scholarly journals Determination of Essential Amino Acids Involved in the CD4-Independent Tropism of the X4 Human Immunodeficiency Virus Type 1 m7NDK Isolate: Role of Potential N Glycosylations in the C2 and V3 Regions of gp120

2001 ◽  
Vol 75 (11) ◽  
pp. 5425-5428 ◽  
Author(s):  
Julie Dumonceaux ◽  
Caroline Goujon ◽  
Veronique Joliot ◽  
Pascale Briand ◽  
Uriel Hazan
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Essential Amino Acids ◽  
Site Directed Mutagenesis ◽  
Immunodeficiency Virus

ABSTRACT Seven mutations in the C2, V3, and C3 regions of gp120 are implicated in the tropism of the first CD4-independent human immunodeficiency virus type 1 isolate, m7NDK. Site-directed mutagenesis revealed that three amino acids are essential to maintain this tropism, one in the C2 region and two in the V3 loop. Two mutations implied N glycosylation modifications.

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 Cellular Membrane-Binding Ability of the C-Terminal Cytoplasmic Domain of Human Immunodeficiency Virus Type 1 Envelope Transmembrane Protein gp41

2001 ◽  
Vol 75 (20) ◽  
pp. 9925-9938 ◽  
Author(s):  
Steve S.-L. Chen ◽  
Sheau-Fen Lee ◽  
Chin-Tien Wang
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Membrane Binding ◽  
Cytoplasmic Tail ◽  
Cellular Membrane ◽  
C Terminus ◽  
Immunodeficiency Virus

ABSTRACT The amphipathic α-helices located in the cytoplasmic tail of the envelope (Env) transmembrane glycoprotein gp41 of human immunodeficiency virus type 1 have been implicated in membrane association and cytopathicity. Deletion of the last 12 amino acids in the C terminus of this domain severely impairs infectivity. However, the nature of the involvement of the cytoplasmic tail in Env-membrane interactions in cells and the molecular basis for the defect in infectivity of this mutant virus are still poorly understood. In this study we examined the interaction of the cytoplasmic tail with membranes in living mammalian cells by expressing a recombinant cytoplasmic tail fragment and an Escherichia coli β-galactosidase/cytoplasmic tail fusion protein, both of them lacking gp120, the gp41 ectodomain, and the transmembrane region. We found through cell fractionation, in vivo membrane flotation, and confocal immunofluorescence studies that the cytoplasmic tail contained determinants to be routed to a perinuclear membrane region in cells. Further mapping showed that each of the three lentivirus lytic peptide (LLP-1, LLP-2, and LLP-3) sequences conferred this cellular membrane-targeting ability. Deletion of the last 12 amino acids from the C terminus abolished the ability of the LLP-1 motif to bind to membranes. High salt extraction, in vitro transcription and translation, and posttranslational membrane binding analyses indicated that the β-galactosidase/LLP fusion proteins were inserted into membranes via the LLP sequences. Subcellular fractionation and confocal microscopy studies revealed that each of the LLP motifs, acting in a position-independent manner, targeted non-endoplasmic reticulum (ER)-associated β-galactosidase and enhanced green fluorescence protein to the ER. Our study provides a basis for the involvement of the gp41 cytoplasmic tail during Env maturation and also supports the notion that the membrane apposition of the C-terminal cytoplasmic tail plays a crucial role in virus-host interaction.

scholarly journals Determination of Coreceptor Usage of Human Immunodeficiency Virus Type 1 from Patient Plasma Samples by Using a Recombinant Phenotypic Assay

2001 ◽  
Vol 75 (1) ◽  
pp. 251-259 ◽  
Author(s):  
Virginie Trouplin ◽  
Francesca Salvatori ◽  
Fanny Cappello ◽  
Veronique Obry ◽  
Anne Brelot ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Coreceptor Usage ◽  
Plasma Samples ◽  
Immunodeficiency Virus ◽  
Semiquantitative Evaluation ◽  
Convenient Technique

ABSTRACT We developed a recombinant virus technique to determine the coreceptor usage of human immunodeficiency virus type 1 (HIV-1) from plasma samples, the source expected to represent the most actively replicating virus population in infected subjects. This method is not subject to selective bias associated with virus isolation in culture, a step required for conventional tropism determination procedures. The addition of a simple subcloning step allowed semiquantitative evaluation of virus populations with a different coreceptor (CCR5 or CXCR4) usage specificity present in each plasma sample. This procedure detected mixtures of CCR5- and CXCR4-exclusive virus populations as well as dualtropic viral variants, in variable proportions. Sequence analysis of dualtropic clones indicated that changes in the V3 loop are necessary for the use of CXCR4 as a coreceptor, but the overall context of the V1-V3 region is important to preserve the capacity to use CCR5. This convenient technique can greatly assist the study of virus evolution and compartmentalization in infected individuals.

scholarly journals Evolution of Envelope Sequences from the Genital Tract and Peripheral Blood of Women Infected with Clade A Human Immunodeficiency Virus Type 1

1998 ◽  
Vol 72 (10) ◽  
pp. 8240-8251 ◽  
Author(s):  
Mary Poss ◽  
Allen G. Rodrigo ◽  
John J. Gosink ◽  
Gerald H. Learn ◽  
Dana de Vange Panteleeff ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Peripheral Blood ◽  
Tissue Compartment ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Over Time

ABSTRACT The development of viral diversity during the course of human immunodeficiency virus type 1 (HIV-1) infection may significantly influence viral pathogenesis. The paradigm for HIV-1 evolution is based primarily on studies of male cohorts in which individuals were presumably infected with a single virus variant of subtype B HIV-1. In this study, we evaluated virus evolution based on sequence information of the V1, V2, and V3 portions of HIV-1 clade A envelope genes obtained from peripheral blood and cervical secretions of three women with genetically heterogeneous viral populations near seroconversion. At the first sample following seroconversion, the number of nonsynonymous substitutions per potential nonsynonymous site (dn) significantly exceeded substitutions at potential synonymous sites (ds) in plasma viral sequences from all individuals. Generally, values of dn remained higher than values of ds as sequences from blood or mucosa evolved. Mutations affected each of the three variable regions of the envelope gene differently; insertions and deletions dominated changes in V1, substitutions involving charged amino acids occurred in V2, and sequential replacement of amino acids over time at a small subset of positions distinguished V3. The relationship among envelope nucleotide sequences obtained from peripheral blood mononuclear cells, plasma, and cervical secretions was evaluated for each individual by both phylogenetic and phenetic analyses. In all subjects, sequences from within each tissue compartment were more closely related to each other than to sequences from other tissues (phylogenetic tissue compartmentalization). At time points after seroconversion in two individuals, there was also greater genetic identity among sequences from the same tissue compartment than among sequences from different tissue compartments (phenetic tissue compartmentalization). Over time, temporal phylogenetic and phenetic structure was detectable in mucosal and plasma viral samples from all three women, suggesting a continual process of migration of one or a few infected cells into each compartment followed by localized expansion and evolution of that population.

scholarly journals A Mutation in Human Immunodeficiency Virus Type 1 Protease, N88S, That Causes In Vitro Hypersensitivity to Amprenavir

2000 ◽  
Vol 74 (9) ◽  
pp. 4414-4419 ◽  
Author(s):  
Rainer Ziermann ◽  
Kay Limoli ◽  
Kalyan Das ◽  
Edward Arnold ◽  
Christos J. Petropoulos ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Salvage Therapy ◽  
The United States ◽  
Site Directed Mutagenesis ◽  
Cross Resistance ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Amprenavir (Agenerase, 141-W94, VX-478) is a human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PRI) recently approved for the treatment of HIV-1 infection in the United States. A major cause of treatment failure is the development of resistance to PRIs. One potential use for amprenavir is as salvage therapy for patients for whom treatment that includes one (or more) of the other four currently approved PRIs—saquinavir, indinavir, ritonavir, and nelfinavir—has failed. We evaluated the cross-resistance to amprenavir of viruses that evolved during treatment with the two most commonly prescribed PRIs, nelfinavir and indinavir. Unexpectedly, a dramatic increase in susceptibility (2.5- to 12.5-fold) was observed with 20 of 312 (6.4%) patient viruses analyzed. The most pronounced increases in susceptibility were strongly associated with an N88S mutation in protease. All viruses that carried the N88S mutation were hypersensitive to amprenavir. Site-directed mutagenesis studies confirmed the causal role of N88S in determining amprenavir hypersensitivity. The presence of the N88S mutation and associated amprenavir hypersensitivity may be useful in predicting an improved clinical response to amprenavir salvage therapy.

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