Herpesviral mRNP formation: identification of cellular co-factors and target specificity

• Applicant: Privatdozent Dr. Jens Bohne
• Subject Area: Virology
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 403670311

Viruses are obligate cellular parasites and thus their gene expression is intimately linked to cellular machineries. Herpesviruses are complex DNA viruses, which establish a lifelong persistence in their hosts characterized by oscillating states named latent and lytic phases. During the latent phase the virus is silent. In contrast, the lytic phase is characterized by a massive up-regulation of viral gene expression and the formation of progeny virus. The genome of Kaposi’s sarcoma-associated herpesvirus (KSHV) is 165 kb in length and only 25% of its genes contain introns. However, splicing of introns enhances all later steps in the life of mRNAs. To substitute for splicing all herpesviruses encode a post-transcriptional regulator. In KSHV ORF57 ensures the formation of an export-competent mRNA-protein complex (mRNP). While various interactions with cellular factors have been described, the ORF57 targeting mechanism for viral RNA remains obscure. The low affinity of ORF57 to RNA and the large portion of disordered regions made analyses difficult. We took a different approach and asked: what are the determinants on the viral RNA leading to ORF57-dependency? We developed the concept of a distinct viral mRNP code based on the sequence composition of KSHV lytic RNAs. We hypothesize that motifs for specific cellular RNA-binding proteins (RBPs) are encoded in the viral RNA sequence possibly embedded in secondary structures. To identify the putative cellular partner of ORF57, we will use a recently described mRNP precipitation technique called 'LNA mRNP capture'. The method is able to purify specific mRNPs based on selection via oligonucleotides probes. As an alternative hypothesis we compare the RNA recognition by ORF57 to a bacterial RBP, which relies on a 'hidden specificity' rather than conserved motifs. In parallel, the Mandel-Gutfreund laboratory will develop new algorithms and machine learning approaches to find sequence attributes in ORF57-dependent vs. non-dependent genes. In addition, a new prediction tool for unstructured protein domains will be developed to retrieve more information on the N-terminal region of ORF57 most probably involved in RNA recognition. Finally, we plan to determine the in-vivo structure of KSHV lytic RNAs by DMSseq, a genome-wide applicable combination of structural probing and deep sequencing. The structural information will be integrated into the algorithms. This output is also the basis for probing putative ORF57 recognition sites in-vitro. In the end, a proximity ligation assay using putative target sites will demonstrate in-vivo binding of ORF57. The results obtained will be expanded to other herpesviruses. So far we could already demonstrate that one of our reporter systems is also activated by the ORF57 homologues of Epstein-Barr virus and Varicella Zoster Virus. The data will also help to further decipher the cellular mRNP code, an important task in the post-genome era.

• DFG Programme: Research Grants
• International Connection: Israel
• International Co-Applicant: Professorin Dr. Yael Mandel-Gutfreund