A bipartite late-budding domain in human immunodeficiency virus type 1 - PubMed (original) (raw)

A bipartite late-budding domain in human immunodeficiency virus type 1

Juan Martin-Serrano et al. J Virol. 2003 Nov.

Abstract

Human immunodeficiency virus type 1 (HIV-1) encodes a PTAP motif within the p6 domain of Gag that recruits Tsg101 and associated factors to facilitate virion budding. In this study, we use trans-complementation assays to demonstrate that the PTAP motif acts synergistically with additional p6 sequences to mediate the formation of infectious extracellular HIV-1 virions. These studies suggest that Tsg101 recruitment is necessary but not sufficient to account for late-budding activity exhibited by HIV-1 p6.

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Figures

FIG. 1.

FIG. 1.

Tsg101 recruitment is not sufficient to account for the L-domain activity exhibited by HIV-1 p6. (A) The complementation assay. A schematic representation of the complementing Gagδp6 protein is shown. It contains MA, CA, and NC domains and the N-terminal 4 residues of p6 (LQSR) to permit cleavage by the HIV-1 protease. The synthetic linker sequence (LEFGGRLE) also encodes _Eco_RI, _Not_I, and _Xho_I restriction sites (underlined) for reinsertion of full-length or truncated versions of p6, the PTAP sequence, or Tsg101. For the assay itself, Gag-Pol and mutant Gag proteins are expressed by the defective proviral construct and are depicted in black; the complementing fusion protein, in this case Gagδp6-Tsg101, is shown in white. Tsg101 is recruited to sites of virion production by virtue of Gag multimerization. (B) Infectious virus production by defective provirus (NL[LTAL], black bars; or NLδp6, white bars) complemented by Gagδp6-Tsg101. The chart shows β-galactosidase activity in HeLa P4/R5 cells following inoculation with supernatant harvested from HOS cells transfected with defective provirus constructs and the indicated complementing expression plasmid. RLU, relative light units. (C) Same information as that given for panel B applies, except that only the pNLδp6 proviral plasmid was used, complemented with either pGagδp6-p6 or pGagδp6-Tsg101, as indicated. (D) Western blot analysis, with an anti-CA monoclonal antibody, of cell lysates and virion pellets following transfection of HOS cells with the pNLδp6 provirus plus either pGagδp6 (lane 1), pGagδp6-p6 (lane 2), or pGagδp6-Tsg101 (lane 3).

FIG. 2.

FIG. 2.

The PTAP motif and additional p6 sequences can act synergistically and in trans to mediate infectious HIV-1 virion production. (A) Schematic representation of the complementation assay. Gag-Pol and Gagδp6 proteins expressed by the defective proviral construct (NLδp6) are depicted in black; the complementing fusion proteins, in this case Gagδp6-PTAP and Gagδp6-p6 (15-51), are shown in white. (B) Infectious virion production measured as for Fig. 1 by using HeLa P4/R5 cells. Forty-eight hours of transfection of 293T cells with 300 ng of NLδp6 was carried out, and the indicated quantities (in nanograms) of pGagδp6, pGagδp6-p6, pGagδp6-PTAP and/or pGagδp6-p6 (15-51) are indicated. RLU, relative light units. (C and D) The same information as that given for panel A applies, except that HOS (C) or TE671 (D) was transfected.

FIG. 3.

FIG. 3.

Western blot analysis of the effect of PTAP and p6 (15-51) on Gag processing and particle release. 293T cells were transfected with 300 ng of pNLδp6 and the indicated complementing pGagδp6-fusion protein expression plasmids, as was done for Fig. 2. Forty-eight hours after transfection, cell lysates and pelleted extracellular virions (harvested by ultracentrifugation through a 20% sucrose cushion) were analyzed by Western blotting with an anti-p24CA monoclonal antibody. The number(s) below each lane indicates the levels of mature p24 CA protein and the incompletely processed p25 CA-p2 intermediate in virion pellets, quantitated by analysis of the blots with NIH Image. ND, not detectable.

FIG. 4.

FIG. 4.

Electron microscopy analysis of p6- and PTAP-mediated virion budding. 293T cells were transfected with pNLδp6 and pGagδp6 (A), pNLδp6 and pGagδp6-p6 (B), or pNLδp6 and pGagδp6-PTAP (C), in each case in a 3:2 molar ratio. Cells were harvested and processed for microscopy 48 h after transfection. Each panel shows an overview of part of a transfected cell (left) as well as a higher magnification of budding structures and virions (right).

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