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

Abstract

12Human cytomegalovirus (HCMV) latency is an active process which remodels the latently infected 13 cell to optimise latent carriage and reactivation. This is achieved, in part, through the expression of 14 viral genes, including the G-protein coupled receptor US28. Here, we use an unbiased proteomic 15 screen to assess changes in host proteins induced by US28, revealing that interferon-inducible genes 16 are downregulated by US28. We validate that MHC Class II and two PYHIN proteins, MNDA and 17 IFI16, are downregulated during experimental latency in primary human CD14 + monocytes. We find 18 that IFI16 is targeted rapidly during the establishment of latency in a US28-dependent manner, but 19 only in undifferentiated myeloid cells, a natural site of latent carriage. Finally, by overexpressing 20 IFI16, we show that IFI16 can activate the viral major immediate early promoter and immediate early 21 2 gene expression during latency via NF-κB, a function which explains why downregulation of IFI16 22 during latency is advantageous for the virus. 23 Importance 24 Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus which infects 50-100% of humans 25 worldwide. HCMV causes a lifelong subclinical infection in immunocompetent individuals, but is a 26 serious cause of mortality and morbidity in the immunocompromised and in neonates. In particular, 27 reactivation of HCMV in the transplant setting is a major cause of transplant failure and related 28 disease. Therefore, a molecular understanding of HCMV latency and reactivation could provide 29 insights into potential ways to target the latent viral reservoir in at-risk patient populations. 30 31 both host and viral factors 18 . One viral factor that suppresses MIEP activity is the G-protein coupled 46 receptor (GPCR) US28, a virally encoded chemokine receptor homologue, which is expressed de 47 novo during latency as well as being delivered to cells with the incoming virion 19-24 and this 48 incoming viral US28 is functional 25 . US28 modulates the signalling pathways of early myeloid cells; it 49 attenuates MAP kinases, NF-κB, and c-fos, whilst activating STAT3 and iNOS 21,23,25 . All of these 50 contribute to the repression of MIEP activity. This US28-mediated signalling is so critical to latency 51 that US28-deleted viruses, or the loss of G-protein coupling by the US28 mutant R129A, result in lytic 52 infection of undifferentiated myeloid cells 19,21,23,25 . Furthermore, when examined, these US28-53 mediated effects on cell signalling did not occur during lytic infection or in permissive cells 21 , 54 implying that US28 represses the MIEP during latency but does not impair reactivation following 55 cellular differentiation. This is reflective of the cell type-specific nature of US28-mediated signalling 56 21,26 . 57Since US28 can modulate all these pathways and control the MIEP, we hypothesised that US28 58 would also cause changes in host protein expression. Here, we perform a proteomic screen 59 comparing host cell protein abundance in myelomonocytic TH...

Understanding HCMV Latency Using Unbiased Proteomic Analyses

“…Some transcriptome analyses during HCMV latency have identified changes of genes involved in cell survival and immune regulation such as PEA-15 [ 23 ], which has since been validated and a mechanism defined [ 32 , 33 ]. Changes in MHC class II [ 23 ] during latency have also been identified and at least one mechanism for this change has been defined [ 34 , 35 ]. These studies have been informative but one way to obtain a more comprehensive understanding of changes in protein expression during latency is to analyse virally induced changes in the total proteome in an unbiased manner.…”

“…However, GFP expression can tend to wane over time and so alternative fluorescent tags to mark latently infected cells have also been derived such as SV40-mCherry or GATA2-mCherry in which mCherry is under the control of the GATA2 transcription factor, which is known to activate a number of viral latency-associated genes in undifferentiated myeloid cells. Proteomic studies on such cells have led to the identification of a number of cellular processes which are affected by HCMV latent infection of myeloid cells [ 35 , 40 , 41 ].…”

“…In all but one study [ 7 , 8 , 25 , 35 , 41 , 48 , 50 , 51 ], US28 has been shown to be essential for the establishment and maintenance of latency in the myeloid lineage. One reported mechanism by which US28 regulates latency involves activation of STAT3 in the iNOS and NO pathway [ 25 ].…”

“…Changes in the total cell proteome resulting from US28 expression in undifferentiated THP1 cells have also been analysed [ 35 ]. Of particular interest was the downregulation of a number of PYHIN family proteins by US28 which included MNDA (myeloid cell nuclear differentiation antigen) and IFI16 (Interferon-Inducible Protein 16).…”

Control of Cytokines in Latent Cytomegalovirus Infection

“…As noted, suppression of the MIEP is necessary to block the lytic cascade and allow the establishment of HCMV latency in myeloid cells. One factor known to mediate MIEP repression is US28 [ 18 ], a viral chemokine receptor that also suppresses the expression of ISGs during latency [ 19 ] (discussed below). Conversely, IE1 and IE2 play a role in thwarting IFNs early in lytic infection, which helps to delay the host antiviral immune response, allowing time for the establishment of latency and facilitating immune escape during reactivation.…”