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. 1998 Aug;72(8):6389-97.
doi: 10.1128/JVI.72.8.6389-6397.1998.

The cytomegalovirus-encoded chemokine receptor US28 can enhance cell-cell fusion mediated by different viral proteins

O Pleskoff  1 , C Tréboute, M Alizon
Affiliations

The cytomegalovirus-encoded chemokine receptor US28 can enhance cell-cell fusion mediated by different viral proteins

O Pleskoff et al. J Virol. 1998 Aug.

Abstract

The human cytomegalovirus (CMV) US28 gene encodes a functional CC chemokine receptor. However, this activity was observed in cells transfected to express US28 and might not correspond to the actual role of the protein in the CMV life cycle. Expression of US28 allows human immunodeficiency virus type 1 (HIV-1) entry into certain CD4(+) cells and their fusion with cells expressing HIV-1 envelope (Env) proteins. Such properties were initially reported for the cellular chemokine receptors CCR5 and CXCR4, which behave as CD4-associated HIV-1 coreceptors. We found that coexpression of US28 and either CXCR4 or CCR5 in CD4(+) cells resulted in enhanced synctium formation with HIV-1 Env+ cells. This positive effect of US28 on cell fusion seems to be distinct from its HIV-1 coreceptor activity. Indeed, enhancement of cell fusion was also observed when US28 was expressed on the HIV-1 Env+ cells instead of an CD4(+) target cells. Furthermore, US28 could enhance cell fusion mediated by other viral proteins, in particular, the G protein of vesicular stomatitis virus (VSV-G). The HIV-1 coreceptor and fusion-enhancing activities could be affected by mutations in different domains of US28. The fusion-enhancing activity of US28 seems to be cell type dependent. Indeed, cells coexpressing VSV-G and US28 fused more efficiently with human, simian, or feline target cells, while US28 had no apparent effect on fusion with the three mouse or rat cell lines tested. The positive effect of US28 on cell fusion might therefore require its interaction with a cell-specific factor. We discuss a possible role for US28 in the fusion of the CMV envelope with target cells and CMV entry.

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Figures

FIG. 1
FIG. 1
Enhancement of HIV-1 Env-mediated cell-cell fusion by US28. HeLa- CD4-LTRlacZ cells (A and B) or U373MG-CD4-LTRlacZ cells (C and D) were transfected with Rc/CMV (Mock) or with CXCR4, CCR5, and US28 expression vectors, as indicated. Cocultures were performed with HeLa cells stably expressing Tat and Env from cell line-adapted HIV-1LAI (A and C) or from M-tropic HIV-1ADA (B and D). Transfections were performed in six-well trays with 4 μg of vector, or with 2 μg of each vector when chemokine receptors were coexpressed. Cells from a subconfluent well were detached with trypsin 24 h posttransfection. Half of them were seeded with an equivalent number of Env+ cells in one well from a 12-well tray. Cells were fixed and stained with X-Gal after a 24-h coculture. Bars represent mean numbers (with standard deviations) of blue-stained foci, indicating cell fusion events, in duplicate wells.
FIG. 2
FIG. 2
Flow cytometry analysis of cell surface markers in HeLa P4 cells transfected with different chemokine receptor expression vectors. (A) Surface expression of CD4, MHC-1, or CXCR4 after transfection with two vectors, i.e., EGFP-N1 (GFP expression vector) and either Rc/CMV-US28, Rc/CMV-CCR5, Rc/CMV-CCR1, or Rc/CMV (control), in a 1:6 ratio. (B) Surface expression of CCR5 after transfection with three vectors, i.e., EGFP-N1, Rc/CMV-CCR5, and either Rc/CMV-US28, Rc/CMV-CXCR4, or Rc/CMV (control), in a 1:2:2 ratio. Cells were stained with PE-coupled antibodies (red fluorescence) 36 h after transfection and analyzed as indicated in Materials and Methods. The graphs show red fluorescence intensity (x axis, arbitrary units, log scale) and numbers of cells (y axis) among GFP-positive cells (transfected cells). Thick lines, transfections with chemokine receptor expression vectors; thin lines and gray areas, control transfections with Rc/CMV.
FIG. 3
FIG. 3
Coexpression of US28 and viral fusiogenic proteins. HeLa-Tat cells were cotransfected with expression vector for HIV-1LAI Env (A), HTLV-1 Env (C), or VSV-G (D) and with either Rc/CMV (mock), Rc/CMV-CCR5, or Rc/CMV-US28, as indicated. Each of these Rc/CMV vectors was also transfected in cells stably expressing Env from HIV-1ADA (B). Cocultures (six-well trays) were initiated 24 h later by adding an equivalent number of HeLa P4 cells (A, C, and D) or their CCR5+ derivatives, HeLa P5 cells (B). Cells were fixed and stained with X-Gal after a 24-h coculture. Bars represent mean numbers (with standard deviations) of blue-stained foci in triplicate wells.
FIG. 4
FIG. 4
Coexpression of VSV-G and chemokine receptors or virally encoded GCRs. Transfections of HeLa-Tat cells with expression vectors for VSV-G and for the indicated GCRs and cocultures with HeLa P4 cells were performed as described for Fig. 3. US27 and UL33 are putative GCRs encoded by human CMV; M33 is a putative GCR from MCMV. Bars represent mean numbers (with standard deviations) of blue-stained foci in triplicate wells.
FIG. 5
FIG. 5
Cell type restriction of the fusion-enhancing activity of US28. HeLa P4 cells were cotransfected in six-well trays with expression vectors for VSV-G and for either US28, CCR5, or CCR1. An equivalent number of the indicated target cells, stably expressing HIV-1 Tat or transiently transfected with Rc/CMV-Tat (CrFK), was added 24 h later. Cells were fixed and stained with X-Gal after a 24-h coculture. Bars represent mean numbers (with standard deviations) of blue-stained foci in triplicate wells.
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