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Journal of Virology, April 2001, p. 3435-3443, Vol. 75, No. 7
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.7.3435-3443.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Loss of a Single N-Linked Glycan Allows CD4-Independent Human
Immunodeficiency Virus Type 1 Infection by Altering the Position of
the gp120 V1/V2 Variable Loops
Peter
Kolchinsky,1
Enko
Kiprilov,1
Peter
Bartley,1,
Roee
Rubinstein,1 and
Joseph
Sodroski1,2,*
Department of Cancer Immunology and AIDS,
Dana-Farber Cancer Institute, and Department of Pathology, Harvard
Medical School,1 and Department of
Immunology and Infectious Diseases, Harvard School of Public
Health,2 Boston, Massachusetts 02115
Received 8 June 2000/Accepted 5 January 2001
The gp120 envelope glycoprotein of primary human immunodeficiency
virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4
and the CCR5 chemokine receptor on the target cell. Previously, we
adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative
canine cells expressing human CCR5. The gp120 changes responsible for
CD4-independent replication were limited to the V2 loop-V1/V2 stem.
Here we show that elimination of a single glycosylation site at
asparagine 197 in the V1/V2 stem is sufficient for CD4-independent
gp120 binding to CCR5 and for HIV-1 entry into CD4-negative cells
expressing CCR5. Deletion of the V1/V2 loops also allowed
CD4-independent viral entry and gp120 binding to CCR5. The binding of
the wild-type ADA gp120 to CCR5 was less dependent upon CD4 at 4°C
than at 37°C. In the absence of the V1/V2 loops, neither removal of
the N-linked carbohydrate at asparagine 197 nor lowering of the
temperature increased the CD4-independent phenotypes. A CCR5-binding
conformation of gp120, achieved by CD4 interaction or by modification
of temperature, glycosylation, or variable loops, was preferentially
recognized by the monoclonal antibody 48d. These results suggest that
the CCR5-binding region of gp120 is occluded by the V1/V2 variable loops, the position of which can be modulated by temperature, CD4
binding, or an N-linked glycan in the V1/V2 stem.
*
Corresponding author. Mailing address: Dana-Farber
Cancer Institute, 44 Binney St., FB 824, Boston, MA 02115. Phone: (617) 632-3371. Fax: (617) 632-4338. E-mail:
joseph_sodroski{at}dfci.harvard.edu.

Present address: University of Glasgow, Glasgow,
Scotland.
Journal of Virology, April 2001, p. 3435-3443, Vol. 75, No. 7
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.7.3435-3443.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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