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 (DeltaMA). By using several methods, the particle-producing capabilities of each mutant were examined. Our analysis indicated that truncated Gag precursors lacking most of C-terminal gag gene products assembled and were released from 293T cells. Additionally, a mutant with a combined deletion of the MA (DeltaMA) and p6 domains even produced particles at levels comparable to that of the wild-type (wt) virus. However, most mutants derived from combination of the DeltaMA 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 retrovirus particle density. Exceptions to this rule were mutants with an intact MA domain but deleted downstream of the p2 domains. These C-terminal 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 domain, which have been shown to be dispensable for core assembly, became critical when most of the MA domain was deleted, suggesting a requirement for an intact CA domain to assemble and release particles.
Wang
C T
CT
Institute of Clinical Medicine, National Yang-Ming University, and Department of Medical Research, Veterans General Hospital-Taipei, Taipei, Taiwan 11217, Republic of China. ctwang@vghtpe.gov.tw
Lai
H Y
HY
Li
J J
JJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D017931:DNA Primers; D019084:Fluorescent Antibody Technique, Indirect; D015683:Gene Products, gag / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D009154:Mutation; D014771:Virion / Q000502:physiology*; D019065:Virus Assembly / Q000502:physiology*
D017931:DNA Primers; D015683:Gene Products, gag