Human immunodeficiency virus type 1 protease inhibitors irreversibly block infectivity of purified virions from chronically infected cells

Abstract

Synthetic peptide analog inhibitors of human immunodeficiency virus type 1 (HIV-1) protease were used to study the effects of inhibition of polyprotein processing on the assembly, structure, and infectivity of virions released from a T-celi line chronically infected with HIV-1. Inhibition of proteolytic processing of both Pr558w9 and Pr1609g W' was observed in purified virions from infected T cells after treatment. Protease inhibition was evident by the accumulation of precursors and processing intermediates of Pr655"' and by corresponding decreases in mature protein products. Electron microscopy revealed that the majority of the virion particles released from inhibitor-treated cells after a 24-h treatment had an immature or aberrant capsid morphology.This morphological change correlated with the inhibition of polyprotein processing and a loss of infectivity. The infectivity of virion particles purified from these chronically infected cell cultures was assessed following treatment with the inhibitor for 1 to 3 days. Virions purified from cultures treated with inhibitor for 1 or 2 days demonstrated a 95-to 100-fold reduction in virus titers, and treatment for 3 days resulted in complete loss of detectable infectivity. The fact that virions from treated cultures were unable to establish infection over the 7-to 10-day incubation period in the titration experiments strongly suggests that particles produced by inhibitor-treated cells were unable to reactivate to an infectious form when they were purified away from exogenous protease inhibitor. Thus, a block of HIV-1 protease processing of viral polyproteins by specific inhibitors results in a potent antiviral effect characterized by the production of noninfectious virions with altered protein structures and immature morphologies.

Regulation of Human Immunodeficiency Virus Type 1 Env-Mediated Membrane Fusion by Viral Protease Activity

“…This conclusion is in agreement with that of a previous biochemical report (35) and with morphological and structural analyses of both mature and immature HIV-1 virions by cryoelectron microscopy, which revealed a comparable density of Env proteins on both types of virions (4,53). Next, we examined whether Gag processing affects virus binding to target CD4 ϩ T cells.…”

Inhibition of Human Endogenous Retrovirus-K10 Protease in Cell-free and Cell-based Assays

“…Similar results obtained with viral RNA isolation from the particulate material of NCCIT cell culture medium and its subsequent quantification with RT-PCR amplification support this observation (data not shown). These data are consistent with the observation that HIV-1 protease inhibitors block the processing of Gag and Gag-Pol precursor polyproteins in HIV-1-infected cells but do not markedly alter either the number of particles released from the infected cells (44,45) or the amount of packaged viral RNA (46,47). In addition to using antigenspecific immunoblotting, we verified the identity of NCCIT released virions by checking the nucleotide sequence of packaged RNA.…”

In vitro Sequential Selection and Characterization of Human Immunodeficiency Virus Type 1 Variants with Reduced Sensitivity to Hydroxyethylurea Protease Inhibitors

“…Disruption of protease activity through site-directed mutagenesis (Kohl et al, 1988;Le Grice et al, 1988;Mous et al, 1988;Gottlinger et al, 1989) or inhibitor interaction (Kotler et al, 1988;Seelmeier et al, 1988) has been shown to result in improper processing of the viral polyproteins and the production of non-infectious virus particles. Since the protease functions at a late stage in the HIV life-cycle, protease inhibitors, unlike inhibitors of HIV reverse transcriptase (RT) , are effective against both productively infected cells and newly infected cells (Lambert et al, 1992).…”