Date of Award

2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Carolina B. López

Abstract

Paramyxoviruses are negative-sense single-stranded RNA viruses that comprise many important human and animal pathogens. During viral replication, paramyxoviruses produce defective viral genomes (DVGs), truncated genomic products that are unable to replicate in the absence of standard virus. DVGs influence the outcomes of infection through interference with standard viral replication and by inducing antiviral immunity. Using the model paramyxovirus, Sendai virus (SeV), we found that full-length (FL) and DVG viral RNA (vRNA) accumulated heterogeneously in cells during infection, with some cells accumulating predominantly full-length genomes (FL-high) and some accumulating predominantly DVGs (DVG-high). Interestingly, in FL-high cells genomes accumulated in a perinuclear region while viral genomes in DVG-high cells remained diffusely distributed throughout the cytoplasm. We sought to address the mechanisms and consequences of the differential intracellular distributions of viral RNA in the presence of DVGs. We found that vRNA in FL-high cells interacts with the host GTPase Rab11a and uses the recycling endosome system for particle production, while viral RNA in DVG-high cells does not interact with the host cell in this way. Consequently, FL-high cells produce both standard virions and defective particles, while DVG-high cells do not produce virions. We next addressed the determinants of this distinct intracellular localization. We reasoned that DVG-high cells, which robustly replicate vRNA but do not progress to virion assembly, fail to accumulate the viral proteins required for interaction between vRNA and Rab11a. We found that neither SeV matrix nor nucleoproteins are sufficient to drive this interaction. We identified the viral polymerase protein L and the accessory protein C as differentiating factors in cells that engage with Rab11a, and found C proteins to be the most enriched proteins in Rab11a immunoprecipitation followed by mass spectrometry. These data suggest that the polymerase complex proteins L and its cofactor C are critical in regulating initial steps in SeV assembly. Overall, this work investigated the intracellular distributions of viral genomes in the presence of DVGs to understand the impact of DVGs on the dynamics of full length and defective particle production, as well as to gain insights into viral proteins required to initiate viral assembly.

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