Natural Occurring Polymorphisms in HIV-1 Integrase and RNase H Regulate Viral Release and Autoprocessing

2021 ◽  
Author(s):  
Tomozumi Imamichi ◽  
John G. Bernbaum ◽  
Sylvain Laverdure ◽  
Jun Yang ◽  
Qian Chen ◽  
...  

Recently, a genome-wide association study using plasma HIV RNA from antiretroviral therapy naïve patients reported that 14 naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) in HIV derived from anti-retrovirus drugs naïve patients were associated with virus load (VL). Those SNPs were detected in reverse transcriptase, RNase H, integrase, envelope, and Nef. However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP and examined their replicative abilities. An HIV variant containing Met-to-Ile change at codon 50 in integrase (HIV(IN:M50I)) was found as an impaired virus. Despite the mutation being in integrase, the virus release was significantly suppressed (P<0.001). Transmission electron microscopy analysis revealed that abnormal bud accumulation on the plasma membrane and the released virus particles retained immature forms. Western blot analysis demonstrated a defect in autoprocessing of GagPol and Gag polyproteins' autoprocessing in the HIV(IN:M50I) particles, although Förster Resonance Energy Transfer (FRET) assay displayed that GagPol containing IN:M50I forms homodimer with a similar efficiency with GagPol (WT). The impaired maturation and replication were rescued by two other VL-associated SNPs, Ser-to-Asn change at codon 17 of integrase or Asn-to-Ser change at codon 79 of RNase H. These data demonstrate that Gag and GagPol assembly, virus release, and autoprocessing are not only regulated by integrase but also RNase H. Importance A nascent HIV-1 is a noninfectious viral particle. Cleaving Gag and GagPol polyproteins in the particle by mature HIV protease (PR), the nascent virus becomes an infectious virus. PR is initially translated as an inactive embedded enzyme in a GagPol polyprotein. The embedded PR in homodimerized GagPol polyproteins catalyzes a proteolytic reaction to release the mature PR. This excision step by a self-cleavage is called autoprocessing. Here, during the evaluation of the roles of naturally emerging non-synonymous SNPs in HIV RNA, we found that autoprocessing is inhibited by Met-to-Ile change at codon 50 in integrase GagPol. Co-existing other SNPs, Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser mutation at codon 79 in RNase H, recovered this defect, suggesting that autoprocessing is regulated by not only integrase but also RNase H in GagPol polyprotein.

2021 ◽  
Author(s):  
Tomozumi Imamichi ◽  
John G. Bernbaum ◽  
Sylvain Laverdure ◽  
Jun Yang ◽  
Qian Chen ◽  
...  

AbstractRecently, a genome-wide association study using plasma HIV RNA reported that 14 naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) in HIV derived from anti-retrovirus naïve patients were associated with virus load (VL). However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP using site-directed mutagenesis and examined their replicative abilities and biological properties. An HIV variant containing Met-to-Ile change at codon 50 in integrase (HIV(IN:M50I)) was found an impaired virus. Despite the mutation being in integrase, a quantification assay demonstrated that the virus release was significantly suppressed (P<0.001). Transmission electron microscopy analyses revealed that the accumulation of abnormal shapes of buds on the plasma membrane and the released virus particles retained immature forms. Western blot analysis demonstrated a defect in autoprocessing of GagPol and Gag polyproteins in the HIV(IN:M50I) particles. Förster Resonance Energy Transfer (FRET) assay displayed that GagPol containing IN:M50I (GagPol(IN:M50I)) significantly increased the efficiency of homodimerization (P<0.05) and heterodimerization with Gag (P<0.001), compared to GagPol(WT). HIV replication assay using a series of variants of HIV(IN:M50I) elucidated that the C-terminus residues, Asn at codon 288, plays a key role in the defect and the impaired maturation and replication capability was rescued by two other VL-associated SNPs, Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser change at codon 79 in RNase H. These data demonstrate that Gag and GagPol assembly, virus release and autoprocessing are not only regulated by integrase but also RNase H.ImportanceA nascent HIV-1 is noninfectious. To become an infectious virus, Gag and GagPol polyproteins in the particles need to be cleaved by mature HIV protease (PR). PR is initially translated as an inactive embedded enzyme in a GagPol polyprotein. The embedded PR in homodimerized GagPol polyproteins catalyzes a proteolytic reaction to release the mature PR. This excision step by a self-cleavage is called autoprocessing. Here, during the evaluation of roles of naturally emerging non-synonymous SNPs in HIV RNA, we found that autoprocessing is inhibited by Met-to-Ile change at codon 50 in integrase in GagPol which increases the efficiency of heterodimerization with Gag. This defect was recovered by co-existing of other SNPs: Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser mutation at codon 79 in RNase H, suggesting that autoprocessing is regulated by not only integrase but also RNase H in GagPol polyprotein.


2012 ◽  
Vol 86 (16) ◽  
pp. 8422-8431 ◽  
Author(s):  
Hong-Tao Xu ◽  
Maureen Oliveira ◽  
Peter K. Quashie ◽  
Matthew McCallum ◽  
Yingshan Han ◽  
...  

The emergence of HIV-1 drug resistance remains a major obstacle in antiviral therapy. M184I/V and E138K are signature mutations of clinical relevance in HIV-1 reverse transcriptase (RT) for the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine (3TC) and emtricitabine (FTC) and the second-generation (new) nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV), respectively, and the E138K mutation has also been shown to be selected by etravirine in cell culture. The E138K mutation was recently shown to compensate for the low enzyme processivity and viral fitness associated with the M184I/V mutations through enhanced deoxynucleoside triphosphate (dNTP) usage, while the M184I/V mutations compensated for defects in polymerization rates associated with the E138K mutations under conditions of high dNTP concentrations. The M184I mutation was also shown to enhance resistance to RPV and ETR when present together with the E138K mutation. These mutual compensatory effects might also enhance transmission rates of viruses containing these two mutations. Therefore, we performed tissue culture studies to investigate the evolutionary dynamics of these viruses. Through experiments in which E138K-containing viruses were selected with 3TC-FTC and in which M184I/V viruses were selected with ETR, we demonstrated that ETR was able to select for the E138K mutation in viruses containing the M184I/V mutations and that the M184I/V mutations consistently emerged when E138K viruses were selected with 3TC-FTC. We also performed biochemical subunit-selective mutational analyses to investigate the impact of the E138K mutation on RT function and interactions with the M184I mutation. We now show that the E138K mutation decreased rates of polymerization, impaired RNase H activity, and conferred ETR resistance through the p51 subunit of RT, while an enhancement of dNTP usage as a result of the simultaneous presence of both mutations E138K and M184I occurred via both subunits.


2014 ◽  
Vol 58 (8) ◽  
pp. 4515-4526 ◽  
Author(s):  
Hong-Tao Xu ◽  
Susan P. Colby-Germinario ◽  
Maureen Oliveira ◽  
Daniel Rajotte ◽  
Richard Bethell ◽  
...  

ABSTRACTA W153L substitution in HIV-1 reverse transcriptase (RT) was recently identified by selection with a novel nucleotide-competing RT inhibitor (NcRTI) termed compound A that is a member of the benzo[4,5]furo[3,2,d]pyrimidin-2-one NcRTI family of drugs. To investigate the impact of W153L, alone or in combination with the clinically relevant RT resistance substitutions K65R (change of Lys to Arg at position 65), M184I, K101E, K103N, E138K, and Y181C, on HIV-1 phenotypic susceptibility, viral replication, and RT enzymatic function, we generated recombinant RT enzymes and viruses containing each of these substitutions or various combinations of them. We found that W153L-containing viruses were impaired in viral replicative capacity and were hypersusceptible to tenofovir (TFV) while retaining susceptibility to most nonnucleoside RT inhibitors. The nucleoside 3TC retained potency against W153L-containing viruses but not when the M184I substitution was also present. W153L was also able to reverse the effects of the K65R substitution on resistance to TFV, and K65R conferred hypersusceptibility to compound A. Biochemical assays demonstrated that W153L alone or in combination with K65R, M184I, K101E, K103N, E138K, and Y181C impaired enzyme processivity and polymerization efficiency but did not diminish RNase H activity, providing mechanistic insights into the low replicative fitness associated with these substitutions. We show that the mechanism of the TFV hypersusceptibility conferred by W153L is mainly due to increased efficiency of TFV-diphosphate incorporation. These results demonstrate that compound A and/or derivatives thereof have the potential to be important antiretroviral agents that may be combined with tenofovir to achieve synergistic results.


2017 ◽  
Vol 114 (18) ◽  
pp. E3659-E3668 ◽  
Author(s):  
Ann Wiegand ◽  
Jonathan Spindler ◽  
Feiyu F. Hong ◽  
Wei Shao ◽  
Joshua C. Cyktor ◽  
...  

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2–18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.


Author(s):  
Toby E. Newman ◽  
Silke Jacques ◽  
Christy Grime ◽  
Fiona L. Kamphuis ◽  
Robert C. Lee ◽  
...  

Chickpea production is constrained worldwide by the necrotrophic fungal pathogen Ascochyta rabiei, the causal agent of ascochyta blight (AB). In order to reduce the impact of this disease, novel sources of resistance are required in chickpea cultivars. Here, we screened a new collection of wild Cicer accessions for AB resistance and identified accessions resistant to multiple, highly pathogenic isolates. In addition to this, analyses demonstrated that some collection sites of Cicer echinospermum harbour predominantly resistant accessions, knowledge that can inform future collection missions. Furthermore, a genome-wide association study identified regions of the Cicer reticulatum genome associated with AB resistance and investigation of these regions identified candidate resistance genes. Taken together, these results can be utilised to enhance the resistance of chickpea cultivars to this globally yield-limiting disease.


Retrovirology ◽  
2009 ◽  
Vol 6 (S2) ◽  
Author(s):  
Julien Guergnon ◽  
◽  
Cyril Dalmasso ◽  
Ioannis Theodorou ◽  
Agostino Riva

PLoS ONE ◽  
2015 ◽  
Vol 10 (5) ◽  
pp. e0129671
Author(s):  
Eric O. Johnson ◽  
Dana B. Hancock ◽  
Nathan C. Gaddis ◽  
Joshua L. Levy ◽  
Grier Page ◽  
...  

2016 ◽  
Vol 54 (6) ◽  
pp. 1641-1643 ◽  
Author(s):  
Sylvie Zida ◽  
Edouard Tuaillon ◽  
Makoura Barro ◽  
Arnaud Kwimatouo Lekpa Franchard ◽  
Thérèse Kagoné ◽  
...  

The impact of HIV-1 DNA coamplification during HIV-1 RNA quantification on dried blood spots (DBS) was explored. False-positive HIV RNA detection (22/62, 35%) was associated with high HIV-1 DNA levels. Specificity of HIV-1 RNA assays on DBS should be evaluated following manufacturer protocols on samples with HIV-1 DNA levels of ≥1,000 copies/106peripheral blood mononuclear cells.


2008 ◽  
Vol 52 (12) ◽  
pp. 4251-4257 ◽  
Author(s):  
Anne-Geneviève Marcelin ◽  
Philippe Flandre ◽  
Jean-Michel Molina ◽  
Christine Katlama ◽  
Patrick Yeni ◽  
...  

ABSTRACT The aim of this study was to identify human immunodeficiency virus (HIV) protease mutations associated with virological response (VR) to fosamprenavir-ritonavir (FPV/r) in 113 protease inhibitor (PI)-experienced patients randomized in both CONTEXT and TRIAD clinical trials and receiving the same dose (700/100 mg twice daily) of FPV/r. The impact of each protease mutation on the VR to FPV/r, defined as the decrease in HIV RNA at week 12, was investigated with nonparametric analyses. A step-by-step procedure was done using a Jonckheere-Terpstra (JT) test that retains the group of mutations most strongly associated with the VR. Mutations at the following 14 codons were associated with a reduced VR to FPV/r: 10, 15, 33, 46, 54, 60, 62, 63, 72, 73, 82, 84, 89, and 90. The JT procedure led to selecting the CONTEXT/TRIAD genotypic set of mutations, I15V, M46I/L, I54L/M/V, D60E, L63P/T, and I84V, as providing the strongest association with the VR (P = 1.45 × 10−11). In the nine patients with zero mutations within this set, the median decrease in HIV RNA was −2.63 log copies/ml, and was −2.22 (n = 45), −1.50 (n = 26), −0.58 (n = 23), −0.47 (n = 6), −0.13 (n = 3), and 0.04 (n = 1) log copies/ml in those with one, two, three, four, five, and six mutations, respectively. This study identified six mutations associated with VR to FPV/r. Some of these mutations are shared with the current FPV/r Agence Nationale de Recherches sur le SIDA (ANRS) resistance score, which has been cross-validated in the CONTEXT/TRIAD data set, suggesting that the current ANRS FPV/r score is a useful tool for the prediction of VR to FPV/r in PI-experienced patients.


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