Murine cytomegalovirus major immediate-early protein 3 interacts with cellular and viral proteins in viral DNA replication compartments and is important for early gene activation

Abstract

Murine cytomegalovirus (MCMV) immediate-early protein 3 (IE3) is essential for successful viral infection. This study developed MCMVs with an EGFP-fused IE3 gene in order to study IE3 gene expression, subnuclear distribution and biological function, as well as to examine the interaction of IE3 with cellular and viral proteins. The generated viruses included MCMVIE3gfp, in which IE1 was completely removed by the in-frame fusion of exons 3 and 5 and the C terminus of IE3 was tagged with EGFP, and MCMVIE1/3gfp, in which IE1 was kept intact and EGFP was also fused to the C terminus of IE3. Unlike human CMV (HCMV), whose growth was significantly reduced when IE2 (the HCMV homologue of IE3 in MCMV) was tagged with EGFP, MCMVs with IE3–EGFP presented an unchanged replication profile. Using these new constructs, the distribution of IE3 was revealed as well as its interaction with viral and cellular proteins, especially proteins pertaining to DNA replication (M44 and E1) and cellular intrinsic defence [promyelocytic leukemia protein and histone deacetylases (HDACs)]. It was also shown that IE3 domains co-localize with DNA replication domains, and IE3 attracted other required proteins into IE3 domains via protein–protein interactions. In addition, IE3 was shown to interact with HDAC2 and to eliminate the inhibitory effect of HDAC2 on early viral gene production. Together, these results suggest that IE3 acts as a key protein for viral DNA replication by establishing pre-replication domains via recruitment of the required viral and cellular proteins, and by reducing host defences.

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

“…Our data could have considerable implications for several studies assigning defined MCMV phenotypes and functions to pIE3 (11,(26)(27)(28), e.g., the analysis of cell cycle regulation. Although wt MCMV infection results in G 1 and G 2 arrest and the ⌬ie3 virus has lost this ability, ectopically expressed pIE611 exhibits cell cycle arrest activity only in G 1 and not G 2 (29), exemplifying the occasion for reevaluation.…”

Transplant-induced reactivation of murine cytomegalovirus immediate early gene expression is associated with recruitment of NF-κB and AP-1 to the major immediate early promoter

“…IE-3 is the MCMV functional homologue of HCMV IE-2, which is the major transactivator of early gene expression (Angulo et al, 2000;Keil et al, 1987aKeil et al, , 1987bMartínez et al, 2010;Messerle et al, 1992). We previously showed that allogeneic transplantation induced reactivation of IE gene expression 48 h after transplant, and that RNA polymerase phosphorylated at Ser2 (Pol II pS2) was recruited to both the MIEP and the IE-3 coding region (Hummel et al, 2001;Liu et al, 2013).…”

A Short cis -Acting Motif in the M112-113 Promoter Region Is Essential for IE3 To Activate M112-113 Gene Expression and Is Important for Murine Cytomegalovirus Replication

“…Plasmid pBB5.5, containing the whole M112-113 gene and promoter, was generously provided by M. Messerle (5,7,8); pET (expressing GFP) and pgfie3 (expressing GFPtagged IE3) were reported previously (6). A diagram of part of the M112-113 gene and the constructed M112-113-expressing vectors are shown in Fig.…”

“…M112-113 proteins are detectable at almost the same time as IE3, but M112-113 gene expression is upregulated strongly by IE3 (6). Compared to HCMV genes, M112-113 is the equivalent of HCMV UL112-113, and MCMV IE3 is the counterpart to HCMV IE2 (9).…”

“…Accumulated studies have demonstrated that the immediate-early 3 (IE3) protein of the major immediate-early (MIE) gene is essential for MCMV replication (3)(4)(5). We recently demonstrated that IE3 has a strong effect on early gene regulation and is critical to the formation of viral prereplication compartments (pre-RC) via interactions with viral and cellular proteins (6,7). However, the detailed mechanisms used by MCMV IE3 to regulate viral early gene expression and to counter cellular defenses still remain enigmatic.…”