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Journal of Virology, May 2003, p. 5547-5556, Vol. 77, No. 10
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.10.5547-5556.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Elimination of Protease Activity Restores Efficient Virion Production to a Human Immunodeficiency Virus Type 1 Nucleocapsid Deletion Mutant

David E. Ott,1* Lori V. Coren,1 Elena N. Chertova,1 Tracy D. Gagliardi,1 Kunio Nagashima,2 Raymond C. Sowder II,1 Dexter T. K. Poon,1 and Robert J. Gorelick1

AIDS Vaccine Program,1 Research Technology Program, SAIC-Frederick Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702-12012

Received 25 October 2002/ Accepted 11 February 2003

The nucleocapsid (NC) region of human immunodeficiency virus type 1 (HIV-1) Gag is required for specific genomic RNA packaging. To determine if NC is absolutely required for virion formation, we deleted all but seven amino acids from NC in a full-length NL4-3 proviral clone. This construct, DelNC, produced approximately four- to sixfold fewer virions than did the wild type, and these virions were noninfectious (less than 10-6 relative to the wild type) and severely genomic RNA deficient. Immunoblot and high-pressure liquid chromatography analyses showed that all of the mature Gag proteins except NC were present in the mutant virion preparations, although there was a modest decrease in Gag processing. DelNC virions had lower densities and were more heterogeneous than wild-type particles, consistent with a defect in the interaction assembly or I domain. Electron microscopy showed that the DelNC virions displayed a variety of aberrant morphological forms. Inactivating the protease activity of DelNC by mutation or protease inhibitor treatment restored virion production to wild-type levels. DelNC-protease mutants formed immature-appearing particles that were as dense as wild-type virions without incorporating genomic RNA. Therefore, protease activity combined with the absence of NC causes the defect in DelNC virion production, suggesting that premature processing of Gag during assembly causes this effect. These results show that HIV-1 can form particles efficiently without NC.

* Corresponding author. Mailing address: AIDS Vaccine Program, SAIC-Frederick Inc., National Cancer Institute at Frederick, Frederick, MD 21702-1201. Phone: (301) 846-5723. Fax: (301) 846-5588. E-mail: ott{at}ncifcrf.gov.

Journal of Virology, May 2003, p. 5547-5556, Vol. 77, No. 10
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.10.5547-5556.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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