title: 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.
authors.affiliation: Department of Microbiology/RCMI Program, Ponce School of Medicine, Ponce, PR 00716, Puerto Rico.
authors.forename: Francisco Puerta
authors.initials: FP
authors.lastname: Martínez
authors.forename: Ruth S Cruz
authors.initials: RS
authors.lastname: Cosme
authors.forename: Qiyi
authors.initials: Q
authors.lastname: Tang
mesh_terms: D019677:Artificial Gene Fusion; D004279:DNA, Viral / Q000096:biosynthesis*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D018146:Muromegalovirus / Q000472:pathogenicity / Q000502:physiology*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; C088646:IE3 protein, cytomegalovirus; D017874:Immediate-Early Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins