title: Cell-based screening assay for antiviral compounds targeting the ability of herpesvirus posttranscriptional regulatory proteins to stabilize viral mRNAs.
abstract: Each human herpesvirus expresses a multifunctional regulatory protein that is essential for lytic viral replication. A cell-based assay targeting the function of these proteins was developed based on the finding that Epstein-Barr virus (EBV) SM and Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 stabilize specific target mRNAs. Both proteins facilitate the accumulation of lytic transcripts by incompletely characterized posttranscriptional mechanisms. SM and ORF57 exhibit target gene specificity and enhance the accumulation of certain EBV and KSHV mRNAs that are poorly expressed in their absence. Conversely, SM- and ORF57-independent viral and cellular transcripts accumulate efficiently, and their expression does not respond to SM or ORF57. Fusion of an ORF57-responsive transcript to ORF57-independent transcripts demonstrated that ORF57 dependence is cis-dominant. EBV SM also enhanced the accumulation of such fused mRNA transcripts. These data suggest that the coding regions of specific viral transcripts confer instability even when fused to heterologous genes. The findings were used to develop a reporter assay that measures EBV SM function in rescuing the expression of poorly expressed transcripts by posttranscriptional mechanisms. The assay represents a method for the screening of small interfering RNAs (siRNAs) and compounds to investigate the mechanism of action of SM and its homologs and potentially to aid in the discovery of novel antiviral agents.
authors.affiliation: Division of Infectious Diseases, Department of Internal Medicine.
authors.forename: Dinesh
authors.initials: D
authors.lastname: Verma
authors.forename: Eun A
authors.initials: EA
authors.lastname: Kim
authors.forename: Sankar
authors.initials: S
authors.lastname: Swaminathan
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D001331:Automation; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D006566:Herpesviridae Infections / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D019288:Herpesvirus 8, Human / Q000502:physiology; D057166:High-Throughput Screening Assays; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D000998:Antiviral Agents; C068382:BRLF1 protein, Human herpesvirus 4; D017874:Immediate-Early Proteins; C415247:ORF57 protein, Herpesvirus saimiri; D012333:RNA, Messenger; D012367:RNA, Viral; D012097:Repressor Proteins; D015534:Trans-Activators
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