A Direct Interaction with RNA Dramatically Enhances the Catalytic Activity of the HIV-1 Protease In Vitro

Abstract

Though the steps of human immunodeficiency virus (HIV)-1 virion maturation are well documented, the mechanisms regulating the proteolysis of the Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) remain obscure. One proposed mechanism argues that the maturation intermediate p15NC must interact with RNA for efficient cleavage by the PR. We investigated this phenomenon and found processing of multiple substrates by the HIV-1 PR was enhanced in the presence of RNA. The acceleration of proteolysis occurred independently from the substrate's ability to interact with nucleic acid, indicating that a direct interaction between substrate and RNA is not necessary for enhancement. Gel-shift assays demonstrated the HIV-1 PR is capable of interacting with nucleic acids, suggesting RNA accelerates processing reactions by interacting with the PR rather than the substrate. All HIV-1 PRs examined have this ability; however, the HIV-2 PR does not interact with RNA, and does not exhibit enhanced catalytic activity in the presence of RNA. No specific sequence or structure was required in the RNA for a productive interaction with the HIV-1 PR, which appears to be principally, though not exclusively, driven by electrostatic forces. For a peptide substrate, RNA increased the kinetic efficiency of the HIV-1 PR by an order of magnitude, affecting both the turnover rate (kcat) and substrate affinity (Km). These results suggest an allosteric binding site exists on the HIV-1 PR, and that HIV-1 PR activity during maturation could be regulated in part by the juxtaposition of the enzyme with virion-packaged RNA.

An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation

“…We observed that ELISA and Western blot can give differing results for Gag protein levels in the presence of RNA binding agents or RNA structural deletion mutants. These findings resonate with the known effects of RNA on structural stability of HIV virions [ 35 ] and may possibly relate to effects of RNA on Gag processing [ 36 ]. This observation has implications for investigation of viral processes that involve different structural changes in Gag and suggests that results should be validated by Western blots, or by alternative analyses relying on quantification of denatured proteins rather than relying only on methods detecting the native state.…”

Effect of tRNA on the Maturation of HIV-1 Reverse Transcriptase

“…3a). Since both p66/p66 dimer formation and PR activity may be influenced by ionic strength 36; 37 , and tRNA may act as a polyanion 38–40 , we investigated the impact of different salt concentrations on p66 processing. In the absence of tRNA, at physiological NaCl concentrations (50–150 mM), aberrant non-specific cleavage products (Fig.…”

Coordination of Genomic RNA Packaging with Viral Assembly in HIV-1

“…This finding suggests that packaging of gRNA is important to ensure that the majority of particles adopt a mature, infectious form. It has also recently been demonstrated in vitro that the activity of the HIV-1 protease, itself, is markedly increased in the presence of RNA, although, curiously, the same effect is not observed for the HIV-2 protease [ 106 ].…”