HIV-1 Gag Processing Intermediates Trans-dominantly Interfere with HIV-1 Infectivity

Abstract

Protease inhibitors (PI) act by blocking human immunodeficiency virus (HIV) polyprotein processing, but there is no direct quantitative correlation between the degree of impairment of Gag processing and virion infectivity at low PI concentrations.To analyze the consequences of partial processing, virus particles were produced in the presence of limiting PI concentrations or by co-transfection of wild-type proviral plasmids with constructs carrying mutations in one or more cleavage sites. Low PI concentrations caused subtle changes in polyprotein processing associated with a pronounced reduction of particle infectivity. Dissection of individual stages of viral entry indicated a block in accumulation of reverse transcriptase products, whereas virus entry, enzymatic reverse transcriptase activity, and replication steps following reverse transcription were not affected. Co-expression of low amounts of partially processed forms of Gag together with wild-type HIV generally exerted a trans-dominant effect, which was most prominent for a construct carrying mutations at both cleavage sites flanking the CA domain. Interestingly, co-expression of low amounts of Gag mutated at the CA-SP1 cleavage site also affected processing activity at this site in the wild-type virus. The results indicate that low amounts (<5%) of Gag processing intermediates can display a trans-dominant effect on HIV particle maturation, with the maturation cleavage between CA and SP1 being of particular importance. These effects are likely to be important for the strong activity of PI at concentrations achieved in vivo and also bear relevance for the mechanism of action of the antiviral drug bevirimat.

The PTAP Sequence within the p6 Domain of Human Immunodeficiency Virus Type 1 Gag Regulates Its Ubiquitination and MHC Class I Antigen Presentation

“…DRiPs could interfere with virus release by disturbing the ordered assembly of Gag molecules in a prion-like manner (91). Moreover, misfolding of the Gag precursor could impair the accessibility of cleavage sites for the viral protease, contributing to the accumulation of processing intermediates, which have been shown to trans-dominantly interfere with viral infectivity (92).…”

Multi-step inhibition explains HIV-1 protease inhibitor pharmacodynamics and resistance

“…Due to the significant contribution of entry inhibition to the overall inhibition produced by PIs (Figure 4), we hypothesized that drug-resistance mutations would alter dose-response curves for both the entry and postentry events. To test this hypothesis, we introduced 2 LPV resistance mutations, V82A and V82F (15), into the NL4-3ΔEnv provirus by site-directed mutagenesis to create NL4-3ΔEnvV82A and NL4-3ΔEnvV82F, respectively. 293T cells were cotransfected with NL4-3ΔEnvV82A or NL4-3ΔEnvV82F, along with either a VSV-G expression vector or an HIV-1 Env expression vector and the BLAM-Vpr construct.…”

“…Virus maturation is generally considered to be important for early postentry steps including uncoating and reverse transcription (13)(14)(15). Both the RT and integrase (IN) enzymes are generated from Pr160 Gag-Pol by cleavages carried out by HIV-1 protease.…”

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.…”