive ART for more than six months, a precaution not taken by Lorenzo-Redondo et al.7. Brodin et al.12 suggested that the decay of labile populations may produce false signals of evolution during treatment, even in the absence of viral replication. We used computer simulations of viral populations during acute infection and treatment to confirm this hypothesis. Simulated virus replicated and seeded subpopulations for four months, and treatment then blocked replication for six months. During treatment, labile subpopulations decayed, while a stable reservoir persisted, as in Fig. 1. Nearly 12,000 simulations were subjected to the tests performed by Lorenzo-Redondo et al.7: genetic divergence from start to end of therapy, evolutionary rate calculations, and measurement of clock-like signal in maximum-likelihood trees (Supplementary Tables 1, 2).

Evaluation of global HIV/SIV envelope gp120 RNA structure and evolution within and among infected hosts

Abstract Lentiviral RNA genomes contain structural elements that play critical roles in viral replication. Although structural features of 5′-untranslated regions have been well characterized, attempts to identify important structures in other genomic regions by Selective 2′-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) have led to conflicting structural and mechanistic conclusions. Previous approaches accounted neither for sequence heterogeneity that is ubiquitous in viral populations, nor for selective constraints operating at the protein level. We developed an approach that augments SHAPE with phylogenetic analyses and applied it to investigate structure in coding regions (cRNA) within the HIV and SIV envelope genes. Analysis of single-genome SHAPE data with phylogenetic information from diverse lentiviral sequences argues against the conservation of a putative global gp120 RNA structure but points to the existence of core RNA sub-structures. Our findings establish a framework for considering sequence heterogeneity and protein function in de novo RNA structure inference approaches.

Selection analyses of paired HIV-1 gag and gp41 sequences obtained before and after antiretroviral therapy

Most HIV-1-infected individuals with virological failure on a pharmacologically-boosted protease inhibitor (PI) regimen do not develop PI-resistance protease mutations. One proposed explanation is that HIV-1 gag or gp41 cytoplasmic domain mutations might also reduce PI susceptibility. In a recent study of paired gag and gp41 sequences from individuals with virological failure on a PI regimen, we did not identify PI-selected mutations and concluded that if such mutations existed, larger numbers of paired sequences from multiple studies would be needed for their identification. In this study, we generated site-specific amino acid profiles using gag and gp41 published sequences from 5,338 and 4,242 ART-naïve individuals, respectively, to assist researchers identify unusual mutations arising during therapy and to provide scripts for performing established and novel maximal likelihood estimates of dN/dS substitution rates in paired sequences. The pipelines used to generate the curated sequences, amino acid profiles, and dN/dS analyses will facilitate the application of consistent methods to paired gag and gp41 sequence datasets and expedite the identification of potential sites under PI-selection pressure.