scholarly journals In vitro induction of human immunodeficiency virus type 1 variants resistant to 2'-beta-Fluoro-2',3'-dideoxyadenosine.

1997 ◽  
Vol 41 (6) ◽  
pp. 1313-1318 ◽  
Author(s):  
M Tanaka ◽  
R V Srinivas ◽  
T Ueno ◽  
M F Kavlick ◽  
F K Hui ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Amino Acid Substitutions ◽  
Infectious Clones ◽  
Immunodeficiency Virus ◽  
Hiv 1

2'-beta-Fluoro-2',3'-dideoxyadenosine (F-ddA) is an acid-stable purine dideoxynucleoside analog active against a wide spectrum of human immunodeficiency virus type 1 (HIV-1) and HIV-2 strains in vitro. F-ddA is presently undergoing a phase I clinical trial at the National Cancer Institute. We induced HIV-1 variants resistant to F-ddA by exposing wild-type HIV-1 (HIV-1LAI) to increasing concentrations of F-ddA in vitro. After 18 passages, the virus was fourfold less sensitive to F-ddA than HIV-1LAI. Sequence analyses of the passage 18 virus revealed changes in three amino acids in the reverse transcriptase (RT)-encoding region of the pol gene: P to S at codon 119 (P119S; present in 3 of 13 and 28 of 28 molecular clones before and after F-ddA exposure, respectively), V179D (0 of 13 and 9 of 28, respectively), and L214F (9 of 13 and 28 of 28, respectively). Drug sensitivity assays using recombinant infectious clones confirmed that P119S was directly responsible for the reduced sensitivity of HIV-1 to F-ddA. Various infectious clones with single or multiple amino acid substitutions conferring viral resistance against nucleoside RT inhibitors, including HIV-1 variants with multi-dideoxynucleoside resistance, were generally sensitive to F-ddA. The moderate level of resistance of HIV-1 to F-ddA, together with the lack of conferment of significant cross-resistance by the F-ddA-associated amino acid substitutions, warrants further investigation of F-ddA as a potential antiviral agent for use in treatment of HIV-1 infection.

scholarly journals Impact of Human Immunodeficiency Virus Type 1 gp41 Amino Acid Substitutions Selected during Enfuvirtide Treatment on gp41 Binding and Antiviral Potency of Enfuvirtide In Vitro

2005 ◽  
Vol 79 (19) ◽  
pp. 12447-12454 ◽  
Author(s):  
M. Mink ◽  
S. M. Mosier ◽  
S. Janumpalli ◽  
D. Davison ◽  
L. Jin ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Binding Affinity ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Fusion Inhibitor ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Enfuvirtide (ENF), a novel human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, has potent antiviral activity against HIV-1 both in vitro and in vivo. Resistance to ENF observed after in vitro passaging was associated with changes in a three-amino-acid (aa) motif, GIV, at positions 36 to 38 of gp41. Patients with ongoing viral replication while receiving ENF during clinical trials acquired substitutions within gp41 aa 36 to 45 in the first heptad repeat (HR-1) of gp41 in both population-based plasma virus sequences and proviral DNA sequences from isolates showing reduced susceptibilities to ENF. To investigate their impact on ENF susceptibility, substitutions were introduced into a modified pNL4-3 strain by site-directed mutagenesis, and the susceptibilities of mutant viruses and patient-derived isolates to ENF were tested. In general, susceptibility decreases for single substitutions were lower than those for double substitutions, and the levels of ENF resistance seen for clinical isolates were higher than those observed for the site-directed mutant viruses. The mechanism of ENF resistance was explored for a subset of the substitutions by expressing them in the context of a maltose binding protein chimera containing a portion of the gp41 ectodomain and measuring their binding affinity to fluorescein-labeled ENF. Changes in binding affinity for the mutant gp41 fusion proteins correlated with the ENF susceptibilities of viruses containing the same substitutions. The combined results support the key role of gp41 aa 36 to 45 in the development of resistance to ENF and illustrate that additional envelope regions contribute to the ENF susceptibility of fusion inhibitor-naïve viruses and resistance to ENF.

scholarly journals Human Immunodeficiency Virus Type 1 Resistance to the Small Molecule Maturation Inhibitor 3-O-(3′,3′-Dimethylsuccinyl)-Betulinic Acid Is Conferred by a Variety of Single Amino Acid Substitutions at the CA-SP1 Cleavage Site in Gag

2006 ◽  
Vol 80 (24) ◽  
pp. 12095-12101 ◽  
Author(s):  
Jing Zhou ◽  
Chin Ho Chen ◽  
Christopher Aiken
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Betulinic Acid ◽  
Cleavage Site ◽  
Amino Acid Substitutions ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The compound 3-O-(3′,3′-dimethylsuccinyl)-betulinic acid (DSB) potently and specifically inhibits human immunodeficiency virus type 1 (HIV-1) replication by delaying the cleavage of the CA-SP1 junction in Gag, leading to impaired maturation of the viral core. In this study, we investigated HIV-1 resistance to DSB by analyzing HIV-1 mutants encoding a variety of individual amino acid substitutions in the CA-SP1 cleavage site. Three of the substitutions were lethal to HIV-1 replication owing to a deleterious effect on particle assembly. The remaining mutants exhibited a range of replication efficiencies; however, each mutant was capable of replicating in the presence of concentrations of DSB that effectively inhibited wild-type HIV-1. Mutations conferring resistance to DSB also led to impaired binding of the compound to immature HIV-1 virions and loss of DSB-mediated inhibition of cleavage of Gag. Surprisingly, two of the DSB-resistant mutants retained an intermediate ability to bind the compound, suggesting that binding of DSB to immature HIV-1 particles may not be sufficient for antiviral activity. Overall, our results indicate that Gag amino acids L363 and A364 are critical for inhibition of HIV-1 replication by DSB and suggest that these residues form key contacts with the drug in the context of the assembling HIV-1 particle. These results have implications for the design of and screening for novel inhibitors of HIV-1 maturation.

scholarly journals Resistance to the Anti-Human Immunodeficiency Virus Type 1 Compound l-Chicoric Acid Results from a Single Mutation at Amino Acid 140 of Integrase

1998 ◽  
Vol 72 (10) ◽  
pp. 8420-8424 ◽  
Author(s):  
Peter J. King ◽  
W. Edward Robinson
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Single Mutation ◽  
Chicoric Acid ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT l-Chicoric acid is an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase in vitro and of HIV-1 replication in tissue culture. Following 3 months of selection in the presence of increasing concentrations of l-chicoric acid, HIV-1 was completely resistant to the compound. Introduction of the mutant integrase containing a single glycine-to-serine amino acid change at position 140 into the native, l-chicoric acid-sensitive virus demonstrated that this change was sufficient to confer resistance to l-chicoric acid. These results confirm through natural selection previous biochemical studies showing thatl-chicoric acid inhibits integrase and that the drug is likely to interact at residues near the catalytic triad in the integrase active site.

scholarly journals Impact of V2 Mutations on Escape from a Potent Neutralizing Anti-V3 Monoclonal Antibody during In Vitro Selection of a Primary Human Immunodeficiency Virus Type 1 Isolate

2007 ◽  
Vol 81 (8) ◽  
pp. 3757-3768 ◽  
Author(s):  
Junji Shibata ◽  
Kazuhisa Yoshimura ◽  
Akiko Honda ◽  
Atsushi Koito ◽  
Toshio Murakami ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Immunodeficiency Virus ◽  
Amino Acid Mutations ◽  
Hiv 1

ABSTRACT KD-247, a humanized monoclonal antibody to an epitope of gp120-V3 tip, has potent cross-neutralizing activity against subtype B primary human immunodeficiency virus type 1 (HIV-1) isolates. To assess how KD-247 escape mutants can be generated, we induced escape variants by exposing bulked primary R5 virus, MOKW, to increasing concentrations of KD-247 in vitro. In the presence of relatively low concentrations of KD-247, viruses with two amino acid mutations (R166K/D167N) in V2 expanded, and under high KD-247 pressure, a V3 tip substitution (P313L) emerged in addition to the V2 mutations. However, a virus with a V2 175P mutation dominated during passaging in the absence of KD-247. Using domain swapping analysis, we demonstrated that the V2 mutations and the P313L mutation in V3 contribute to partial and complete resistance phenotypes against KD-247, respectively. To identify the V2 mutation responsible for the resistance to KD-247, we constructed pseudoviruses with single or double amino acid mutations in V2 and measured their sensitivity to neutralization. Interestingly, the neutralization phenotypes were switched, so that amino acid residue 175 (Pro or Leu) located in the center of V2 was exchanged, indicating that the amino acid at position 175 has a crucial role, dramatically changing the Env oligomeric state on the membrane surface and affecting the neutralization phenotype against not only anti-V3 antibody but also recombinant soluble CD4. These data suggested that HIV-1 can escape from anti-V3 antibody attack by changing the conformation of the functional envelope oligomer by acquiring mutations in the V2 region in environments with relatively low antibody concentrations.

scholarly journals Construction of a Human Immunodeficiency Virus Type 1 (HIV-1) Library Containing Random Combinations of Amino Acid Substitutions in the HIV-1 Protease due to Resistance by Protease Inhibitors

2002 ◽  
Vol 76 (6) ◽  
pp. 3031-3037 ◽  
Author(s):  
Keisuke Yusa ◽  
Wei Song ◽  
Matthias Bartelmann ◽  
Shinji Harada
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Amino Acid Substitutions ◽  
In Vitro System ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) heterogeneity contributes to the emergence of drug-resistant virus, escape from host defense systems, and/or conversion of the cellular tropism. To establish an in vitro system to address a heterogeneous virus population, we constructed a library of HIV-1 molecular clones containing a set of random combinations of zero to 11 amino acid substitutions associated with resistance to protease inhibitors by the HIV-1 protease. The complexity (2.1 × 105) of the HIV-1 library pNG-PRL was large enough to cover all of the possible combinations of zero to 11 amino acid substitutions (a total of 4,096 substitutions possible). The T-cell line MT-2 was infected with the HIV-1 library, and resistant viruses were selected after treatment by the protease inhibitor ritonavir (0.03 to 0.30 μM). The viruses that contained three to eight amino acid substitutions could be selected within 2 weeks. These results demonstrate that this HIV-1 library could serve as an alternative in vitro system to analyze the emergence of drug resistance and to evaluate the antiviral activity of novel compounds against multidrug-resistant viruses.

scholarly journals Replacement of the P1 Amino Acid of Human Immunodeficiency Virus Type 1 Gag Processing Sites Can Inhibit or Enhance the Rate of Cleavage by the Viral Protease

2002 ◽  
Vol 76 (20) ◽  
pp. 10226-10233 ◽  
Author(s):  
Steve C. Pettit ◽  
Gavin J. Henderson ◽  
Celia A. Schiffer ◽  
Ronald Swanstrom
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Site Directed Mutagenesis ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Processing of the human immunodeficiency virus type 1 (HIV-1) Gag precursor is highly regulated, with differential rates of cleavage at the five major processing sites to give characteristic processing intermediates. We examined the role of the P1 amino acid in determining the rate of cleavage at each of these five sites by using libraries of mutants generated by site-directed mutagenesis. Between 12 and 17 substitution mutants were tested at each P1 position in Gag, using recombinant HIV-1 protease (PR) in an in vitro processing reaction of radiolabeled Gag substrate. There were three sites in Gag (MA/CA, CA/p2, NC/p1) where one or more substitutions mediated enhanced rates of cleavage, with an enhancement greater than 60-fold in the case of NC/p1. For the other two sites (p2/NC, p1/p6), the wild-type amino acid conferred optimal cleavage. The order of the relative rates of cleavage with the P1 amino acids Tyr, Met, and Leu suggests that processing sites can be placed into two groups and that the two groups are defined by the size of the P1′ amino acid. These results point to a trans effect between the P1 and P1′ amino acids that is likely to be a major determinant of the rate of cleavage at the individual sites and therefore also a determinant of the ordered cleavage of the Gag precursor.

scholarly journals Identification of Biased Amino Acid Substitution Patterns in Human Immunodeficiency Virus Type 1 Isolates from Patients Treated with Protease Inhibitors

1999 ◽  
Vol 73 (7) ◽  
pp. 6197-6202 ◽  
Author(s):  
Robert W. Shafer ◽  
Phillip Hsu ◽  
Amy K. Patick ◽  
Charles Craig ◽  
Volker Brendel
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Amino Acid Substitution ◽  
Sampling Error ◽  
Amino Acid Substitutions ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) amino acid substitutions observed during antiretroviral drug therapy may be caused by drug selection, non-drug-related evolution, or sampling error introduced by the sequencing process. We analyzed HIV-1 sequences from 371 untreated patients and from 178 patients receiving a single protease inhibitor. Amino acid substitution patterns during treatment were compared with inferred substitution patterns arising evolutionarily without treatment. Our results suggest that most treatment-associated amino acid substitutions are caused by selective drug pressure, including substitutions not previously associated with drug resistance.

scholarly journals The Late-Domain-Containing Protein p6 Is the Predominant Phosphoprotein of Human Immunodeficiency Virus Type 1 Particles

2002 ◽  
Vol 76 (3) ◽  
pp. 1015-1024 ◽  
Author(s):  
Barbara Müller ◽  
Tilo Patschinsky ◽  
Hans-Georg Kräusslich
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Metabolic Labeling ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
A Minor ◽  
Hiv 1

ABSTRACT The Gag-derived protein p6 of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the release of virions from the membranes of infected cells. It is presumed that p6 and functionally related proteins from other viruses act as adapters, recruiting cellular factors to the budding site. This interaction is mediated by so-called late domains within the viral proteins. Previous studies had suggested that virus release from the plasma membrane shares elements with the cellular endocytosis machinery. Since protein phosphorylation is known to be a regulatory mechanism in these processes, we have investigated the phosphorylation of HIV-1 structural proteins. Here we show that p6 is the major phosphoprotein of HIV-1 particles. After metabolic labeling of infected cells with [ortho- 32P]phosphate, we found that phosphorylated p6 from infected cells and from virus particles consisted of several forms, suggesting differential phosphorylation at multiple sites. Apparently, phosphorylation occurred shortly before or after the release of p6 from Gag and involved only a minor fraction of the total virion-associated p6 molecules. Phosphoamino acid analysis indicated phosphorylation at Ser and Thr, as well as a trace of Tyr phosphorylation, supporting the conclusion that multiple phosphorylation events do occur. In vitro experiments using purified virus revealed that endogenous or exogenously added p6 was efficiently phosphorylated by virion-associated cellular kinase(s). Inhibition experiments suggested that a cyclin-dependent kinase or a related kinase, most likely ERK2, was involved in p6 phosphorylation by virion-associated enzymes.

scholarly journals Ontogeny and Specificities of Mucosal and Blood Human Immunodeficiency Virus Type 1-Specific CD8+ Cytotoxic T Lymphocytes

2003 ◽  
Vol 77 (1) ◽  
pp. 291-300 ◽  
Author(s):  
L. Musey ◽  
Y. Ding ◽  
J. Cao ◽  
J. Lee ◽  
C. Galloway ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cytotoxic T Lymphocyte ◽  
Infected Individual ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Induction of adaptive immunity to human immunodeficiency virus type 1 (HIV-1) at the mucosal site of transmission is poorly understood but crucial in devising strategies to control and prevent infection. To gain further understanding of HIV-1-specific T-cell mucosal immunity, we established HIV-1-specific CD8+ cytotoxic T-lymphocyte (CTL) cell lines and clones from the blood, cervix, rectum, and semen of 12 HIV-1-infected individuals and compared their specificities, cytolytic function, and T-cell receptor (TCR) clonotypes. Blood and mucosal CD8+ CTL had common HIV-1 epitope specificities and major histocompatibility complex restriction patterns. Moreover, both systemic and mucosal CTL lysed targets with similar efficiency, primarily through the perforin-dependent pathway in in vitro studies. Sequence analysis of the TCRβ VDJ region revealed in some cases identical HIV-1-specific CTL clones in different compartments in the same HIV-1-infected individual. These results clearly establish that a subset of blood and mucosal HIV-1-specific CTL can have a common origin and can traffic between anatomically distinct compartments. Thus, these effectors can provide immune surveillance at the mucosa, where rapid responses are needed to contain HIV-1 infection.

scholarly journals In Vitro Selection and Characterization of Human Immunodeficiency Virus Type 2 with Decreased Susceptibility to Lopinavir

2007 ◽  
Vol 51 (9) ◽  
pp. 3075-3080 ◽  
Author(s):  
Sherie Masse ◽  
Xiaozhi Lu ◽  
Tatyana Dekhtyar ◽  
Liangjun Lu ◽  
Gennadiy Koev ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
In Vitro Selection ◽  
Substantial Reduction ◽  
Protease Gene ◽  
Infectious Clones ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Lopinavir (LPV)-ritonavir has demonstrated durable antiviral activity in human immunodeficiency virus type 1 (HIV-1)-infected antiretroviral-naïve and protease inhibitor (PI)-experienced patients. However, information on LPV activity against HIV-2 and the patterns of mutations in HIV-2 in response to selection by LPV is limited. The activity of LPV against three strains of HIV-2 was assessed and compared to activity against a reference HIV-1 strain. LPV demonstrated activity similar to that observed against HIV-1 in two HIV-2 strains (HIV-2MS and HIV-2CBL-23) tested. On the other hand, approximately 10-fold-reduced susceptibility was observed with the third HIV-2 strain, HIV-2CDC310319. Passage of HIV-2MS with increasing concentrations of LPV selected mutations V47A and D17N in the HIV-2 protease gene. The introduction of both 17N and 47A either individually or together into HIV-2ROD molecular infectious clones showed that the single V47A substitution in HIV-2 resulted in a substantial reduction in susceptibility to LPV. In contrast, this mutant retained wild-type susceptibility to other PIs and appeared to be hypersusceptible to atazanavir and saquinavir.

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