DEFINING THE LEVEL OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 (HIV-1) PROTEASE ACTIVITY REQUIRED FOR HIV-1 PARTICLE MATURATION AND INFECTIVITY

The human immunodeficiency virus type 1 (HIV-1) protease is the enzyme required for processing of the Gag and Gag-Pol polyproteins to yield mature, infectious virions. Although the complete absence of proteolyt ic activity prevents maturation, the level of activity sufficient for maturation and subsequent. infectivity has not been determined. Amino acid substitutions that reduce Catalytic activity without affecting su bstrate recognition have been engineered into the active site of the H IV-1 protease. The catalytic efficiency (k(cat)) of the HIV-1 protease is decreased 4-fold when threonine 26 is replaced by serine (T26S) an d approximately 50-fold when alanine 28 is replaced by serine (A28S). Genes containing these mutations were cloned into a proviral vector fo r analysis of their effects on virion maturation and infectivity. The results show that virions containing the T26S protease variant, in whi ch only 25% of the protease is active, are very similar to wild-type v irions, although slight reductions in infectivity are observed. Virion s containing the A28S protease variant are not infectious, even though a limited amount of polyprotein processing does occur. There appears to be a linear correlation between the level of protease activity and particle infectivity. Our observations suggest that a threshold of pro tease activity exists between a 4-fold and 50 fold reduction, below wh ich processing is insufficient to yield infectious particles. Our data also suggest that a reduction of protease activity by 50-fold or grea ter is sufficient to prevent the formation of infectious particles.