Understanding genome packaging and the function of vimentin during mammalian orthoreovirus infection.

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2023-05
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DHAR, DEBARPAN
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Miller, Cathy CLM
Miller, W. Allen
Blitvich, Bradley
Moss, Walter
Sashital, Dipali
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Veterinary Microbiology and Preventative Medicine
Abstract
Mammalian orthoreovirus (MRV) is a member of the order Reovirales, which includes viruses having a linear and segmented double stranded RNA (dsRNA) genome. Viruses within this order can infect a diverse range of hosts, causing disease symptoms. In contrast, MRV is clinically benign in immunocompetent human adults and therefore is a tractable model to study the pathogenesis of viruses within the Reovirales order. Additionally, MRV is an oncolytic virus that has undergone numerous clinical trials against different tumor types and has a strong potential to be used as a gene delivery vehicle. Therefore, studying the fundamental biology of MRV is also crucial from a translational research standpoint as it is likely to help us improve its anti-tumor potential. But despite decades of research, critical knowledge gaps still exist in our understanding of the MRV life cycle. One such gap lies in our understanding of how one copy of each MRV gene segment gets faithfully packaged into the mature virion. MRV has ten linear dsRNA segments (L1, L2, L3, M1, M2, M3, S1, S2, S3, S4), and with my research, I have determined the significance of terminal nucleotide sequences in driving the packaging of the S gene segments (S1, S2, S3 and S4). Furthermore, I have identified the minimum nucleotide stretches at the 5′ and 3′ end of the S1 gene that are sufficient for its packaging. Within this work, I have also experimentally determined the significance of a predicted RNA secondary structure and conserved sequences within that structure in driving the packaging of S1. With the design of recombinant MRV containing long length of foreign gene sequences proving to be extremely challenging, I have shown with that 850-900 additional nucleotides can be incorporated into the L2, M2, and M3 gene segments of replicating MRV using an approach of termini duplication, wobble mutation and juxtaposition. As viruses utilize multiple components of the host cellular machinery, identifying such host factors is essential to better understand the viral life cycle. Using proximity labeling approaches, I have determined the close association of cytoskeletal proteins with NS. MRV protein NS forms the structural matrix of viral factories (VF), and prior research has shown that the formation of VF is indispensable in completing the MRV infection cycle. In my research, I have determined the importance of an intermediate cytoskeletal protein, vimentin, in regulating VF morphology and size, viral replication, and egress. Altogether, my work has generated significant novel information that will lead to a better understanding of MRV life cycle, which can help further developments towards boosting the therapeutic potential of MRV.
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