Mutational analysis of the hydrophobic tail of the human immunodeficiency virus type 1 p6(Gag) protein produces a mutant that fails to package its envelope protein

The p6(Gag) protein of human immunodeficiency virus type I (HIV-1) is produ ced as the carboxyl-terminal sequence within the Gag polyprotein. The amino acid composition of this protein is high in hydrophilic and polar residues except for a patch of relatively hydrophobic amino acids found in the carb oxyl-terminal 16 amino acids. Internal cleavage of p6(Gag) between Y-36 and P-37, apparently by the HIV-1 protease, removes this hydrophobic tail regi on from approximately 30% of the mature p6(Gag) proteins in HIV-1(MN). To i nvestigate the importance of this cleavage and the hydrophobic nature of th is portion of p6(Gap), site-directed mutations were made at the minor prote ase cleavage site and within the hydrophobic tail. The results showed that all of the single-amino-acid replacement mutants exhibited either reduced o r undetectable cleavage at the site Set almost all were nearly as infectiou s as wild-type virus, demonstrating that processing at this site is not imp ortant for viral replication. However, one exception, Y36F, was 300-fold as infectious the wild type. In contrast to the single-substitution mutants, a virus with two substitutions in this region of p6(Gap), Y36S-L41P, could not infect susceptible cells. Protein analysis showed that while the proces sing of the Gag precursor was normal, the double mutant did not incorporate Env into virus particles. This mutant could be complemented with surface g lycoproteins from vesicular stomatitis virus and murine leukemia virus, sho wing that the inability to incorporate Env was the lethal defect for the Y3 6S-L41P virus, However, this mutant was not rescued by an HIV-1 Env with a truncated gp41(TM) cytoplasmic domain, showing that it is phenotypically di fferent from the previously described MA mutants that do not incorporate th eir full-length Env proteins. Cotransfection experiments with Y36S-L41P and wild-type proviral DNAs revealed that the mutant Gag dominantly blocked th e incorporation of Env by wild-type Gag. These results show that the Y36S-L 41P p6(Gag) mutation dramatically blacks the incorporation of HIV-1 Env, pr esumably acting late in assembly and early during budding.