West Central Africa has been implicated as the epicenter of the HIV-1 epidemic, and almost all group M subtypes can be found there. Previous analysis of early HIV-1 group M sequences from Kinshasa in the Democratic Republic of Congo, formerly Zaire, revealed that isolates from a number of individuals fall in different positions in phylogenetic trees constructed from sequences from opposite ends of the genome as a result of recombination between viruses of different subtypes. Here, we use discrete ancestral trait mapping to develop a procedure for quantifying HIV-1 group M intersubtype recombination across phylogenies, using individuals' gag (p17) and env (gp41) subtypes. The method was applied to previously described HIV-1 group M sequences from samples obtained in Kinshasa early in the global radiation of HIV. Nine different p17 and gp41 intersubtype recombinant combinations were present in the data set. The mean number of excess ancestral subtype transitions (NEST) required to map individuals' p17 subtypes onto the gp14 phylogeny samples, compared to the number required to map them onto the p17 phylogenies, and vice versa, indicated that excess subtype transitions occurred at a rate of approximately 7 × 10−3 to 8 × 10−3 per lineage per year as a result of intersubtype recombination. Our results imply that intersubtype recombination may have occurred in approximately 20% of lineages evolving over a period of 30 years and confirm intersubtype recombination as a substantial force in generating HIV-1 group M diversity.

Viruses commonly use host cell survival mechanisms to their own advantage. We show that Akt, an important signaling kinase involved in cell survival, phosphorylates the RNA-dependent RNA polymerase (RdRp) from norovirus, the major cause of gastroenteritis outbreaks worldwide. The Akt phosphorylation of RdRp appears to be a feature unique to the more prevalent norovirus genotypes such as GII.4 and GII.b. This phosphorylation event occurs at a residue (Thr33) located at the interface where the RdRp finger and thumb domains interact and decreases de novo activity of the polymerase. This finding provides fresh insights into virus-host cell interactions.

The directional migration of neutrophils towards inflammatory mediators, such as chemokines and cannabinoids, occurs via the activation of seven transmembrane G protein coupled receptors (7TM/GPCRs) and is a highly organized process. A crucial role for controlling neutrophil migration has been ascribed to the cannabinoid CB2 receptor (CB2R), but additional modulatory sites distinct from CB2R have recently been suggested to impact CB2R-mediated effector functions in neutrophils. Here, we provide evidence that the recently de-orphanized 7TM/GPCR GPR55 potently modulates CB2R-mediated responses. We show that GPR55 is expressed in human blood neutrophils and its activation augments the migratory response towards the CB2R agonist 2-arachidonoylglycerol (2-AG), while inhibiting neutrophil degranulation and reactive oxygen species (ROS) production. Using HEK293 and HL60 cell lines, along with primary neutrophils, we show that GPR55 and CB2R interfere with each other's signaling pathways at the level of small GTPases, such as Rac2 and Cdc42. This ultimately leads to cellular polarization and efficient migration as well as abrogation of degranulation and ROS formation in neutrophils. Therefore, GPR55 limits the tissue-injuring inflammatory responses mediated by CB2R, while it synergizes with CB2R in recruiting neutrophils to sites of inflammation.

Background. Many studies of sexual behavior have shown that individuals vary greatly in their number of sexual partners over time, but it has proved difficult to obtain parameter estimates relating to the dynamics of human immunodeficiency virus (HIV) transmission except in small-scale contact tracing studies. Recent developments in molecular phylodynamics have provided new routes to obtain these parameter estimates, and current clinical practice provides suitable data for entire infected populations.

Methods. A phylodynamic analysis was performed on partial pol gene sequences obtained for routine clinical care from 14 560 individuals, representing approximately 60% of the HIV-positive men who have sex with men (MSM) under care in the United Kingdom.

Results. Among individuals linked to others in the data set, 29% are linked to only 1 individual, 41% are linked to 2–10 individuals, and 29% are linked to ≥10 individuals. The right-skewed degree distribution can be approximated by a power law, but the data are best fitted by a Waring distribution for all time depths. For time depths of 5–7 years, the distribution parameter ρ lies within the range that indicates infinite variance.

Conclusions. The transmission network among UK MSM is characterized by preferential association such that a randomly distributed intervention would not be expected to stop the epidemic.

The HIV epidemic in higher-income nations is driven by receptive anal intercourse, injection drug use through needle/syringe sharing, and, less efficiently, vaginal intercourse. Alcohol and noninjecting drug use increase sexual HIV vulnerability. Appropriate diagnostic screening has nearly eliminated blood/blood product-related transmissions and, with antiretroviral therapy, has reduced mother-to-child transmission radically. Affected subgroups have changed over time (e.g., increasing numbers of Black and minority ethnic men who have sex with men). Molecular phylogenetic approaches have established historical links between HIV strains from central Africa to those in the United States and thence to Europe. However, Europe did not just receive virus from the United States, as it was also imported from Africa directly. Initial introductions led to epidemics in different risk groups in Western Europe distinguished by viral clades/sequences, and likewise, more recent explosive epidemics linked to injection drug use in Eastern Europe are associated with specific strains. Recent developments in phylodynamic approaches have made it possible to obtain estimates of sequence evolution rates and network parameters for epidemics.

Molecular phylogenetic approaches have traced the evolutionary history of HIV strains, showing that HIV spread from central Africa to the United States and then to Europe, as well as directly from Africa to Europe.

The directional migration of neutrophils towards inflammatory mediators, such as chemokines and cannabinoids, occurs via the activation of seven transmembrane spanning/G protein coupled receptors (7TM/GPCRs) and is a highly organized process. A crucial role for controlling neutrophil migration has been ascribed to the cannabinoid CB2 receptor (CB2R), but additional modulatory sites distinct from CB2R have recently been suggested to impact CB2R-mediated effector functions in neutrophils. Here, we provide evidence that the recently de-orphanized 7TM/GPCR GPR55 potently modulates CB2R-mediated responses. We show that GPR55 is expressed in human blood neutrophils and its activation augments the migratory response towards the CB2R agonist 2-arachidonoylglycerol (2-AG), whilst inhibiting the degranulation and reactive oxygen species (ROS) production. Using HEK293 and HL60 cell lines, along with primary neutrophils, we show that the GPR55 and the cannabinoid 2 receptor (CB2R) interfere with each other’s signaling pathways at the level of small GTPases, such as Rac2 and Cdc42. This ultimately leads to cellular polarization and efficient migration but abrogation of degranulation and ROS formation in neutrophils. Therefore, GPR55 limits the tissue-injuring inflammatory responses mediated by CB2R, while it synergizes with CB2R in recruiting neutrophils to sites of inflammation.

Lipid droplets (LDs) are important cellular organelles that govern the storage and turnover of lipids. Little is known about how the size of LDs is controlled, although LDs of diverse sizes have been observed in different tissues and under different (patho)physiological conditions. Recent studies have indicated that the size of LDs may influence adipogenesis, the rate of lipolysis and the oxidation of fatty acids. Here, a genome-wide screen identifies ten yeast mutants producing “supersized” LDs that are up to 50 times the volume of those in wild-type cells. The mutated genes include: FLD1, which encodes a homologue of mammalian seipin; five genes (CDS1, INO2, INO4, CHO2, and OPI3) that are known to regulate phospholipid metabolism; two genes (CKB1 and CKB2) encoding subunits of the casein kinase 2; and two genes (MRPS35 and RTC2) of unknown function. Biochemical and genetic analyses reveal that a common feature of these mutants is an increase in the level of cellular phosphatidic acid (PA). Results from in vivo and in vitro analyses indicate that PA may facilitate the coalescence of contacting LDs, resulting in the formation of “supersized” LDs. In summary, our results provide important insights into how the size of LDs is determined and identify novel gene products that regulate phospholipid metabolism.

Author Summary

Lipid droplets (LD) are primary lipid storage structures that also function in membrane and lipid trafficking, protein turnover, and the reproduction of deadly viruses. Increased LD accumulation in liver, skeletal muscle, and adipose tissue is a hallmark of the metabolic syndrome. Enlarged LDs are often found in these tissues under disease conditions. However, little is known about how the size of LDs is controlled in eukaryotic cells. In this study, we use genetic and biochemical methods to identify important gene products that regulate the size of the LDs. Notably, a common feature among these mutants with “supersized” LDs is an increased level of phosphatidic acid (PA). We also show that a small amount of PA can increase the size of artificial LDs in vitro. Overall, our study identifies important lipids and proteins in determining LD size. These results provide valuable insights into how human cells/tissues handle abnormal influx of lipids in today's obesogenic environment.

ORP5 works together with Niemann Pick C-1 to facilitate exit of cholesterol from endosomes and lysosomes.

Oxysterol-binding protein (OSBP) and its related proteins (ORPs) constitute a large and evolutionarily conserved family of lipid-binding proteins that target organelle membranes to mediate sterol signaling and/or transport. Here we characterize ORP5, a tail-anchored ORP protein that localizes to the endoplasmic reticulum. Knocking down ORP5 causes cholesterol accumulation in late endosomes and lysosomes, which is reminiscent of the cholesterol trafficking defect in Niemann Pick C (NPC) fibroblasts. Cholesterol appears to accumulate in the limiting membranes of endosomal compartments in ORP5-depleted cells, whereas depletion of NPC1 or both ORP5 and NPC1 results in luminal accumulation of cholesterol. Moreover, trans-Golgi resident proteins mislocalize to endosomal compartments upon ORP5 depletion, which depends on a functional NPC1. Our results establish the first link between NPC1 and a cytoplasmic sterol carrier, and suggest that ORP5 may cooperate with NPC1 to mediate the exit of cholesterol from endosomes/lysosomes.

Akt is a critical regulator of cell growth, proliferation, and survival that is activated by phosphatidylinositol 3-kinase (PI3K). We investigated the effect of PI3K inhibition on activation of sterol regulatory element binding protein-2 (SREBP-2), a master regulator of cholesterol homeostasis. SREBP-2 processing increased in response to various cholesterol depletion approaches (including statin treatment) and this increase was blunted by treatment with a potent and specific inhibitor of PI3K, LY294002, or when a plasmid encoding a dominant-negative form of Akt (DN-Akt) was expressed. LY294002 also suppressed SREBP-2 processing induced by insulin-like growth factor-1. Furthermore, LY294002 treatment down-regulated SREBP-2 or -1c gene targets and decreased cholesterol and fatty acid synthesis. Fluorescence microscopy studies indicated that LY294002 disrupts transport of the SREBP escort protein, SCAP, from the endoplasmic reticulum to the Golgi. This disruption was also shown by immunofluorescence staining when DN-Akt was expressed. Taken together, our studies indicate that the PI3K/Akt pathway is involved in SREBP-2 transport to the Golgi, contributing to the control of SREBP-2 activation. Our results provide a crucial mechanistic link between the SREBP and PI3K/Akt pathways that may be reconciled teleologically because synthesis of new membrane is an absolute requirement for cell growth and proliferation.

Criminal convictions relying solely on this to establish transmission are unsafe

The heterosexual risk group has become the largest HIV infected group in the United Kingdom during the last 10 years, but little is known of the network structure and dynamics of viral transmission in this group. The overwhelming majority of UK heterosexual infections are of non-B HIV subtypes, indicating viruses originating among immigrants from sub-Saharan Africa. The high rate of HIV evolution, combined with the availability of a very high density sample of viral sequences from routine clinical care has allowed the phylodynamics of the epidemic to be investigated for the first time. Sequences of the viral protease and partial reverse transcriptase coding regions from 11,071 patients infected with HIV of non-B subtypes were studied. Of these, 2774 were closely linked to at least one other sequence by nucleotide distance. Including the closest sequences from the global HIV database identified 296 individuals that were in UK-based groups of 3 or more individuals. There were a total of 8 UK-based clusters of 10 or more, comprising 143/2774 (5%) individuals, much lower than the figure of 25% obtained earlier for men who have sex with men (MSM). Sample dates were incorporated into relaxed clock phylogenetic analyses to estimate the dates of internal nodes. From the resulting time-resolved phylogenies, the internode lengths, used as estimates of maximum transmission intervals, had a median of 27 months overall, over twice as long as obtained for MSM (14 months), with only 2% of transmissions occurring in the first 6 months after infection. This phylodynamic analysis of non-B subtype HIV sequences representing over 40% of the estimated UK HIV-infected heterosexual population has revealed heterosexual HIV transmission in the UK is clustered, but on average in smaller groups and is transmitted with slower dynamics than among MSM. More effective intervention to restrict the epidemic may therefore be feasible, given effective diagnosis programmes.

Author Summary

Since 1995, HIV among heterosexuals in the UK increased to the point where the total number of heterosexuals infected with HIV, predominantly of non-B subtypes, exceeds the number of HIV-positive homosexual men. To understand the dynamics of this epidemic, we have applied the novel technique of phylodynamics to the analysis of viral sequences taken in the course of routine clinical care from approximately 40% of the HIV-infected heterosexual population in the UK. Phylodynamics reconstructs the pattern of viral sequence divergence in time, revealing the size of transmission clusters and the dynamics of transmission within them. Of 11,071 patients studied, 296 were linked to at least two others in the UK. There were 8 clusters comprising 10 or more individuals among these, yielding a total of 143 or 5% of all individuals with links, much lower than seen earlier among homosexual men (25%). Viral transmissions within clusters also occurred less rapidly, only 2% being dated to the first 6 months of infection, compared to 25% among homosexual men. Overall, transmission clusters exist in the UK heterosexual HIV epidemic but they are generally smaller than among homosexuals; onward transmission occurs less rapidly and is not associated with acute HIV infection.

We develop a model-based phylogenetic maximum likelihood test for evidence of preferential substitution toward a given residue at individual positions of a protein alignment—directional evolution of protein sequences (DEPS). DEPS can identify both the target residue and sites evolving toward it, help detect selective sweeps and frequency-dependent selection—scenarios that confound most existing tests for selection, and achieve good power and accuracy on simulated data. We applied DEPS to alignments representing different genomic regions of influenza A virus (IAV), sampled from avian hosts (H5N1 serotype) and human hosts (H3N2 serotype), and identified multiple directionally evolving sites in 5/8 genomic segments of H5N1 and H3N2 IAV. We propose a simple descriptive classification of directionally evolving sites into 5 groups based on the temporal distribution of residue frequencies and document known functional correlates, such as immune escape or host adaptation.

Lipid droplets (LDs) are emerging cellular organelles that are of crucial importance in cell biology and human diseases. In this study, we present our screen of ∼4,700 Saccharomyces cerevisiae mutants for abnormalities in the number and morphology of LDs; we identify 17 fld (few LDs) and 116 mld (many LDs) mutants. One of the fld mutants (fld1) is caused by the deletion of YLR404W, a previously uncharacterized open reading frame. Cells lacking FLD1 contain strikingly enlarged (supersized) LDs, and LDs from fld1Δ cells demonstrate significantly enhanced fusion activities both in vivo and in vitro. Interestingly, the expression of human seipin, whose mutant forms are associated with Berardinelli-Seip congenital lipodystrophy and motoneuron disorders, rescues LD-associated defects in fld1Δ cells. Lipid profiling reveals alterations in acyl chain compositions of major phospholipids in fld1Δ cells. These results suggest that an evolutionally conserved function of seipin in phospholipid metabolism and LD formation may be functionally important in human adipogenesis.

Background

The structure of sexual contact networks plays a key role in the epidemiology of sexually transmitted infections, and their reconstruction from interview data has provided valuable insights into the spread of infection. For HIV, the long period of infectivity has made the interpretation of contact networks more difficult, and major discrepancies have been observed between the contact network and the transmission network revealed by viral phylogenetics. The high rate of HIV evolution in principle allows for detailed reconstruction of links between virus from different individuals, but often sampling has been too sparse to describe the structure of the transmission network. The aim of this study was to analyze a high-density sample of an HIV-infected population using recently developed techniques in phylogenetics to infer the short-term dynamics of the epidemic among men who have sex with men (MSM).

Methods and Findings

Sequences of the protease and reverse transcriptase coding regions from 2,126 patients, predominantly MSM, from London were compared: 402 of these showed a close match to at least one other subtype B sequence. Nine large clusters were identified on the basis of genetic distance; all were confirmed by Bayesian Monte Carlo Markov chain (MCMC) phylogenetic analysis. Overall, 25% of individuals with a close match with one sequence are linked to 10 or more others. Dated phylogenies of the clusters using a relaxed clock indicated that 65% of the transmissions within clusters took place between 1995 and 2000, and 25% occurred within 6 mo after infection. The likelihood that not all members of the clusters have been identified renders the latter observation conservative.

Conclusions

Reconstruction of the HIV transmission network using a dated phylogeny approach has revealed the HIV epidemic among MSM in London to have been episodic, with evidence of multiple clusters of transmissions dating to the late 1990s, a period when HIV prevalence is known to have doubled in this population. The quantitative description of the transmission dynamics among MSM will be important for parameterization of epidemiological models and in designing intervention strategies.

Using viral genotype data from HIV drug resistance testing at a London clinic, Andrew Leigh Brown and colleagues derive the structure of the transmission network through phylogenetic analysis.

Editors' Summary

Background.

Human immunodeficiency virus (HIV), the cause of acquired immunodeficiency syndrome (AIDS), is mainly spread through unprotected sex with an infected partner. Like other sexually transmitted diseases, HIV/AIDS spreads through networks of sexual contacts. The characteristics of these complex networks (which include people who have serial sexual relationships with single partners and people who have concurrent sexual relationships with several partners) affect how quickly diseases spread in the short term and how common the disease is in the long term. For many sexually transmitted diseases, sexual contact networks can be reconstructed from interview data. The information gained in this way can be used for partner notification so that transmitters of the disease and people who may have been unknowingly infected can be identified, treated, and advised about disease prevention. It can also be used to develop effective community-based prevention strategies.

Why Was This Study Done?

Although sexual contact networks have provided valuable information about the spread of many sexually transmitted diseases, they cannot easily be used to understand HIV transmission patterns. This is because the period of infectivity with HIV is long and the risk of infection from a single sexual contact with an infected person is low. Another way to understand the spread of HIV is through phylogenetics, which examines the genetic relatedness of viruses obtained from different individuals. Frequent small changes in the genetic blueprint of HIV allow the virus to avoid the human immune response and to become resistant to antiretroviral drugs. In this study, the researchers use recently developed analytical methods, viral sequences from a large proportion of a specific HIV-infected population, and information on when each sample was taken, to learn about transmission of HIV/AIDS in London among men who have sex with men (MSM; a term that encompasses gay, bisexual, and transgendered men and heterosexual men who sometimes have sex with men). This new approach, which combines information on viral genetic variation and viral population dynamics, is called “molecular phylodynamics.”

What Did the Researchers Do and Find?

The researchers compared the sequences of the genes encoding the HIV-1 protease and reverse transcriptase from more than 2,000 patients, mainly MSM, attending a large London HIV clinic between 1997 and 2003. 402 of these sequences closely matched at least one other subtype B sequence (the HIV/AIDS epidemic among MSM in the UK primarily involves HIV subtype B). Further analysis showed that the patients from whom this subset of sequences came formed six clusters of ten or more individuals, as well as many smaller clusters, based on the genetic relatedness of their HIV viruses. The researchers then used information on the date when each sample was collected and a “relaxed clock” approach (which accounts for the possibility that different sequences evolve at different rates) to determine dated phylogenies (patterns of genetic relatedness that indicate when gene sequences change) for the clusters. These phylogenies indicated that at least in one in four transmissions between the individuals in the large clusters occurred within 6 months of infection, and that most of the transmissions within each cluster occurred over periods of 3–4 years during the late 1990s.

What Do These Findings Mean?

This phylodynamic reconstruction of the HIV transmission network among MSM in a London clinic indicates that the HIV epidemic in this population has been episodic with multiple clusters of transmission occurring during the late 1990s, a time when the number of HIV infections in this population doubled. It also suggests that transmission of the virus during the early stages of HIV infection is likely to be an important driver of the epidemic. Whether these results apply more generally to the MSM population at risk for transmitting or acquiring HIV depends on whether the patients in this study are representative of that group. Additional studies are needed to determine this, but if the patterns revealed here are generalizable, then this quantitative description of HIV transmission dynamics should help in the design of strategies to strengthen HIV prevention among MSM.

Additional Information.

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050050.

Read a related PLoS Medicine Perspective article

Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS

HIV InSite has comprehensive information on all aspects of HIV/AIDS, including a list of organizations that provide information for gay men and MSM

The US Centers for Disease Control and Prevention provides information on HIV/AIDS and on HIV/AIDS among MSM (in English and Spanish)

Information is available from Avert, an international AIDS charity, on HIV, AIDS, and men who have sex with men

The Center for AIDS Prevention Studies (University of California, San Francisco) provides information on sexual networks and HIV prevention

The US National Center for Biotechnology Information provides a science primer on molecular phylogenetics

UK Collaborative Group on HIV Drug Resistance maintains a database of resistance tests

HIV i-Base offers HIV treatment information for health-care professionals and HIV-positive people

The NIH-funded HIV Sequence Database contains data on genetic sequences, resistance, immunology, and vaccine trials

Aim

The effects of 24(S),25-epoxycholesterol (24,25EC) on aspects of cholesterol homeostasis is well-documented. When added to cells, 24,25EC decreases cholesterol synthesis and up-regulates cholesterol efflux genes, including ABCA1. Synthesis of 24,25EC occurs in a shunt of the mevalonate pathway which also produces cholesterol. Therefore, 24,25EC synthesis should be subject to the same negative feedback regulation as cholesterol synthesis. To date, no role has been ascribed to 24,25EC in light of the fact that increased accumulation of cholesterol should decrease formation of this oxysterol through feedback inhibition. This leads to the intriguing paradox: why inhibit production of an apparently important regulator of cholesterol homeostasis when it is needed most?

Methods

We used a combination of pharmacological and genetic approaches in Chinese Hamster Ovary cell-lines to investigate this paradox. Endogenous synthesis of 24,25EC was manipulated using partial inhibition of the enzyme, Oxidosqualene Cyclase. Changes in cholesterol and 24,25EC synthesis were determined using metabolic labelling with [1-14C]-acetate, thin-layer chromatography and phosphorimaging. Transcriptional effects mediated via SREBP and LXR were analysed by luciferase reporter assays.

Results

We showed that cholesterol addition to cells lead to a rapid and preferential inhibition of 24,25EC synthesis. Addition of 24,25EC resulted in parallel inhibition of 24,25EC and cholesterol synthesis. Furthermore, we used a variety of approaches to examine the relationship between cholesterol and 24,25EC synthesis, including cell-lines with different rates of cholesterol synthesis, varying cholesterol synthetic rates by pre-treatment with a statin, or lipoprotein cholesterol loading of macrophages. In all cases, we showed that 24,25EC synthesis faithfully tracked cholesterol synthesis. Moreover, changes in 24,25EC synthesis exerted downstream effects, reducing SREBP transcriptional activity whilst increasing ABCA1 and LXR transcriptional activity.

Conclusion

Our results show that 24,25EC synthesis parallels cholesterol synthesis, consistent with this oxysterol functioning as a safety valve to protect against the accumulation of newly-synthesised cholesterol (as opposed to exogenously-derived cholesterol). Considering that 24,25EC is capable of being produced in all cholesterogenic cells, we propose that production of 24,25EC may represent a ubiquitous defence mechanism.

CD8+ cytotoxic T-lymphocytes (CTLs) perform a critical role in the immune control of viral infections, including those caused by human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV). As a result, genetic variation at CTL epitopes is strongly influenced by host-specific selection for either escape from the immune response, or reversion due to the replicative costs of escape mutations in the absence of CTL recognition. Under strong CTL-mediated selection, codon positions within epitopes may immediately “toggle” in response to each host, such that genetic variation in the circulating virus population is shaped by rapid adaptation to immune variation in the host population. However, this hypothesis neglects the substantial genetic variation that accumulates in virus populations within hosts. Here, we evaluate this quantity for a large number of HIV-1– (n ≥ 3,000) and HCV-infected patients (n ≥ 2,600) by screening bulk RT-PCR sequences for sequencing “mixtures” (i.e., ambiguous nucleotides), which act as site-specific markers of genetic variation within each host. We find that nonsynonymous mixtures are abundant and significantly associated with codon positions under host-specific CTL selection, which should deplete within-host variation by driving the fixation of the favored variant. Using a simple model, we demonstrate that this apparently contradictory outcome can be explained by the transmission of unfavorable variants to new hosts before they are removed by selection, which occurs more frequently when selection and transmission occur on similar time scales. Consequently, the circulating virus population is shaped by the transmission rate and the disparity in selection intensities for escape or reversion as much as it is shaped by the immune diversity of the host population, with potentially serious implications for vaccine design.

Author Summary

The rapid accumulation of genetic variation in human viruses, such as human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV), enables these pathogens to elude the immune system and forestalls the development of effective vaccines. This variation may be shaped by selection due to host-specific immune responses, such that the total virus population mirrors the immune diversity of the host population. However, the often-neglected viral genetic variation within hosts may also play an important role in shaping variation in the total virus population. We carry out an innovative analysis of bulk HIV-1 and HCV sequences isolated from over 4,000 human patients, exploiting “mixtures” (i.e., ambiguous nucleotides) as a site-specific marker of within-host genetic variation. We find that nonsynonymous mixtures are disproportionately abundant at codon positions under strong host-specific immune selection. Because existing models of virus evolution provide no explanation for this outcome, we have formulated a new model supplemented with stochastic simulations to demonstrate that the rapid transmission of viruses through diverse selective environments creates a positive correlation between nonsynonymous variation within and among hosts.

Several codon-based methods are available for detecting adaptive evolution in protein-coding sequences, but to date none specifically identify sites that are selected differentially in two populations, although such comparisons between populations have been historically useful in identifying the action of natural selection. We have developed two fixed effects maximum likelihood methods: one for identifying codon positions showing selection patterns that persist in a population and another for detecting whether selection is operating differentially on individual codons of a gene sampled from two different populations. Applying these methods to two HIV populations infecting genetically distinct human hosts, we have found that few of the positively selected amino acid sites persist in the population; the other changes are detected only at the tips of the phylogenetic tree and appear deleterious in the long term. Additionally, we have identified seven amino acid sites in protease and reverse transcriptase that are selected differentially in the two samples, demonstrating specific population-level adaptation of HIV to human populations.

Synopsis

Despite the efforts devoted to surveying HIV genetic diversity and the development of an effective vaccine, there is still no consensus on the extent to which the former prejudices the latter. Experimental studies show that escape from cell-mediated immunity is selected for in HIV and SIV, and sometimes very quickly. Conversely, escape mutants may be selected against at transmission, so how much does this selection within individuals influence the genotype of the circulating HIV population overall? Kosakovsky Pond, Leigh Brown, and colleagues have developed a new statistical approach to address this question. Using sequences from the globally most abundant HIV subtype (subtype C), the authors directly compared virus of the same subtype infecting genetically different human populations. They show at least half of the amino acid sites selected within individuals are not selected at a population level, and they identify six amino acid sites in the RT gene that are selected differentially between populations. We can now partition molecular adaptation between individual and population components for whatever genes may be included in candidate vaccines, in the target populations themselves. The methods are also important beyond the HIV world, where analogous issues arise in the more general question of the evolution of virulence in pathogens.

Increased susceptibility to the protease inhibitors saquinavir and amprenavir has been observed in human immunodeficiency virus type 1 (HIV-1) with specific mutations in protease (V82T and N88S). Increased susceptibility to ritonavir has also been described in some viruses from antiretroviral agent-naïve patients with primary HIV-1 infection in association with combinations of amino acid changes at polymorphic sites in the protease. Many of the viruses displaying increased susceptibility to protease inhibitors also had low replication capacity. In this retrospective study, we analyze the drug susceptibility phenotype and the replication capacity of virus isolates obtained at the peaks of viremia during five consecutive structured treatment interruptions in 12 chronically HIV-1-infected patients. Ten out of 12 patients had at least one sample with protease inhibitor hypersusceptibility (change ≤0.4-fold) to one or more protease inhibitor. Hypersusceptibility to different protease inhibitors was observed at variable frequency, ranging from 38% to amprenavir to 11% to nelfinavir. Pairwise comparisons between susceptibilities for the protease inhibitors showed a consistent correlation among all pairs. There was also a significant relationship between susceptibility to protease inhibitors and replication capacity in all patients. Replication capacity remained stable over the course of repetitive cycles of structured treatment interruptions. We could find no association between in vitro replication capacity and in vivo plasma viral load doubling time and CD4+ and CD8+ T-cell counts at each treatment interruption. Several mutations were associated with hypersusceptibility to each protease inhibitor in a univariate analysis. This study extends the association between hypersusceptibility to protease inhibitors and low replication capacity to virus isolated from chronically infected patients and highlights the complexity of determining the genetic basis of this phenomenon. The potential clinical relevance of protease inhibitor hypersusceptibility and low replication capacity to virologic response to protease inhibitor-based therapies deserves to be investigated further.

Sequential infection with different strains of human immunodeficiency virus type 1 (HIV-1) is a rarely identified phenomenon with important implications for immunopathogenesis and vaccine development. Here, we identify an individual whose good initial control of viremia was lost in association with reduced containment of a superinfecting strain. Subject 2030 presented with acute symptoms of HIV-1 infection with high viremia and an incomplete seroconversion as shown by Western blotting. A low set point of viremia (∼1,000 HIV-1 copies/ml) was initially established without drug therapy, but a new higher set point (∼40,000 HIV-1 copies/ml) manifested about 5 months after infection. Drug susceptibility testing demonstrated a multidrug-resistant virus initially but a fully sensitive virus after 5 months, and an analysis of pol genotypes showed that these were two phylogenetically distinct strains of virus (strains A and B). Replication capacity assays suggested that the outgrowth of strain B was not due to higher fitness conferred by pol, and env sequences indicated that the two strains had the same R5 coreceptor phenotype. Delineation of CD8+-T-lymphocyte responses against HIV-1 showed a striking pattern of decay of the initial cellular immune responses after superinfection, followed by some adaptation of targeting to new epitopes. An examination of targeted sequences suggested that differences in the recognized epitopes contributed to the poor immune containment of strain B. In conclusion, the rapid overgrowth of a superinfecting strain of HIV-1 of the same subtype raises major concerns for effective vaccine development.

The initial virus strains from as many as 12% of individuals with primary human immunodeficiency virus (HIV) infection have a 50% inhibitory concentration ≤0.4-fold that of HIV type 1NL4-3 (HIV-1NL4-3) to ritonavir (hypersusceptibility [HS]). There is also substantial variation in replicative capacity (RC) or an in vitro assay of the contributions of protease (PR) and reverse transcriptase to viral fitness. In chronically infected antiretrovirally treated patients, amprenavir HS has been associated with the mutation N88S in PR, but this mutation is not seen in untreated patients. In this study, virus strains from 182 cases of primary HIV infection were analyzed, and a highly significant association between HS and low RC (≤10% that of HIV-1NL4-3) was observed (P < 10−6). Multivariate analysis was used to determine the genotypic basis of ritonavir HS, analyzing all polymorphic amino acid sites and insertions from p7gag through PR. Decision tree models developed on the entire Gag-plus-PR data set and on PR alone gave overall correct classifications of 73 and 72%, respectively, on cross-validation. They were also able to predict low RC, with sensitivities of 69 and 62% and specificities of 84 and 70%, respectively. The analysis shows that ritonavir HS in untreated primary HIV infection is not associated with single mutations but with combinations of amino acids at polymorphic sites and that the same genotypes which confer HS to PR inhibitors confer low RC. This supports the view that variation in PR function is directly responsible for variation in fitness among strains in primary infection.

Although antiviral agents which block human immunodeficiency virus (HIV) replication can result in long-term suppression of viral loads to undetectable levels in plasma, long-term therapy fails to eradicate virus, which generally rebounds after a single treatment interruption. Multiple structured treatment interruptions (STIs) have been suggested as a possible strategy that may boost HIV-specific immune responses and control viral replication. We analyze viral dynamics during four consecutive STI cycles in 12 chronically infected patients with a history (>2 years) of viral suppression under highly active antiretroviral therapy. We fitted a simple model of viral rebound to the viral load data from each patient by using a novel statistical approach that allows us to overcome problems of estimating viral dynamics parameters when there are many viral load measurements below the limit of detection. There is an approximate halving of the average viral growth rate between the first and fourth STI cycles, yet the average time between treatment interruption and detection of viral loads in the plasma is approximately the same in the first and fourth interruptions. We hypothesize that reseeding of viral reservoirs during treatment interruptions can account for this discrepancy, although factors such as stochastic effects and the strength of HIV-specific immune responses may also affect the time to viral rebound. We also demonstrate spontaneous drops in viral load in later STIs, which reflect fluctuations in the rates of viral production and/or clearance that may be caused by a complex interaction between virus and target cells and/or immune responses.

Recently, significant numbers of individuals with primary human immunodeficiency virus (HIV) infection have been found to harbor viral strains with reduced susceptibility to antiretroviral drugs. In one study, HIV from 16% of such antiretroviral-naive individuals was shown to have a susceptibility to nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) between 2.5- and 10-fold lower than that of a wild-type control. Mutations in the RT domain that had previously been associated with antiretroviral resistance were not shared by these strains. We have analyzed by logistic regression 46 variable amino acid sites in RT for their effect on susceptibility and have identified two novel sites influencing susceptibility to NNRTIs: amino acids 135 and 283 in RT. Eight different combinations of amino acids at these sites were observed among these patients. These combinations showed a 14-fold range in mean susceptibility to both nevirapine and delavirdine. In vitro mutagenesis of the control strain combined with a phenotypic assay confirmed the significance of amino acid variation at these sites for susceptibility to NNRTIs.

Human immunodeficiency virus type 1 (HIV-1) rapidly develops resistance to lamivudine during monotherapy, typically resulting in the appearance at position 184 in reverse transcriptase (RT) of isoleucine instead of the wild-type methionine (M184I) early in therapy, which is later replaced by valine (M184V). M184V reduces viral susceptibility to drug in vitro by approximately 100-fold, but also results in a lower processivity of RT. We show that a drop in absolute viral fitness associated with the outgrowth of M184V results in a drop in viral load only in individuals with high CD4+ counts, from whom we estimate the relative fitness of M184V in the presence of drug to be approximately 10% of that of the wild type prior to therapy. The timing of emergence of the M184V mutant varies widely between infected individuals. From analysis of the frequency of M184I and M184V mutants determined at multiple time points in seven individuals during lamivudine therapy, we estimated the fitness advantage of M184V over M184I during therapy to be approximately 23% on average. We have also estimated the average ratio of the frequencies of the two mutants prior to therapy to be 0.2:1, with a range from 0.12:1 to 0.33:1. We have found that the differences between individuals in the rate of evolution of lamivudine resistance arise due to genetic drift affecting the relative frequency of M184I and M184V prior to therapy. These results show that stochastic effects can be significant in HIV evolution, even when there is large fitness difference between mutant and wild-type variants.