V3 recombinants indicate a central role for CCR5 as a coreceptor in tissue infection by human immunodeficiency virus type 1

Abstract

Binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 to both CD4 and one of several chemokine receptors (coreceptors) permits entry of virus into target cells. Infection of tissues may establish latent viral reservoirs as well as cause direct pathologic effects that manifest as clinical disease such as HIV-associated dementia. We sought to identify the critical coreceptors recognized by HIV-1 tissue-derived strains as well as to correlate these coreceptor preferences with site of infection and dementia diagnosis. To reconstitute coreceptor use, we cloned HIV-1 envelope V3 sequences encoding the primary determinants of coreceptor specificity from 13 brain-derived and 6 colon-derived viruses into an isogenic (NL4-3) viral background. All V3 recombinants utilized the chemokine receptor CCR5 uniformly and efficiently as a coreceptor but not CXCR4, BOB/GPR15, or Bonzo/STRL33. Other receptors such as CCR3, CCR8, and US28 were inefficiently and variably used as coreceptors by various envelopes. CCR5 without CD4 present did not allow for detectable infection by any of the tested recombinants. In contrast to the pathogenic switch in coreceptor specificity frequently observed in comparisons of blood-derived viruses early after HIV-1 seroconversion and after onset of AIDS, the characteristics of these V3 recombinants suggest that CCR5 is a primary coreceptor for brain- and colon-derived viruses regardless of tissue source or diagnosis of dementia. Therefore, tissue infection may not depend significantly on viral envelope quasispeciation to broaden coreceptor range but rather selects for CCR5 use throughout disease progression.

FIG. 1

Three groups of V3 loop amino acid sequences, isolated by postmortem PCR from brain tissue and colon tissue of AIDS patients, show homology to a macrophage-tropic consensus sequence (11) deduced from blood-borne strains. A period denotes identity with the consensus, while a dash denotes deletion. Bullets denote V3 positions 13 and 25 in the consensus sequence. Sequences of colon-derived viruses and demented brain-derived virus (5-1) were isolated from three AIDS patients (27). Remaining brain-derived virus sequences were isolated from 11 AIDS patients diagnosed with HIVD (76). Recombinant virus 59-1 was previously termed 59-3. Recombinant viruses 26-4 and 26-5 (previously termed 26-1 and 26-2, respectively) have identical V3 sequences as noted above but vary at gp120 position 335 in the C3 region, as described elsewhere (76).

FIG. 2

Efficient and uniform use of CCR5 but not CXCR4 as a coreceptor by all V3 recombinant viruses. HIV-1 YU-2 (CCR5 dependent) and NL4-3 (CXCR4 dependent) were used as controls. Displayed values are representative of day 8 supernatant p24 concentrations seen in two separate infections performed with these recombinants.

FIG. 3

Variable use of CCR3, CCR8, and US28 as coreceptors by all V3 recombinant viruses compared to use of CCR5. (A) Comparison of infections by pseudotype V3 recombinant viruses of COS-7 cells transiently expressing CCR5 with and without CD4. No tested recombinant displayed infection mediated by CCR5 in the absence of CD4. (B) Infection of COS-7 cells transiently expressing CD4 and CCR3. Recombinant virus 10-1 displayed no detectable infection mediated by CCR3. (C) Infection of COS-7 cells transiently expressing CD4 and CCR8. Recombinant viruses 17-2, 54-1, 15-2, 19-1, 59-1, and 4-9 displayed no detectable infection mediated by CCR8. (D) Infection of COS-7 cells transiently expressing CD4 and US28. Recombinant viruses 10-1, 54-1, 15-2, 19-1, 59-1, and 4-9 displayed no detectable infection mediated by US28. In all studies, Luc⁺ pseudotype HIV-1 carrying the ADA envelope was used as a positive control. Background readings were obtained from infection of COS-7 cells transiently expressing CD4 without coreceptor and ranged from 0 to 5,000 light units/mg of protein. Displayed values were normalized by subtraction of background signals, and they are representative of the relative ratios of coreceptor specificity seen in two to three separate infections performed with Luc⁺ pseudotype recombinant viruses.