Adaptation of a CCR5-using, primary human immunodeficiency virus type 1 isolate for CD4-independent replication

Abstract

The gp120 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4 and chemokine receptors on the target cell. Primary, clinical HIV-1 isolates require interaction with CD4 to allow gp120 to bind the CCR5 chemokine receptor efficiently. We adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative canine cells expressing human CCR5. The gp120 changes responsible for the adaptation were limited to alteration of glycosylation addition sites in the V2 loop-V1-V2 stem. The gp120 glycoproteins of the adapted viruses bound CCR5 directly, without prior interaction with CD4. Thus, a major function of CD4 binding in the entry of primary HIV-1 isolates can be bypassed by changes in the gp120 V1-V2 elements, which allow the envelope glycoproteins to assume a conformation competent for CCR5 binding.

FIG. 1

Replication of adapted viruses in CD4-negative and CD4-positive cells expressing CCR5. Virus production in the supernatants of Cf2Th-CCR5 (A) or Cf2Th-CD4/CCR5 (B) cells following infection by the ADA, ADA-P1, or ADA-P2 virus is shown. The results of a single experiment are shown. The experiment was repeated with similar results.

FIG. 2

Envelope glycoprotein changes observed for the ADA-P1 and ADA-P2 viruses. The organization of the HIV-1 envelope glycoproteins is shown, with the signal peptide (S) and transmembrane region (T) indicated. The gp120 regions conserved among different viral strains are black, and the variable regions are white and labeled. The sequences shown are of the wild-type (w.t.) ADA, ADA-P1, and ADA-P2 envelope glycoproteins in the two regions in which differences were observed. In the wild-type ADA sequence, the relevant residues are underlined and numbered according to the prototypic HXBc2 sequence (37). In the characterized ADA-P1 and ADA-P2 clones, the altered residue is in boldface. The observed frequency of each sequence is indicated at the right. Sites of N-linked glycosylation are indicated by circles atop vertical lines.

FIG. 3

Influence of envelope glycoprotein changes on infection of CD4-negative and CD4-positive cells. An env-deficient, CAT-expressing HIV-1 isolate was complemented by the wild-type (w.t.) ADA or mutant envelope glycoproteins. The envelope glycoproteins exhibit either the VQ, EQ, or ER sequence at gp41 positions 539 and 540, as indicated. CAT activity observed for equivalent amounts of cell lysates derived from Cf2Th-CCR5 (A) or Cf2Th-CD4/CCR5 (B) cells incubated with the recombinant virions is shown. The mean values derived from three independent experiments are shown.

FIG. 4

CCR5-binding ability of gp120 glycoproteins from the adapted virus. The binding of radiolabeled, soluble gp120 glycoproteins to Cf2Th-synCCR5 cells, in the absence or presence of 6 μg of sCD4 per ml, is shown. The gp120 glycoproteins were also incubated with Cf2Th cells not expressing CCR5, in the presence of 6 μg of sCD4 per ml. Equivalent levels of all three gp120 glycoproteins were incubated with the cells, as described in Materials and Methods. w.t., wild type.