Mutational Analysis of the C-Terminal Gag Cleavage Sites in Human Immunodeficiency Virus Type 1

Abstract

Human immunodeficiency virus type 1 (HIV-1) Gag is expressed as a polyprotein that is cleaved into six proteins by the viral protease in a maturation process that begins during assembly and budding. While processing of the N terminus of Gag is strictly required for virion maturation and infectivity, the necessity for the C-terminal cleavages of Gag is less well defined. To examine the importance of this process, we introduced a series of mutations into the C terminus of Gag that interrupted the cleavage sites that normally produce in the nucleocapsid (NC), spacer 2 (SP2), or p6Gag proteins. Protein analysis showed that all of the mutant constructs produced virions efficiently upon transfection of cells and appropriately processed Gag polyprotein at the nonmutated sites. Mutants that produced a p9 NC/SP2 protein exhibited only minor effects on HIV-1 infectivity and replication. In contrast, mutants that produced only the p8 SP2/p6 or p15 NC/SP2/p6 protein had severe defects in infectivity and replication. To identify the key defective step, we quantified reverse transcription and integration products isolated from infected cells by PCR. All mutants tested produced levels of reverse transcription products either similar to or only somewhat lower than that of wild type. In contrast, mutants that failed to cleave the SP2-p6Gag site produced drastically less provirus than the wild type. Together, our results show that processing of the SP2-p6Gag and not the NC-SP2 cleavage site is important for efficient viral DNA integration during infection in vitro. In turn, this finding suggests an important role for the p9 NC/SP2

A Strongly Transdominant Mutation in the Human Immunodeficiency Virus Type 1 gag Gene Defines an Achilles Heel in the Virus Life Cycle

“…1B). The N432I mutant showed infectivity at about 30% of wildtype infectivity, and this is consistent with the phenotype described previously (10). Western analysis of virus particles (probed with an anti-CA antibody or an anti-NC antibody) are shown in Fig.…”

Importance of Protease Cleavage Sites within and Flanking Human Immunodeficiency Virus Type 1 Transframe Protein p6* for Spatiotemporal Regulation of Protease Activation

“…Furthermore, we observed NCassociated processing defects in csM1 particles likely associated with a substitution in the NC-p1 cleavage site introduced by amino-terminal p6* mutation. Albeit hydrolysis of the NC-p1 site has been reported to be rate limiting in virus maturation (16,62), recent analyses by Coren et al (10) have provided evidence that NC-p1 cleavage is not required for viral replication and infectivity. Together, these findings suggest that the aberrant csM1 phenotype is mainly due to compromised frameshifting at the altered slippery site.…”

“…These sequential processing steps driving maturation of Gag and Gag-Pol precursors have largely been clarified (10,30,43,44,57), and kinetic analyses revealed significantly differing hydrolysis rates for individual PR cleavage events (15,25,42,43). It is widely accepted that cleavage of the Gag precursor is initiated at the carboxyl terminus of the spacer peptide p2, separating the MA-CA-p2 polypeptide from the NC-p1-p6 moiety (39,43).…”

Induced Maturation of Human Immunodeficiency Virus

“…Open circles, DMSO control; filled triangles, APV; filled circles, SQV. errant capsid structures, even though the degree of CA processing is not impaired (20,21,44). We therefore performed a more comprehensive immunoblot analysis of Gag processing upon inhibitor washout using antibodies against MA and NC in addition to anti-CA (Fig.…”

“…Inhibition of PR activity and premature processing due to enhanced activation of the enzyme are detrimental for virus replication (16). An ordered sequence of PR cleavage events has been deduced from in vitro analyses (17)(18)(19); accordingly, mutational analyses revealed that even partial blocking of processing at individual sites impairs viral infectivity, and an ordered series of cleavage events within Gag is important for formation of a mature viral core (20)(21)(22); reviewed in reference 23).…”