These data indicate that enhanced polyprotein processing occurs in the absence of Gag and Gag-Pol localization to the plasma membrane as previously described for myr (-) mutants of HIV-1 [34]. The impact of potent NNRTIs on the inhibition of viral PIM-1 Inhibitor 2 particle production was observed at concentrations of inhibitor (5 M) that are two to three orders of magnitude higher than levels required to inhibit HIV-1 replication in cell culture based assays (0.001C0.084 M) [15,23,25]. cells. The increased polyprotein processing is consistent PIM-1 Inhibitor 2 with premature activation of the HIV-1 protease by NNRTI-enhanced Gag-Pol multimerization through the embedded RT sequence. These findings support the view that Gag-Pol multimerization is an important step in viral assembly and demonstrate that regulation of Gag-Pol/Gag-Pol interactions is a novel target for small molecule inhibitors of HIV-1 production. Furthermore, these drugs can serve as useful probes to further understand processes involved in HIV-1 particle assembly and maturation. Synopsis HIV-1 encodes reverse transcriptase (RT), an enzyme that is essential for disease replication. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are allosteric inhibitors of the HIV-1 RT. In HIV-1-infected cells NNRTIs block the RT-catalyzed synthesis of a double-stranded DNA copy of the viral genomic RNA, which is an early step in the disease life cycle. Potent NNRTIs have the novel feature of advertising the interaction between the two RT subunits. However, the importance of this effect on the inhibition of HIV-1 replication has not been defined. In this study, the authors display that potent NNRTIs block an additional step in the disease life cycle. NNRTIs increase the intracellular processing of viral polyproteins called Gag and Gag-Pol that communicate the HIV-1 structural proteins and viral enzymes. Enhanced polyprotein processing is associated with a decrease in viral particles released from NNRTI-treated cells. NNRTI enhanced polyprotein processing is likely due to the drug binding to RT, indicated as part of the Gag-Pol polyprotein and advertising the connection between independent Gag-Pol polyproteins. This prospects to premature activation of the Gag-Pol inlayed HIV-1 protease, resulting in a decrease in full-length viral polyproteins available for assembly and budding from your sponsor cell membrane. This study provides proof-of-concept that small molecules can modulate the relationships between Gag-Pol polyproteins and suggests a new target for the development of HIV-1 antiviral medicines. Intro The HIV-1 reverse transcriptase (RT) is Rabbit Polyclonal to TSC2 (phospho-Tyr1571) responsible for the conversion of the viral single-stranded genomic RNA into a double-stranded proviral DNA precursor. This process is catalyzed from the RNA- and DNA-dependent polymerase and ribonuclease H activities of the enzyme. HIV-1 RT is an asymmetric dimer that consists of a 66- (p66) and a p66-derived 51-kDa (p51) subunit [1]. The RT heterodimer is the biologically active form of the enzyme; monomeric subunits are devoid of polymerase activity [2,3]. The HIV-1 RT is definitely translated as part of a 160-kDa Gag-Pol polyprotein (Pr160open reading frame partially overlaps with and is translated by a ribosomal frameshifting mechanism, which occurs in one out of 20 Gag translation events [5]. This ensures the stringent maintenance of a 20:1 percentage of Gag to Gag-Pol that is important for viral assembly, replication, and the production of infectious virions [6]. During or subsequent to disease budding, the viral PR auto-activates and cleaves Gag and Gag-Pol into the structural and viral proteins, which results in the maturation of immature particles to form infectious virions [7]. While HIV-1 PR activation is definitely a critical step in the viral existence cycle, the processes required for PR activation in HIV-1-infected cells is not well defined [7,8]. It is thought that Gag-Pol multimerization during viral assembly prospects to activation of the HIV-1 PR by dimerization of PR areas on independent Gag-Pol polyproteins, followed by the autocatalytic cleavage and launch of a functionally active PR homodimer [7]. Although direct multimerization of Gag-Pol has not been shown biochemically, several domains within Gag-Pol have been shown to influence PR activation including areas that are proximal to the C- and N-termini of PR [9C13]. If Gag-Pol dimerizes, as expected, then HIV-1 RT, due to its size and propensity to dimerize, is likely to contribute to Gag-Pol dimerization and promote PR activation. In support of this notion, deletions or C-terminal truncations of the RT in PIM-1 Inhibitor 2 the context of Gag-Pol prospects to decreased control of Gag and Gag-Pol and impaired disease maturation [9,11,14]. Consequently, the proper rules of Gag and Gag-Pol processing is an essential step in the production of adult viral particles. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a chemically varied group of lipophilic compounds that comprise over 30 different classes and specifically inhibit HIV-1, but not HIV-2 RT [15]. NNRTIs bind to an allosteric pocket in the p66 subunit of the RT and inhibit DNA synthesis reactions by a.