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Structure and Assembly

Hydrogen Bonding at a Conserved Threonine in Lentivirus Capsid Is Required for Virus Replication

Sarah M. Rue, Jason W. Roos, L. Mario Amzel, Janice E. Clements, Sheila A. Barber
Sarah M. Rue
1Department of Comparative Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287
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Jason W. Roos
1Department of Comparative Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287
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L. Mario Amzel
2Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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Janice E. Clements
1Department of Comparative Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287
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Sheila A. Barber
1Department of Comparative Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287
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  • For correspondence: sabarber@jhmi.edu
DOI: 10.1128/JVI.77.14.8009-8018.2003
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  • FIG. 1.
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    FIG. 1.

    Growth kinetics and infectivity of mutants with substitutions of SIVmac239 CA threonines in consensus CK2 sites. (A) Cell-free supernatants of CEM×174 cells transfected with infectious viral DNA were analyzed at various times posttransfection with a standard RT assay. (B) Virus derived from transfection of 293T cells was normalized according to RT activity and analyzed with the LuSIV assay. Induction values are relative to background at day 2 postinfection.

  • FIG. 2.
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    FIG. 2.

    Sequence alignment of lentivirus CA proteins. Amino acid sequences for the aligned viruses were obtained from the National Center for Biotechnology Information Entrez protein database. Gag proteins were aligned by ClustalW (www2.ebi.ac.uk/clustalw ) (52) by using the BLOSUM matrix and visualized with BOXSHADE (www.ch.embnet.org/software/BOX_form.html ). Black shading indicates sequence identity; gray shading indicates sequence similarity. The solid arrow indicates the position of T(47)CA of SIV and homologous residues; the dashed arrow indicates the position of the aspartate or glutamate that participates in the salt bridge. The SIV sequence is representative of SIVmac, SIVsmm, SIVstm, and SIVagmver strains (34). EIAV, equine infectious anemia virus; FIV, feline immunodeficiency virus; VISNA, visna virus; CAEV, caprine arthritis encephalitis virus; BIV, bovine immunodeficiency virus.

  • FIG. 3.
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    FIG. 3.

    Growth kinetics of a panel of SIVmac239 CA mutants with polar and nonpolar substitutions. RT activity in cell-free supernatants of CEM×174 cells transfected with infectious viral DNA was assayed at various times posttransfection. The D(50)ACA growth curve is not typical (arrow) and is representative of only one of three independent transfections.

  • FIG. 4.
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    FIG. 4.

    TCID50 and infectivity of wild-type and mutant SIVmac239. (A) TCID50 of virus derived from 293T cells at day 7 postinfection. (B) TCID50 of virus derived from COS-1 cells at day 7 postinfection. (C) LuSIV assay for infectivity of virus derived from 293T cells. Values are the fold induction over the background at day 2 postinfection. Results are representative of three independent experiments.

  • FIG. 5.
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    FIG. 5.

    Virion release and analysis of viral proteins. (A) Virus lysates and cell lysates from metabolically labeled transfected 293T cells were immunoprecipitated with IgG-purified SIV CA antiserum, resolved by SDS-PAGE, and visualized by autoradiography. (B) Relative virion release efficiency was calculated as the total amount of Gag in the virus lysate divided by the sum of the total amount of Gag in the cell and virus lysates, based on densitometric analysis of the gels in panel A. (C) Virus lysates produced as for panel A were immunoprecipitated with IgG-purified SIV antiserum, resolved by SDS-PAGE, and visualized by autoradiography. Env, envelope; INT, integrase.

  • FIG. 6.
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    FIG. 6.

    Transmission electron micrographs of wild-type and CA mutant SIVmac239 virions. 293T cells transfected with wild-type (WT) or mutant infectious viral DNA were harvested 1 day posttransfection, ultrathin sectioned, and analyzed by transmission EM. Labeled arrows indicate virions representative of typical virion morphologies: Ce, centric; I, immature; A, acentric; Co, conical. Unlabeled arrows show tethered immature virions (G), doublet and triplet particles (J), tethered virion chains (K), and accumulation of Gag at the plasma membrane (L). Bars, 100 nm.

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    FIG. 7.

    Models of the HIV-1 and SIVmac239 CA proteins. Models were generated with QUANTA and rendered with SETOR (12). (A) Region surrounding HIV-1 T(48)CA modeled according to the reported crystal structure (15). (B) Region surrounding SIV T(47)CA modeled by homology to the HIV CA structure used for panel A. Color coding: blue, nitrogen; red, oxygen; gray, carbon.

Tables

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  • TABLE 1.

    Proportion of acentric mature cores observed for wild-type and capsid mutant SIVmac239 viruses

    Genotype% Acentric mature coresP valuea
    WT46.60.002
    D(50)A1000.02
    T(47)A73.7NA
    • ↵ a P values were obtained by using a one-sided test for differences in the proportion of acentric cores relative to T(47)ACA SIVmac239. NA, not applicable.

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Hydrogen Bonding at a Conserved Threonine in Lentivirus Capsid Is Required for Virus Replication
Sarah M. Rue, Jason W. Roos, L. Mario Amzel, Janice E. Clements, Sheila A. Barber
Journal of Virology Jul 2003, 77 (14) 8009-8018; DOI: 10.1128/JVI.77.14.8009-8018.2003

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Hydrogen Bonding at a Conserved Threonine in Lentivirus Capsid Is Required for Virus Replication
Sarah M. Rue, Jason W. Roos, L. Mario Amzel, Janice E. Clements, Sheila A. Barber
Journal of Virology Jul 2003, 77 (14) 8009-8018; DOI: 10.1128/JVI.77.14.8009-8018.2003
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KEYWORDS

Capsid Proteins
simian immunodeficiency virus
Threonine
virus replication

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