High-Sequence Diversity and Rapid Virus Turnover Contribute to Higher Rates of Coreceptor Switching in Treatment-Experienced Subjects with HIV-1 Viremia.

Coreceptor switching from CCR5 to CXCR4 is common during chronic HIV-1 infection, but is even more common in individuals who have failed antiretroviral therapy (ART). Prior studies have suggested rapid mutation and/or recombination of HIV-1 envelope (env) genes during coreceptor switching. We compared the functional and genotypic changes in env of viruses from viremic subjects who had failed ART just before and after coreceptor switching and compared those to viruses from matched subjects without coreceptor switching. Analysis of multiple unique functional env clones from each subject revealed extensive diversity at both sample time points and rapid diversification of sequences during the 4-month interval in viruses from both 9 subjects with coreceptor switching and 15 control subjects. Only two subjects had envs with evidence of recombination. Three findings distinguished env clones from subjects with coreceptor switching from controls: (1) lower entry efficiency via CCR5; (2) longer V1/V2 regions; and (3), lower nadir CD4 T cell counts during prior years of infection. Most of these subjects harbored virus with lower replicative capacity associated with protease (PR) and/or reverse transcriptase inhibitor resistance mutations, and the extensive diversification tended to lead either to improved entry efficiency via CCR5 or the gain of entry function via CXCR4. These results suggest that R5X4 or X4 variants emerge from a diverse, low-fitness landscape shaped by chronic infection, multiple ART resistance mutations, the availability of target cells, and reduced entry efficiency via CCR5.

Efficiency of bridging-sheet recruitment explains HIV-1 R5 envelope glycoprotein sensitivity to soluble CD4 and macrophage tropism.

HIV-1 R5 viruses vary extensively in their capacity to infect macrophages. R5 viruses that confer efficient infection of macrophages are able to exploit low levels of CD4 for infection and predominate in brain tissue, where macrophages are a major target for infection. HIV-1 R5 founder viruses that are transmitted were reported to be non-macrophage-tropic. Here, we investigated the sensitivities of macrophage-tropic and non-macrophage-tropic R5 envelopes to neutralizing antibodies. We observed striking differences in the sensitivities of Env(+) pseudovirions to soluble CD4 (sCD4) and to neutralizing monoclonal antibodies (MAbs) that target the CD4 binding site. Macrophage-tropic R5 Envs were sensitive to sCD4, while non-macrophage-tropic Envs were significantly more resistant. In contrast, all Envs were sensitive to VRC01 regardless of tropism, while MAb b12 conferred an intermediate neutralization pattern where all the macrophage-tropic and about half of the non-macrophage-tropic Envs were sensitive. CD4, b12, and VRC01 share binding specificities on the outer domain of gp120. However, these antibodies differ in their ability to induce conformational changes on the trimeric envelope and in specificity for residues on the V1V2 loop stem and ß20-21 junction that are targets for CD4 in recruiting the bridging sheet. These distinct specificities of CD4, b12, and VRC01 likely explain the observed differences in Env sensitivity to inhibition by these reagents and provide an insight into the envelope mechanisms that control macrophage tropism. We present a model where the efficiency of bridging-sheet recruitment by CD4 is a major determinant of HIV-1 R5 envelope sensitivity to soluble CD4 and macrophage tropism.

Drug resistance and coreceptor usage in HIV type 1 subtype C-infected children initiating or failing highly active antiretroviral therapy in South Africa.

HIV-1 drug resistance monitoring in resource-poor settings is crucial due to limited drug alternatives. Recent reports of the increased prevalence of CXCR4 usage in subtype C infections may have implications for CCR5 antagonists in therapy. We investigated the prevalence of drug resistance mutations and CXCR4 coreceptor utilization of viruses from HIV-1 subtype C-infected children. Fifty-one children with virological failure during highly active antiretroviral therapy (HAART) and 43 HAART-naive children were recruited. Drug resistance genotyping and coreceptor utilization assessment by phenotypic and genotypic methods were performed. At least one significant drug resistance mutation was present in 85.4% of HAART-failing children. Thymidine analogue mutations (TAMs) were detected in 58.5% of HAART-failing children and 39.0% had ≥3 TAMs. CXCR4 (X4) or dual (R5X4)/mixed (R5, X4) (D/M)-tropic viruses were found in 54.3% of HAART-failing and 9.4% of HAART-naive children (p<0.0001); however, the HAART-failing children were significantly older (p<0.0001). In multivariate logistic regression, significant predictors of CXCR4 usage included antiretroviral treatment, older age, and lower percent CD4(+) T cell counts. The majority of genotypic prediction tools had low sensitivity (≤65.0%) and high specificity (≥87.5%) for predicting CXCR4 usage. Extensive drug resistance, including the high percentage of TAMs found, may compromise future drug choices for children, highlighting the need for improved treatment monitoring and adherence counseling. Additionally, the increased prevalence of X4/D/M viruses in HAART-failing children suggests limited use of CCR5 antagonists in salvage therapy. Enhanced genotypic prediction tools are needed as current tools are not sensitive enough for predicting CXCR4 usage.

Drug resistance and viral tropism in HIV-1 subtype C-infected patients in KwaZulu-Natal, South Africa: implications for future treatment options.

BACKGROUND: Drug resistance poses a significant challenge for the successful application of highly active antiretroviral therapy (HAART) globally. Furthermore, emergence of HIV-1 isolates that preferentially use CXCR4 as a coreceptor for cell entry, either as a consequence of natural viral evolution or HAART use, may compromise the efficacy of CCR5 antagonists as alternative antiviral therapy. METHODS: We sequenced the pol gene of viruses from 45 individuals failing at least 6 months of HAART in Durban, South Africa, to determine the prevalence and patterns of drug-resistance mutations. Coreceptor use profiles of these viruses and those from 45 HAART-naive individuals were analyzed using phenotypic and genotypic approaches. RESULTS: Ninety-five percent of HAART-failing patients had at least one drug-resistant mutation. Thymidine analog mutations (TAMs) were present in 55% of patients with 9% of individuals possessing mutations indicative of the TAM1 pathway, 44% had TAM2, whereas 7% had mutations common to both pathways. Sixty percent of HAART-failing subjects had X4/dual//mixed-tropic viruses compared with 30% of HAART-naïve subjects (P < 0.02). Genetic coreceptor use prediction algorithms correlated with phenotypic results with 60% of samples from HAART-failing subjects predicted to possess CXCR4-using (X4/dual/mixed viruses) versus 15% of HAART-naïve patients. CONCLUSIONS: The high proportion of TAMs and X4/dual/mixed HIV-1 viruses among patients failing therapy highlight the need for intensified monitoring of patients taking HAART and the problem of diminished drug options (including CCR5 antagonists) for patients failing therapy in resource-poor settings.

Comprehensive analysis of transcriptional promoter structure and function in 1% of the human genome.

Transcriptional promoters comprise one of many classes of eukaryotic transcriptional regulatory elements. Identification and characterization of these elements are vital to understanding the complex network of human gene regulation. Using full-length cDNA sequences to identify transcription start sites (TSS), we predicted more than 900 putative human transcriptional promoters in the ENCODE regions, representing a comprehensive sampling of promoters in 1% of the genome. We identified 387 fragments that function as promoters in at least one of 16 cell lines by measuring promoter activity in high-throughput transient transfection reporter assays. These positive functional results demonstrate widespread use of alternative promoters. We show a strong correlation between promoter activity and the corresponding endogenous RNA transcript levels, providing the first experimental quantitative estimate of promoter contribution to gene regulation. Finally, we identified functional regions within a randomly selected subset of 45 promoters using deletion analyses. These experiments showed that, on average, the sequence -300 to -50 bp of the TSS positively contributes to core promoter activity. Interestingly, putative negative elements were identified -1000 to -500 bp upstream of the TSS for 55% of genes tested. These data provide the largest and most comprehensive view of promoter function in the human genome.