Generation of infectious virus particles by transient co-expression of human immunodeficiency virus type 1 gag mutants.

Abstract

We have demonstrated that COS7 cells transiently co-expressing myristylation-defective (Myr N ) and protease-defective (PR N ) human immunodeficiency virus (HIV) mutants can release infectious virions when co-transfected with an amphotropic murine leukaemia virus envelope protein expression plasmid (SV-A-MLV-env). In contrast, no infectious virions were detected when a PR N , noninfectious HIV gag mutant was co-expressed with the Myr N mutant, although the Myr N mutant could still process the immature core particles in trans. This result indicates that generation of functionally normal Gag proteins is required for virus infectivity in our complementation system. A mutant with a 56-amino-acid deletion in the N-terminal region of the capsid (CA) domain could still complement the PR N mutant to generate infectious virions, suggesting that the deletion mutant could provide a functional protease for processing in the PR N mutant. This result is consistent with the concept that mutations within the N-terminal region of the CA domain have no major effects on Gag-Pol incorporation into particles.

Human Cytomegalovirus Exploits ESCRT Machinery in the Process of Virion Maturation

“…Viral progeny is released within 48 to 72 h from CMV-infected cells (44), reducing the likelihood that nonspecific or long-term toxicity of DN-ESCRT proteins would impact our analysis. This assay has been effectively used earlier for both immediate-early gene (54) and late gene (2, 62) mutants, and similar complementation assay results have been reported in diverse systems (8,49,73). This assay further provided an opportunity to determine when inhibition occurred relative to the viral replication cycle.…”

A Single Amino Acid Substitution in HIV-1 Reverse Transcriptase Significantly Reduces Virion Release

“…For infection, 10 g of wild-type (wt) or mutant HIVgpt was cotransfected with 5 g of the vesicular stomatitis virus G (VSV-G) protein expression vector pHCMV-G (5). HeLa cell infection and drug-resistant colony selection were performed as previously described (8). Numbers of drug-resistant colonies were converted into titers (CFU/ml).…”

Mutations in the α-helix Directly C-terminal to the Major Homology Region of Human Immunodeficiency Virus Type 1 Capsid Protein Disrupt Gag Multimerization and Markedly Impair Virus Particle Production

“…The PR-mediated processing of virus particles is not required for virus particle assembly, but is essential for virus infectivity [8][9][10][11]. Besides PR, the enzymes required for virus replication are also encoded by pol.…”