"Project 1" KSHV short and long noncoding RNAs and alteration of host IncRNA expression

PROJECT SUMMARY
Kaposi's sarcoma-associated herpesvirus (KSHV), a human gamma-herpesvirus, is the causative agent of AIDS
malignancies like KS and primary effusion lymphomas (PEL). In recent years it became clear that pathogenic
herpesviruses including EBV, KSHV, and MHV68 express numerous long non-coding RNAs (lncRNAs) many of
which are in antisense orientation to protein coding transcripts. The function and structure of these RNAs is
largely unknown. In addition, these viruses express microRNAs (miRNAs). While characterizing the KSHV
miRNA targetomes using a modified Crosslinking and Sequencing of Hybrids (qCLASH) protocol, we identified
several hundred host cellular lncRNAs as putative miRNA targets. These data strongly suggest that both KSHV
encoded proteins and miRNAs contribute to dysregulation of host lncRNAs. Importantly, 34 lncRNAs that are
perturbed following KSHV infection, including MALAT1, HOTTIP, ANRIL, Meg3, UCA1 and GAS-5 are reported
to be associated with human cancers. We also linked both mRNA and lncRNA targets to cancer hallmark
phenotypes such as proliferation, migration, angiogenesis, and glucose metabolism, and started to identify
signaling pathways that are perturbed by KSHV miRNAs in human endothelial cells as a model for KS. We also
identified aberrant splicing as an additional cancer hallmark phenotype. Here we propose to extend our studies
by integrating multi-omics data sets from qCLASH, RNAseq and miRNAseq data to comprehensively analyze
miRNA-regulated gene regulatory networks in KSHV infected endothelial cells. Moreover, we propose to validate
our findings in a significant number of human tumor samples and by generating the first KS tumor miRNA
targetome by qCLASH. These studies will be performed in a comparative fashion with Projects 2 and 3 and
furthermore will be supported by Cores B, C, and D. In addition, we are functionally studying the role of the
antisense LANA transcript (ALT) for which we have identified 81 putative binding proteins using a highly
innovative RNA-pulldown assay. Moreover, we will study the role of a newly identified class of viral circular RNAs
that has been discovered by this program. In summary, the goal of this project is to delineate the role of viral
lncRNAs and host cellular lncRNAs that are perturbed by viral infection in AIDS malignancies.