Latency-Associated Expression of Human Cytomegalovirus US28 Attenuates Cell Signaling Pathways To Maintain Latent Infection

Abstract

Reactivation of human cytomegalovirus (HCMV) latent infection from early myeloid lineage cells constitutes a threat to immunocompromised or immune-suppressed individuals. Consequently, understanding the control of latency and reactivation to allow targeting and killing of latently infected cells could have far-reaching clinical benefits. US28 is one of the few viral genes that is expressed during latency and encodes a cell surface G protein-coupled receptor (GPCR), which, during lytic infection, is a constitutive cell-signaling activator. Here we now show that in monocytes, which are recognized sites of HCMV latency in vivo, US28 attenuates multiple cell signaling pathways, including mitogen-activated protein (MAP) kinase and NF-κB, and that this is required to establish a latent infection; viruses deleted for US28 initiate a lytic infection in infected monocytes. We also show that these monocytes then become potent targets for the HCMV-specific host immune response and that latently infected cells treated with an inverse agonist of US28 also reactivate lytic infection and similarly become immune targets. Consequently, we suggest that the use of inhibitors of US28 could be a novel immunotherapeutic strategy to reactivate the latent viral reservoir, allowing it to be targeted by preexisting HCMV-specific T cells.

Interferon-responsive genes are targeted during the establishment of human cytomegalovirus latency

“…US28-mediated signalling is critical to latency: US28-deleted viruses, or the loss of G-protein coupling by the US28 mutant R129A, result in lytic infection of undifferentiated myeloid cells 19,21,23,25 . Similarly, infection of US28-WT-expressing THP-1 cells with US28-deletion viruses leads to complementation and the establishment of latency; both the US28-R129A and empty vector cell lines fail to establish latent infection under these conditions 21 . Therefore, to understand how US28-WT alters the host cell environment to support latency, we analysed the total proteomes of myelomonocytic THP-1 cell lines which express either US28-WT (sequence derived from the VHL/E strain of HCMV), signalling mutant US28-R129A, or the empty vector (EV) control (Figures 1A, 1B, 1C, File S5).…”

“…The lentiviral vectors encoding US28 from the VHL/E strain of HCMV have been described previously 21 ; US28 is expressed in these vectors via the SFFV promoter. The lentiviral vectors pHRSIN UbEm and pHRsin SV40blast were a kind gift from D. van den Boomen, University of Cambridge, and were based upon a previously published lentiviral system 44,45 .…”

“…Furthermore, when examined, these US28-mediated effects on cell signalling did not occur during lytic infection or in permissive cells 21 , implying that US28 represses the MIEP during latency but does not impair reactivation following cellular differentiation. This is reflective of the cell type-specific nature of US28-mediated signalling 21,26 .…”

“…One viral factor that suppresses MIEP activity is the G-protein coupled receptor (GPCR) US28, a virally encoded chemokine receptor homologue, which is expressed de novo during latency as well as being delivered to cells with the incoming virion 19–24 and this incoming viral US28 is functional 25 . US28 modulates the signalling pathways of early myeloid cells; it attenuates MAP kinases, NF-κB, and c-fos, whilst activating STAT3 and iNOS 21,23,25 . All of these contribute to the repression of MIEP activity.…”

“…This US28-mediated signalling is so critical to latency that US28-deleted viruses, or the loss of G-protein coupling by the US28 mutant R129A, result in lytic infection of undifferentiated myeloid cells 19,21,23,25 . Furthermore, when examined, these US28-mediated effects on cell signalling did not occur during lytic infection or in permissive cells 21 , implying that US28 represses the MIEP during latency but does not impair reactivation following cellular differentiation. This is reflective of the cell type-specific nature of US28-mediated signalling 21,26 .…”