Replicative fitness costs of nonnucleoside reverse transcriptase inhibitor drug resistance mutations on HIV subtype C.

Single-dose nevirapine (NVP) is quite effective in preventing transmission of the human immunodeficiency virus (HIV) from mother to child; however, many women develop resistance to NVP in this setting. Comparing outcomes of clinical studies reveals an increased amount of resistance in subtype C relative to that in other subtypes. This study investigates how nonnucleoside reverse transcriptase inhibitor (NNRTI) drug resistance mutations of subtype C affect replication capacity. The 103N, 106A, 106M, 181C, 188C, 188L, and 190A drug resistance mutations were placed in a reverse transcriptase (RT) that matches the consensus subtype C sequence as well as the HXB2 RT, as a subtype B reference. The replicative fitness of each mutant was compared with that of the wild type in a head-to-head competition assay. The 106A mutant of subtype C would not grow in the competition assay, making it the weakest virus tested. The effect of the 106M mutation was weaker than those of the 181C and 188C mutations in the consensus C RT, but in subtype B, this difference was not seen. To see if the 106A mutation in a different subtype C background would have a different replicative profile, the same NNRTI resistance mutations were added to the MJ4 RT, a reference subtype C molecular clone. In the context of MJ4 RT, the 106A mutant was not the only mutant that showed poor replicative fitness; the 106M, 188C, and 190A mutants also failed to replicate. These results suggest that NNRTIs may be a cost-effective alternative for salvage therapy if deleterious mutations are present in a subtype C setting.

Replicative capacity differences of thymidine analog resistance mutations in subtype B and C human immunodeficiency virus type 1.

In order to understand the impact of zidovudine resistance and thymidine analog mutations (TAMs) on subtype C human immunodeficiency virus type 1, we created mutants in subtype C reverse transcriptase (RT). The subtype B RT was placed in a subtype C backbone to act as a control. Mutants and wild-type (WT) virus were competed in a head-to-head competition assay to determine how different clones grew in the same culture. Different viruses were distinguished by sequence tags in nef and a quantitative-PCR assay. The 67N and 70R accessory mutations gave an advantage over the WT in subtype C, but these mutations in subtype B had replication capacities similar to that of the WT. Of the triple mutants examined, the TAM-1 types, 41L210W215Y, were the most fit in both subtypes, but only in subtype C was the replication capacity the same as that of the WT. The TAM-2 mutants, 67N70R215F, had the slowest replication in both clones. The mixed TAM pathway mutant, 67N70R215Y, in subtype C had a significant advantage over the TAM-2 mutant, but this was not seen in subtype B. When the WT viruses were competed with each other, the subtype B RT had enhanced replication relative to subtype C. The increased capacities of the 67N and 70R mutations may indicate that there will be greater transmitted resistance and persistence in a subtype C setting than what is known for subtype B.

Double-stranded RNA adenosine deaminases enhance expression of human immunodeficiency virus type 1 proteins.

ADARs (adenosine deaminases that act on double-stranded RNA) are RNA editing enzymes that catalyze a change from adenosine to inosine, which is then recognized as guanosine by translational machinery. We demonstrate here that overexpression of ADARs but not of an ADAR mutant lacking editing activity could upregulate human immunodeficiency virus type 1 (HIV-1) structural protein expression and viral production. Knockdown of ADAR1 by RNA silencing inhibited HIV-1 production. Viral RNA harvested from transfected ADAR1-knocked-down cells showed a decrease in the level of unspliced RNA transcripts. Overexpression of ADAR1 induced editing at a specific site in the env gene, and a mutant with the edited sequence was expressed more efficiently than the wild-type viral genome. These data suggested the role of ADAR in modulation of HIV-1 replication. Our data demonstrate a novel mechanism in which HIV-1 employs host RNA modification machinery for posttranscriptional regulation of viral protein expression.

Genome-Wide Analyses Reveal Gene Influence on HIV Disease Progression and HIV-1C Acquisition in Southern Africa.

Sub-Saharan Africans infected with HIV-1C make up the largest AIDS patient population in the world and exhibit large heterogeneity in disease progression before initiating antiretroviral therapy. To identify host variants associated with HIV disease progression, we performed genome-wide association studies on a total of 556 treatment-naive HIV-infected individuals in Botswana. We characterized the pattern of HIV disease progression using a novel functional principal component analysis, which can better capture longitudinal CD4 and viral load (VL) trajectories. Two single-nucleotide polymorphisms (SNPs) near HCG22 (chr6, peak variant rs2535307, combined p = 3.72 × 10-7, minor allele as risky allele) and CCNG1 (chr5, peak variant kgp22385164, combined p = 1.88 × 10-6, minor allele as risky allele) were significantly associated with CD4 and VL dynamics. Inspection of SNPs in these gene regions in a third Botswana cohort (using GWATCH) also revealed a strong association of HCG22 with HIV-1C acquisition, suggesting that this region is associated with infection as well as disease progression. Our study uncovered two genetic regions that are significant and have specific effects on HIV-1C acquisition or progression in sub-Saharan Africans, and the result suggested new potential targets for AIDS prevention and treatment. In addition, our results also indicate the possibility of using genetic markers as HIV disease progression indicators in sub-Saharan Africans to prioritize fast progressors for antiretroviral treatment.

The Effect of a Single Nucleotide Substitution in the Splicing Silencer in the tat/rev Intron on HIV Type 1 Envelope Expression.

A complex mRNA splicing pattern, which remains to be fully characterized, influences HIV-1 gene expression. In this study, poor envelope expression of a primary HIV-1 isolate was observed and linked to increased splicing of the two coding exons of tat/rev. The substitution of a nucleotide G, located 28 nucleotides upstream of the splice acceptor site SA7 in the recently identified intron splicing silencer sequence, was found to be responsible for the poor envelope expression. A single nucleotide substitution of G with A at this position results in a poor envelope expression phenotype. Moreover, substitution of the nucleotide G with any other nucleotide in an infectious HIV-1 proviral clone, HXB2RU3, results in poor envelope expression. The substitution of this nucleotide reduces the hnRNP A1 binding affinity but increases the splicing of env mRNA. The nucleotide G at this position is highly conserved among HIV-1 isolates and appears to play a critical role in HIV-1 splicing.

Breastfeeding plus infant zidovudine prophylaxis for 6 months vs formula feeding plus infant zidovudine for 1 month to reduce mother-to-child HIV transmission in Botswana: a randomized trial: the Mashi Study.

CONTEXT: Postnatal transmission of human immunodeficiency virus-1 (HIV) via breastfeeding reverses gains achieved by perinatal antiretroviral interventions. OBJECTIVE: To compare the efficacy and safety of 2 infant feeding strategies for the prevention of postnatal mother-to-child HIV transmission. DESIGN, SETTING, AND PATIENTS: A 2 x 2 factorial randomized clinical trial with peripartum (single-dose nevirapine vs placebo) and postpartum infant feeding (formula vs breastfeeding with infant zidovudine prophylaxis) interventions. In Botswana between March 27, 2001, and October 29, 2003, 1200 HIV-positive pregnant women were randomized from 4 district hospitals. Infants were evaluated at birth, monthly until age 7 months, at age 9 months, then every third month through age 18 months. INTERVENTION: All of the mothers received zidovudine 300 mg orally twice daily from 34 weeks' gestation and during labor. Mothers and infants were randomized to receive single-dose nevirapine or placebo. Infants were randomized to 6 months of breastfeeding plus prophylactic infant zidovudine (breastfed plus zidovudine), or formula feeding plus 1 month of infant zidovudine (formula fed). MAIN OUTCOME MEASURES: Primary efficacy (HIV infection by age 7 months and HIV-free survival by age 18 months) and safety (occurrence of infant adverse events by 7 months of age) end points were evaluated in 1179 infants. RESULTS: The 7-month HIV infection rates were 5.6% (32 infants in the formula-fed group) vs 9.0% (51 infants in the breastfed plus zidovudine group) (P = .04; 95% confidence interval for difference, -6.4% to -0.4%). Cumulative mortality or HIV infection rates at 18 months were 80 infants (13.9%, formula fed) vs 86 infants (15.1% breastfed plus zidovudine) (P = .60; 95% confidence interval for difference, -5.3% to 2.9%). Cumulative infant mortality at 7 months was significantly higher for the formula-fed group than for the breastfed plus zidovudine group (9.3% vs 4.9%; P = .003), but this difference diminished beyond month 7 such that the time-to-mortality distributions through age 18 months were not significantly different (P = .21). CONCLUSIONS: Breastfeeding with zidovudine prophylaxis was not as effective as formula feeding in preventing postnatal HIV transmission, but was associated with a lower mortality rate at 7 months. Both strategies had comparable HIV-free survival at 18 months. These results demonstrate the risk of formula feeding to infants in sub-Saharan Africa, and the need for studies of alternative strategies. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00197587.

Conserved determinants of enhanced CCR5 binding in the human immunodeficiency virus subtype D envelope third variable loop.

Human immunodeficiency virus 1 subtype D (HIV-1D) contributes to a significant portion of the HIV-1 disease burden in eastern and central Africa, and is associated with more rapid disease progression. Its viral envelope sequences, particularly in the third variable region (V3), are highly divergent from other major subtypes yet have rarely been studied to date. We evaluated the V3 and select bridging sheet residues of the HIV-1D 94UG114 envelope by alanine-scanning mutagenesis to determine the residues involved in CCR5 usage conservation in the face of sequence variability. We found most single alanine mutations capable of abolishing CCR5 binding, suggesting binding contacts that are highly sensitive to mutation. Despite drastic binding defects across the board, most mutants mediated fusion at or near wild-type levels, demonstrating an ability to accommodate changes in CCR5 affinity while maintaining the ability to complete entry. Three of the alanine mutations did not abolish CCR5 binding but rather resulted in enhanced CCR5 binding. The positions of these residues were found to be conserved between strains of two subtypes, revealing similar V3 elements that suggest a conservation of constraints in V3 loop conformation.

Relative concordance of human immunodeficiency virus oligomeric and monomeric envelope in CCR5 coreceptor usage.

A major difference between binding and fusion assays commonly used to study the human immunodeficiency virus (HIV) envelope is the use of monomeric envelope for the former assay and oligomeric envelope for the latter. Due to discrepancies in their readouts for some mutants, envelope regions involved in CCR5 coreceptor usage were systematically studied to determine whether the discordance is due to inherent differences between the two assays or whether it genuinely reflects functional differences at each entry step. By adding the binding inhibitor TAK-779 to delay coreceptor binding kinetics in the fusion assay, the readouts were found comparable between the assays for the mutants analysed in this study. Our finding indicates that monomeric binding reflects oligomeric envelope-CCR5 interaction, thus discordant results between binding and fusion assays do not necessarily indicate differences in coreceptor usage by oligomeric envelope and monomeric gp120.

Efficacy of antiretroviral drugs in reducing mother-to-child transmission of HIV in Africa: a meta-analysis of published clinical trials.

Antiretroviral drugs (ARVs) have been shown to be efficacious in decreasing mother-to-child transmission (MTCT) of HIV. A summary estimate of the efficacy of ARVs in reducing MTCT is important for modeling and policy decisions. However, no one has hitherto attempted to generate this summary estimate for Africa, the continent with the greatest HIV/AIDS burden. This study estimates the efficacy of ARVs in reducing MTCT in Africa through a meta-analysis of published studies conducted in Africa. Using an a priori protocol, Medline, EMBASE, and the Cochrane Library were searched for primary studies that measured MTCT of HIV, had ARVs as the exposure to the mother, and were conducted in Africa. Extracted data included characteristics of the study, population, quality, exposure, and results. The data were analyzed using a random effects model with each trial arm as a data point. Ten randomized clinical trials conducted in West, East, and Southern Africa published from 1999 to 2007 satisfied the inclusion criteria. They ranged in sample size from 139 to 1797, and used different ARV regimens as the exposure to the mother antepartum, intrapartum, or postpartum, and to the baby. The combined effect estimate of using ARVs is 10.6% (95% CI: 8.6-13.1) transmission at 4-6 weeks and 21.0% (95% CI: 15.5-27.7) transmission for placebo. This represents approximately 50% efficacy. The result is stable and not driven by any single study. All regimens were well tolerated. We conclude that ARV use to reduce MTCT of HIV in Africa is efficacious and well tolerated.

Estimating the lost benefits of antiretroviral drug use in South Africa.

South Africa is one of the countries most severely affected by HIV/AIDS. At the peak of the epidemic, the government, going against consensus scientific opinion, argued that HIV was not the cause of AIDS and that antiretroviral (ARV) drugs were not useful for patients and declined to accept freely donated nevirapine and grants from the Global Fund. Using modeling, we compared the number of persons who received ARVs for treatment and prevention of mother-to-child HIV transmission between 2000 and 2005 with an alternative of what was reasonably feasible in the country during that period. More than 330,000 lives or approximately 2.2 million person-years were lost because a feasible and timely ARV treatment program was not implemented in South Africa. Thirty-five thousand babies were born with HIV resulting in 1.6 million person-years lost by not implementing a mother-to-child transmission prophylaxis program using nevirapine. The total lost benefits of ARVs are at least 3.8 million person-years for the period 2000-2005.

Mutations in the V3 stem versus the V3 crown and C4 region have different effects on the binding and fusion steps of human immunodeficiency virus type 1 gp120 interaction with the CCR5 coreceptor.

The current model for HIV-1 envelope-coreceptor interaction depicts the V3 stem and bridging sheet binding to the CCR5 N-terminus while the V3 crown interacts with the second extracellular loop, which is the coreceptor domain that appears to be relatively more important for fusion and infection. Our prediction based on this model is that mutations in the V3 crown might consequently have more effects on cell-cell fusion and virus entry than mutations introduced in the V3 stem and C4 region. We performed alanine-scanning of the V3 loop and selected C4 residues in the JRFL envelope and tested the capacity of the resulting mutants for CCR5 binding, cell-cell fusion, and virus infection. Our cross comparison analysis revealed that residues in C4 and in both the V3 stem and crown were important for CCR5 binding of gp120 subunits. Contrary to our prediction, mutations in the V3 crown had less effect on membrane fusion than mutations in the V3 stem. The V3 stem thus appears to be the most important region for CCR5 utilization since it affected both coreceptor binding and subsequent fusion and viral entry. Our data raises the possibility that some residues in the V3 crown and in C4 may play distinct roles in the binding and fusion steps of envelope-coreceptor interaction.

Helminth infection suppresses T-cell immune response to HIV-DNA-based vaccine in mice.

A number of HIV-1 vaccines are in various phases of clinical trials and many more are in the developmental pipeline. Vaccines are especially needed for developing countries where morbidity and mortality due to HIV/AIDS is most severe, the prevalence of HIV infection is highest, and its incidence is often still rising dramatically. Individuals living in these regions are often infected with one or more helminth parasites which systemically bias the immune system towards Th2-type as well as drive immune anergy. The goal of this study was to develop a multi-T-cell epitope DNA-based vaccine for HIV-1 subtype C and to determine the impact of helminth infection on the immune response to this vaccine. We found that vaccination of naïve mice with the multi-epitope vaccine, designated TD158, induced a strong HIV-1C-specific T-cell immune response, and that the addition of the Igkappa leader sequence to the TD158 vaccine construct significantly increased the frequencies of IFN-gamma secreting CD8+ T cells. However, the TD158 vaccine specific response of mice infected with the human helminth Schistosoma mansoni was significantly suppressed. The impact of schistosome infection on suppressing the virus-specific immune response was the same whether mice were vaccinated with the TD158 vaccine or with the Igkappa enhanced TD158. The results of this study suggest that helminth infection may pose a serious problem for vaccination with the DNA-based HIV-1 vaccine in developing country populations, and that the prevalence of helminth infections in the vaccine cohorts should be taken into account for HIV-1 vaccine trial design.

Interactive association of proviral load and IFN-gamma-secreting T cell responses in HIV-1C infection.

We investigated the interactive relationship between proviral DNA load and virus-specific IFN-gamma-secreting T cell responses in HIV-1C infection. The presence or absence of correlation, and inverse or direct type of correlation, if any, were dependent on targeted viral gene product. Responses to Gag p24 or to Pol were associated with lower proviral DNA load. Associations between proviral DNA load and T cell responses did not necessarily mirror relationships between plasma RNA load and T cell responses. An interaction analysis showed a synergy in that lower proviral DNA and lower plasma RNA load were associated with high Gag p24-specific IFN-gamma-secreting T cell response (interaction test P = 0.0003). Our findings support the idea that HIV proteins have differential value for vaccine design, and suggest that, for HIV-1C, Gag p24 may be one of the most attractive regions to include in vaccine designs to control both plasma RNA load and cell-associated proviral DNA load.

HIV-1 Cis Enhancing Sequence (CES) enhances CTE-dependent Gag expression.

In order to export intron-containing RNA from nucleus, retroviruses use either viral trans-acting factors or constitutive cellular factors interacting with cis-elements in their intron-containing RNA. We have previously identified a Cis Enhancing Sequence (CES) in HIV-1 env region that could co-operate with Rev and RRE to enhance Gag expression by promoting RNA stabilization and exportation. In this study, we found that CES could function in a Rev-independent manner by co-operating with a Constitutive Transport Element (CTE) of Mason-Pfizer monkey viruses (MPMV). RRE and CTE promote intron-containing RNA exportation through different pathways. The fact that CES could function in both pathways of RNA export suggested that CES might function at a common step either up- or downstream to Rev/RRE or CTE functions. Known hnRNP-A1-binding sites as well as other 3 highly conserved sequences in the CES were found to be required for its activity.

A highly conserved arginine in gp120 governs HIV-1 binding to both syndecans and CCR5 via sulfated motifs.

HIV-1 has maximized its utilization of syndecans. It uses them as in cis receptors to infect macrophages and as in trans receptors to infect T-lymphocytes. In this study, we investigated at a molecular level the mechanisms that control HIV-1-syndecan interactions. We found that a single conserved arginine (Arg-298) in the V3 region of gp120 governs HIV-1 binding to syndecans. We found that an amine group on the side chain of this residue is necessary for syndecan utilization by HIV-1. Furthermore, we showed that HIV-1 binds syndecans via a 6-O sulfation, demonstrating that this binding is not the result of random interactions between basic residues and negative charges, but the result of specific contacts between gp120 and a well defined sulfation in syndecans. Surprisingly, we found that Arg-298, which mediates HIV-1 binding to syndecans, also mediates HIV-1 binding to CCR5. We postulated that HIV-1 recognizes similar motifs on syndecans and CCR5. Supporting this hypothesis, we obtained several lines of evidence that suggest that the 6-O sulfation recognized by HIV-1 on syndecans mimics the sulfated tyrosines recognized by HIV-1 in the N terminus of CCR5. Our finding that CCR5 and syndecans are exploited by HIV-1 via a single determinant echoes the mechanisms by which chemokines utilize these two disparate receptors and suggests that the gp120/chemokine mimicry may represent a common strategy in microbial pathogenesis.

Intragenic HIV-1 env sequences that enhance gag expression.

Expression of HIV-1 genes is regulated at multiple levels including the complex RNA splicing and transport mechanisms. Multiple cis-acting elements involved in these regulations have been previously identified in various regions of HIV-1 genome. Here we show that another cis-acting element was present in HIV-1 env region. This element enhanced the expression of Gag when inserted together with Rev response element (RRE) into a truncated HIV-1 genome in the presence of Rev. The enhancing activity was mapped to a 263-bp fragment in the gp41 region downstream to RRE. RNA analysis showed that it might function by promoting RNA stability and Rev-dependent RNA export. The enhancement was specific to Rev-dependent expression, since it did not enhance Gag expression driven by Sam68, a cellular protein that has been shown to be able to substitute for Rev in RNA export function.

Effect of amino acid substitution of the V3 and bridging sheet residues in human immunodeficiency virus type 1 subtype C gp120 on CCR5 utilization.

The V3 loop and the bridging sheet domain of human immunodeficiency virus type 1 (HIV-1) subtype B envelope glycoprotein gp120 have been implicated in CCR5 coreceptor utilization. In this study, mutant envelope glycoproteins of a subtype C isolate containing substitutions in the V3 or C4 region were generated to determine which are required for efficient CCR5-dependent cell fusion and viral entry. We found that the V3 crown and C4 residues are relatively dispensable for cell-cell fusion, although some residues may be involved in the regulation of early postentry steps in viral replication. In contrast, seven highly conserved residues located in the V3 stem are critical for CCR5 utilization, which can explain the apparent paradox that the functional convergence in CCR5 usage by genetically divergent HIV-1 strains involves a variable region. The finding that C4 residues do not have a critical role may appear to contradict the current model that bridging sheet residues are involved in the gp120-CCR5 interaction. However, a plausible interpretation is that these C4 residues may have a distinct role in the binding and fusion steps of the gp120-CCR5 interaction.

Comparative prediction of perinatal human immunodeficiency virus type 1 transmission, using multiple virus load markers.

Maternal plasma human immunodeficiency virus (HIV) type 1 RNA load has a role in perinatal transmission, but significant overlap in the range of plasma virus loads among transmitters and nontransmitters is often observed, which makes it difficult to predict transmission outcome. We measured several virus markers in a drug-naive population of HIV-1-infected mothers in Botswana. Maternal plasma HIV-1 RNA load, peripheral blood mononuclear cell-associated blood HIV-1 DNA load, and cervicovaginal fluid (CVF) HIV-1 DNA load were determined using quantitative real-time polymerase chain reaction. The overall rate of transmission among these mother-infant pairs was 35.7%. Median infant age was 2.5 months. An association between increased plasma HIV-1 RNA load and perinatal transmission was observed (odds ratio [OR], 2.20/1-log virus load; 95% confidence interval [CI], 1.15-4.18). However, the association between increased blood HIV-1 DNA load and perinatal transmission was stronger (OR, 10.30; 95% CI, 2.11-50.38). When blood HIV-1 DNA load was combined with CVF HIV-1 DNA load, the association with transmission increased (OR, 25.0; 95% CI 2.73-228.60).