Reversible S-nitrosation and inhibition of HIV-1 protease.
Nitric oxide (*NO) is a short-lived free radical with many functions including vasoregulation, synaptic plasticity, and immune modulation and has recently been associated with AIDS pathology. Various pathophysiological conditions, such as viral infection, trigger inducible nitric oxide synthase (iNOS) to synthesize NO in the cell. NO-derived species can react with thiols of proteins and form nitrosothiol adducts. HIV-1 protease (HIV-PR) contains two cysteine residues, Cys67 and Cys95, which are believed to serve a regulatory function. We have found that HIV-PR is inactivated by nitric oxide produced in vitro by NO donors and by iNOS. Sodium nitroprusside inhibited HIV-PR by 70%, and S-nitroso-N-acetylpenicillamine completely inhibited the enzyme. Furthermore, iNOS generated sufficient NO to inhibit HIV-PR activity by almost 90%. This inactivation was reversed by the addition of reducing agents. Treatment of HIV-PR with NO donors and ritonavir (a competitive peptide inhibitor) indicates that NO exerts its effect through a site independent of the active site of HIV-PR. Using electrospray ionization mass spectrometry, we found that NO forms S-nitrosothiols on Cys67 and Cys95 of HIV-PR which directly correlate with a loss of activity. These data indicate that NO may suppress HIV-1 replication by directly inhibiting HIV-PR.
Sehajpal
P K
PK
Department of Biochemistry, Weill Medical College of Cornell University, New York 10021, USA.
Basu
A
A
Ogiste
J S
JS
Lander
H M
HM
D001667:Binding, Competitive; D004789:Enzyme Activation / Q000187:drug effects; D005033:Ethylmaleimide / Q000737:chemistry; D016333:HIV Protease / Q000737:chemistry / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D009566:Nitrates / Q000737:chemistry / Q000378:metabolism*; D009569:Nitric Oxide / Q000737:chemistry; D015538:Nitrosation; D009603:Nitroso Compounds / Q000737:chemistry / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism
D017320:HIV Protease Inhibitors; D009566:Nitrates; D009603:Nitroso Compounds; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D009569:Nitric Oxide; D016333:HIV Protease; D005033:Ethylmaleimide