Elimination of retroviral infectivity by N-ethylmaleimide with preservation of functional envelope glycoproteins.
The zinc finger motifs in retroviral nucleocapsid (NC) proteins are essential for viral replication. Disruption of these Cys-X2-Cys-X4-His-X4-Cys zinc-binding structures eliminates infectivity. To determine if N-ethylmaleimide (NEM) can inactivate human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) preparations by alkylating cysteines of NC zinc fingers, we treated infectious virus with NEM and evaluated inactivation of infectivity in cell-based assays. Inactivation was rapid and proportional to the NEM concentration. NEM treatment of HIV-1 or SIV resulted in extensive covalent modification of NC and other internal virion proteins. In contrast, viral envelope glycoproteins, in which the cysteines are disulfide bonded, remained intact and functional, as assayed by high-performance liquid chromatography, fusion-from-without analyses, and dendritic cell capture. Quantitative PCR assays for reverse transcription intermediates showed that NEM and 2,2'-dipyridyl disulfide (aldrithiol-2), a reagent which inactivates retroviruses through oxidation of cysteines in internal virion proteins such as NC, blocked HIV-1 reverse transcription prior to the formation of minus-strand strong-stop products. However, the reverse transcriptase from NEM-treated virions remained active in exogenous template assays, consistent with a role for NC in reverse transcription. Since disruption of NC zinc finger structures by NEM blocks early postentry steps in the retroviral infection cycle, virus preparations with modified NC proteins may be useful as vaccine immunogens and probes of the role of NC in viral replication.
Morcock
David R
DR
AIDS Vaccine Program, SAIC Frederick, NCI-Frederick, Building 535, 5th Floor, PO Box B, Frederick, MD 21702, USA.
Thomas
James A
JA
Gagliardi
Tracy D
TD
Gorelick
Robert J
RJ
Roser
J David
JD
Chertova
Elena N
EN
Bess
Julian W
JW
Ott
David E
DE
Sattentau
Quentin J
QJ
Frank
Ines
I
Pope
Melissa
M
Lifson
Jeffrey D
JD
Henderson
Louis E
LE
Crise
Bruce J
BJ
D002460:Cell Line; D003713:Dendritic Cells / Q000166:cytology / Q000821:virology; D005033:Ethylmaleimide / Q000494:pharmacology*; D015686:Gene Products, env / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects* / Q000472:pathogenicity*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D019251:Nucleocapsid / Q000378:metabolism; D015302:Simian Immunodeficiency Virus / Q000187:drug effects* / Q000472:pathogenicity*; D016335:Zinc Fingers
D015686:Gene Products, env; D005033:Ethylmaleimide