HIV-1 protease (PR) mediates the proteolytic processing of computer virus particles

HIV-1 protease (PR) mediates the proteolytic processing of computer virus particles during or after computer virus budding. the PR activation process. Here we demonstrate that due to enhanced Gag cleavage efficiency W402 alanine or leucine substitution significantly reduces BRL-49653 computer virus production. However W402 replacement with phenylalanine does not significantly affect computer virus particle assembly or processing nonetheless it will markedly impair viral infectivity within a single-cycle infections assay. Our outcomes demonstrate a one amino acidity substitution at HIV-1 RT can radically have an effect on pathogen assembly by improving Gag cleavage performance suggesting that furthermore to adding to RT natural function through the first stages of pathogen replication the HIV-1 RT tryptophan do it again motif within a Gag-Pol framework may play a significant function in suppressing the early activation of PR during late-stage pathogen replication. In the past due stage of individual immunodeficiency pathogen type 1 (HIV-1) replication a large number of viral capsid precursor (Pr55is cleaved by viral protease (PR) into four main items: matrix (MA) (p17) capsid (CA) BRL-49653 (p24) nucleocapsid (NC) (p7) as well as the C-terminal p6 area (42). PR is usually encoded by is usually incorporated into assembling virions via conversation with Pr55through its N-terminal Gag determinants (10 11 19 22 40 41 However some researchers have exhibited that HIV-1 and murine leukemia computer virus (MLV) Pol can be packaged into virions at a reasonable efficiency despite an absence of Gag-Pol formation (3 7 The proteolytic processing of Pr160gives rise to PR reverse transcriptase BRL-49653 (RT) and integrase (IN) in addition to Gag cleavage products. Blocking PR activity does not significantly affect computer virus particle assembly BRL-49653 and release but it does eliminate viral infectivity (17 27 36 The reading frame of HIV-1 partially overlaps that of to Pr55(24). An artificial overexpression of Pr160or PR-containing chimeric proteins results in a significant reduction in virion release presumably due to the premature cleavage of Pr55as mediated by PR (1 20 28 35 38 39 48 Accordingly both the PR expression level and PR activation timing with respect to the proteolytic processing of Gag and Gag-Pol are crucial to computer virus assembly. The molecular mechanism behind PR activation is not entirely obvious. It is generally accepted that Pr160dimerization or multimerization triggers PR activation and therefore sequences upstream or downstream of PR may impact PR-mediated computer virus maturation by interfering with Gag-Pol multimerization. Consistent with this suggestion deletions of sequences upstream of PR (11 52 or mutations in downstream sequences can significantly affect PR-directed computer virus particle maturation (4 30 37 Biologically active RT is usually assumed to be present in the form of a p66/p51 heterodimer (12 31 A hydrophobic cluster consisting of six tryptophan (Trp) residues has been identified in the connection subdomain of the HIV-1 RT subunit (codons 398 to 414). This Trp repeat motif is highly conserved among primate lentiviral reverse transcriptases (2). It has been shown that substitution mutations of HIV-1 RT Trp repeat motif residues can markedly impair RT dimerization in vitro (33 44 Rabbit polyclonal to CCNB1. suggesting a motif role in RT-RT interactions. Although the extent to which the RT domain name contributes to Gag-Pol multimerization is usually unknown some data support the idea that this RT sequence (the BRL-49653 Trp repeat motif in particular) may impact PR activation by favoring Gag-Pol multimerization. First RT truncation mutations involving the Trp repeat motif significantly impair PR-mediated Gag processing (30). Second efavirenz (EFV)-a nonnucleoside reverse transcriptase inhibitor (NNRTI) that greatly enhances HIV-1 RT dimerization in vitro (46 47 capable of suppressing virion production by enhancing the efficiency of PR-mediated Gag and Gag-Pol cleavage (14 45 However a W401A alanine substitution mutation that abrogates RT dimerization in vitro (49) almost completely negates the EFV inhibitory effect on virion production (9). The lack of W401A susceptibility to this inhibitory effect is most likely due to a defect in Gag-Pol/Gag-Pol conversation that impedes the.