Unconventional Sequence Requirement for Viral Late Gene Core Promoters of Murine Gammaherpesvirus 68

Expression of the human cytomegalovirus (HCMV) IE1 and IE2 proteins is critical for the establishment of lytic infection and reactivation from viral latency. Defining the mechanisms controlling IE1 and IE2 expression is therefore important for understanding how HCMV regulates its replicative cycle. Here we identify several novel transcripts encoding full-length IE1 and IE2 proteins during HCMV lytic replication. Two of the alternative major immediate early (MIE) transcripts initiate in the first intron, intron A, of the previously defined MIE transcript, while others extend the 5= untranslated region. Each of the MIE transcripts associates with polyribosomes in infected cells and therefore contributes to IE1 and IE2 protein levels. Surprisingly, deletion of the core promoter region of the major immediate early promoter (MIEP) from a plasmid containing the MIE genomic locus did not completely abrogate IE1 and IE2 expression. Instead, deletion of the MIEP core promoter resulted in increased expression of alternative MIE transcripts, suggesting that the MIEP suppresses the activity of the alternative MIE promoters. While the canonical MIE mRNA was the most abundant transcript at immediate early times, the novel MIE transcripts accumulated to levels equivalent to that of the known MIE transcript later in infection. Using two HCMV recombinants, we found that sequences in intron A of the previously defined MIE transcript are required for efficient IE1 and IE2 expression and viral replication. Together, our results identify new regulatory sequences controlling IE1 and IE2 expression and suggest that multiple transcription units act in concert to regulate IE1 and IE2 expression during lytic infection. IMPORTANCEThe HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and reactivation from viral latency. This study expands our understanding of the sequences controlling IE1 and IE2 expression by defining novel transcriptional units controlling the expression of full-length IE1 and IE2 proteins. Our results suggest that alternative promoters may allow for IE1 and IE2 expression when MIEP activity is limiting, as occurs in latently infected cells. The human cytomegalovirus (HCMV) IE1 and IE2 proteins are critical regulators of the viral replicative cycle. Both proteins are immediately expressed upon infection and together stimulate the expression of host and viral genes necessary for virus replication (1). IE2 acts as a general transcription factor that broadly transactivates host genes and viral early and late genes to facilitate virus replication (2-12). IE1 promotes transcription from the HCMV genome by inhibiting histone deactylases (HDACs) (13-15), which otherwise limit virus transcription by forming inhibitory chromatin structures on the viral genome. Reexpression of IE1 and IE2 is also thought to be critical for the reactivation of quiescent HCMV genomes from latent infection. Thus, understanding the regulatory mechanisms controlling IE1 and IE2 expression is im...

Section: Discussionmentioning

confidence: 73%

Multiple Transcripts Encode Full-Length Human Cytomegalovirus IE1 and IE2 Proteins during Lytic Infection

Arend

Ziehr

Vincent

et al. 2016

“…Murine gammaherpesvirus 68 (MHV-68), a member of the related Rhadinovirus (RHV) genus, contains approximately 80 ORFs, 80% of which are homologues to genes of KSHV (genus RHV) and EBV (genus LCV) (62). MHV-68 is also closely related in its biological function to both viruses (63). Despite this high similarity to LCVs, neither MHV-68 nor KSHV encodes a BILF1 receptor, neither at the expected genomic position nor elsewhere.…”

Section: Discussionmentioning

confidence: 99%

Identification and Functional Comparison of Seven-Transmembrane G-Protein-Coupled BILF1 Receptors in Recently Discovered Nonhuman Primate Lymphocryptoviruses

Spieß

Fares

Sparre-Ulrich

et al. 2015

Coevolution of herpesviruses with their respective host has resulted in a delicate balance between virus-encoded immune evasion mechanisms and host antiviral immunity. BILF1 encoded by human Epstein-Barr virus (EBV) is a 7-transmembrane (7TM) G-proteincoupled receptor (GPCR) with multiple immunomodulatory functions, including attenuation of PKR phosphorylation, activation of G-protein signaling, and downregulation of major histocompatibility complex (MHC) class I surface expression. In this study, we explored the evolutionary and functional relationships between BILF1 receptor family members from EBV and 12 previously uncharacterized nonhuman primate (NHP) lymphocryptoviruses (LCVs). Phylogenetic analysis defined 3 BILF1 clades, corresponding to LCVs of New World monkeys (clade A) or Old World monkeys and great apes (clades B and C). Common functional properties were suggested by a high degree of sequence conservation in functionally important regions of the BILF1 molecules. A subset of BILF1 receptors from EBV and LCVs from NHPs (chimpanzee, orangutan, marmoset, and siamang) were selected for multifunctional analysis. All receptors exhibited constitutive signaling activity via G protein G␣i and induced activation of the NF-B transcription factor. In contrast, only 3 of 5 were able to activate NFAT (nuclear factor of activated T cells); chimpanzee and orangutan BILF1 molecules were unable to activate NFAT. Similarly, although all receptors were internalized, BILF1 from the chimpanzee and orangutan displayed an altered cellular localization pattern with predominant cell surface expression. This study shows how biochemical characterization of functionally important orthologous viral proteins can be used to complement phylogenetic analysis to provide further insight into diverse microbial evolutionary relationships and immune evasion function. IMPORTANCE Epstein-Barr virus (EBV), known as an oncovirus, is the only human herpesvirus in the genus Lymphocryptovirus (LCV). EBV uses multiple strategies to hijack infected host cells, establish persistent infection in B cells, and evade antiviral immune responses. As part of EBV's immune evasion strategy, the virus encodes a multifunctional 7-transmembrane (7TM) G-protein-coupled receptor (GPCR), EBV BILF1. In addition to multiple immune evasion-associated functions, EBV BILF1 has transforming properties, which are linked to its high constitutive activity. We identified BILF1 receptor orthologues in 12 previously uncharacterized LCVs from nonhuman primates (NHPs) of Old and New World origin. As 7TM receptors are excellent drug targets, our unique insight into the molecular mechanism of action of the BILF1 family and into the evolution of primate LCVs may enable validation of EBV BILF1 as a drug target for EBV-mediated diseases, as well as facilitating the design of drugs targeting EBV BILF1.

“…Both BcRF1 and UL87 were postulated to be a potential viral TATA-binding protein (TBP) based on bioinformatics analysis (51). Recently, BcRF1 of EBV and MHV-68 ORF24 were both shown to be capable of binding the core promoter sequence of the viral late gene in vitro (52,53). Previously, we carried out extensive mutagenesis of the viral late promoters of MHV-68 and found that the viral TATA boxes have unconventional requirements for the TATA sequence, supporting a virus-specific recognition of the TATA box (53).…”

Section: Discussionmentioning

confidence: 99%

Kaposi's Sarcoma-Associated Herpesvirus ORF18 and ORF30 Are Essential for Late Gene Expression during Lytic Replication

Gong

Xie

et al. 2014

Self Cite

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several human malignances. As saliva is likely the major vehicle for KSHV transmission, we studied in vitro KSHV infection of oral epithelial cells. Through infection of two types of oral epithelial cells, normal human oral keratinocytes (NHOKs) and papilloma-immortalized human oral keratinocyte (HOK16B) cells, we found that KSHV can undergo robust lytic replication in oral epithelial cells. By employing de novo lytic infection of HOK16B cells, we studied the functions of two previously uncharacterized genes, ORF18 and ORF30, during the KSHV lytic cycle. For this purpose, an ORF18-deficient virus and an ORF30-deficient virus were generated using a mutagenesis strategy based on bacterial artificial chromosome (BAC) technology. We found that neither ORF18 nor ORF30 is required for immediately early or early gene expression or viral DNA replication, but each is essential for late gene expression during both de novo lytic replication and reactivation. This critical role of ORF18 and ORF30 in late gene expression was also observed during KSHV reactivation. In addition, global analysis of viral transcripts by RNA sequencing indicated that ORF18 and ORF30 control the same set of viral genes. Therefore, we suggest that these two viral ORFs are involved in the same mechanism or pathway that coregulates the viral late genes as a group. IMPORTANCEWhile KSHV can infect multiple cell types in vitro, only a few can support a full lytic replication cycle with progeny virions produced. Consequently, KSHV lytic replication is mostly studied through reactivation, which requires chemicals to induce the lytic cycle or overexpression of the viral transcriptional activator, RTA. In this study, we present a robust de novo lytic infection system based on oral epithelial cells. Using this system, we demonstrate the role of two viral ORFs, ORF18 and ORF30, in regulating viral gene expression during KSHV lytic replication. As the major route of KSHV transmission is thought to be via saliva, this new KSHV lytic replication system will have important utility in the field.