Assembly and release of human immunodeficiency virus type 1 Gag proteins containing tandem repeats of the matrix protein coding sequences in the matrix domain.
We have constructed human immunodeficiency virus (HIV) gag mutants by increasing the matrix protein (MA) sequences via tandemly repeated duplication of the central 107-MA codons. Instead of a total of 132 amino acid residues for the wild-type MA, the resultant mutants designated as MA2, MA3, and MA4 contained a total of 242, 352, and 462 codons in the MA domains, respectively. Analysis indicated that the addition of 110 or 220 amino acid residues to the MA did not significantly affect the assembly, release, and processing of particles; however, particle production was markedly reduced when another copy of 110 residues was added to the MA. Subcellular fractionation analysis suggested that the MA tandem repeat mutations enhanced the Gag membrane affinity, in a manner which correlated with the copy number of MA sequences. The effects of enhanced membrane affinity were substantially reduced when sequences downstream of the capsid (CA) domain were deleted. Sucrose density gradient fractionation analysis showed that particles produced by the large insertion mutants possessed wild-type (wt) HIV particle density. Truncation of sequences downstream of the nucleocapsid (NC) domains of the mutants did not influence the budding of particles. In contrast, particle budding was severely impaired when sequences downstream of the CA domain were truncated. Particle densities for the large Gag proteins, which were truncated at the C-terminus of CA, were about 1.12-1.14 g/ml lower than that for wt. Our results suggest that the HIV MA domain could adopt insertions of large protein sequences, and strongly support the proposal that the NC and p2 domains play a crucial role in the process of correct Gag protein packing.
Wang
C T
CT
Department of Medical Research and Education, Institute of Clinical Medicine, Taipei, Taiwan.
Chen
S S
SS
Chiang
C C
CC
D002460:Cell Line; D005591:Chemical Fractionation; D015683:Gene Products, gag / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D015497:HIV-1 / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D016254:Mutagenesis, Insertional; D011995:Recombination, Genetic; D013395:Sucrose; D020080:Tandem Repeat Sequences; D014763:Viral Matrix Proteins / Q000235:genetics; D019065:Virus Assembly
D015683:Gene Products, gag; D014763:Viral Matrix Proteins; D013395:Sucrose