Dissertation/Thesis Abstract

Functional Studies on Polyadenylated Nuclear RNA in Kaposi's Sarcoma- Associated Herpesvirus-lnfected Cells
by Vallery, Tenaya K., Ph.D., Yale University, 2018, 127; 13851924
Abstract (Summary)

Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the known human cancer viruses, causing Kaposi's sarcoma and primary effusion lymphoma in immunosuppressed patients. Although of medical concern, the mechanisms through which the virus causes cancer remain poorly understood. Researchers speculate that the lytic phase contributes to the development of human cancers by this virus.

KSHV, like other herpesviruses, is predominantly latent in the human host, but undergoes lytic activation to produce infectious viral particles. In the process, the virus hijacks the host machinery to express large quantities of viral genes via a process known as the host shutoff effect. The virus then replicates its DNA and assembles viral capsids within nuclear viral replication compartments. Viral proteins act in various locations within the cell depending on their function. However little is known about the location of viral transcripts and how their localization relates to their function. Thus I sought to understand the localization of viral transcripts to gain insight into the spatiotemporal regulation of the lytic phase.

Using fluorescence in situ hybridization (FISH) and immunofluorescence (IF), I observed that particular viral transcripts accumulate within the nucleus in or near replication compartments. This occurs late in the lytic phase coinciding with viral DNA replication. My findings indicate that the mechanism is independent of the host shutoff effect and splicing, but dependent on active viral DNA synthesis and in part on the viral noncoding RNA, polyadenylated nuclear (PAN) RNA. PAN RNA is essential for the viral life cycle and its contribution to the nuclear accumulation of viral messages may facilitate propagation of the virus.

One key regulator of the KSHV lifecycle is a long noncoding RNA (IncRNA) called the polyadenylated nuclear (PAN) RNA. PAN RNA is an early gene product comprising nearly 80% of total polyadenylated cellular transcripts in lytic infected cells. Studies on its function demonstrate that PAN RNA is a regulator of virion production through modulation of viral genes.

A glimpse into the mechanism comes from recent chromatin isolation by RNA purification (CHIRP) studies on lytic KSHV-infected B lymphocytes. The studies revealed widespread binding of PAN RNA to viral and host chromatin, but could not identify an underlying mechanism. The most feasible approach to study function is a genetic knockout. However, a complete PAN RNA gene deletion is unachievable in the KSHV genome due to an overlapping open reading frame, K7. In a related gammaherpesvirus, rhesus rhadinovirus (RRV), a computational search uncovered a PAN RNA homologue, whose sequence does not overlap with any known genes. I found that RRV PAN RNA is present at about 150,000 copies per cell and organized the purchase of RRV ΔPAN RNA constructs to facilitate study of PAN RNA's mechanism.

Capitalizing on the RRV homolog, Dr. Johanna B. Withers and I compared changes in chromatin association by PAN RNA between homologs and over the lytic phase with CHART (capture hybridization analysis of RNA targets). After careful analysis, the data suggest that chromatin-association by PAN RNA is nonspecific and that the mechanism of regulation by PAN RNA is not primarily related to chromatin remodeling. With this is mind, I looked to another potential mechanism, one related to binding by nuclear relocalized cytoplasmic polyAbinding protein (PABPC).

Both PAN RNA homologs associate with several host proteins, one of which is cytoplasmic polyA-binding protein (PABPC). Upon lytic induction, SOX, the host shutoff mediator, facilitates degradation of messages in the cytoplasm, causing the PABPC to relocalize to the nucleus. Nuclear relocalized PABPC binds KSHV PAN RNA at ~8-10 proteins per RNA molecule. I hypothesize that a function of PAN RNA is to act as nuclear relocalized PABPC sponge to facilitate preferential expression of viral genes and assembly of virions.

I designed mutant to capitalize on a unique feature of PAN RNA, the triple helical stabilization element (ENE). At the 3' end a triple helix (ENE) forms with polyA tail, protecting PAN RNA from deadenylation, stabilizing it. I reduced the length of the polyA-tail to eight adenylates, which permits formation of the triple helix, but falls below the 20-adenylate footprint of PABPC. The RRV PAN constructs would supplement RRV ΔPAN RNA virus to examine if the tailless PAN RNA mutants rescue the loss of virion production seen previously during the downregulation of KSHV PAN RNA. The results from these experiments would yield a deeper understanding of host-virus interactions and will provide insights into the importance of PABP-binding for the function of a nuclear noncoding RNA.

Indexing (document details)
Advisor: Steitz, Joan A.
Commitee:
School: Yale University
School Location: United States -- Connecticut
Source: DAI-B 80/07(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Biochemistry, Virology
Keywords: Herpesvirus, Kaposi's Sarcoma-Associated Herpesvirus, Noncoding RNA, PAN RNA, Replication Compartments, Rhesus Rhadinovirus
Publication Number: 13851924
ISBN: 9780438983571
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