Virological outcomes of various first-line ART regimens in patients harbouring HIV-1 E157Q integrase polymorphism: a multicentre retrospective study.

BACKGROUND: Integrase strand transfer inhibitors (INSTIs) are recommended as first-line ART for people living with HIV (PLWH) in most guidelines. The INSTI-resistance-associated mutation E157Q, a highly prevalent (2%-5%) polymorphism of the HIV-1 (human immunodeficiency virus type 1) integrase gene, has limited data on optimal first-line ART regimens. We assessed the virological outcomes of various first-line ART regimens in PLWH with E157Q in real-world settings. METHODS: A multicentre retrospective observational study was conducted on PLWH who underwent integrase genotypic drug-resistance testing before ART initiation between 2008 and 2019 and were found to have E157Q. Viral suppression (<50 copies/mL) rate at 24 and 48 weeks, time to viral suppression and time to viral rebound (≥100 copies/mL) were compared among the first-line ART regimens. RESULTS: E157Q was detected in 167 (4.1%) of 4043 ART-naïve PLWH. Among them, 144 had available clinical data after ART initiation with a median follow-up of 1888 days. Forty-five started protease inhibitors + 2 NRTIs (PI group), 33 started first-generation INSTI (raltegravir or elvitegravir/cobicistat) + 2 NRTIs (INSTI-1 group), 58 started once-daily second-generation INSTI (dolutegravir or bictegravir) + 2 NRTIs (INSTI-2 group) and eight started other regimens. In the multivariate analysis, the INSTI-2 group showed similar or favourable outcomes compared with the PI group for viral suppression rates, time to viral suppression and time to viral rebound. Two cases in the INSTI-1 group experienced virological failure. CONCLUSIONS: The general guideline recommendation of second-generation INSTI-based first-line ART for most PLWH is also applicable to PLWH harbouring E157Q.

Specific mutations in the HIV-1 G-tract of the 3'-polypurine tract cause resistance to integrase strand transfer inhibitors.

BACKGROUND: In vitro selection experiments identified viruses resistant to integrase strand transfer inhibitors (INSTIs) carrying mutations in the G-tract (six guanosines) of the 3'-polypurine tract (3'-PPT). A clinical study also reported that mutations in the 3'-PPT were observed in a patient receiving dolutegravir monotherapy. However, recombinant viruses with the 3'-PPT mutations that were found in the clinical study were recently shown to be susceptible to INSTIs. OBJECTIVES: To identify the specific mutation(s) in the G-tract of the 3'-PPT for acquiring INSTI resistance, we constructed infectious clones bearing single or multiple mutations and systematically characterized the susceptibility of these clones to both first- and second-generation INSTIs. METHODS: The infectious clones were tested for their infectivity and susceptibility to INSTIs in a single-cycle assay using TZM-bl cells. RESULTS: A single mutation of thymidine (T) at the fifth position (GGG GTG) in the G-tract of the 3'-PPT had no effect on INSTI resistance. A double mutation, cytidine (C) or 'T' at the second position and 'T' at the fifth position (GCG GTG and GTG GTG), increased resistance to INSTIs, with the appearance of a plateau in the maximal percentage inhibition (MPI) of the dose-response curves, consistent with a non-competitive mechanism of inhibition. CONCLUSIONS: Mutations at the second and fifth positions in the G-tract of the 3'-PPT may result in complex resistance mechanism(s), rather than simply affecting INSTI binding at the IN active site.

Structural basis of chimpanzee APOBEC3H dimerization stabilized by double-stranded RNA.

APOBEC3H (A3H) is a mammal-specific cytidine deaminase that potently restricts the replication of retroviruses. Primate A3Hs are known to exert key selective pressures against the cross-species transmission of primate immunodeficiency viruses from chimpanzees to humans. Despite recent advances, the molecular structures underlying the functional mechanisms of primate A3Hs have not been fully understood. Here, we reveal the 2.20-Å crystal structure of the chimpanzee A3H (cpzA3H) dimer bound to a short double-stranded RNA (dsRNA), which appears to be similar to two recently reported structures of pig-tailed macaque A3H and human A3H. In the structure, the dsRNA-binding interface forms a specialized architecture with unique features. The analysis of the dsRNA nucleotides in the cpzA3H complex revealed the GC-rich palindrome-like sequence preference for dsRNA interaction, which is largely determined by arginine residues in loop 1. In cells, alterations of the cpzA3H residues critical for the dsRNA interaction severely reduce intracellular protein stability due to proteasomal degradation. This suggests that cpzA3H stability is regulated by the dsRNA-mediated dimerization as well as by unknown cellular machinery through proteasomal degradation in cells. Taken together, these findings highlight unique structural features of primate A3Hs that are important to further understand their cellular functions and regulation.

Pre-treatment and acquired HIV drug resistance in Dar es Salaam, Tanzania in the era of tenofovir and routine viral load monitoring.

OBJECTIVES: We investigated the prevalence and patterns of pre-treatment and acquired HIV drug resistance mutations (DRMs) in Tanzania as a 'treat all' strategy, virological monitoring and the progressive increase in usage of tenofovir are being implemented in HIV treatment programmes. METHODS: Viral RNA was isolated from plasma of 60 ART-naive and 166 treated-but-viraemic (>400 copies/mL) HIV-1-infected adults attending a care and treatment clinic at Muhimbili National Hospital, Dar es Salaam, Tanzania, between June and October 2017. Viral genes encoding protease and reverse transcriptase were amplified by PCR and directly sequenced. RESULTS: Viral genotyping of successfully amplified samples revealed pre-treatment DRMs in 14/47 (29.8%) ART-naive subjects. Of these, 7/47 (14.9%) harboured mutations that confer high-level resistance to at least one drug of the default first-line regimen. In treated-but-viraemic subjects, DRMs were found in 100/111 (90%), where DRMs against NNRTI, NRTI and PI were observed in 95/100 (95%), 92/100 (92%) and 13/100 (13%), respectively. Tenofovir-resistance mutations K65R and K70G/E or ≥3 thymidine analogue resistance mutations including M41L and L210W were found in 18/36 (50%) subjects on a tenofovir-containing regimen at failure. Four patients harboured multiple DRMs, which can confer resistance to all available ART regimens in Tanzania. CONCLUSIONS: Taken together, pre-treatment and acquired DRMs were highly prevalent, which represents a major risk for the efficacy of ART programmes in Tanzania. Availability of a newer generation of antiretroviral drugs with a higher genetic barrier to resistance and robust treatment monitoring is warranted for effective and sustainable HIV treatment.

Impact of HIV-1 Integrase L74F and V75I Mutations in a Clinical Isolate on Resistance to Second-Generation Integrase Strand Transfer Inhibitors.

A novel HIV-1 integrase mutation pattern, L74F V75I, which conferred resistance to first-generation integrase strand transfer inhibitors (INSTIs), was identified in a clinical case with virological failure under a raltegravir-based regimen. Addition of L74F V75I to N155H or G140S Q148H increased resistance levels to the second-generation INSTIs dolutegravir (>385- and 100-fold, respectively) and cabotegravir (153- and 197-fold, respectively). These findings are important for the development of an accurate system for interpretation of INSTI resistance and the rational design of next-generation INSTIs.

Structural Insights into HIV-1 Vif-APOBEC3F Interaction.

UNLABELLED: The HIV-1 Vif protein inactivates the cellular antiviral cytidine deaminase APOBEC3F (A3F) in virus-infected cells by specifically targeting it for proteasomal degradation. Several studies identified Vif sequence motifs involved in A3F interaction, whereas a Vif-binding A3F interface was proposed based on our analysis of highly similar APOBEC3C (A3C). However, the structural mechanism of specific Vif-A3F recognition is still poorly understood. Here we report structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Alanine-scanning analysis of Vif revealed that six residues located within the conserved Vif F1-, F2-, and F3-box motifs are essential for both A3C and A3F degradation, and an additional four residues are uniquely required for A3F degradation. Modeling of the Vif structure on an HIV-1 Vif crystal structure revealed that three discontinuous flexible loops of Vif F1-, F2-, and F3-box motifs sterically cluster to form a flexible A3F interaction interface, which represents hydrophobic and positively charged surfaces. We found that the basic Vif interface patch (R17, E171, and R173) involved in the interactions with A3C and A3F differs. Furthermore, our crystal structure determination and extensive mutational analysis of the A3F C-terminal domain demonstrated that the A3F interface includes a unique acidic stretch (L291, A292, R293, and E324) crucial for Vif interaction, suggesting additional electrostatic complementarity to the Vif interface compared with the A3C interface. Taken together, these findings provide structural insights into the A3F-Vif interaction mechanism, which will provide an important basis for development of novel anti-HIV-1 drugs using cellular cytidine deaminases. IMPORTANCE: HIV-1 Vif targets cellular antiviral APOBEC3F (A3F) enzyme for degradation. However, the details on the structural mechanism for specific A3F recognition remain unclear. This study reports structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Three discontinuous sequence motifs of Vif, F1, F2, and F3 boxes, assemble to form an A3F interaction interface. In addition, we determined a crystal structure of the wild-type A3F C-terminal domain responsible for the Vif interaction. These results demonstrated that both electrostatic and hydrophobic interactions are the key force driving Vif-A3F binding and that the Vif-A3F interfaces are larger than the Vif-A3C interfaces. These findings will allow us to determine the configurations of the Vif-A3F complex and to construct a structural model of the complex, which will provide an important basis for inhibitor development.

Molecular epidemiology of co-infection with hepatitis B virus and human immunodeficiency virus (HIV) among adult patients in Harare, Zimbabwe.

The objective of this study was to determine the prevalence of co-infection with hepatitis B virus (HBV) and human immunodeficiency virus (HIV) and the genetic characteristics of both viruses among pre-HIV-treatment patients in Harare, Zimbabwe. This cross-sectional survey involved 176 remnant plasma samples collected from consenting HIV patients (median age 35 [18-74]) between June and September 2014. HBV seromarkers were determined by high-sensitivity chemiluminescence assays. Molecular evolutionary analyses were conducted on the basal core promoter/precore (BCP/PC) and S regions of HBV, as well as part of the HIV pol region. Of the 176 participants (65.7% female), 19 (10.8%) were positive for HBsAg (median 0.033 IU/ml (IQR 0.01-415). The HBsAg incidence was higher in men than women (P = 0.009). HBsAg-positive subjects had lower median CD4 counts (P = 0.016). HBV DNA was detectable in 12 HBsAg-positive samples (median 3.36 log cp/ml (2.86-4.51), seven being amplified and sequenced. All isolates were subgenotype A1 without HBV drug resistance mutations but each had at least one BCP/PC mutation. PreS deletion mutants and small S antigen variants M133I/T and D144G were identified. Of the 164 HIV isolates successfully genotyped, 163 (99.4%) were HIV-1 subtype C and only one was HIV-1 subtype F1. Sixteen (9.8%) had at least one drug resistance mutation, predominantly non-nucleoside reverse transcriptase inhibitor-related mutations, observed mostly among female participants. This study shows that co-infection with HBV is present among HIV patients enrolling into HIV care in Zimbabwe, suggesting that HBV screening and monitoring programmes be strengthened in this context. J. Med. Virol. 89:257-266, 2017. © 2016 Wiley Periodicals, Inc.

Fusobacterium nucleatum detected simultaneously in a pyogenic liver abscess and advanced sigmoid colon cancer.

Fusobacterium nucleatum is an invasive, adherent, and pro-inflammatory anaerobic bacterium involved in various infections and colorectal cancer. We report a case with pyogenic liver abscess, diagnosed with advanced sigmoid colon cancer, in whom F. nucleatum was simultaneously detected. In this patient, F. nucleatum was systematically analyzed using the molecular biological techniques of metagenome analysis, conventional PCR, and microbial fluorescence in situ hybridization.

[Investigation of the Pathophysiology and Diagnostic Methods Through a Case of Acute HIV Infection in which the Western Blot Method Remained Negative Over the Long Term and the 4th Generation Screening Assay Became Negative for a Certain Period of Time after Initiating Antiretroviral Therapy].

Confirmatory tests using Western blot (WB) and HIV-1 nucleic acid testing (HIV-1 RNA) following a positive screening test are required for the diagnosis of HIV-1 infection according to the current Japanese guidelines for HIV-1/2 diagnosis. We report herein on a rare case in a patient who remained negative for WB over 10 months in spite of being positive by fourth-generation immunoassays (4thGIA) and who subsequently seroreverted by 4thGIA for three months after initiating antiretroviral therapy. Case: A man in his early twenties previously visited a hospital because of fever in October 2012. Laboratory data revealed leukocytopenia, thrombocytopenia and increased serum ferritin, suggesting hemophagocytic syndrome (HPS). During that visit, he tested positive for a 4thGIA, but negative for HIV-1 WB and his result of HIV-1 RNA result was detected invalid because of the presence of some inhibitory material in his RNA preparation. Thereafter, he was diagnosed as having cytomegalovirus-associated HPS treatment was for which initiated. In January 2013, he developed Pneumocystis jirovecii pneumonia, and his HIV-1 RNA viral load was 7.7 × 105 copies/mL in February 2013. Acute HIV infection was suspected, because the HIV-1 WB remained negative. He was started on antiretroviral therapy in April 2013. His 4thGIA was converted to negative in May 2013 and was reconverted to positive in August 2013. HIV-1 WB, however, continued to be indeterminant until February 2014, in which it turned positive for the first time according to the CDC criteria. Methods and Results: The genetic analyses of HIV-1 were done on the gag, env, nef and pol region of the HIV-1 gene from the patient. There was no clear element to delay antibody production on the virus side. Preserved specimens of the patient were measured with eight kinds of HIV screening assay. It was thought that the fourth generation assay was positive only by the presence of the antigen until March 2013 because the antibody had not been detected. Discussion: We encountered a case of acute HIV infection in which the WB result was negative for 10 months after the first positive response of the 4thGIA. The 4thGIA is essential for the early diagnosis and early treatment of HIV infection; therefore, the 4thGIA should be strictly recommended to avoid the use of older generations of immunoassay in the diagnostic guidelines. The role of the WB test should be examined closely from various aspects for use as a confirmatory test under recent laboratory situations in which highly sensitive and specific methods, e.g. the 4th GIA, have become available. In addition, unnecessary confusion due to the diversities of antibody formation should be avoided. The antibody detection tests for HIV are still necessary and indispensable for the confirmation of the disease or the diagnosis of the acute infection stage. Therefore development of a newer antibody measuring method which could achieve an easier operation and should have a higher sensitivity and specificity for HIV confirmation is strongly expected.

Rilpivirine resistance mutation E138K in HIV-1 reverse transcriptase predisposed by prevalent polymorphic mutations.

BACKGROUND: Rilpivirine is listed as a recommended or alternative key drug in the current ART guidelines. E138K in HIV-1 reverse transcriptase (RT) is a primary mutation in resistance to rilpivirine, although in vitro experiments showed it confers only <3-fold resistance. An unidentified mechanism could amplify resistance to rilpivirine conferred by E138K. OBJECTIVES: The objective of this study was to reveal the mechanism amplifying rilpivirine resistance conferred by E138K. PATIENTS AND METHODS: HIV-1 RT sequences were compared in patients who failed rilpivirine-containing ART virologically. The effects of mutations commonly identified with E138K on rilpivirine susceptibility were analysed by using recombinant HIV-1 variants. RESULTS: Rilpivirine-containing ART was introduced in 162 HIV-1-infected patients at the outpatient clinic of the AIDS Clinical Center (National Center for Global Health and Medicine, Tokyo, Japan) between May 2012 and June 2015. Virological treatment failure occurred in six of these patients. E138K emerged in three patients while other rilpivirine resistance mutations emerged in the other three patients. I135T/L were identified in only three patients with E138K and existed before the introduction of rilpivirine-containing ART. Analysis of recombinant HIV-1 variants indicated that E138K conferred low-level rilpivirine resistance and that coexistence of I135T/L with E138K amplified the resistance. CONCLUSIONS: I135T/L, escape mutations from HLA-B*51/52-restricted cytotoxic T lymphocytes, which are prevalent in Japan, may predispose HIV-1 to harbour E138K upon failure of rilpivirine-containing ART. The mutation patterns of drug resistance may vary due to baseline polymorphic mutations.

Structural basis of clade-specific HIV-1 neutralization by humanized anti-V3 monoclonal antibody KD-247.

Humanized monoclonal antibody KD-247 targets the Gly(312)-Pro(313)-Gly(314)-Arg(315) arch of the third hypervariable (V3) loop of the HIV-1 surface glycoprotein. It potently neutralizes many HIV-1 clade B isolates, but not of other clades. To understand the molecular basis of this specificity, we solved a high-resolution (1.55 Å) crystal structure of the KD-247 antigen binding fragment and examined the potential interactions with various V3 loop targets. Unlike most antibodies, KD-247 appears to interact with its target primarily through light chain residues. Several of these interactions involve Arg(315) of the V3 loop. To evaluate the role of light chain residues in the recognition of the V3 loop, we generated 20 variants of KD-247 single-chain variable fragments with mutations in the antigen-binding site. Purified proteins were assessed for V3 loop binding using AlphaScreen technology and for HIV-1 neutralization. Our data revealed that recognition of the clade-specificity defining residue Arg(315) of the V3 loop is based on a network of interactions that involve Tyr(L32), Tyr(L92), and Asn(L27d) that directly interact with Arg(315), thus elucidating the molecular interactions of KD-247 with its V3 loop target.

Adaptation of HIV-1 to human leukocyte antigen class I.

The rapid and extensive spread of the human immunodeficiency virus (HIV) epidemic provides a rare opportunity to witness host-pathogen co-evolution involving humans. A focal point is the interaction between genes encoding human leukocyte antigen (HLA) and those encoding HIV proteins. HLA molecules present fragments (epitopes) of HIV proteins on the surface of infected cells to enable immune recognition and killing by CD8(+) T cells; particular HLA molecules, such as HLA-B*57, HLA-B*27 and HLA-B*51, are more likely to mediate successful control of HIV infection. Mutation within these epitopes can allow viral escape from CD8(+) T-cell recognition. Here we analysed viral sequences and HLA alleles from >2,800 subjects, drawn from 9 distinct study cohorts spanning 5 continents. Initial analysis of the HLA-B*51-restricted epitope, TAFTIPSI (reverse transcriptase residues 128-135), showed a strong correlation between the frequency of the escape mutation I135X and HLA-B*51 prevalence in the 9 study cohorts (P = 0.0001). Extending these analyses to incorporate other well-defined CD8(+) T-cell epitopes, including those restricted by HLA-B*57 and HLA-B*27, showed that the frequency of these epitope variants (n = 14) was consistently correlated with the prevalence of the restricting HLA allele in the different cohorts (together, P < 0.0001), demonstrating strong evidence of HIV adaptation to HLA at a population level. This process of viral adaptation may dismantle the well-established HLA associations with control of HIV infection that are linked to the availability of key epitopes, and highlights the challenge for a vaccine to keep pace with the changing immunological landscape presented by HIV.

Biochemical, inhibition and inhibitor resistance studies of xenotropic murine leukemia virus-related virus reverse transcriptase.

We report key mechanistic differences between the reverse transcriptases (RT) of human immunodeficiency virus type-1 (HIV-1) and of xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus that can infect human cells. Steady and pre-steady state kinetics demonstrated that XMRV RT is significantly less efficient in DNA synthesis and in unblocking chain-terminated primers. Surface plasmon resonance experiments showed that the gammaretroviral enzyme has a remarkably higher dissociation rate (k(off)) from DNA, which also results in lower processivity than HIV-1 RT. Transient kinetics of mismatch incorporation revealed that XMRV RT has higher fidelity than HIV-1 RT. We identified RNA aptamers that potently inhibit XMRV, but not HIV-1 RT. XMRV RT is highly susceptible to some nucleoside RT inhibitors, including Translocation Deficient RT inhibitors, but not to non-nucleoside RT inhibitors. We demonstrated that XMRV RT mutants K103R and Q190M, which are equivalent to HIV-1 mutants that are resistant to tenofovir (K65R) and AZT (Q151M), are also resistant to the respective drugs, suggesting that XMRV can acquire resistance to these compounds through the decreased incorporation mechanism reported in HIV-1.

HIV-1 reverse transcriptase (RT) polymorphism 172K suppresses the effect of clinically relevant drug resistance mutations to both nucleoside and non-nucleoside RT inhibitors.

Polymorphisms have poorly understood effects on drug susceptibility and may affect the outcome of HIV treatment. We have discovered that an HIV-1 reverse transcriptase (RT) polymorphism (RT(172K)) is present in clinical samples and in widely used laboratory strains (BH10), and it profoundly affects HIV-1 susceptibility to both nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) when combined with certain mutations. Polymorphism 172K significantly suppressed zidovudine resistance caused by excision (e.g. thymidine-associated mutations) and not by discrimination mechanism mutations (e.g. Q151M complex). Moreover, it attenuated resistance to nevirapine or efavirenz imparted by NNRTI mutations. Although 172K favored RT-DNA binding at an excisable pre-translocation conformation, it decreased excision by thymidine-associated mutation-containing RT. 172K affected DNA handling and decreased RT processivity without significantly affecting the k(cat)/K(m) values for dNTP. Surface plasmon resonance experiments revealed that RT(172K) decreased DNA binding by increasing the dissociation rate. Hence, the increased zidovudine susceptibility of RT(172K) results from its increased dissociation from the chain-terminated DNA and reduced primer unblocking. We solved a high resolution (2.15 Å) crystal structure of RT mutated at 172 and compared crystal structures of RT(172R) and RT(172K) bound to NNRTIs or DNA/dNTP. Our structural analyses highlight differences in the interactions between α-helix E (where 172 resides) and the active site ß9-strand that involve the YMDD loop and the NNRTI binding pocket. Such changes may increase dissociation of DNA, thus suppressing excision-based NRTI resistance and also offset the effect of NNRTI resistance mutations thereby restoring NNRTI binding.

Biochemical mechanism of HIV-1 resistance to rilpivirine.

Rilpivirine (RPV) is a second generation nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) that efficiently inhibits HIV-1 resistant to first generation NNRTIs. Virological failure during therapy with RPV and emtricitabine is associated with the appearance of E138K and M184I mutations in RT. Here we investigate the biochemical mechanism of RT inhibition and resistance to RPV. We used two transient kinetics approaches (quench-flow and stopped-flow) to determine how subunit-specific mutations in RT p66 or p51 affect association and dissociation of RPV to RT as well as their impact on binding of dNTP and DNA and the catalytic incorporation of nucleotide. We compared WT with four subunit-specific RT mutants, p66(M184I)/p51(WT), p66(E138K)/p51(E138K), p66(E138K/M184I)/p51(E138K), and p66(M184I)/p51(E138K). Ile-184 in p66 (p66(184I)) decreased the catalytic efficiency of RT (k(pol)/K(d)(.dNTP)), primarily through a decrease in dNTP binding (K(d)(.dNTP)). Lys-138 either in both subunits or in p51 alone abrogated the negative effect of p66(184I) by restoring dNTP binding. Furthermore, p51(138K) reduced RPV susceptibility by altering the ratio of RPV dissociation to RPV association, resulting in a net reduction in RPV equilibrium binding affinity (K(d)(.RPV) = k(off.RPV)/k(on.RPV)). Quantum mechanics/molecular mechanics hybrid molecular modeling revealed that p51(E138K) affects access to the RPV binding site by disrupting the salt bridge between p51(E138) and p66(K101). p66(184I) caused repositioning of the Tyr-183 active site residue and decreased the efficiency of RT, whereas the addition of p51(138K) restored Tyr-183 to a WT-like conformation, thus abrogating the Ile-184-induced functional defects.

Naturally selected rilpivirine-resistant HIV-1 variants by host cellular immunity.

BACKGROUND: Rilpivirine is listed as an alternative key drug in current antiretroviral therapy (ART) guidelines. E138G/A/K in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are rilpivirine resistance-associated mutations and can be identified in a few ART-naive patients, although at low frequency. The 138th position in HIV-1 RT is located in one of the putative epitopes of human leukocyte antigen (HLA)-B*18-restricted cytotoxic T lymphocytes (CTLs). CTL-mediated immune pressure selects escape mutations within the CTL epitope. Here we tested whether E138G/A/K could be selected by HLA-B*18-restricted CTLs. METHODS: The amino acid variation at the 138th position was compared between ART-naive HIV-1-infected patients with and without HLA-B*18. The optimal epitope containing the 138th position was determined and the impact of E138G/A/K on CTL response was analyzed by epitope-specific CTLs. The effect of E138G/A/K on drug susceptibility was determined by constructing recombinant HIV-1 variants. RESULTS: The prevalence of E138G/A/K was 21% and 0.37% in 19 and 1088 patients with and without HLA-B*18, respectively (odds ratio, 72.3; P = 4.9 × 10(-25)). The CTL response was completely abolished by the substitution of E138G/A/K in the epitope peptide. E138G/A/K conferred 5.1-, 7.1-, and 2.7-fold resistance to rilpivirine, respectively. CONCLUSIONS: E138G/A/K can be selected by HLA-B*18-restricted CTLs and confer significant rilpivirine resistance. We recommend drug resistance testing before the introduction of rilpivirine-based ART in HLA-B*18-positive patients.

Hypersusceptibility mechanism of Tenofovir-resistant HIV to EFdA.

BACKGROUND: The K65R substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is the major resistance mutation selected in patients treated with first-line antiretroviral tenofovir disoproxil fumarate (TDF). 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), is the most potent nucleoside analog RT inhibitor (NRTI) that unlike all approved NRTIs retains a 3'-hydroxyl group and has remarkable potency against wild-type (WT) and drug-resistant HIVs. EFdA acts primarily as a chain terminator by blocking translocation following its incorporation into the nascent DNA chain. EFdA is in preclinical development and its effect on clinically relevant drug resistant HIV strains is critically important for the design of optimal regimens prior to initiation of clinical trials. RESULTS: Here we report that the K65R RT mutation causes hypersusceptibility to EFdA. Specifically, in single replication cycle experiments we found that EFdA blocks WT HIV ten times more efficiently than TDF. Under the same conditions K65R HIV was inhibited over 70 times more efficiently by EFdA than TDF. We determined the molecular mechanism of this hypersensitivity using enzymatic studies with WT and K65R RT. This substitution causes minor changes in the efficiency of EFdA incorporation with respect to the natural dATP substrate and also in the efficiency of RT translocation following incorporation of the inhibitor into the nascent DNA. However, a significant decrease in the excision efficiency of EFdA-MP from the 3' primer terminus appears to be the primary cause of increased susceptibility to the inhibitor. Notably, the effects of the mutation are DNA-sequence dependent. CONCLUSION: We have elucidated the mechanism of K65R HIV hypersusceptibility to EFdA. Our findings highlight the potential of EFdA to improve combination strategies against TDF-resistant HIV-1 strains.

Evaluation of Combinations of 4'-Ethynyl-2-Fluoro-2'-Deoxyadenosine with Clinically Used Antiretroviral Drugs.

Drug combination studies of 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) with FDA-approved drugs were evaluated by two different methods, MacSynergy II and CalcuSyn. Most of the combinations, including the combination of the two adenosine analogs EFdA and tenofovir, were essentially additive, without substantial antagonism or synergism. The combination of EFdA and rilpivirine showed apparent synergism. These studies provide information that may be useful for the design of EFdA combination regimens for initial and salvage therapy assessment.

Association of demographics, HCV co-infection, HIV-1 subtypes and genetic clustering with late HIV diagnosis: a retrospective analysis from the Japanese Drug Resistance HIV-1 Surveillance Network.

INTRODUCTION: Late diagnosis of the human immunodeficiency virus (HIV) is a major concern epidemiologically, socially and for national healthcare systems. Although the association of certain demographics with late HIV diagnosis has been reported in several studies, the association of other factors, including clinical and phylogenetic factors, remains unclear. In the present study, we conducted a nationwide analysis to explore the association of demographics, clinical factors, HIV-1 subtypes/circulating recombinant form (CRFs) and genetic clustering with late HIV diagnosis in Japan, where new infections mainly occur among young men who have sex with men (MSM) in urban areas. METHODS: Anonymized data on demographics, clinical factors and HIV genetic sequences from 39.8% of people newly diagnosed with HIV in Japan were collected by the Japanese Drug Resistance HIV-1 Surveillance Network from 2003 to 2019. Factors associated with late HIV diagnosis (defined as HIV diagnosis with a CD4 count <350 cells/µl) were identified using logistic regression. Clusters were identified by HIV-TRACE with a genetic distance threshold of 1.5%. RESULTS: Of the 9422 people newly diagnosed with HIV enrolled in the surveillance network between 2003 and 2019, 7752 individuals with available CD4 count at diagnosis were included. Late HIV diagnosis was observed in 5522 (71.2%) participants. The overall median CD4 count at diagnosis was 221 (IQR: 62-373) cells/µl. Variables independently associated with late HIV diagnosis included age (adjusted odds ratio [aOR] 2.21, 95% CI 1.88-2.59, ≥45 vs. ≤29 years), heterosexual transmission (aOR 1.34, 95% CI 1.11-1.62, vs. MSM), living outside of Tokyo (aOR 1.18, 95% CI 1.05-1.32), hepatitis C virus (HCV) co-infection (aOR 1.42, 95% CI 1.01-1.98) and not belonging to a cluster (aOR 1.30, 95% CI 1.12-1.51). CRF07_BC (aOR 0.34, 95% CI 0.18-0.65, vs. subtype B) was negatively associated with late HIV diagnosis. CONCLUSIONS: In addition to demographic factors, HCV co-infection, HIV-1 subtypes/CRFs and not belonging to a cluster were independently associated with late HIV diagnosis in Japan. These results imply the need for public health programmes aimed at the general population, including but not limited to key populations, to encourage HIV testing.

Structural and inhibition studies of the RNase H function of xenotropic murine leukemia virus-related virus reverse transcriptase.

RNase H inhibitors (RNHIs) have gained attention as potential HIV-1 therapeutics. Although several RNHIs have been studied in the context of HIV-1 reverse transcriptase (RT) RNase H, there is no information on inhibitors that might affect the RNase H activity of other RTs. We performed biochemical, virological, crystallographic, and molecular modeling studies to compare the RNase H function and inhibition profiles of the gammaretroviral xenotropic murine leukemia virus-related virus (XMRV) and Moloney murine leukemia virus (MoMLV) RTs to those of HIV-1 RT. The RNase H activity of XMRV RT is significantly lower than that of HIV-1 RT and comparable to that of MoMLV RT. XMRV and MoMLV, but not HIV-1 RT, had optimal RNase H activities in the presence of Mn²âº and not Mg²âº. Using hydroxyl-radical footprinting assays, we demonstrated that the distance between the polymerase and RNase H domains in the MoMLV and XMRV RTs is longer than that in the HIV-1 RT by ∼3.4 Å. We identified one naphthyridinone and one hydroxyisoquinolinedione as potent inhibitors of HIV-1 and XMRV RT RNases H with 50% inhibitory concentrations ranging from ∼0.8 to 0.02 µM. Two acylhydrazones effective against HIV-1 RT RNase H were less potent against the XMRV enzyme. We also solved the crystal structure of an XMRV RNase H fragment at high resolution (1.5 Å) and determined the molecular details of the XMRV RNase H active site, thus providing a framework that would be useful for the design of antivirals that target RNase H.