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. 2014 Feb 10;6(2):661-82.
doi: 10.3390/v6020661.

Intracellular trafficking of the human cytomegalovirus-encoded 7-trans-membrane protein homologs pUS27 and pUL78 during viral infection: a comparative analysis

Ina Niemann  1 , Anna Reichel  2 , Thomas Stamminger  3
Affiliations

Intracellular trafficking of the human cytomegalovirus-encoded 7-trans-membrane protein homologs pUS27 and pUL78 during viral infection: a comparative analysis

Ina Niemann et al. Viruses. .

Abstract

Human cytomegalovirus (HCMV) encodes four G protein-coupled receptor (GPCR) homologs, termed pUS27, pUS28, pUL33, and pUL78. In contrast to the extensively characterized vGPCRs pUS28 and pUL33, knowledge concerning pUS27 and pUL78 is limited. Previous studies already demonstrated constitutive internalization of pUS27 and pUL78, as well as an association with the endosomal machinery, however, these results were mainly obtained using transiently transfected cells. To explore the subcellular localization of both receptors during viral infection, we constructed recombinant HCMVs expressing tagged vGPCRs. Colocalization analyses revealed a predominant association of pUS27 or pUL78 with the trans-Golgi network or the endoplasmic reticulum, respectively. Intriguingly, our data emphasize that protein sorting is highly regulated by viral functions as we detected dramatic changes in the colocalization of pUS27 and pUL78 with endosomal markers during progression of HCMV replication. Furthermore, we observed cell type-dependent differences in trafficking of both vGPCRs between fibroblasts and epithelial cells. Most importantly, infection experiments with a recombinant HCMV carrying tagged versions of pUS27 and pUL78 simultaneously, revealed that these two proteins do not colocalize during viral infection. This contrasts to results of transient expression experiments. In conclusion, our results highlight the importance to investigate vGPCR trafficking in a viral context.

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Figures

Figure 1
Figure 1
Colocalization analysis of FLAG-US27 and UL78-GFP in transiently transfected HeLa cells. HeLa cells were transfected with 1 μg of an expression plasmid encoding N-terminally tagged wild-type TB40/E FLAG-US27 (b), C-terminally tagged wild-type TB40/E UL78-GFP (c), or both plasmids together (d). pcDNA3 was used as a negative control (a). Cells were fixed 24 h post transfection and permeabilized with Triton X-100. The primary antibody mAb-FLAG in combination with a secondary anti-mouse antibody coupled to Alexa-555 were used to detect pUS27. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI).
Figure 2
Figure 2
Construction and characterization of recombinant HCMVs expressing tagged US27 and/or UL78. (a) Schematic illustration of the US27 and UL78 genomic loci of HCMV strain TB40/E. Recombinant viruses were generated with C-terminal EYFP (US27 or UL78, upper panel) or N-terminal FLAG-tag (US27, lower panel); (b) Growth kinetics of recombinant TB40/E-derived viruses. HFF cells were seeded and infected three days later with wild-type and recombinant viruses at an MOI of 0.1. Virus supernatant samples were harvested at indicated times (dpi, days post infection) and digested with proteinase K. Viral DNA was quantitated via Taqman PCR to determine the release of viral particles from infected cells. Each infection was performed in triplicate, and the standard deviations are shown.
Figure 3
Figure 3
Subcellular localization of US27-EYFP and UL78-EYFP in infected HFFs and ARPE-19 cells. HFF (left and middle panel) or ARPE-19 cells (right panel) were infected with recombinant TB40/E viruses (MOI: 0.5 or 1) expressing fusion proteins of pUS27 (left panel) or pUL78 (middle and right panel) with EYFP and fixed at different time points during HCMV infection (6–96 hpi). Cells were stained with primary antibody mAb-IE1 (p63-27), and a secondary anti-mouse antibody coupled to Alexa-555. Cell nuclei were stained with DAPI.
Figure 4
Figure 4
Colocalization of US27-EYFP and UL78-EYFP with TGN46 and calreticulin in infected HFFs. HFFs were infected with recombinant TB40/E viruses (MOI: 0.5) expressing fusion proteins of pUS27 (left panel) or pUL78 (right panel) with EYFP and fixed at different time points during HCMV infection (24–96 hpi). Cells were stained with primary antibody (a) pAb-TGN46, diluted 1:300, or (b) pAb-Calreticulin (PA3-900), diluted 1:100, and the respective secondary anti-sheep antibody or anti-rabbit antibody coupled to Alexa-555 (1:400). Cell nuclei were stained with DAPI.
Figure 5
Figure 5
EEA1 staining of US27-EYFP and UL78-EYFP infected HFFs and ARPE-19 cells. HFF (left and right panel) or ARPE-19 cells (middle panel) were infected with recombinant TB40/E viruses (MOI: 0.5 or 1) expressing fusion proteins of pUS27 (left and middle panel) or pUL78 (right panel) with EYFP and fixed at different time points during HCMV infection (24–96 hpi). Cells were stained with primary antibody pAb-EEA1 (H-300), diluted 1:200, and the secondary anti-rabbit antibody coupled to Alexa-555 (1:400). Cell nuclei were stained with DAPI.
Figure 6
Figure 6
CD63 and LAMP1 staining of US27-EYFP and UL78-EYFP infected HFFs and ARPE-19. HFF (left and middle panel) or ARPE-19 cells (right panel) were infected with recombinant TB40/E viruses (MOI: 0.5 or 1) expressing fusion proteins of pUS27 (left panel) or pUL78 (middle and right panel) with EYFP and fixed at different time points during HCMV infection (24–96 hpi). Cells were stained with primary antibody (a) mAb-CD63 (MX-49.129.5) or (b) mAb-LAMP-1 (H5G11), each diluted 1:50, and the secondary anti-mouse antibody coupled to Alexa-555 (1:400). Cell nuclei were stained with DAPI.
Figure 7
Figure 7
Colocalization analysis of Flag-US27 and UL78-EYFP in infected HFFs and ARPE-19 cells. HFF (left panel) or ARPE-19 cells (right panel) were infected with a recombinant TB40/E virus (MOI: 0.5 or 1) expressing fusion proteins of both pUS27 (red) and pUL78 (green) with Flag or EYFP, respectively, and fixed at different time points during HCMV infection (24–96 hpi). Cells were stained with primary antibody mAb-Flag, diluted 1:400, in combination with the secondary anti-mouse antibody coupled to Alexa-555 (1:400) in order to detect pUS27. Cell nuclei were stained with DAPI.
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