Limited SHIV env diversification in macaques failing oral antiretroviral pre-exposure prophylaxis

BACKGROUND: Pre-exposure prophylaxis (PrEP) with daily Truvada [a combination of emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF)] is a novel HIV prevention strategy recently found to prevent HIV transmission among men who have sex with men and heterosexual couples. Acute infection in adherent persons who fail PrEP will inevitably occur under concurrent antiretroviral therapy, thus raising questions regarding the potential impact of PrEP on early viral dynamics. We investigated viral evolution dynamics in a macaque model of PrEP consisting of repeated rectal exposures to SHIV(162P3) in the presence of PrEP. RESULTS: Four macaques were infected during daily or intermittent PrEP with FTC or FTC/TDF, and five were untreated controls. SHIV env sequence evolution was monitored by single genome amplification with phylogenetic and sequence analysis. Mean nucleotide divergence from transmitted founder viruses calculated 17 weeks (range = 12–20) post peak viremia was significantly lower in PrEP failures than in control animals (7.2 × 10(-3) compared to 1.6 × 10(-2) nucleotide substitutions per site per year, respectively, p < 0.0001). Mean virus diversity was also lower in PrEP failures after 17 weeks (0.13% vs. 0.53% in controls, p < 0.0001). CONCLUSIONS: Our results in a macaque model of acute HIV infection suggest that infection during PrEP limits early virus evolution likely because of a direct antiviral effect of PrEP and/or reduced target cell availability. Reduced virus diversification during early infection might enhance immune control by slowing the selection of escape mutants

Detection of Novel SARS-like and Other Coronaviruses in Bats from Kenya

Diverse coronaviruses have been identified in bats from several continents but not from Africa. We identified group 1 and 2 coronaviruses in bats in Kenya, including SARS-related coronaviruses. The sequence diversity suggests that bats are well-established reservoirs for and likely sources of coronaviruses for many species, including humans

Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6

The Saccharomyces cerevisiae Spt16/Cdc68, Pob3, and Nhp6 proteins (SPN or yFACT) bind to and alter nucleosomes in vitro, providing a potential explanation for their importance in both transcription and replication in vivo. We show that nucleosomes bound by either Nhp6 alone or the yFACT complex remain largely intact and immobile but are significantly reorganized, as indicated by changes in the pattern of sensitivity to DNase I and enhanced digestion by some restriction endonucleases. In contrast, yFACT enhanced access to exonuclease III only at very high levels of enzyme, suggesting that the DNA near the entry and exit sites of nucleosomes is largely unperturbed and that the position of the histone octamers relative to the DNA is not altered during reorganization. DNase I sensitivity was enhanced at sites clustered near the center of the nucleosomal DNA, away from the entry and exit points, and the pattern of nuclease sensitivity was only mildly affected by the configuration of linker extensions, further indicating that linkers play only a minor role in the reorganization of nucleosomes by yFACT. The DNA in contact with H2A-H2B dimers is therefore the region whose nuclease sensitivity was the least affected by yFACT reorganization. The most dramatic changes in nucleosome structure occurred when Spt16-Pob3 and the HMG box protein Nhp6 were both present, but Nhp6 alone altered DNase I sensitivity at some specific sites, supporting an independent role for this class of proteins in the general management of chromatin properties. yFACT activity does not require ATP hydrolysis and does not alter the position of nucleosomes, indicating that it acts through a mechanism distinct from chromatin remodeling. The results presented here suggest instead that yFACT promotes polymerase progression by reorganizing nucleosome cores into a less inhibitory conformation in which the properties of DNA sequences near the center of the nucleosomes are altered

Role of the Human T-Cell Leukemia Virus Type 1 PTAP Motif in Gag Targeting and Particle Release

Human T-cell leukemia virus type 1 (HTLV-1) Gag is targeted to the plasma membrane for particle assembly and release. How HTLV-1 Gag targeting occurs is not well understood. The PPPY and PTAP motifs were previously shown to be involved in HTLV-1 particle release with PTAP playing a more subtle role in virus budding. These L domains function through the interaction with host cellular proteins normally involved in multivesicular body (MVB) morphogenesis. The plasma membrane pathway rather than the MVB pathway was found to be the primary pathway for HTLV-1 particle release in HeLa cells. Intriguingly, disruption of the PTAP motif led to a defect in the targeting of Gag from the plasma membrane to CD63-positive MVBs. Particles or particle buds were observed to be associated with MVBs by electron microscopy, implying that Gag targeting to the MVB resulted in particle budding. Blocking clathrin-dependent endocytosis was found not to influence localization of the HTLV-1 Gag PTAP mutant, indicating that Gag did not reach the MVBs through clathrin-dependent endocytosis. Our observations imply that the interaction between Gag and TSG101 is not required for Gag targeting to the MVB. Overexpression of dynamitin p50 increased particle release, suggesting that there was an increase in the intracellular transport of MVBs to the cell periphery by the utilization of the dynein-dynactin motor complex. Intriguingly, virus particle release with this mutant was reduced by 20-fold compared to that of wild type in HeLa cells, which is in marked contrast to the less-than-twofold defect observed for particle production of the HTLV-1 Gag PTAP mutant from 293T cells. These results indicate that the role of the PTAP motif in L domain function is cell type dependent

Defects in SPT16 or POB3 (yFACT) in Saccharomyces cerevisiae cause dependence on the Hir/Hpc pathway: polymerase passage may degrade chromatin structure.

Spt16/Cdc68, Pob3, and Nhp6 collaborate in vitro and in vivo as the yeast factor SPN, which is homologous to human FACT. SPN/FACT complexes mediate passage of polymerases through nucleosomes and are important for both transcription and replication. An spt16 mutation was found to be intolerable when combined with a mutation in any member of the set of functionally related genes HIR1, HIR2/SPT1, HIR3/HPC1, or HPC2. Mutations in POB3, but not in NHP6A/B, also display strong synthetic defects with hir/hpc mutations. A screen for other mutations that cause dependence on HIR/HPC genes revealed genes encoding members of the Paf1 complex, which also promotes transcriptional elongation. The Hir/Hpc proteins affect the expression of histone genes and also promote normal deposition of nucleosomes; either role could explain an interaction with elongation factors. We show that both spt16 and pob3 mutants respond to changes in histone gene numbers, but in opposite ways, suggesting that Spt16 and Pob3 each interact with histones but perhaps with different subsets of these proteins. Supporting this, spt16 and pob3 mutants also display different sensitivities to mutations in the N-terminal tails of histones H3 and H4 and to mutations in enzymes that modulate acetylation of these tails. Our results support a model in which SPN/FACT has two functions: it disrupts nucleosomes to allow polymerases to access DNA, and it reassembles the nucleosomes afterward. Mutations that impair the reassembly activity cause chromatin to accumulate in an abnormally disrupted state, imposing a requirement for a nucleosome reassembly function that we propose is provided by Hir/Hpc proteins

Training rhesus macaques to take daily oral antiretroviral therapy for preclinical evaluation of HIV prevention and treatment strategies.

BackgroundMacaque models of simian or simian/human immunodeficiency virus (SIV or SHIV) infection are critical for the evaluation of antiretroviral (ARV)-based HIV treatment and prevention strategies. However, modelling human oral ARV administration is logistically challenging and fraught by limited adherence. Here, we developed a protocol for administering daily oral doses of ARVs to macaques with a high rate of compliance.MethodsParameters of positive reinforcement training (PRT), behavioral responses and optimal drug delivery foods were defined in 7 male rhesus macaques (Macaca mulatta). Animals were trained to sit in a specified cage location prior to receiving ARVs, emtricitabine (FTC) and tenofovir alafenamide (TAF), in a blended food mixture, which was followed immediately with a juice chaser. Consistency of daily oral adherence was evaluated in 4 trained macaques receiving clinically equivalent doses of FTC and TAF (20 and 1.5 mg/kg, respectively) in a short-term (1 month) and an extended (6 month) trial. Adherence was monitored using medication diaries and by quantifying intracellular FTC-triphosphate (FTC-TP) and tenofovir-diphosphate (TFV-DP) concentrations in peripheral mononuclear blood cells (PBMCs).ResultsTrained macaques quickly and consistently took daily oral ARVs for 1 month with an average 99.8% observed adherence. Intracellular concentrations of TFV-DP (median = 845.8 fmol/million cells [range, 620.8-1031.3]) and FTC-TP (median = 367.0 fmol/million cells [range, 289.5-413.5) in PBMCs were consistent with high adherence. Extended treatment with select subjects yielded similar observations for three months (99.5% adherence, 352/356 complete doses taken), although a sudden drop in adherence was observed after splenic biopsy surgery.ConclusionsWe demonstrate that trained macaques reliably adhere to a daily oral ARV regimen, although unexpected adherence issues are possible. Our approach, using clinical doses of oral FTC and TAF daily, further refines macaque models of HIV treatment and prevention by mimicking the human route and timing of ARV administration

Weekly Oral Tenofovir Alafenamide Protects Macaques from Vaginal and Rectal Simian HIV Infection

Pre-exposure prophylaxis (PrEP) with a weekly oral regimen of antiretroviral drugs could be a suitable preventative option for individuals who struggle with daily PrEP or prefer not to use long-acting injectables. We assessed in macaques the efficacy of weekly oral tenofovir alafenamide (TAF) at doses of 13.7 or 27.4 mg/kg. Macaques received weekly oral TAF for six weeks and were exposed twice-weekly to SHIV vaginally or rectally on day 3 and 6 after each dose. Median TFV-DP levels in PBMCs following the 13.7 mg/kg dose were 3110 and 1137 fmols/106 cells on day 3 and 6, respectively. With the 27.4 mg/kg dose, TFV-DP levels were increased (~2-fold) on day 3 and 6 (6095 and 3290 fmols/106 cells, respectively). Both TAF doses (13.7 and 27.4 mg/kg) conferred high efficacy (94.1% and 93.9%, respectively) against vaginal SHIV infection. Efficacy of the 27.4 mg/kg dose against rectal SHIV infection was 80.7%. We estimate that macaque doses of 13.7 and 27.4 mg/kg are equivalent to approximately 230 and 450 mg of TAF in humans, respectively. Our findings demonstrate the effectiveness of a weekly oral PrEP regimen and suggest that a clinically achievable oral TAF dose could be a promising option for non-daily PrEP