Viral RNPs, mRNA Stability and Export

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The roles of non-coding nuclear RNAs in lymphoid cells harboring three transforming herpesviruses are being investigated. Epstein-Barr virus (EBV) infects and transforms human B cells and is the causative agent of infectious mononucleosis, as well as being associated with several human cancers. Herpesvirus saimiri induces fatal lymphomas and leukemias in New World monkeys and transforms human and monkey T lymphocytes in culture, resulting in a mature, activated phenotype. Kaposi sarcoma-associated herpesvirus (KSHV) afflicts AIDS and other immunocompromised patients and, like other herpesviruses, persists in a latent form until activation of the lytic phase. Among the few viral gene products expressed in transformed cells are the two EBV-encoded EBERs and the seven H. saimiri-encoded HSURS; yet, they are not essential for viral growth or transformation. Upon induction, KSHV produces PAN, a capped, polyadenylated RNA that does not leave the nucleus. These viral RNAs are all relatively small (<1.1 kb), abundant, conserved, and associate with host proteins that - in the case of EBERs and HSURs - are autoantigens. They are therefore excellent probes for dissecting host cell pathways, as well as mechanisms of viral transformation or lytic growth. Proposed experiments will test both the hypothesis that EBERs, HSURs and PAN may perturb cellular metabolism by sequestering important host proteins, and the possibility that their RNPs perform some function critical for the virus. The molecular details of the interactions of protein L22, PKR and La with EBER1 will be probed by structural studies and nucleotide analog interference mapping. Components of the EBER2 RNP will be identified. EBERs will be examined for possible shuttling between the nucleus and cytoplasm, and their contribution to host gene expression assessed by microarray analyses, Microarray evidence that HSURs 1 and 2 enhance the activated state of transformed T cells will be confirmed in rescue experiments using lentiviral vectors, their interaction with TTP and its effects examined, and the level at which HSURs modulate host gene expression determined. RNA elements and trans-acting factors essential for PAN accumulation in the nucleus will be characterized. PAN function in KSHV lytic infection will be investigated by siRNA knockdown and/or genomic PAN deletion, followed by complementation experiments.