Abstract
BackgroundThe mature HIV-1 conical core formation proceeds through highly regulated protease cleavage of the Gag precursor, which ultimately leads to substantial rearrangements of the capsid (CAp24) molecule involving both inter- and intra-molecular contacts of the CAp24 molecules. In this aspect, Asp51 which is located in the N-terminal domain of HIV-1 CAp24 plays an important role by forming a salt-bridge with the free imino terminus Pro1 following proteolytic cleavage and liberation of the CAp24 protein from the Pr55Gag precursor. Thus, previous substitution mutation of Asp51 to alanine (D51A) has shown to be lethal and that this invariable residue was found essential for tube formation in vitro, virus replication and virus capsid formation.ResultsWe extended the above investigation by introducing three different D51 substitution mutations (D51N, D51E, and D51Q) into both prokaryotic and eukaryotic expression systems and studied their effects on in vitro capsid assembly and virus infectivity. Two substitution mutations (D51E and D51N) had no substantial effect on in vitro capsid assembly, yet they impaired viral infectivity and particle production. In contrast, the D51Q mutant was defective both for in vitro capsid assembly and for virus replication in cell culture.ConclusionThese results show that substitutions of D51 with glutamate, glutamine, or asparagine, three amino acid residues that are structurally related to aspartate, could partially rescue both in vitro capsid assembly and intra-cellular CAp24 production but not replication of the virus in cultured cells.
Highlights
The mature HIV-1 conical core formation proceeds through highly regulated protease cleavage of the Gag precursor, which leads to substantial rearrangements of the capsid (CAp24) molecule involving both inter- and intra-molecular contacts of the CAp24 molecules
Viral protein expression of HIV-1 CAp24 mutants We investigated the effects of three HIV-1 CAp24 mutants carrying the D51N, D51 with glutamate (D51E), and D51Q mutations for viral protein expression by initially transfecting HeLa-tat cells
Whereas the D51Q mutant displayed a slightly reduced amount of CAp24, the level of processed CAp24 proteins in the D51N mutant was significantly reduced relative to the wild type and the D51E CAp24 mutant
Summary
The mature HIV-1 conical core formation proceeds through highly regulated protease cleavage of the Gag precursor, which leads to substantial rearrangements of the capsid (CAp24) molecule involving both inter- and intra-molecular contacts of the CAp24 molecules In this aspect, Asp which is located in the N-terminal domain of HIV-1 CAp24 plays an important role by forming a salt-bridge with the free imino terminus Pro following proteolytic cleavage and liberation of the CAp24 protein from the Pr55Gag precursor. During proteolysis of the Pr55Gag and maturation of CAp24, the NTD of CAp24 refolds into a β-hairpin structure which is stabilized by formation of a salt-bridge between Pro and Asp of the processed NTD (Fig. 1) The fact that this structure is not formed in immature virus-like structures [7] indicates that this motif does not form in an immature particle. In multiple sequence alignment analysis of 4198 HIV-1 CAp24 sequences found in the HIV database (May 7, 2007), we found only 11 exceptions to the highly conserved Asp among all HIV-1 strains, demonstrating that this residue is conserved among various retroviruses and in HIV strains
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