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.
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 degrees C than at 37 degrees 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.
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
P
P
Kolchinsky
E
E
Kiprilov
P
P
Bartley
R
R
Rubinstein
J
J
Sodroski
D000911:Antibodies, Monoclonal / Q000276:immunology; D015704:CD4 Antigens / Q000502:physiology*; D002460:Cell Line; D006031:Glycosylation; D015699:HIV Envelope Protein gp120 / Q000737:chemistry* / Q000276:immunology / Q000502:physiology; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D019713:Receptors, CCR5 / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D019713:Receptors, CCR5