scholarly journals Extended nucleocapsid protein is cleaved from the Gag–Pol precursor of human immunodeficiency virus type 1

2001 ◽  
Vol 82 (3) ◽  
pp. 581-590 ◽  
Author(s):  
Nissim Chen ◽  
Abraham Morag ◽  
Nava Almog ◽  
Immanuel Blumenzweig ◽  
Orna Dreazin ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cleavage Site ◽  
Amino Acid Residues ◽  
Site Mutation ◽  
Reading Frame ◽  
C Terminus ◽  
Immunodeficiency Virus

Human immunodeficiency virus type 1 Gag and Gag–Pol precursors are translated from an mRNA which is indistinguishable from the full-length genomic RNA. The ratio of Gag to Gag–Pol polyproteins is approximately 20:1 and is controlled by a frameshift of the reading frame, which takes place downstream of the p7 nucleocapsid (NC) in the N terminus of the p1 peptide. The viral precursors Gag and Gag–Pol are cleaved by the virus-encoded protease (PR) into the structural proteins, and into p6Pol, PR, reverse transcriptase and integrase. Due to the frameshift event, the cleavage site at the C terminus of NC coded in the Gag frame (ERQAN-FLGKI) changes either to ERQANFLRED or ERQANFFRED. The results presented in this report demonstrate that the NC released from the Gag–Pol precursor is 8 amino acid residues longer than the NC cleaved from the Gag polyprotein. Our results also show that truncated Gag–Pol precursors bearing cleavage site mutation at the NC/p6Pol, and/or p6Pol/PR junctions, undergo autoprocessing in bacterial and eukaryotic cells, indicating that PR is active when part of the precursor.

scholarly journals Novel Gag-Pol Frameshift Site in Human Immunodeficiency Virus Type 1 Variants Resistant to Protease Inhibitors

1998 ◽  
Vol 72 (7) ◽  
pp. 6146-6150 ◽  
Author(s):  
Louise Doyon ◽  
Catherine Payant ◽  
Léa Brakier-Gingras ◽  
Daniel Lamarre
Keyword(s):  
Human Immunodeficiency Virus ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cleavage Site ◽  
Messenger Rna ◽  
Site Mutation ◽  
Ribosomal Frameshifting ◽  
Immunodeficiency Virus

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease inhibitors have been shown to contain a mutation in the p1/p6 Gag precursor cleavage site. At the messenger RNA level, this mutation generates a U UUU UUU sequence that is reminiscent of the U UUU UUA sequence required for ribosomal frameshifting and Gag-Pol synthesis. To test whether the p1/p6 cleavage site mutation was generating a novel frameshift site, HIV sequences were inserted in translation vectors containing a chloramphenicol acetyltransferase (CAT) reporter gene requiring −1 frameshifting for expression. All sequences containing the original HIV frameshift site supported the synthesis of CAT but expression was increased 3- to 11-fold in the presence of the mutant p1/p6 sequence. When the original frameshift site was abolished by mutation, expression remained unchanged when using constructs containing the mutant p1/p6 sequence, whereas it was decreased 2- to 4.5-fold when using wild-type p1/p6 constructs. Similarly, when introduced into HIV molecular clones, the p1/p6 mutant sequence supported Gag-Pol synthesis and protease activity in the absence of the original frameshift site, indicating that this sequence could also promote ribosomal frameshifting in virus-expressing cells.

scholarly journals Repair of a Rev-Minus Human Immunodeficiency Virus Type 1 Mutant by Activation of a Cryptic Splice Site

2001 ◽  
Vol 75 (7) ◽  
pp. 3495-3500 ◽  
Author(s):  
Koen Verhoef ◽  
Patricia S. Bilodeau ◽  
Jeroen L. B. van Wamel ◽  
Jørgen Kjems ◽  
C. Martin Stoltzfus ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Splice Site ◽  
Site Mutation ◽  
Reading Frame ◽  
Splicing Silencer ◽  
Immunodeficiency Virus ◽  
Revertant Virus

ABSTRACT We isolated a revertant virus after prolonged culturing of a replication-impaired human immunodeficiency virus type 1 (HIV-1) mutant of which the Rev open reading frame was inactivated by mutation of the AUG translation initiation codon. Sequencing of the tat-rev region of this revertant virus identified a second-site mutation in tat that restored virus replication in the mutant background. This mutation activated a cryptic 5′ splice site (ss) that, when used in conjunction with the regular HIV 3′ ss #5, fuses the tat and revreading frames to encode a novel T-Rev fusion protein that rescues Rev function. We also demonstrate an alternative route to indirectly activate this cryptic 5′ ss by mutational inactivation of an adjacent exon splicing silencer element.

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 In Vitro Resistance to the Human Immunodeficiency Virus Type 1 Maturation Inhibitor PA-457 (Bevirimat)

2006 ◽  
Vol 80 (22) ◽  
pp. 10957-10971 ◽  
Author(s):  
Catherine S. Adamson ◽  
Sherimay D. Ablan ◽  
Ioana Boeras ◽  
Ritu Goila-Gaur ◽  
Ferri Soheilian ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Single Amino Acid ◽  
Compensatory Mutation ◽  
C Terminus ◽  
Immunodeficiency Virus ◽  
Maturation Inhibitor ◽  
Hiv 1

ABSTRACT 3-O-(3′,3′-dimethylsuccinyl)betulinic acid (PA-457 or bevirimat) potently inhibits human immunodeficiency virus type 1 (HIV-1) maturation by blocking a late step in the Gag processing pathway, specifically the cleavage of SP1 from the C terminus of capsid (CA). To gain insights into the mechanism(s) by which HIV-1 could evolve resistance to PA-457 and to evaluate the likelihood of such resistance arising in PA-457-treated patients, we sought to identify and characterize a broad spectrum of HIV-1 variants capable of conferring resistance to this compound. Numerous independent rounds of selection repeatedly identified six single-amino-acid substitutions that independently confer PA-457 resistance: three at or near the C terminus of CA (CA-H226Y, -L231F, and -L231M) and three at the first and third residues of SP1 (SP1-A1V, -A3T, and -A3V). We determined that mutations CA-H226Y, CA-L231F, CA-L231M, and SP1-A1V do not impose a significant replication defect on HIV-1 in culture. In contrast, mutations SP1-A3V and -A3T severely impaired virus replication and inhibited virion core condensation. The replication defect imposed by SP1-A3V was reversed by a second-site compensatory mutation in CA (CA-G225S). Intriguingly, high concentrations of PA-457 enhanced the maturation of SP1 residue 3 mutants. The different phenotypes associated with mutations that confer PA-457 resistance suggest the existence of multiple mechanisms by which HIV-1 can evolve resistance to this maturation inhibitor. These findings have implications for the ongoing development of PA-457 to treat HIV-1 infection in vivo.

scholarly journals Characterization of Stable, Soluble Trimers Containing Complete Ectodomains of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins

2000 ◽  
Vol 74 (12) ◽  
pp. 5716-5725 ◽  
Author(s):  
Xinzhen Yang ◽  
Michael Farzan ◽  
Richard Wyatt ◽  
Joseph Sodroski
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cleavage Site ◽  
Neutralizing Antibodies ◽  
Coiled Coil ◽  
Envelope Glycoproteins ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins function as a membrane-anchored trimer of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. Previously, we reported three approaches to stabilize soluble trimers containing parts of the gp41 ectodomains: addition of GCN4 trimeric helices, disruption of the cleavage site between gp120 and gp41, and introduction of cysteines in the gp41 coiled coil to form intersubunit disulfide bonds. Here, we applied similar approaches to stabilize soluble gp140 trimers including the complete gp120 and gp41 ectodomains. A combination of fusion with the GCN4 trimeric sequences and disruption of the gp120-gp41 cleavage site resulted in relatively homogeneous gp140 trimers with exceptional stability. The gp120 epitopes recognized by neutralizing antibodies are intact and exposed on these gp140 trimers. By contrast, the nonneutralizing antibody epitopes on the gp120 subunits of the soluble trimers are relatively occluded compared with those on monomeric gp120 preparations. This antigenic similarity to the functional HIV-1 envelope glycoproteins and the presence of the complete gp41 ectodomain should make the soluble gp140 trimers useful tools for structural and immunologic studies.

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 The Conserved Carboxy Terminus of the Capsid Domain of Human Immunodeficiency Virus Type 1 Gag Protein Is Important for Virion Assembly and Release

2004 ◽  
Vol 78 (18) ◽  
pp. 9675-9688 ◽  
Author(s):  
Daniel Melamed ◽  
Michal Mark-Danieli ◽  
Michal Kenan-Eichler ◽  
Osnat Kraus ◽  
Asher Castiel ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Hinge Region ◽  
Virion Assembly ◽  
C Terminus ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Ca Protein

ABSTRACT The retroviral Gag precursor plays an important role in the assembly of virion particles. The capsid (CA) protein of the Gag molecule makes a major contribution to this process. In the crystal structure of the free CA protein of the human immunodeficiency virus type 1 (HIV-1), 11 residues of the C terminus were found to be unstructured, and to date no information exists on the structure of these residues in the context of the Gag precursor molecule. We performed phylogenetic analysis and demonstrated a high degree of conservation of these 11 amino acids. Deletion of this cluster or introduction of various point mutations into these residues resulted in significant impairment of particle infectivity. In this cluster, two putative structural regions were identified, residues that form a hinge region (353-VGGP-356) and those that contribute to an α-helix (357-GHKARVL-363). Overall, mutations in these regions resulted in inhibition of virion production, but mutations in the hinge region demonstrated the most significant reduction. Although all the Gag mutants appeared to have normal Gag-Gag and Gag-RNA interactions, the hinge mutants were characterized by abnormal formation of cytoplasmic Gag complexes. Gag proteins with mutations in the hinge region demonstrated normal membrane association but aberrant rod-like membrane structures. More detailed analysis of these structures in one of the mutants demonstrated abnormal trapped Gag assemblies. These data suggest that the conserved CA C terminus is important for HIV-1 virion assembly and release and define a putative target for drug design geared to inhibit the HIV-1 assembly process.

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