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. 1998 May;72(5):4243-9.
doi: 10.1128/JVI.72.5.4243-4249.1998.

Chemokine receptor utilization by human immunodeficiency virus type 1 isolates that replicate in microglia

J T Shieh  1 , A V Albright, M Sharron, S Gartner, J Strizki, R W Doms, F González-Scarano
Affiliations

Chemokine receptor utilization by human immunodeficiency virus type 1 isolates that replicate in microglia

J T Shieh et al. J Virol. 1998 May.

Abstract

The role of human immunodeficiency virus (HIV) strain variability remains a key unanswered question in HIV dementia, a condition affecting around 20% of infected individuals. Several groups have shown that viruses within the central nervous system (CNS) of infected patients constitute an independently evolving subset of HIV strains. A potential explanation for the replication and sequestration of viruses within the CNS is the preferential use of certain chemokine receptors present in microglia. To determine the role of specific chemokine coreceptors in infection of adult microglial cells, we obtained a small panel of HIV type 1 brain isolates, as well as other HIV strains that replicate well in cultured microglial cells. These viruses and molecular clones of their envelopes were used in infections, in cell-to-cell fusion assays, and in the construction of pseudotypes. The results demonstrate the predominant use of CCR5, at least among the major coreceptors, with minor use of CCR3 and CXCR4 by some of the isolates or their envelope clones.

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Figures

FIG. 1
FIG. 1
Multinucleated giant cell formation in uninfected microglial cells (A) and in microglial cultures infected with HIV-1KJ-br (B), HIV-1DS-br (C), or HIV-1RC-br (D). Microglial cultures were infected as described in Materials and Methods, and syncytium formation was monitored 12 days after infection. Syncytium formation was also quantified by counting the number of nuclei and the number of cells and was consistent with these photomicrographs, which are representative of seven experiments with each of the illustrated isolates.
FIG. 2
FIG. 2
Use of chemokine coreceptors in a cell-to-cell fusion assay. (A) The env genes from isolates HIV-1BORI and HIV-1BORI-15 were PCR amplified and cloned in an expression vector as described in the text. Fusion assays were then performed with QT6 cells transiently expressing several chemokine coreceptors as previously described (13). Note that the CCR3 construct (CCR3P) was designed to increase surface expression. In this assay, cell-to-cell fusion results in expression of the luciferase gene, which is monitored by chemiluminescence (relative light units). Three HIV-1BORI-15 envelope clones [BORI-15 (4C), BORI-15 (5C), and BORI-15 (7A)] and one HIV-1BORI envelope clone [BORI (11A)] were used. Envelopes from HIV-1JRFL and HIV-1YU-2, two viruses obtained directly from the brain, were used as controls for expression of CCR3. The envelope genes from brain isolates DS-br and RC-br were amplified and cloned as described in the text, and a fusion assay was performed as described previously (13). The envelope from the dualtropic isolate HIV-189.6 was used as a control for expression of CCR3. These experiments are representative of two to three assays performed with these envelopes.
FIG. 3
FIG. 3
Western blot of selected env clones. Selected env clones from the HIV-1RC-br and HIV-1DS-br isolates were resolved by sodium dodecyl sulfate–7.5% polyacrylamide gel electrophoresis transferred to a polyvinylidene difluoride membrane, and probed with a polyclonal rabbit anti-gp120 serum followed by peroxidase-conjugated goat anti-rabbit serum, and the bound antibody was detected by enhanced chemiluminescence (Amersham). Recombinant gp120 prepared in a baculovirus system was used as a positive control (+ lane); the slight difference in mobility may be due to differences in glycosylation. “None” represents a lysate from cells transfected with pNL4-3-LucR+E− only. All of the env clones analyzed resulted in proteins that were processed into gp120.
FIG. 4
FIG. 4
Blocking microglial infection with antibodies against coreceptors. Monoclonal antibodies against CCR3, CCR5, and CXCR4 and a control anti-human herpesvirus 6 p41 antibody (see Materials and Methods for details) were used to block infection of cultured microglial cells with pseudotyped virus expressing the HIV-1BaL env. Three days after infection, the cells were lysed, and levels of chemiluminescence were determined and expressed as relative light units/10 s. The anti-CCR5 antibody was the only one that had an effect on infection.
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