Induced Maturation of Human Immunodeficiency Virus

Abstract

HIV-1 assembles at the plasma membrane of virus-producing cells as an immature, noninfectious particle. Processing of the Gag and Gag-Pol polyproteins by the viral protease (PR) activates the viral enzymes and results in dramatic structural rearrangements within the virion-termed maturation-that are a prerequisite for infectivity. Despite its fundamental importance for viral replication, little is currently known about the regulation of proteolysis and about the dynamics and structural intermediates of maturation. This is due mainly to the fact that HIV-1 release and maturation occur asynchronously both at the level of individual cells and at the level of particle release from a single cell. Here, we report a method to synchronize HIV-1 proteolysis in vitro based on protease inhibitor (PI) washout from purified immature virions, thereby temporally uncoupling virus assembly and maturation. Drug washout resulted in the induction of proteolysis with cleavage efficiencies correlating with the off-rate of the respective PR-PI complex. Proteolysis of Gag was nearly complete and yielded the correct products with an optimal halflife (t 1/2 ) of ϳ5 h, but viral infectivity was not recovered. Failure to gain infectivity following PI washout may be explained by the observed formation of aberrant viral capsids and/or by pronounced defects in processing of the reverse transcriptase (RT) heterodimer associated with a lack of RT activity. Based on our results, we hypothesize that both the polyprotein processing dynamics and the tight temporal coupling of immature particle assembly and PR activation are essential for correct polyprotein processing and morphological maturation and thus for HIV-1 infectivity. IMPORTANCECleavage of the Gag and Gag-Pol HIV-1 polyproteins into their functional subunits by the viral protease activates the viral enzymes and causes major structural rearrangements essential for HIV-1 infectivity. This proteolytic maturation occurs concomitant with virus release, and investigation of its dynamics is hampered by the fact that virus populations in tissue culture contain particles at all stages of assembly and maturation. Here, we developed an inhibitor washout strategy to synchronize activation of protease in wild-type virus. We demonstrated that nearly complete Gag processing and resolution of the immature virus architecture are accomplished under optimized conditions. Nevertheless, most of the resulting particles displayed irregular morphologies, Gag-Pol processing was not faithfully reconstituted, and infectivity was not recovered. These data show that HIV-1 maturation is sensitive to the dynamics of processing and also that a tight temporal link between virus assembly and PR activation is required for correct polyprotein processing.

Highly Resistant HIV-1 Proteases and Strategies for Their Inhibition

“…This essential process in the viral life cycle must occur in a precise order to produce infectious virus [74,75]. An inhibitor of maturation, bevirimat, was developed to target the final processing step for the Gag precursor (Figure 1).…”

RNA and Nucleocapsid Are Dispensable for Mature HIV-1 Capsid Assembly

“…Morphology was classified as wt (the typical conical or tubular cores found in the wt viruses), mildly aberrant (cores with a slightly deformed structure with some similarity to the conical or tubular ones), or severely aberrant (cores with a spherical or highly disordered core morphology). Core morphology data for the wt preparation were derived from the data set collected for and described by Mattei et al (28).…”

“Coarse-grained simulation reveals key features of HIV-1 capsid self-assembly”

“…Molecular crowding effects are known to influence protein behaviours 29 and yet, remarkably, virion maturation consistently produces similar capsid morphologies. The relatively fast maturation process 30,31 makes in vivo study of capsid self--assembly pathways difficult. Nonetheless, Woodward et al 32 show capsid formation proceeding via a hook--shaped precursor to the broad end of the capsid, in contrast with models of capsid assembly that begin with the narrow end of the cone 33 .…”