AN ACTIVE-SITE MUTATION IN THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROTEINASE (PR) CAUSES REDUCED PR ACTIVITY AND LOSS OF PR-MEDIATED CYTOTOXICITY WITHOUT APPARENT EFFECT ON VIRUS MATURATION AND INFECTIVITY
J. Konvalinka et al., AN ACTIVE-SITE MUTATION IN THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROTEINASE (PR) CAUSES REDUCED PR ACTIVITY AND LOSS OF PR-MEDIATED CYTOTOXICITY WITHOUT APPARENT EFFECT ON VIRUS MATURATION AND INFECTIVITY, Journal of virology, 69(11), 1995, pp. 7180-7186
Infectious retrovirus particles are derived from structural polyprotei
ns which are cleaved by the viral proteinase (PR) during virion morpho
genesis. Besides cleaving viral polyproteins, which is essential for i
nfectivity, PR of human immunodeficiency virus (HIV) also cleaves cell
ular proteins and PR expression causes a pronounced cytotoxic effect,
Retroviral PRs are aspartic proteases and contain two copies of the tr
iplet Asp-Thr-GLy in the active center with the threonine adjacent to
the catalytic aspartic acid presumed to have an important structural r
ole, We have changed this threonine in HIV type 1 PR to a serine, The
purified mutant enzyme had an approximately 5- to l0-fold Lower activi
ty against HIV type 1 polyprotein and peptide substrates compared with
the wild-type enzyme. It did not induce toxicity on bacterial express
ion and yielded significantly reduced cleavage of cytoskeletal protein
s in vitro. Cleavage of vimentin in mutant-infected T-cell lines was a
lso markedly reduced, Mutant virus did, however, elicit productive inf
ection of several T-cell lines and of primary human lymphocytes with n
o significant difference in polyprotein cleavage and with similar infe
ction kinetics and titer compared with wild-type virus, The discrepanc
y between reduced processing in vitro and normal virion maturation can
be explained by the observation that reduced activity was due to an i
ncrease in K-m which may not be relevant at the high substrate concent
ration in the virus particle, This mutation enables us therefore to di
ssociate the essential function of PR in viral maturation from its cyt
otoxic effect.