title: The multifunctional ORF57 protein of Kaposi's sarcoma associated herpesvirus
Creators: name: Malik, Poonam
Publisher: name: University of Glasgow
Year: 2002

description: The aim of this work was to identify proteins, both cellular and viral, that interact in infected cells with the Kaposi's sarcoma-associated herpesvirus (KSHV) regulatory protein ORF57. In the light of this information, functional assays were performed to characterise various activities of the ORF57 protein. KSHV, the most recently identified herpesvirus to infect humans and associated with cancer, encodes ORP57, an immediate early (IE) regulatory protein. ORF57 is the only KSHV IE gene that has a counterpart in all herpesviruses sequenced so far. Studies of this protein, that may have similar functions throughout the Herpesviridae, are important to answer key questions about herpesvirus biology and the development of effective therapies. During lytic virus infection, expression of different classes of KHSV genes is temporally regulated. Two of the IE proteins, ORJF50 and ORF57, are central to this regulation and are essential for viral growth. ORF50 causes the switch from the viral latent to lytic cycle and is required for establishment of latency. ORF57 regulates viral gene expression at the transcriptional and post-transcriptional levels. Transcriptionally, it is capable of acting on its own or in combination with ORF50; post-transcriptionally, ORF57 enhances expression of viral genes and represses expression of some intron-containing genes. This study shows that ORF57 (like its herpes simplex virus ICP27 counterpart) in fusion protein pull down assays and coimmunoprecipitation assays interacts with four cellular proteins using extracts from KSHV-infected cells. These are: heterogeneous nuclear ribonucleoprotein K (hnRNP K), protein kinase casein kinase 2 (CK2), RNA export factor binding protein (REF) and splicing associated protein 145 (SAP 145). CK2 was present in complexes with hnRNPK or SAP 145, only in the presence of ORF57. Interestingly, the CK2 activity that came down with anti-ORF57 antibody could phosphorylate ORF57 protein itself and certain other proteins, including hnRNPK present in the coimmunoprecipitated complex. ORF57 phosphorylation by CK2 increased its affinity for hnRNP K. CK2 activity could be important for regulation of the various interactions, and act to control them depending on the phosphorylation status of ORF57 and its partners. A further ORF57 interaction was identified with the viral transcriptional transactivator ORF50 protein, and immunofluorescence on virus-infected cells showed that ORF57 partially colocalised with ORF50, in accordance with the involvement of these two IE proteins in activities together. In functional assays, ORF57 protein was shown to augment activation of the ORF50 promoter by ORF50 protein, and this activity may reflect a requirement for ORF57 at an early stage of lytic infection to amplify environmental stimuli thereby facilitating viral replication. The interaction of ORF57 with several proteins known to be involved at several stages of gene expression point to it being a multifunctional protein; and how these partner proteins contribute to ORF57's various functions can be inferred from a knowledge of their activities. An association with ImRNP K may account for ORF57's transcriptional regulation of viral gene expression acting either on its own or with ORF50. Much of the ORF57 activity is seen at the post-transcriptional level which also could be attributed to the action of hnRNP K. Immunofluorescence data demonstrated that ORF57 colocalises in punctate spots in the nucleus with hnRNP K, and reporter assays have shown that ORF57 and hnRNP K are functionally linked. Finally, the multiple interactions of ORF57 are likely to be facilitated by its ability to self interact, enabling the formation of large complexes. These protein:protein and protein:nucleic interactions could however be dynamic, changing at different intracellular locations or times post-infection to facilitate the various functions of ORF57. The present study has allowed insights as to how ORF57 exerts its various activities and will help in the understanding of how viral transcription, RNA processing and RNA export are interlinked in KHSV-infected cells.