HIV-1 Gag shares a signature motif with annexin (Anx7), which is required for virus replication

Abstract

Genetic and biochemical analyses of the Gag protein of HIV-1 indicate a crucial role for this protein in several functions related to viral replication, including viral assembly. It has been suggested that Gag may fulfill some of the functions by recruiting host cellular protein(s). In our effort to identify structural and functional homologies between Gag and cellular cytoskeletal and secretory proteins involved in transport, we observed that HIV-1 Gag contains a unique PGQM motif in the capsid region. This motif was initially noted in the regulatory domain of synexin the membrane fusion protein of Xenopus laevis. To evaluate the functional significance of the highly conserved PGQM motif, we introduced alanine (A) in place of individual residues of the PGQM and deleted the motif altogether in a Gag expression plasmid and in an HIV-1 proviral DNA. The proviral DNA containing mutations in the PGQM motif showed altered expression, assembly, and release of viral particles in comparison to parental (NL4-3) DNA. When tested in multiple- and single-round replication assays, the mutant viruses exhibited distinct replication phenotypes; the viruses containing the A for the G and Q residues failed to replicate, whereas A in place of the P and M residues did not inhibit viral replication. Deletion of the tetrapeptide also resulted in the inhibition of replication. These results suggest that the PGQM motif may play an important role in the infection process of HIV-1 by facilitating protein-protein interactions between viral and/or viral and cellular proteins.

Figure 1

The conservation of PGQM motif among HIV-1 isolates of different clades. The predicted amino acid sequences of Gag were according to Myers et al. (25). The designation of the clades is indicated on the right.

Figure 2

Generation of HIV-1 proviral DNA with alteration for individual residues in PGQM motif. Schematic representation of full-length HIV-1 proviral DNA and the changes in the PGQM motif are indicated.

Figure 3

Analysis of Gag protein. (A) Radioimmunoprecipitation assay analysis of in vitro-transcribed and -translated Gag. Antiserum to Flag epitope was used. Lane 1, pCDNA3 vector; lane 2, P93A; lane 3, G94A; lane 4, Q95A; lane 5, M96A; lane 6; ΔPGQM. (B) Cell lysate. (C) Culture supernatant of HeLa cells transfected with Gag plasmid. Immunoblot analysis was used. Details as above.

Figure 4

Radioimmunoprecipitation assay analysis of viral proteins. RD cells were transfected with 10 μg of HIV-1 proviral DNA by using the calcium phosphate precipitation method. Cells were labeled with [³⁵S]methionine for 12 hours. Both cells and culture supernatants were processed as described in Materials and Methods. (A) Cells. (B) Culture supernatant. Lane 1, NL-P93-A; lane 2, NL-G94A; lane 3, NL-Q95A; lane 4, NL-M96A; lane 5, NL-ΔPGQM; lane 6, NL4-3. Immunoprecipitation with normal sera did not show reactivity with viral proteins (data not shown).