Site-Directed Mutagenesis of HIV-1 Protease: Generation of Mutant Proteases with Increased Stability to Autodigestion

Tomasselli, Alfredo G.; Mildner, Ana M.; Rothrock, Donna J.; Sarcich, Jean L.; Lull, June M.; Leone, Joseph W.; Heinrikson, Robert L.

doi:10.1007/978-1-4615-1871-6_62

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“…KS is the most common cancer in AIDS patients, and the viruses responsible for both of these devastating diseases, although structurally unrelated, share similar functional regulatory mechanisms. HIV protease is also regulated by dimerization and contains inactivating internal autolysis sites ( − ). Regulation of HIV protease is necessary for virion formation.…”

Section: Discussionmentioning

confidence: 99%

One Functional Switch Mediates Reversible and Irreversible Inactivation of a Herpesvirus Protease

et al. 2006

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Distinct mechanisms have evolved to regulate the function of proteolytic enzymes. Viral proteases in particular have developed novel regulatory mechanisms, presumably due to their comparatively rapid life cycles and responses to constant evolutionary pressure. Herpesviruses are a family of human pathogens that require a viral protease with a concentration-dependent zymogen activation involving folding of two R-helices and activation of the catalytic machinery, which results in formation of infectious virions. Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) is unique among the herpesvirus proteases in possessing an autolysis site in the dimer interface, which removes the carboxyl-terminal 27 amino acids comprising an R-helix adjacent to the active site. Truncation results in the irreversible loss of dimerization and concomitant inactivation. We characterized the conformational and functional differences between the active dimer, inactive monomer, and inactive truncated protease to determine the different protease regulatory mechanisms that control the KSHV lytic cycle. Circular dichroism revealed a loss of 31% R-helicity upon dimer dissociation. Comparison of the full-length and truncated monomers by NMR showed differences in 21% of the protein structure, mainly located adjacent to the dimer interface, with little perturbation of the overall protein upon truncation. Fluorescence polarization and active site labeling, with a transition state mimetic, characterized the functional effects of these conformational changes. Substrate turnover is abolished in both the full-length and truncated monomers; however, substrate binding remained intact. Disruption of the helix 6 interaction with the active site oxyanion loop is therefore used in two independent regulatory mechanisms of proteolytic activity.The nine known human herpesviruses (HHVs) 1 are responsible for diseases ranging from herpes labialis, caused by Herpes simplex virus-1, to Kaposi's sarcoma (KS), caused by Kaposi's sarcoma-associated herpesvirus (KSHV, HHV8) (1-3). These nine HHVs are grouped into three families, based on phenotypic and genomic similarities. Although the R-, the -, and the γ-herpesviruses (including KSHV) vary in tissue distribution and disease states, all herpesviruses possess homologous structural and enzymatic proteins involved in their lytic cycles. Among these proteins are the protease (Pr) and assembly protein (AP), which are necessary for infectious virion formation (4-8). Pr is expressed as a Pr/AP fusion (9-13), AP binds the major capsid protein, and the complex translocates to the nucleus where the immature capsid is assembled around the AP scaffold (14). Pr cleaves the Pr/AP fusion at the release site (R-site) and the maturation site (M-site) (10,11,13,15,16), allowing DNA packaging, formation of the infectious virion, and completion of the lytic cycle. Additional internal cleavage sites have been identified in multiple herpesvirus proteases; however, KSHV Pr is unique in that autolysis at the D-site (dimer d...

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Section: Discussionmentioning

confidence: 99%