An efficient phage plaque screen for the random mutational analysis of the interaction of HIV-1 gp120 with human CD4.

ABSTRACT Mutational analysis of the four conserved proline residues in human immunodeficiency virus type 1 (HIV-1) Vpr reveals that only Pro-35 is required for efficient replication of R5-tropic, but not of X4-tropic, viruses in human lymphoid tissue (HLT) cultivated ex vivo. While Vpr-mediated apoptosis and G2 cell cycle arrest, as well as the expression and subcellular localization of Vpr, were independent, the capacity for encapsidation of Vpr into budding virions was dependent on Pro-35. 1H nuclear magnetic resonance data suggest that mutation of Pro-35 causes a conformational change in the hydrophobic core of the molecule, whose integrity is required for the encapsidation of Vpr, and thus, Pro-35 supports the replication of R5-tropic HIV-1 in HLT.

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Mutations in the Spacer Peptide and Adjoining Sequences in Rous Sarcoma Virus Gag Lead to Tubular Budding

Journal of Virology ◽

10.1128/jvi.00213-08 ◽

2008 ◽

Vol 82 (14) ◽

pp. 6788-6797 ◽

Cited By ~ 30

Author(s):

Paul W. Keller ◽

Marc C. Johnson ◽

Volker M. Vogt

Keyword(s):

Rous Sarcoma Virus ◽

Mutational Analysis ◽

Leukemia Virus ◽

Alpha Helix ◽

Insertion Mutagenesis ◽

Structural Protein ◽

Enveloped Virus ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Hiv 1

ABSTRACT All orthoretroviruses encode a single structural protein, Gag, which is necessary and sufficient for the assembly and budding of enveloped virus-like particles from the cell. The Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus type 1 (HIV-1) contain a short spacer peptide (SP or SP1, respectively) separating the capsid (CA) and nucleocapsid (NC) domains. SP or SP1 and the residues immediately upstream are known to be critical for proper assembly. Using mutagenesis and electron microscopy analysis of insect cells or chicken cells overexpressing RSV Gag, we defined the SP assembly domain to include the last 8 residues of CA, all 12 residues of SP, and the first 4 residues of NC. Five- or two-amino acid glycine-rich insertions or substitutions in this critical region uniformly resulted in the budding of abnormal, long tubular particles. The equivalent SP1-containing HIV-1 Gag sequence was unable to functionally replace the RSV sequence in supporting normal RSV spherical assembly. According to secondary structure predictions, RSV and HIV-1 SP/SP1 and adjoining residues may form an alpha helix, and what is likely the functionally equivalent sequence in murine leukemia virus Gag has been inferred by mutational analysis to form an amphipathic alpha helix. However, our alanine insertion mutagenesis did not provide evidence for an amphipathic helix in RSV Gag. Taken together, these results define a short assembly domain between the folded portions of CA and NC, which is essential for formation of the immature Gag shell.

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Mutational Analysis of HIV-1 gp160-Mediated Receptor Interference: Intracellular Complex Formation

Virology ◽

10.1006/viro.1996.0333 ◽

1996 ◽

Vol 220 (2) ◽

pp. 461-472 ◽

Cited By ~ 6

Author(s):

ROBERT A. MARTIN ◽

DEBI P. NAYAK

Keyword(s):

Complex Formation ◽

Mutational Analysis ◽

Hiv 1 ◽

Receptor Interference

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Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase

FEBS Letters ◽

10.1016/0014-5793(91)80484-k ◽

1991 ◽

Vol 282 (2) ◽

pp. 231-234 ◽

Cited By ~ 46

Author(s):

Denise M. Lowe ◽

Vanita Parmar ◽

Sharon D. Kemp ◽

Brendan A. Larder

Keyword(s):

Reverse Transcriptase ◽

Mutational Analysis ◽

Sequence Motifs ◽

Conserved Sequence ◽

Conserved Sequence Motifs ◽

Hiv 1

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The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage

Journal of Virology ◽

10.1128/jvi.77.1.366-374.2003 ◽

2003 ◽

Vol 77 (1) ◽

pp. 366-374 ◽

Cited By ~ 46

Author(s):

Steven C. Pettit ◽

Sergei Gulnik ◽

Lori Everitt ◽

Andrew H. Kaplan

Keyword(s):

Mutational Analysis ◽

Expression System ◽

Enzyme Activation ◽

Nucleocapsid Proteins ◽

Dimer Interface ◽

Late Event ◽

In The Wild ◽

Retroviral Proteases ◽

Hiv 1

ABSTRACT Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

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HIV-1 Gag release from yeast reveals ESCRT interaction with the Gag N-terminal protein region

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014710 ◽

2020 ◽

Vol 295 (52) ◽

pp. 17950-17972

Author(s):

Birgit Meusser ◽

Bettina Purfuerst ◽

Friedrich C. Luft

Keyword(s):

Plasma Membrane ◽

Mutational Analysis ◽

Genetic Manipulation ◽

Membrane Binding ◽

Hek293 Cells ◽

Binding Studies ◽

Site Mutation ◽

Terminal Protein ◽

Hiv 1 ◽

Plasma Membrane Binding

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT-independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag-plasma membrane–binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared with p6 in GST-pulldown experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly.

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