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A genome-to-genome analysis of associations between human genetic variation, HIV-1 sequence diversity, and viral control

HIV-1 sequence diversity is affected by selection pressures arising from host genomic factors. Using paired human and viral data from 1071 individuals, we ran >3000 genome-wide scans, testing for associations between host DNA polymorphisms, HIV-1 sequence variation and plasma viral load (VL), while considering human and viral population structure. We observed significant human SNP associations to a total of 48 HIV-1 amino acid variants (p<2.4 × 10−12). All associated SNPs mapped to the HLA class I region. Clinical relevance of host and pathogen variation was assessed using VL results. We identified two critical advantages to the use of viral variation for identifying host factors: (1) association signals are much stronger for HIV-1 sequence variants than VL, reflecting the ‘intermediate phenotype’ nature of viral variation; (2) association testing can be run without any clinical data. The proposed genome-to-genome approach highlights sites of genomic conflict and is a strategy generally applicable to studies of host–pathogen interaction. DOI: http://dx.doi.org/10.7554/eLife.01123.001

Host and viral genetic correlates of clinical definitions of HIV-1 disease progression.

Background Various patterns of HIV-1 disease progression are described in clinical practice and in research. There is a need to assess the specificity of commonly used definitions of long term non-progressor (LTNP) elite controllers (LTNP-EC), viremic controllers (LTNP-VC), and viremic non controllers (LTNP-NC), as well as of chronic progressors (P) and rapid progressors (RP). Methodology and Principal Findings We re-evaluated the HIV-1 clinical definitions, summarized in Table 1, using the information provided by a selected number of host genetic markers and viral factors. There is a continuous decrease of protective factors and an accumulation of risk factors from LTNP-EC to RP. Statistical differences in frequency of protective HLA-B alleles (p-0.01), HLA-C rs9264942 (p-0.06), and protective CCR5/CCR2 haplotypes (p-0.02) across groups, and the presence of viruses with an ancestral genotype in the “viral dating” (i.e., nucleotide sequences with low viral divergence from the most recent common ancestor) support the differences among principal clinical groups of HIV-1 infected individuals. Conclusions A combination of host genetic and viral factors supports current clinical definitions that discriminate among patterns of HIV-1 progression. The study also emphasizes the need to apply a standardized and accepted set of clinical definitions for the purpose of disease stratification and research.

A dual superinfection and recombination within HIV-1 subtype B 12 years after primoinfection.

Summary: To analyze superinfection in an HIV-1-infected patient showing high-risk practices, viral quasispecies were analyzed in pol and env genes in several plasma samples. Phylogenetic analysis in the reverse transcriptase fragment in pol gene identified a single virus in the first 3 samples analyzed, but 12 years after primoinfection, 3 different viral strains were detected in the patient quasispecies. This result suggests a superinfection with 2 HIV-1 strains, one of which showed the T215Y + M184V resistance mutations. The analysis in the env gene confirmed the existence of 3 different strains in the viral population, one of them a recombinant. This study illustrates that events of superinfection and recombination contribute to the viral genetic variability observed in HIV-1-infected individuals.

In vitro analysis of human immunodeficiency virus type 1 resistance to nevirapine and fitness determination of resistant variants.

Nevirapine-resistant variants were generated by serial passages in MT-2 cells in the presence of increasing drug concentrations. In passage 5, mutations V106A, Y181C and G190A were detected in the global population, associated with a 100-fold susceptibility decrease. Sequence analysis of biological clones obtained from passage 5 and subsequent passages showed that single mutants, detected in first passages, were progressively replaced in passage 15 by double mutants, correlating with a 500-fold increase in phenotypic resistance. Fitness determination of single mutants confirmed that, in the presence of nevirapine, every variant was more fit than wild-type with a fitness order Y181C>V106A>G190A>wild-type. Unexpectedly, in the absence of the drug, the Y181C resistant mutant was more fit than wild-type, with a fitness gradient Y181C>wild-type >G106A>or=V190A. Using a molecular clone in which the Y181C mutation was introduced by in vitro mutagenesis, the greater fitness of the Y181C mutant was confirmed in new competition cultures. These data exemplify the role of resistance mutations on virus phenotype but also on virus evolution leading, occasionally, to resistant variants fitter than the wild-type in the absence of the drug.

Coinfection and Superinfection in Patients with Long-Term, Nonprogressive HIV-1 Disease

Human immunodeficiency virus 1 (HIV-1) dual infections are considered important because they have been related to AIDS progression. We identified dual infections in 2 patients with long-term, nonprogressive HIV-1 disease; the first patient was diagnosed as being already coinfected, on the basis of the first sample analyzed, but a previous superinfection could not be excluded; the second patient was diagnosed as having a superinfection, on the basis of the 9-year difference between the viral dating of the 2 strains. Dual infections occur in patients with long-term, nonprogressive disease, with no immediate clinical manifestations. Such occurrences could indicate a general phenomenon in natural HIV-1 infections.

Initial Fitness Recovery of HIV-1 Is Associated with Quasispecies Heterogeneity and Can Occur without Modifications in the Consensus Sequence

Background Fitness recovery of HIV-1 “in vitro” was studied using viral clones that had their fitness decreased as a result of plaque-to-plaque passages. Principal Findings After ten large population passages, the viral populations showed an average increase of fitness, although with wide variations among clones. While 5 clones showed significant fitness increases, 3 clones showed increases that were only marginally significant (p<0.1), and 4 clones did not show any change. Fitness recovery was not accompanied by an increase in p24 production, but was associated with an increase in viral titer. Few mutations (an average of 2 mutations per genome) were detected in the consensus nucleotide sequence of the entire genome in all viral populations. Five of the populations did not fix any mutation, and three of them displayed marginally significant fitness increases, illustrating that fitness recovery can occur without detectable alterations of the consensus genomic sequence. The investigation of other possible viral factors associated with the initial steps of fitness recovery, showed that viral quasispecies heterogeneity increased between the initial clones and the passaged populations. A direct statistical correlation between viral heterogeneity and viral fitness was obtained. Conclusions Thus, the initial fitness recovery of debilitated HIV-1 clones was mediated by an increase in quasispecies heterogeneity. This observation, together with the invariance of the consensus sequence despite fitness increases demonstrates the relevance of quasispecies heterogeneity in the evolution of HIV-1 in cell culture.

Different evolutionary patterns are found within human immunodeficiency virus type 1-infected patients.

In order to study the evolution in vivo of human immunodeficiency virus type 1 (HIV-1) in patients with normal clinical evolution, six individuals were selected from a group of 46 patients followed for 1 to 4 years. Patients were selected not by clinical progression characteristics but on the basis of virus genetic variability, as analysed by heteroduplex mobility assay and RNase A mismatch cleavage method. Two patients displayed a homogeneous virus population, two showed very heterogeneous quasispecies and two presented two distinct variants within the virus population. Virus quasispecies were studied by nucleotide sequencing of the C2-fusion domain of the env gene. Virus evolution was approached by analysing the distribution of genetic distances, calculation of divergence and heterogeneity as well as the K(a)/K(s) ratio and by the construction of the phylogenetic trees. Three patients displayed the same tree topology, characterized by the presence of independent clades supported by high bootstrap values, whereas this pattern was not present in the other three patients. In the three patients displaying independent clades, a recombination analysis was carried out between distinct subpopulations and recombinant variants were identified. In one patient of this group, different selective pressures were detected in distinct virus clades, measured by their corresponding K(a)/K(s) ratios, revealing that different evolutionary forces are occurring at the same time within the same patient. These results show that multiple evolutionary patterns can be found in typical HIV-1-infected patients.

Genetic analysis of HIV-1 samples from Spain.

Summary: To characterize the viruses responsible for the HIV‐1 epidemic in Spain, we genetically characterized 79 samples obtained from Spanish patients with different risk practices (injecting drug users and male homosexuals) in two regions (Madrid and Navarra). Genetic characterization was carried out by nucleotide sequencing in the C2‐V3‐C3 region and by phylogenetic analysis. All samples were of subtype B except one that clustered with clade F. Because no segregation of samples was determined according to the risk group of patients nor to their geographic origin, the Spanish samples analyzed constitute a single group of viruses. These data, along with the starlike topology of the phylogenetic tree, support the existence of a single introduction of HIV‐1 subtype B in Spain. The mean genetic distance among subtype B sequences was of 13.9% ± 0.06% (range, 5%‐25%), suggesting an epidemic of long evolution. Analysis of sequences in relation to isolation dates revealed an increase in the heterogeneity of the nucleotide sequences with time. According to these data, a divergence rate of 0.49% ± 0.11% per year was calculated for the Spanish samples during the period analyzed.

Different distribution of HIV type 1 genetic variants in European patients with distinct risk practices.

The use of two genetic markers has permitted the analysis of the distribution of two different human immunodeficiency virus type 1 (HIV-1) variants in patients of the homosexual (HO) and intravenous drug user (IDU) groups in distinct European countries. In Germany, Holland, and Italy the variants circulating in each risk group of HO and IDU patients were genetically distinguishable according to the genetic markers used. In contrast, in France and Spain, the same variant has been recovered from patients with different risk practices. These data highlight the diversity of the HIV-1 epidemic in Europe and the different patterns of HIV-1 variant distribution in European countries.

Identification of a new HIV Type 1 circulating recombinant form (CRF38_BF1) in Uruguay.

Recombination has been shown to be an important force in HIV-1 evolution. Recombination contributes to an increase in genetic variation and hinders vaccine design efforts. Several molecular epidemiology studies in South America described the circulation of subtypes B, F, and C as well as several B/F1 recombinants. This study performed by nucleotide sequencing in at least two genes of 89 samples from Uruguay has shown a complex HIV-1 epidemic characterized by the cocirculation of subtype B, and subtype C strains, together with an important group of BF1 recombinant viruses, including isolates similar to CRF12_BF. In addition we identified a new circulating recombinant form, designated CRF38_BF(1), which was dominant in the recombinant virus group.