ANALYSIS OF MINIMAL HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 GAG CODING SEQUENCES CAPABLE OF VIRUS-LIKE PARTICLE ASSEMBLY AND RELEASE

We have constructed a series of human immunodeficiency virus (HIV) gag mutants by progressive truncation of the gag coding sequence from the C terminus and have combined these mutants with an assembly-competent matrix domain deletion mutation (Delta MA). By using several methods, the particle-producing capabilities of each mutant were examined. Our analysis indicated that truncated Gag precursors lacking most of C-te rminal gag gene products assembled and were released from 293T cells. Additionally, a mutant with a combined deletion of the MA (hMA) and p6 domains even produced particles at levels comparable to that of the w ild-type (Wt) virus. However, most mutants derived from combination of the Delta MA and the C-terminal truncation mutations did not release particles as well as the wt. Our smallest HIV gag gene product capable of virus-like particle formation was a 28-kDa protein which consists of a few MA amino acids and the CA-p2 domain. Sucrose density gradient fractionation analysis indicated that most mutants exhibited a wt ret rovirus particle density. Exceptions to this rule were mutants with an intact MA domain but deleted downstream of the p2 domains. These C-te rminal truncation mutants possessed particle densities of 1.13 to 1.15 g/ml, lower than that of the wt. The N-terminal portions of the CA do main, which have been shown to be dispensable for core assembly, becam e critical when most of the. MA domain was deleted, suggesting a requi rement for an intact CA domain to assemble and release particles.