AEGiS-12IAC: Identification of a minimum HIV-1 gag sequence required for particle assembly.

12th International AIDS Conference

Geneva, Switzerland - June 28-July 3, 1998

Identification of a minimum HIV-1 gag sequence required for particle assembly.

Int Conf AIDS 1998 Jun 28-Jul 3; 12:9 (abstract no. 11134)

Wang CT;;;

OBJECTIVES: To identify a minimum HIV-1 gag coding sequence capable of particle assembly and release from mammalian cells.

METHODS: We constructed a series of C-terminally truncated HIV gag mutants and mutants derived from combination of the C-terminal truncation mutants and a MA deletion (delta MA) mutant. The abilities of these mutants to assemble and release virus particles were assessed by Western immunoblotting and sucrose density gradient fractionation experiments. Localization of the mutant Gag proteins in expressing cells were revealed by indirect immunofluorescence experiments, and mutant particle-associated RT activities were tested by in vitro RT assays.

RESULTS: Our analysis indicated that truncated Gag precursors lacking most of the C-terminal gag gene products assembled and were released from 293T cells. A mutant with a combined deletion of the MA (delta MA) and p6 domain even produced particles at levels comparable to that of wild-type virus. Furthermore, most mutants derived from combination of the delta MA and the C-terminal truncation mutations did not prevent particle production completely, provided the CA-p2 region remained intact. Sucrose density gradient fractionation analysis indicated that most mutants exhibited a wt Retrovirus particle density. Exceptions to this rule were mutants with an intact MA domain, by deleted downstream of the p2 domains. These C-terminal truncations mutants possessed particle densities of 1.13-1.15 g/ml, lower than that of wt.

CONCLUSION: Our smallest HIV gag gene product capable of particle formation was a 28 kDa protein which consists of a few MA amino acids and the CA-p2 domain. The N-terminal portions of the CA domain, which have been shown to be dispensable for core assembly, became critical when most of the MA domain was deleted, suggesting a requirement of an intact CA to assemble and release particles.

Keywords: AEGIS, Gene Products, gag, HIV-1, Virion, HIV-1 Reverse Transcriptase, Glycosaminoglycans, Mutation, Sucrose, In Vitro, genetics, ICA12
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