Human immunodeficiency virus type 1 nucleocapsid p1 confers ESCRT pathway dependence

Abstract

To facilitate the release of infectious progeny virions, human immunodeficiency virus type 1 (HIV-1) exploits the Endosomal Sorting Complex Required for Transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in the C-terminal p6 domain of Gag. However, the L domains in p6 are known to be dispensable for efficient particle production by certain HIV-1 Gag constructs that have the nucleocapsid (NC) domain replaced by a foreign dimerization domain to substitute for the assembly function of NC. We now show that one such L domain-independent HIV-1 Gag construct (termed Z(WT)) that has NC-p1-p6 replaced by a leucine zipper domain is resistant to dominant-negative inhibitors of the ESCRT pathway that block HIV-1 particle production. However, Z(WT) became dependent on the presence of an L domain when NC-p1-p6 was restored to its C terminus. Furthermore, when the NC domain was replaced by a leucine zipper, the p1-p6 region, but not p6 alone, conferred sensitivity to inhibition of the ESCRT pathway. In an authentic HIV-1 Gag context, the effect of an inhibitor of the ESCRT pathway on particle production could be alleviated by deleting a portion of the NC domain together with p1. Together, these results indicate that the ESCRT pathway dependence of HIV-1 budding is determined, at least in part, by the NC-p1 region of Gag.

FIG. 1.

Replacement of NC-p1-p6 by the GCN4 leucine zipper confers resistance to dominant-negative CHMP3. 293T cells were transfected with 1.5 μg of WT HIV-1 proviral DNA, of a PR-defective HIV-1 provirus, or of the Z_WT proviral construct, along with 0.5 μg of a vector expressing RFP alone or CHMP3-RFP. VLP pellets and the cell lysates were analyzed by Western blotting with anti-CA serum. The GCN4 zipper (Z) domain is indicated by a gray box.

FIG. 2.

The chimeric Z_WT Gag construct exhibits L domain dependence when NC-p1 is fused to its C terminus. (A) 293T cells were transfected with a PR-defective HIV-1 provirus expressing WT Pr55^gag or with the indicated Gag constructs. VLP pellets and the cell lysates were analyzed by Western blotting with anti-CA serum. (B) Thin sections of 293T cells transiently expressing the Z_WT-NCp1p6 or Z_WT-NCp1 proviral construct were examined by transmission electron microscopy. Scale bars, 100 nm.

FIG. 3.

The addition of p1-p6 but not of p6 alone to a chimeric Gag construct confers ESCRT pathway dependence. 293T cells were transfected with 1.5 μg of the indicated Gag constructs together with 0.5 μg of a vector expressing RFP alone or CHMP3-RFP. VLP pellets and the cell lysates were analyzed by Western blotting with anti-CA serum.

FIG. 4.

The addition of p1-p6 but not of p6 alone to a chimeric Gag construct confers L domain dependence. (A) Effect of deleting the Tsg101 or the ALIX binding site from a chimeric Gag construct containing p1. 293T cells were transfected with 1.5 μg of WT Z_IL-p1p6 or the indicated mutants, along with 0.5 μg of a vector expressing RFP alone or CHMP3-RFP. VLP pellets and the cell lysates were analyzed by Western blotting with anti-CA serum. (B) Effect of deleting the Tsg101 binding site from a chimeric Gag construct lacking p1. 293T cells were transfected with 1.5 μg of WT Z_IL-p6 or a ΔPTAPP version.

FIG. 5.

The removal of NC-p1 sequences from authentic HIV-1 Gag alleviates the effect of dominant-negative CHMP3 on particle production. (A) 293T cells were transfected with 1.5 μg of a PR-defective HIV-1 provirus expressing WT Pr55^gag or with the indicated NC-p1 deletion mutants, along with a vector expressing RFP alone or CHMP3-RFP. VLP pellets and the cell lysates were analyzed by Western blotting with anti-CA serum. (B) Effect of CHMP3-RFP on the indicated NC zinc finger mutants. Cross-hatched boxes indicate the positions of the zinc fingers in NC, and arrowheads indicate the positions of residues that were substituted. Western blotting was performed with 183-H12-5C anti-HIV CA antibody.