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. 1999 Jun;73(6):4696-704.
doi: 10.1128/JVI.73.6.4696-4704.1999.

Proline residues in human immunodeficiency virus type 1 p6(Gag) exert a cell type-dependent effect on viral replication and virion incorporation of Pol proteins

M Dettenhofer  1 , X F Yu
Affiliations

Proline residues in human immunodeficiency virus type 1 p6(Gag) exert a cell type-dependent effect on viral replication and virion incorporation of Pol proteins

M Dettenhofer et al. J Virol. 1999 Jun.

Abstract

The C terminus of the HIV-1 Gag protein contains a proline-rich domain termed p6(Gag). This domain has been shown to play a role in efficient virus release and incorporation of Vpr into virions. In a previous study (X. F. Yu, L. Dawson, C. J. Tian, C. Flexner, and M. Dettenhofer, J. Virol. 72:3412-3417, 1998), we observed that the removal of the p6 domain of Gag as well as drastic mutations in the PTAP motif resulted in reduced virion-associated Pol proteins from transfected COS cells. In the present study, amino acid substitutions at residues 5 and 7 of p6(Gag) resulted in a cell type-dependent replication of the mutant virus in CD4(+) T cells; the virus was replication competent in Jurkat cells but restricted in H9 cells and primary blood-derived monocytes. Established Jurkat and H9 cell lines expressing p6(Gag) mutant and parental virus were used to further understand this defect. Mutant virions produced from H9 cells, which displayed no defect in extracellular virion production, showed an approximately 16-fold reduction in Pol protein levels, whereas the levels of Pol proteins were only marginally reduced in mutant virions produced from Jurkat cells. The reduction in the virion-associated Pol proteins could not be accounted for by differences in the levels of intracellular p160(Gag-Pol) or in the interaction between p55(Gag) and p160(Gag-Pol) precursors. Electron microscopic analysis of the p6(Gag) mutant virions showed a predominately immature morphology in the absence of significant defects in Gag proteolytic cleavage. Taken together, these data suggest that the proline-rich motif of p6(Gag) is involved in the late stages of virus maturation, which include the packaging of cleaved Pol proteins in viral particles, a process which may involve cell-type-specific factors.

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Figures

FIG. 1
FIG. 1
(A). Schematic depiction of partial HIV-1 Gag and Pol sequences of the p6Gag mutant (HXB2Pro) and parental construct (HXB2) used in this study. Proline residues at positions 5 and 7 of p6Gag where changed to arginine and glutamine (boldface), respectively, without altering the Pol amino acid sequence. (B to E) Replication growth curve of HXB2 and HXB2Pro viruses as monitored by RT activity from cell-free supernatants. Virions were generated from transfected COS-7 cells and used to initiate infections in Jurkat (B), CEM (C), or H9 (D) cells or PBMC (E). PBMC were additionally infected with a 1,000-fold-reduced concentration of the HXB2 virus [HXB2(1/1000)], to check the relative degree of infectivity of the HXB2Pro virus.
FIG. 2
FIG. 2
Radioimmunoprecipitation of viral proteins from uninfected H9 (Mock) cells, established H9 cell lines expressing p6Gag mutant (HXB2ProBgl), and parental (HXB2Bgl) constructs. The cells were metabolically labeled with [35S]cysteine for 12 h. The proteins were immunoprecipitated with HIV-1-positive patient serum either from cell lysates (A) or from particles pelleted through 20% sucrose (B). The proteins were separated by SDS-PAGE and visualized by autoradiography.
FIG. 3
FIG. 3
Intracellular viral protein profiles derived from established Jurkat and H9 cell lines containing HXB2Bgl (parent) or HXB2ProBgl (p6Gag mutant). Proteins from mock-infected and HXB2Bgl- and HXB2ProBgl-infected cell lysates were separated by 12% (A) or 7.5% (B) polyacrylamide gels and transferred onto nitrocellulose membranes. The blots were reacted with either HIV-1-positive human serum (A) or anti-RT MAb (B).
FIG. 4
FIG. 4
Virion-associated-protein profiles derived from established Jurkat and H9 cell lines containing HXB2Bgl (parent) or HXB2ProBgl (p6Gag mutant). Virion proteins from mock-infected and HXB2Bgl- and HXB2ProBgl-infected cells were separated by 12% polyacrylamide gels and transferred onto nitrocellulose membranes. The blots were reacted with either HIV-1-positive human serum (A), anti-RT MAb (B), or anti-integrase (Int) antiserum (C).
FIG. 5
FIG. 5
Quantitation of virion-associated protein profiles derived from Jurkat (A and B) and H9 (C and D) cell lines. Twofold dilutions of HXB2Bgl virion lysates (as indicated below each corresponding lane) were run side by side with a fixed amount of HXB2ProBgl (furthest-right lane) to compare the relative amounts of Gag proteins and RT proteins in each sample. Virion proteins from mock-infected and HXB2Bgl- and HXB2ProBgl-infected cells were separated by 12% polyacrylamide gels and transferred onto nitrocellulose membranes. The blots were reacted with either HIV-1-positive human serum (A and C) or anti-RT MAb (B and D).
FIG. 6
FIG. 6
Coimmunoprecipitation of intracellular viral complex. Analysis of p55Gag precursor association with p160Gag-Pol precursor- and Pol domain-containing proteins. Proteins from mock-infected HXB2Bgl- and HXB2ProBgl-infected establish Jurkat or H9 cell lysates were immunoprecipitated with anti-p6 antiserum as described in Materials and Methods. The anti-p6 antiserum used specifically recognizes only p55Gag-related proteins containing the p6 domain of Gag. The precipitated proteins were separated on 7.5% polyacrylamide gels and transferred onto nitrocellulose membranes. The Western blots were reacted with either HIV-1-positive human serum (A) or anti-RT MAb (B).
FIG. 7
FIG. 7
Ultrastructure analysis of parental and p6 mutant virions. Viruses produced from HXB2Bgl- (A) and HXB2ProBgl-containing (B) established H9 cells were examined by EM. Representative fields are shown.
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