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. 2014 Mar 12;6(3):1202-18.
doi: 10.3390/v6031202.

Human cytomegalovirus US28 facilitates cell-to-cell viral dissemination

Vanessa M Noriega  1 , Thomas J Gardner  2 , Veronika Redmann  3 , Gerold Bongers  4 , Sergio A Lira  5 , Domenico Tortorella  6
Affiliations

Human cytomegalovirus US28 facilitates cell-to-cell viral dissemination

Vanessa M Noriega et al. Viruses. .

Abstract

Human cytomegalovirus (HCMV) encodes a number of viral proteins with homology to cellular G protein-coupled receptors (GPCRs). These viral GPCRs, including US27, US28, UL33, and UL78, have been ascribed numerous functions during infection, including activating diverse cellular pathways, binding to immunomodulatory chemokines, and impacting virus dissemination. To investigate the role of US28 during virus infection, two variants of the clinical isolate TB40/E were generated: TB40/E-US28(YFP) expressing a C-terminal yellow fluorescent protein tag, and TB40/E-FLAG(YFP) in which a FLAG-YFP cassette replaces the US28 coding region. The TB40/E-US28(YFP) protein localized as large perinuclear fluorescent structures at late times post-infection in fibroblasts, endothelial, and epithelial cells. Interestingly, US28(YFP) is a non-glycosylated membrane protein throughout the course of infection. US28 appears to impact cell-to-cell spread of virus, as the DUS28 virus (TB40/E-FLAG(YFP)) generated a log-greater yield of extracellular progeny whose spread could be significantly neutralized in fibroblasts. Most strikingly, in epithelial cells, where dissemination of virus occurs exclusively by the cell-to-cell route, TB40/E-FLAG(YFP) (DUS28) displayed a significant growth defect. The data demonstrates that HCMV US28 may contribute at a late stage of the viral life cycle to cell-to-cell dissemination of virus.

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Figures

Figure 1
Figure 1
Generation of TB40/E-US28 variants. (a) Using a bacterial artificial chromosome (BAC) recombineering approach Human cytomegalovirus (HCMV) TB40/E variants were generated that express either chimeric US28 containing a carboxy-terminal YFP tag (US28YFP) or a US28 deletion mutant where the US28 ORF has been replaced with an engineered FLAG-YFP cassette (FLAGYFP). YFP sequences are denoted by the diagonally hatched box; FLAG sequences are denoted by the horizontally striped box. TR, terminal repeat; U, unique sequences; IR, inverted repeat; L, long; S, short. (b) Fibroblasts mock-infected or infected (MOI = 5) with TB40/E wt or TB40/E-US28 variants were harvested 48 hours post-infection and subjected to RT-PCR with primers specific to US28 (lanes 1–5) or β-actin (lanes 6–10). A sample lacking RNA ((−)RNA) was included as a negative control. HCMV US28, β-actin, and relative DNA standards are indicated. (c) Fibroblasts mock-infected or infected (MOI = 5) with TB40/E-US28YFP or TB40/E-FLAGYFP were harvested at the indicated times and subjected to SDS-PAGE and immunoblot analysis. US28YFP, FLAGYFP, GAPDH, and molecular weight standards are indicated. (d) Fibroblasts infected (MOI = 5) with TB40/E wt, TB40/E-US28YFP or TB40/E-FLAGYFP were harvested 48 hours post-infection and visualized using the EVOS Cell Imaging Systems at 60× magnification.
Figure 2
Figure 2
Formation of US28-expressing structures late during HCMV infection. (a) Confocal microscopy was performed on fibroblasts either mock-infected or infected with TB40/E-US28YFP (MOI = 5). At various times post-infection cells were fixed and analyzed using an ImageXpress Ultra plate-scanning confocal microscope. (b) Endothelial and epithelial cells were infected with either TB40/E-US28YFP (left) or -FLAGYFP (right) (MOI = 25) and visualized at 4 days post-infection using the EVOS Cell Imaging Systems at 60× magnification.
Figure 3
Figure 3
HCMV US28 is a non-glycosylated membrane protein incorporated into infected cells. Fibroblasts mock-infected or infected (MOI = 5) with either TB40/E-US28YFP or TB40/E-FLAGYFP were subjected to subcellular fractionation at 72 (a) or 24 (b) hours post-infection. Cell pellets from the 15,000 and 120,000 × g centrifugations (15 k × g pellet, 120 k × g pellet) and the 120,000 × g supernatant (120 k × g supe) were resolved by SDS-PAGE and subjected to immunoblot analysis. US28YFP, FLAGYFP, gB, and molecular weight standards are indicated. gB* indicates the mature form of glycoprotein B. (c) Fibroblasts infected with TB40/E-US28YFP (MOI = 5) were harvested at the indicated time points and left non-treated (NT) or treated with EndoH (H) or PNGaseF (F). US28YFP, glycosylated MHC class I heavy chains (HC(+)CHO), deglycosylated MHC class I heavy chains (HC(−)CHO), GAPDH, and molecular weight standards are indicated.
Figure 4
Figure 4
HCMV US28 modulates cell-to-cell spread of virus. Infectious extracellular progeny from fibroblasts infected at 5 PFU/cell (a) or 0.1 PFU/cell (b) with TB40/E wt, TB40/E-US28YFP, or TB40/E-FLAGYFP were measured by TCID50 assay. Viral titers were assayed in duplicate. Error bars represent standard deviation of the mean. (c) ARPE-19 cells were infected at 0.1 PFU/cell with TB40/E-US28YFP or TB40/E-FLAGYFP and at the indicated times post-infection cell-associated virus was determined by TCID50 assay. Viral titers were assayed in duplicate. Error bars represent standard deviation of the mean.
Figure 5
Figure 5
TB40/E ΔUS28 displays a growth defect when required to use the cell-to-cell route of dissemination. Fibroblasts infected (MOI = 0.01) with either TB40/E-US28YFP or TB40/E-FLAGYFP were cultured in the presence of the HCMV neutralizing antibody 14-4b. Two weeks post-infection cells were analyzed by fluorescent microplate reader (a) or by fluorescence microscopy (b). For (a) YFP fluorescence was assayed in triplicate. Error bars represent standard deviation of the mean; * p < 0.05, Student’s one tailed T-test.
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