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

Abstract

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...

Precision mouse models with expanded tropism for human pathogens

“…2l). The expression of IE proteins is consistent with lytic HCMV replication in this model as observed during human primary infection and reactivation 42 . In vivo, HCMV antigen was observed in human mesenchymal cells ( Fig.…”

“…4f,g and Supplementary Table 5). IE1 is an immunoprevalent HCMV protein not found in virions but expressed after infection 42 . The detection of IE1-specific responses is consistent with the in vivo lytic replication of HCMV observed in Fig.…”

Alternative promoters drive human cytomegalovirus reactivation from latency

“…1C). Surprisingly, a primer pair that detects transcripts arising from the MIEP (transcript UTR136) (22) or the 5′ distal promoter (dP) (23) (transcript UTR406) (22) (Fig. 1B, orange arrows) detected few MIEP/dP-derived transcripts at any time after infection or following reactivation ( Fig.…”

“…We recently identified two promoters within an intron 3′ of the MIEP [intronic promoter 1 (iP1) and intronic promoter 2 (iP2)] that drive the expression of alternate transcripts (UTR378 and UTR70, respectively) encoding IE1 and IE2 proteins ( Fig. 2 A and B) (22). The iPderived transcripts lack noncoding exon 1 found in MIEPderived mRNAs but encode the complete set of distal exons Mature mRNAs encoding IE1 and IE2 will also include exons 3 and 4 or 3 and 5, respectively.…”

“…necessary to synthesize full-length IE1 or IE2 proteins. As the intronic promoters are dispensable for viral replication in fibroblasts, and transcripts derived from these promoters are expressed with late kinetics during the replicative infection (22) (SI Appendix, Fig. S2), we hypothesized that the iP1 and iP2 might contribute to re-expression of UL122 and UL123 subsequent to a reactivation stimulus.…”