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.
Highlights
G protein-coupled receptors (GPCRs) act as key regulators of numerous cellular processes via transmitting the response of various signal molecules like chemokines, hormones, or neurotransmitters.it is not surprising that viruses have hijacked mammalian GPCRs during coevolution to ensure efficient viral propagation [1,2]
To analyze the intracellular virally encoded GPCRs (vGPCRs) localization, receptors were transiently expressed in HeLa cells, either alone or in combination
We demonstrate that co-localization of pUL78 signal with that of EEA1 is not restricted to the 48 h post infection time point, but occurs throughout the Human cytomegalovirus (HCMV) replication cycle in human foreskin fibroblasts (HFFs) (Figure 5, right panel) as well as in ARPE-19 cells
Summary
It is not surprising that viruses have hijacked mammalian GPCRs during coevolution to ensure efficient viral propagation [1,2]. Thereby, virally encoded GPCRs (vGPCRs) illustrate an effective means to bypass the immune system, modulate cellular functions, and redirect cellular signaling networks [3]. Human cytomegalovirus (HCMV) encodes four GPCR homologs, termed pUS27, pUS28, pUL33, and pUL78 [4,5]. CMVs, pUL33, and pUL78 are highly conserved among all betaherpesviruses. While their expression is not crucial for virus replication in cell culture [6,7], a deletion leads to a significantly diminished pathogenesis in animal experiments [8,9]
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