Proteolytic processing of the P2/nucleocapsid cleavage site is critical for human immunodeficiency virus type 1 RNA dimer maturation

Differences in virion RNA dimer stability between mature and protease-defec tive (immature) forms of human immunodeficiency virus type 1 (HIV-1) sugges t that maturation of the viral RNA dimer is regulated by the proteolytic pr ocessing of the HIV-1 Gag and Gag-Pol precursor proteins. However, the prot eolytic processing of these proteins occurs in several steps denoted primar y, secondary, and tertiary cleavage events and, to date, the processing ste p associated with formation of stable HIV-1 RNA dimers has not been identif ied. We show here that a mutation in the primary cleavage site (p2/nucleoca psid [NC]) hinders formation of stable virion RNA dimers, while dimer stabi lity is unaffected by mutations in the secondary (matrix/capsid [CA], p1/p6 ) or a tertiary cleavage site (CA/p2). By introducing mutations in a shared cleavage site of either Gag or Gag-Pol, we also show that the cleavage of the p2/NC site in Gag is more important for dimer formation and stability t han p2/NC cleavage in Gag-Pol. Electron microscopy analysis of viral partic les shows that mutations in the primary cleavage site in Gag but not in Gag -Pol inhibit viral particle maturation. We conclude that virion RNA dimer m aturation is dependent on proteolytic processing of the primary cleavage si te and is associated with virion core formation.