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Journal of Virology, July 2001, p. 5998-6006, Vol. 75, No. 13
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.13.5998-6006.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

N-Linked Glycosylation Sites Adjacent to and within the V1/V2 and the V3 Loops of Dualtropic Human Immunodeficiency Virus Type 1 Isolate DH12 gp120 Affect Coreceptor Usage and Cellular Tropism

Robert A. Ogert,Dagger Myung K. Lee,dagger William Ross, Alicia Buckler-White, Malcolm A. Martin, and Michael W. Cho*

Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0460

Received 29 January 2001/Accepted 10 April 2001

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is extensively glycosylated, containing approximately 23 asparagine (N)-linked glycosylation sites on its gp120 subunit. In this study, specific glycosylation sites on gp120 of a dualtropic primary HIV-1 isolate, DH12, were eliminated by site-directed mutagenesis and the properties of the resulting mutant envelopes were evaluated using a recombinant vaccinia virus-based cell-to-cell fusion assay alone or in the context of viral infections. Of the glycosylation sites that were evaluated, those proximal to the V1/V2 loops (N135, N141, N156, N160) and the V3 loops (N301) of gp120 were functionally critical. The glycosylation site mutations near the V1/V2 loop compromised the use of CCR5 and CXCR4 equally. In contrast, a mutation within the V3 loop preferentially inhibited the usage of CCR5; although this mutant protein completely lost its CCR5-dependent fusion activity, it retained 50% of the wild-type fusion activity with CXCR4. The replication of a virus containing this mutation was severely compromised in peripheral blood mononuclear cells, MT-4 cells, and primary monocyte-derived macrophages. A revertant virus, which acquired second site changes in the V3 loop that resulted in an increase in net positive charge, was isolated. The revertant virus fully recovered the usage of CXCR4 but not of CCR5, thereby altering the tropism of the parental virus from dualtropic to T-tropic. These results suggest that carbohydrate moieties near the V1/V2 and the V3 loops play critical roles in maintaining proper conformation of the variable loops for optimal interaction with receptors. Our results, combined with those of previously reported studies, further demonstrate that the function of individual glycans may be virus isolate dependent.

* Corresponding author. Mailing address: Laboratory of Molecular Microbiology, NIAID, NIH, 9000 Rockville Pike, Bldg. 4, Rm. 339, Bethesda, MD 20892-0460. Phone: (301) 496-0576. Fax: (301) 402-0226. E-mail: mcho{at}nih.gov.

dagger Present address: Protein Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-600, Republic of Korea.

Dagger Present address: Purdue BioPharma, Biologics Discovery, Princeton, NJ 08540.

Journal of Virology, July 2001, p. 5998-6006, Vol. 75, No. 13
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.13.5998-6006.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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