Assembly and release of human immunodeficiency virus type 1 Gag proteins containing tandem repeats of the matrix protein coding sequences in the matrix domain
Ct. Wang et al., Assembly and release of human immunodeficiency virus type 1 Gag proteins containing tandem repeats of the matrix protein coding sequences in the matrix domain, VIROLOGY, 278(1), 2000, pp. 289-298
We have constructed human immunodeficiency virus (HIV) gag mutants by incre
asing the matrix protein (MA) sequences via tandemly repeated duplication o
f 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, respectiv
ely. Analysis indicated that the addition of 110 or 220 amino acid residues
to the MA did not significantly affect the assembly, release, and processi
ng of particles; however, particle production was markedly reduced when ano
ther copy of 110 residues was added to the MA. Subcellular fractionation an
alysis suggested that the MA tandem repeat mutations enhanced the Gag membr
ane affinity, in a manner which correlated with the copy number of MA seque
nces. The effects of enhanced membrane affinity were substantially reduced
when sequences downstream of the capsid (CA) domain were deleted. Sucrose d
ensity gradient fractionation analysis showed that particles produced by th
e large insertion mutants possessed wild-type (wt) HIV particle density. Tr
uncation of sequences downstream of the nucleocapsid (NC) domains of the mu
tants did not influence the budding of particles. In contrast, particle bud
ding was severely impaired when sequences downstream of the CA domain were
truncated. Particle densities for the large Gag proteins, which were trunca
ted 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 la
rge 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.
(C) 2000 Academic Press.