title: Identification and Characterization of the Physiological Gene Targets of the Essential Lytic Replicative Epstein-Barr Virus SM Protein.
abstract: Epstein-Barr virus (EBV) SM protein is an essential lytic cycle protein with multiple posttranscriptional mechanisms of action. SM binds RNA and increases accumulation of specific EBV transcripts. Previous studies using microarrays and PCR have shown that SM-null mutants fail to accumulate several lytic cycle mRNAs and proteins at wild-type levels. However, the complete effect of SM on the EBV transcriptome has been incompletely characterized. Here we precisely identify the effects of SM on all EBV transcripts by high-throughput RNA sequencing, quantitative PCR (qPCR), and Northern blotting. The effect of SM on EBV mRNAs was highly skewed and was most evident on 13 late genes, demonstrating why SM is essential for infectious EBV production. EBV DNA replication was also partially impaired in SM mutants, suggesting additional roles for SM in EBV DNA replication. While it has been suggested that SM specificity is based on recognition of either RNA sequence motifs or other sequence properties, no such unifying property of SM-responsive targets was discernible. The binding affinity of mRNAs for SM also did not correlate with SM responsiveness. These data suggest that while target RNA binding by SM may be required for its effect, specific activation by SM is due to differences in inherent properties of individual transcripts. We therefore propose a new model for the mechanism of action and specificity of SM and its homologs in other herpesviruses: that they bind many RNAs but only enhance accumulation of those that are intrinsically unstable and poorly expressed.
  
  
  OBJECTIVE
  This study examines the mechanism of action of EBV SM protein, which is essential for EBV replication and infectious virus production. Since SM protein is not similar to any cellular protein and has homologs in all other human herpesviruses, it has potential importance as a therapeutic target. Here we establish which EBV RNAs are most highly upregulated by SM, allowing us to understand why it is essential for EBV replication. By comparing and characterizing these RNA transcripts, we conclude that the mechanism of specific activity is unlikely to be based simply on preferential recognition of a target motif. Rather, SM binding to its target RNA may be necessary but not sufficient for enhancing accumulation of the RNA. Preferential effects of SM on its most responsive RNA targets may depend on other inherent characteristics of these specific mRNAs that require SM for efficient expression, such as RNA stability.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of Utah School of Medicine, Salt Lake City, Utah, USA.
authors.forename: Jacob
authors.initials: J
authors.lastname: Thompson
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of Utah School of Medicine, Salt Lake City, Utah, USA.
authors.forename: Dinesh
authors.initials: D
authors.lastname: Verma
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of Utah School of Medicine, Salt Lake City, Utah, USA.
authors.forename: DaJiang
authors.initials: D
authors.lastname: Li
authors.affiliation: Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, Utah, USA.
authors.forename: Tim
authors.initials: T
authors.lastname: Mosbruger
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of Utah School of Medicine, Salt Lake City, Utah, USA sankar.swaminathan@hsc.utah.edu.
authors.forename: Sankar
authors.initials: S
authors.lastname: Swaminathan
mesh_terms: D015152:Blotting, Northern; D004854:Herpesvirus 4, Human / Q000502:physiology*; D006801:Humans; D017874:Immediate-Early Proteins; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D015534:Trans-Activators; D014779:Virus Replication
chemical_list: C068382:BRLF1 protein, Human herpesvirus 4; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D015534:Trans-Activators