The Canonical Immediate Early 3 Gene Product pIE611 of Mouse Cytomegalovirus Is Dispensable for Viral Replication but Mediates Transcriptional and Posttranscriptional Regulation of Viral Gene Products

Abstract

Transcription of mouse cytomegalovirus (MCMV) immediate early ie1 and ie3 is controlled by the major immediate early promoter/enhancer (MIEP) and requires differential splicing. Based on complete loss of genome replication of an MCMV mutant carrying a deletion of the ie3-specific exon 5, the multifunctional IE3 protein (611 amino acids; pIE611) is considered essential for viral replication. Our analysis of ie3 transcription resulted in the identification of novel ie3 isoforms derived from alternatively spliced ie3 transcripts. Construction of an IE3-hemagglutinin (IE3-HA) virus by insertion of an in-frame HA epitope sequence allowed detection of the IE3 isoforms in infected cells, verifying that the newly identified transcripts code for proteins. This prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using ⌬ie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication. To determine the role of pIE611 for viral gene expression during MCMV infection in an unbiased global approach, we used label-free quantitative mass spectrometry to delineate pIE611-dependent changes of the MCMV proteome. Interestingly, further analysis revealed transcriptional as well as posttranscriptional regulation of MCMV gene products by pIE611. IMPORTANCECytomegaloviruses are pathogenic betaherpesviruses persisting in a lifelong latency from which reactivation can occur under conditions of immunosuppression, immunoimmaturity, or inflammation. The switch from latency to reactivation requires expression of immediate early genes. Therefore, understanding of immediate early gene regulation might add insights into viral pathogenesis. The mouse cytomegalovirus (MCMV) immediate early 3 protein (611 amino acids; pIE611) is considered essential for viral replication. The identification of novel protein isoforms derived from alternatively spliced ie3 transcripts prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using ⌬ie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication and delineated pIE611-dependent changes of the MCMV proteome. Our findings have fundamental implications for the interpretation of earlier studies on pIE3 functions and highlight the complex orchestration of MCMV gene regulation. Human cytomegalovirus (HCMV) is a ubiquitous humanpathogenic herpesvirus. Primary infection is mostly subclinical and leads to lifelong persistence of the viral genome in a latent state from which reactivation occurs under immune stress or immunosuppression. HCMV reactivation is a major cause of morbidity and mortality in AIDS patients and other immunocompromised individuals (1). Like HCMV, mouse cytomegalovirus (MCMV) represents a prototypical member of the family of betaherpesviruses, and it has been extensively used as a model system to study common aspects of CM...

Mouse Cytomegalovirus M34 Encodes a Non-essential, Nuclear, Early-Late Expressed Protein Required for Efficient Viral Replication

“…This finding is consistent with previous data (Kattenhorn et al, 2004). Under selective IE expression conditions [by infection in the presence of CHX which was washed out at 4 h p. i. in the presence of ActD followed by 4 h in presence of ActD; see (Rattay et al, 2015)], pp89/IE1 was detectable, while pM34HA was not ( Figure 3C). Upon addition of the antiviral drug phosphonoacetic acid (PAA), which prevents viral genome replication and impairs viral late gene expression, pM34HA expression was diminished but not abrogated ( Figure 3D).…”

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