Mutational Analysis of a Native Substrate of the HIV-1 Proteinase
Proteolytic processing of the gag/pol precursor by the human immunodeficiency virus (HIV) type 1 proteinase (PR) is essential for production of infectious viral particles. Although the sites of viral specific cleavages have been determined, the primary amino acid sequence around these scissile bonds are heterogeneous, and the determinants that direct the cleavage specificity exhibited by HIV-1 PR remain largely undefined. We performed mutational analysis of the Tyr/Pro site which produces the amino terminus of the capsid protein (CA) and the Phe/Pro site which produces the amino terminus of PR. Single-amino-acid substitutions were made, and their effects on proteolytic processing were examined by in vitro translation of a synthetic mRNA encoding both the mutated substrate and PR as a truncated gag/pol precursor. We found evidence for sequence determinants of cleavage at the Tyr/Pro site, and structural determinants of cleavage at the Phe/Pro site. We next investigated the role of the polyprotein structure in either impeding or facilitating cleavage at Phe/Pro. Deletion of p6*, the region in pol upstream of PR, resulted in improved processing of the gag cleavage sites. Our results suggest that p6* is involved in the regulation of PR activity and that release of p6* inhibition may be an activation step necessary for infectious particle maturation.
KeywordsHuman Immunodeficiency Virus Human Immunodeficiency Virus Type Amino Terminus Proteolytic Processing Equine Infectious Anemia Virus
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