scholarly journals Long-Term Control of Human Immunodeficiency Virus-1 Replication Despite Extensive Resistance to Current Antiretroviral Regimens: Clonal Analysis of Resistance Mutations in Proviral Deoxyribonucleic Acid

2016 ◽  
Vol 3 (1) ◽  
Author(s):  
Natalia Stella-Ascariz ◽  
Rocio Montejano ◽  
María Martin-Vicente ◽  
Jesús Mingorance ◽  
Ignacio Pérez-Valero ◽  
...  
Keyword(s):  
Antiretroviral Treatment ◽  
Deoxyribonucleic Acid ◽  
Clonal Analysis ◽  
Wild Type Virus ◽  
Wild Type ◽  
Resistance Mutations ◽  
Treatment Interruptions ◽  
Immunodeficiency Virus ◽  
Almost All

Abstract Archived resistance mutations compromise antiretroviral treatment. We have investigated 3 selected aviremic patients who had extensive historical resistance to their current regimen. All 3 patients underwent unstructured treatment interruptions associated to the re-emergence of wild-type virus before starting their current suppressive regimes. Almost all historical resistance mutations detected in plasma were found in circulating proviral deoxyribonucleic acid. None of the clones analyzed was fully resistant to the current antiretroviral regimen.

scholarly journals Changes in Human Immunodeficiency Virus Type 1 Populations after Treatment Interruption in Patients Failing Antiretroviral Therapy

2001 ◽  
Vol 75 (14) ◽  
pp. 6410-6417 ◽  
Author(s):  
Allan J. Hance ◽  
Virginie Lemiale ◽  
Jacques Izopet ◽  
Denise Lecossier ◽  
Véronique Joly ◽  
...  
Keyword(s):  
Treatment Interruption ◽  
Wild Type Virus ◽  
Minority Populations ◽  
Type Virus ◽  
Wild Type ◽  
Resistance Mutations ◽  
Immunodeficiency Virus ◽  
Drug Free ◽  
Hiv 1

ABSTRACT Mutations in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and protease that confer resistance to antiretroviral agents are usually accompanied by a reduction in the viral replicative capacity under drug-free conditions. Consequently, when antiretroviral treatment is interrupted in HIV-1-infected patients harboring drug-resistant virus, resistant quasi-species appear to be most often replaced within several weeks by wild-type virus. Using a real-time PCR-based technique for the selective quantification of resistant viral sequences in plasma, we have studied the kinetics of the switch from mutant to wild-type virus and evaluated the extent to which minority populations of resistant viruses not detected by genotyping persist in these individuals. Among 12 patients with viruses expressing the V82A or L90M resistance mutation who had undergone a 3-month interruption of therapy and for whom conventional genotyping had revealed an apparent total reconversion to wild-type virus, minority populations expressing these mutations, representing 0.1 to 21% of total virus, were still detectable in 9 cases. Kinetic studies demonstrated that viruses expressing resistance mutations could be detected for >5 months after the discontinuation of treatment in some patients. Most of the minority resistant genomes detected more than 3 months after the interruption of therapy carried only part of the mutations present in the resistant viruses prior to treatment interruption and appeared to result from the emergence of existing strains selected at earlier stages in the development of drug resistance. Thus, following the interruption of treatment, viral populations containing resistance mutations can persist for several months after the time when conventional genotyping techniques detect only wild-type virus. These populations include viral strains with only some of the resistance mutations initially present, strains that presumably express better fitness under drug-free conditions.

scholarly journals Viral Evolution in Response to the Broad-Based Retroviral Protease Inhibitor TL-3

2001 ◽  
Vol 75 (19) ◽  
pp. 9502-9508 ◽  
Author(s):  
Bernd Bühler ◽  
Ying-Chuan Lin ◽  
Garrett Morris ◽  
Arthur J. Olson ◽  
Chi-Huey Wong ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Viral Evolution ◽  
Selective Advantage ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Resistance Mutations ◽  
Immunodeficiency Virus ◽  
Retroviral Protease

ABSTRACT TL-3 is a protease inhibitor developed using the feline immunodeficiency virus protease as a model. It has been shown to efficiently inhibit replication of human, simian, and feline immunodeficiency viruses and therefore has broad-based activity. We now demonstrate that TL-3 efficiently inhibits the replication of 6 of 12 isolates with confirmed resistance mutations to known protease inhibitors. To dissect the spectrum of molecular changes in protease and viral properties associated with resistance to TL-3, a panel of chronological in vitro escape variants was generated. We have virologically and biochemically characterized mutants with one (V82A), three (M46I/F53L/V82A), or six (L24I/M46I/F53L/L63P/V77I/V82A) changes in the protease and structurally modeled the protease mutant containing six changes. Virus containing six changes was found to be 17-fold more resistant to TL-3 in cell culture than was wild-type virus but maintained similar in vitro replication kinetics compared to the wild-type virus. Analyses of enzyme activity of protease variants with one, three, and six changes indicated that these enzymes, compared to wild-type protease, retained 40, 47, and 61% activity, respectively. These results suggest that deficient protease enzymatic activity is sufficient for function, and the observed protease restoration might imply a selective advantage, at least in vitro, for increased protease activity.

scholarly journals A Replication-Defective Gammaherpesvirus Efficiently Establishes Long-Term Latency in Macrophages but Not in B Cells In Vivo

2008 ◽  
Vol 82 (17) ◽  
pp. 8500-8508 ◽  
Author(s):  
Haiyan Li ◽  
Kazufumi Ikuta ◽  
John W. Sixbey ◽  
Scott A. Tibbetts
Keyword(s):  
B Cells ◽  
Viral Genome ◽  
Lytic Replication ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Latency

ABSTRACT Murine gammaherpesvirus 68 (γHV68 or MHV68) is genetically related to the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), providing a useful system for in vivo studies of the virus-host relationship. To begin to address fundamental questions about the mechanisms of the establishment of gammaherpesvirus latency, we previously generated a replication-defective γHV68 lacking the expression of the single-stranded DNA binding protein encoded by orf6. In work presented here, we demonstrate that this mutant virus established a long-term infection in vivo that was molecularly identical to wild-type virus latency. Thus, despite the absence of an acute phase of lytic replication, the mutant virus established a chronic infection in which the viral genome (i) was maintained as an episome and (ii) expressed latency-associated, but not lytic replication-associated, genes. Macrophages purified from mice infected with the replication-defective virus harbored viral genome at a frequency that was nearly identical to that of wild-type γHV68; however, the frequency of B cells harboring viral genome was greatly reduced in the absence of lytic replication. Thus, this replication-defective gammaherpesvirus efficiently established in vivo infection in macrophages that was molecularly indistinguishable from wild-type virus latency. These data point to a critical role for lytic replication or reactivation in the establishment or maintenance of latent infection in B cells.

scholarly journals Novel Single-Cell-Level Phenotypic Assay for Residual Drug Susceptibility and Reduced Replication Capacity of Drug-Resistant Human Immunodeficiency Virus Type 1

2004 ◽  
Vol 78 (4) ◽  
pp. 1718-1729 ◽  
Author(s):  
Haili Zhang ◽  
Yan Zhou ◽  
Cecily Alcock ◽  
Tara Kiefer ◽  
Daphne Monie ◽  
...  
Keyword(s):  
Drug Combination ◽  
Wild Type Virus ◽  
Replication Capacity ◽  
Drug Resistant ◽  
Type Virus ◽  
Wild Type ◽  
Cell Level ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-infected individuals who develop drug-resistant virus during antiretroviral therapy may derive benefit from continued treatment for two reasons. First, drug-resistant viruses can retain partial susceptibility to the drug combination. Second, therapy selects for drug-resistant viruses that may have reduced replication capacities relative to archived, drug-sensitive viruses. We developed a novel single-cell-level phenotypic assay that allows these two effects to be distinguished and compared quantitatively. Patient-derived gag-pol sequences were cloned into an HIV-1 reporter virus that expresses an endoplasmic reticulum-retained Env-green fluorescent protein fusion. Flow cytometric analysis of single-round infections allowed a quantitative analysis of viral replication over a 4-log dynamic range. The assay faithfully reproduced known in vivo drug interactions occurring at the level of target cells. Simultaneous analysis of single-round infections by wild-type and resistant viruses in the presence and absence of the relevant drug combination divided the benefit of continued nonsuppressive treatment into two additive components, residual virus susceptibility to the drug combination and selection for drug-resistant variants with diminished replication capacities. In some patients with drug resistance, the dominant circulating viruses retained significant susceptibility to the combination. However, in other cases, the dominant drug-resistant viruses showed no residual susceptibility to the combination but had a reduced replication capacity relative to the wild-type virus. In this case, simplification of the regimen might still allow adequate suppression of the wild-type virus. In a third pattern, the resistant viruses had no residual susceptibility to the relevant drug regimen but nevertheless had a replication capacity equivalent to that of wild-type virus. In such cases, there is no benefit to continued treatment. Thus, the ability to simultaneously analyze residual susceptibility and reduced replication capacity of drug-resistant viruses may provide a basis for rational therapeutic decisions in the setting of treatment failure.

scholarly journals The Thiocarboxanilide Nonnucleoside Inhibitor UC781 Restores Antiviral Activity of 3′-Azido-3′-Deoxythymidine (AZT) against AZT-Resistant Human Immunodeficiency Virus Type 1

1999 ◽  
Vol 43 (2) ◽  
pp. 259-263 ◽  
Author(s):  
Gadi Borkow ◽  
Dominique Arion ◽  
Mark A. Wainberg ◽  
Michael A. Parniak
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Wild Type Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT N-[4-Chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-furancarbothioamide (UC781) is an exceptionally potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. We found that a 1:1 molar combination of UC781 and 3′-azido-3′-deoxythymidine (AZT) showed high-level synergy in inhibiting the replication of AZT-resistant virus, implying that UC781 can restore antiviral activity to AZT against AZT-resistant HIV-1. Neither the nevirapine plus AZT nor the 2′,5′-bis-O-(t-butyldimethylsilyl)-3′-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide plus AZT combinations had this effect. Studies with purified HIV-1 reverse transcriptase (from a wild type and an AZT-resistant mutant) showed that UC781 was a potent inhibitor of the pyrophosphorolytic cleavage of nucleotides from the 3′ end of the DNA polymerization primer, a process that we have proposed to be critical for the phenotypic expression of AZT resistance. Combinations of UC781 plus AZT did not act in synergy to inhibit the replication of either wild-type virus or UC781-resistant HIV-1. Importantly, the time to the development of viral resistance to combinations of UC781 plus AZT is significantly delayed compared to the time to the development of resistance to either drug alone.

scholarly journals Multiple human immunodeficiency virus type 1 Nef functions contribute to efficient replication in primary human macrophages

2004 ◽  
Vol 85 (6) ◽  
pp. 1463-1469 ◽  
Author(s):  
Amanda Brown ◽  
Shaghayegh Moghaddam ◽  
Thomas Kawano ◽  
Cecilia Cheng-Mayer
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Single Point ◽  
Wild Type Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Efficient Replication ◽  
Hiv 1

The human immunodeficiency virus type 1 (HIV-1) Nef protein has been shown to accelerate viral growth kinetics in primary human T-lymphocytes and macrophages; however, the specific function(s) of Nef responsible for this phenotype in macrophages is unknown. To address this issue, mutants of a molecularly cloned macrophage-tropic isolate, HIV-1SF162, were generated expressing single point mutations that abrogate the ability of Nef to interact with cellular kinases or mediate CD4 down-regulation. Infection of primary monocyte-derived macrophages (MDM) with these mutant viruses revealed that residues in the PXXP motif contribute to efficient replication. Interestingly, viruses expressing alleles of Nef defective in CD4 down-modulation activity retain wild-type levels of infectivity in single-round assays but exhibited delayed replication kinetics and grew to lower titres compared to the wild-type virus in MDM. These data suggest that efficient HIV-1 replication is dependent on the ability of Nef to interact with cellular kinases and remove CD4 from the surface of infected macrophages.

scholarly journals Protection of Macaca nemestrina from Disease following Pathogenic Simian Immunodeficiency Virus (SIV) Challenge: Utilization of SIV Nucleocapsid Mutant DNA Vaccines with and without an SIV Protein Boost

2000 ◽  
Vol 74 (24) ◽  
pp. 11935-11949 ◽  
Author(s):  
Robert J. Gorelick ◽  
Raoul E. Benveniste ◽  
Jeffrey D. Lifson ◽  
Jason L. Yovandich ◽  
William R. Morton ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Peak Plasma ◽  
Full Range ◽  
Macaca Nemestrina ◽  
Wild Type Virus ◽  
Term Study ◽  
Live Attenuated Vaccine ◽  
Long Term Study ◽  
Immunodeficiency Virus

ABSTRACT Molecular clones were constructed that express nucleocapsid (NC) deletion mutant simian immunodeficiency viruses (SIVs) that are replication defective but capable of completing virtually all of the steps of a single viral infection cycle. These steps include production of particles that are viral RNA deficient yet contain a full complement of processed viral proteins. The mutant particles are ultrastructurally indistinguishable from wild-type virus. Similar to a live attenuated vaccine, this approach should allow immunological presentation of a full range of viral epitopes, without the safety risks of replicating virus. A total of 11 Macaca nemestrina macaques were inoculated with NC mutant SIV expressing DNA, intramuscularly (i.m.) in one study and i.m. and subcutaneously in another study. Six control animals received vector DNA lacking SIV sequences. Only modest and inconsistent humoral responses and no cellular immune responses were observed prior to challenge. Following intravenous challenge with 20 animal infectious doses of the pathogenic SIV(Mne) in a long-term study, all control animals became infected and three of four animals developed progressive SIV disease leading to death. All 11 NC mutant SIV DNA-immunized animals became infected following challenge but typically showed decreased initial peak plasma SIV RNA levels compared to those of control animals (P = 0.0007). In the long-term study, most of the immunized animals had low or undetectable postacute levels of plasma SIV RNA, and no CD4+ T-cell depletion or clinical evidence of progressive disease, over more than 2 years of observation. Although a subset of immunized and control animals were boosted with SIV(Mne) proteins, no apparent protective benefit was observed. Immunization of macaques with DNA that codes for replication-defective but structurally complete virions appears to protect from or at least delay the onset of AIDS after infection with a pathogenic immunodeficiency virus. With further optimization, this may be a promising approach for vaccine development.

scholarly journals Human Cytomegalovirus Resistance to Deoxyribosylindole Nucleosides Maps to a Transversion Mutation in the Terminase Subunit-Encoding GeneUL89

2014 ◽  
Vol 59 (1) ◽  
pp. 226-232 ◽  
Author(s):  
Brian G. Gentry ◽  
Quang Phan ◽  
Ellie D. Hall ◽  
Julie M. Breitenbach ◽  
Katherine Z. Borysko ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Structural Similarity ◽  
Open Reading Frames ◽  
Wild Type Virus ◽  
Wild Type ◽  
Term Administration ◽  
Transversion Mutation ◽  
Exon 1

ABSTRACTHuman cytomegalovirus (HCMV) infection can cause severe illnesses, including encephalopathy and mental retardation, in immunocompromised and immunologically immature patients. Current pharmacotherapies for treating systemic HCMV infections include ganciclovir, cidofovir, and foscarnet. However, long-term administration of these agents can result in serious adverse effects (myelosuppression and/or nephrotoxicity) and the development of viral strains with reduced susceptibility to drugs. The deoxyribosylindole (indole) nucleosides demonstrate a 20-fold greater activityin vitro(the drug concentration at which 50% of the number of plaques was reduced with the presence of drug compared to the number in the absence of drug [EC50] = 0.34 μM) than ganciclovir (EC50= 7.4 μM) without any observed increase in cytotoxicity. Based on structural similarity to the benzimidazole nucleosides, we hypothesize that the indole nucleosides target the HCMV terminase, an enzyme responsible for packaging viral DNA into capsids and cleaving the DNA into genome-length units. To test this hypothesis, an indole nucleoside-resistant HCMV strain was isolated, the open reading frames of the genes that encode the viral terminase were sequenced, and a G766C mutation in exon 1 ofUL89was identified; this mutation resulted in an E256Q change in the amino acid sequence of the corresponding protein. An HCMV wild-type strain, engineered with this mutation to confirm resistance, demonstrated an 18-fold decrease in susceptibility to the indole nucleosides (EC50= 3.1 ± 0.7 μM) compared to that of wild-type virus (EC50= 0.17 ± 0.04 μM). Interestingly, this mutation did not confer resistance to the benzimidazole nucleosides (EC50for wild-type HCMV = 0.25 ± 0.04 μM, EC50for HCMV pUL89 E256Q = 0.23 ± 0.04 μM). We conclude, therefore, that the G766C mutation that results in the E256Q substitution is unique for indole nucleoside resistance and distinct from previously discovered substitutions that confer both indole and benzimidazole nucleoside resistance (D344E and A355T).

scholarly journals Molecular Mechanisms of Tenofovir Resistance Conferred by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Containing a Diserine Insertion after Residue 69 and Multiple Thymidine Analog-Associated Mutations

2004 ◽  
Vol 48 (3) ◽  
pp. 992-1003 ◽  
Author(s):  
Kirsten L. White ◽  
James M. Chen ◽  
Nicolas A. Margot ◽  
Terri Wrin ◽  
Christos J. Petropoulos ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Insertion Mutation ◽  
Wild Type ◽  
Resistance Mutations ◽  
Thymidine Analog ◽  
Immunodeficiency Virus ◽  
High Level ◽  
Hiv 1

ABSTRACT Two amino acids inserted between residues 69 and 70 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are rare mutations that may develop in viruses containing multiple thymidine analog (zidovudine [AZT], stavudine)-associated mutations and that confer high-level resistance to all currently approved chain-terminating nucleoside and nucleotide RT inhibitors (NRTIs). The two known mechanisms of resistance to NRTIs are decreased incorporation and increased excision. The mechanism used by RT insertion mutants has not been described for tenofovir (TFV), a recently approved agent in this class. A patient-derived HIV-1 strain (strain FS-SSS) that contained an insertion mutation in a background of additional resistance mutations M41L, L74V, L210W, and T215Y was obtained. A second virus (strain FS) was derived from FS-SSS. In strain FS the insertion and T69S were reverted but the other resistance mutations were retained. The FS virus showed strong resistance to AZT but low-level changes in susceptibilities to other NRTIs and TFV. The FS-SSS virus showed reduced susceptibilities to all NRTIs including TFV. Steady-state kinetics demonstrated that the relative binding or incorporation of TFV was slightly decreased for FS-SSS RT compared to those for wild-type RT. However, significant ATP-mediated excision of TFV was detected for both mutant RT enzymes and followed the order FS-SSS RT > FS RT > wild-type RT. The presence of physiological concentrations of the +1 nucleotide inhibited TFV excision by the wild-type RT and slightly inhibited excision by the FS RT, whereas the level of excision by the FS-SSS RT remained high. Computer modeling suggests that the increased mobility of the β3-β4 loop may contribute to the high-level and broad NRTI resistance caused by the T69 insertion mutation.

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