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 proteolytic 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 substrate recognition have been engineered into the active site of the HIV-1 protease. The catalytic efficiency (kcat) of the HIV-1 protease is decreased 4-fold when threonine 26 is replaced by serine (T26S) and approximately 50-fold when alanine 28 is replaced by serine (A28S). Genes containing these mutations were cloned into a proviral vector for analysis of their effects on virion maturation and infectivity. The results show that virions containing the T26S protease variant, in which only 25% of the protease is active, are very similar to wild-type virions, although slight reductions in infectivity are observed. Virions 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 protease activity exists between a 4-fold and 50-fold reduction, below which processing is insufficient to yield infectious particles. Our data also suggest that a reduction of protease activity by 50-fold or greater is sufficient to prevent the formation of infectious particles.
Rosé
J R
JR
Department of Pharmacology, University of California, San Francisco 94143-0446, USA.
Babé
L M
LM
Craik
C S
CS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D015683:Gene Products, gag / Q000378:metabolism; D015684:Gene Products, pol / Q000378:metabolism; D016333:HIV Protease / Q000235:genetics / Q000378:metabolism*; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology* / Q000254:growth & development / Q000472:pathogenicity; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014774:Virulence / Q000235:genetics
D004279:DNA, Viral; D015683:Gene Products, gag; D015684:Gene Products, pol; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016333:HIV Protease