The Requirement For US28 During Cytomegalovirus Latency Is Independent Of US27 And US29 Gene Expression

Abstract

<jats:title>Abstract</jats:title><jats:p>The ability to establish a latent infection with periodic reactivation events ensures herpesviruses, like human cytomegalovirus (HCMV), lifelong infection and serial passage. The host-pathogen relationship throughout HCMV latency is complex, though both cellular and viral factors influence the equilibrium between latent and lytic infection. We and others have shown one of the viral-encoded G protein-coupled receptors, US28, is required for HCMV latency. US28 potentiates signals both constitutively and in response to ligand binding, and we previously showed deletion of the ligand binding domain or mutation of the G protein-coupling domain results in the failure to maintain latency similar to deletion of the entire US28 open reading frame (ORF). Interestingly, a recent publication detailed an altered phenotype from that previously reported, showing US28 is required for viral reactivation rather than latency, suggesting the US28 ORF deletion impacts transcription of the surrounding genes. Here, we show an independently generated US28-stop mutant, like the US28 ORF deletion mutant, fails to maintain latency in hematopoietic cells. Further, we found <jats:italic>US27</jats:italic> and <jats:italic>US29</jats:italic> transcription in each of these mutants was comparable to their expression during wild type infection, suggesting neither US28 mutant alters mRNA levels of the surrounding genes. Finally, infection with a US28 ORF deletion virus expressed US27 protein comparable to its expression following wild type infection. In sum, our new data strongly support previous findings from our lab and others, detailing a requirement for US28 during HCMV latent infection.</jats:p>

Herpesviral Latency—Common Themes

“…US28 is a chemokine receptor (vGPCR)-that has been detected in HCMV latency [93,[100][101][102]. It was shown to affect differentiation of infected cells and to be required for latency establishment in myeloid progenitors [102][103][104][105]. US28 affects several signaling pathways, which results in silencing of the HCMV major immediate early promoter (MIEP) [103].…”

Virion-associated US28 rapidly modulates Akt activity to suppress HCMV lytic replication in monocytes

“…US28 is essential for latency/quiescence in CD34+ HPCs (12,14,15,17), THP1 cells (10,15,35), Kasumi-3 cells (12,14,15), and monocytes (10,36,37); thus, we sought to determine the mechanism through which US28 promotes the establishment of a quiescent infection within monocytes. Herein, we demonstrate that virion-associated US28 is necessary and sufficient to dampen EGFR signaling to limit Akt activity triggered by HCMV entry.…”

“…HCMV G protein-coupled receptor (GPCR) US28 is found within mature virions (12) and expressed during latent (10,(12)(13)(14)(15)(16)(17)(18)(19) as well as lytic infection (e.g. refs (20)(21)(22)).…”

“…US28 also modulates multiple cellular pathways during lytic infection, including calcium signaling (20,21), FAK/Src (29), PLC (20,30), COX-2 (31), STAT3 (32), Akt, ERK1/2, eNOS (33), beta-catenin (34). Importantly, US28 is essential for viral latency/quiescence in cells of the myeloid lineage, including CD34+ HPCs (12,14,15,17), THP1 cells (10,15,35), Kasumi-3 cells (12,14,15), and monocytes (10,36,37). During latency, US28-mediated signaling silences the major immediate-early promoter (MIEP) (10,12,14,15), a key promoter in the latent-to-lytic switch that regulates the expression of immediate-early 1 (IE1) and immediate-early 2 (IE2) viral proteins, encoded by UL123 and UL122, respectively.…”

“…Importantly, US28 is essential for viral latency/quiescence in cells of the myeloid lineage, including CD34+ HPCs (12,14,15,17), THP1 cells (10,15,35), Kasumi-3 cells (12,14,15), and monocytes (10,36,37). During latency, US28-mediated signaling silences the major immediate-early promoter (MIEP) (10,12,14,15), a key promoter in the latent-to-lytic switch that regulates the expression of immediate-early 1 (IE1) and immediate-early 2 (IE2) viral proteins, encoded by UL123 and UL122, respectively. In Kasumi-3 cells and CD34+ HPCs, US28 attenuates cellular fos (c-fos) to prevent AP-1 transcription factor-dependent activation of the MIEP (15).…”