Defining the level of human immunodeficiency virus type 1 (HIV-1) protease activity required for HIV-1 particle maturation and infectivity

Abstract

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 (k cat ) 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.

Systematic mutational analysis of the active‐site threonine of HIV‐1 proteinase: Rethinking the “fireman's grip” hypothesis

“…Taken together, these observations suggest that the Thr r Ser exchange, while maintaining the essential hydrogen bond network of the "fireman's grip," may play an important role in regulating PR activity. This hypothesis is supported by studies concerning a variant of HIV-1 PR with a Thr26 r Ser substitution~Konvalinka et al, 1995b;Rose et al, 1995!. Kinetic analysis of this enzyme showed an eightfold reduction in activity at neutral or slightly acidic pH when compared to wild-type PR.…”

Intracellular expression of human immunodeficiency virus type 1 (HIV-1) protease variants inhibits replication of wild-type and protease inhibitor-resistant HIV-1 strains in human T-cell lines

“…The HIV-gpt plasmid has been described previously (27). Engineered mutations were introduced into the PR gene by site-directed mutagenesis as described previously (32). The frameshift constructions were made with the oligonucleotide 5Ј-CAGGCTAATTTTATTAGGGAAGATCT -3Ј to insert the nucleotide A (in bold) in the phagemid pSPRD25N/KWW at position 1635 of the viral genome.…”

“…Cell culture and transfections. Transient 293T transfections, isolation of viral particles, quantification of p24 CA protein by p24 ELISA, and Western blot (immunoblot) analysis were carried out as previously described (2,22,32).…”

Placement of Leucine Zipper Motifs at the Carboxyl Terminus of HIV-1 Protease Significantly Reduces Virion Production

“…Confluent 293T cells were trypsinized, split 1:10 and seeded onto 10-cm dish plates 24 hours before transfection. For each construct, 293T cells were transfected with 20 mg of plasmid DNA by the calcium phosphate precipitation method (18), with the addition of 50 mM chloroquine to enhance transfection efficiency.…”

“…One proposal is that interaction among Pr160 gagpol molecules triggers the activation of embedded PR, which in homodimeric form mediates Gag and Gag-Pol cleavage following PR autocleavage from Pr160 gag-pol . Maintenance of the Pr55 gag / Pr160 gag-pol expression ratio is critical to virus assembly; the artificial overexpression of Pr160 gag-pol or PR drastically reduces virion production as a result of enhanced Gag processing by overexpressed PR activity [14,15,16,17,18,19,20]. Equally important is the Pr160 gag-pol sequence and structure, since sequence mutations upstream or downstream of PR often result in defective virus maturation or Gag cleavage [4,21,22,23,24].…”