Date of Award

4-30-2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Dr. Richard K. Plemper

Second Advisor

Dr. Sang-Moo Kang

Third Advisor

Dr. Andrew Gewirtz

Abstract

The paramyxovirus replication machinery comprises the viral large (L) and phospho-(P) proteins in addition to the nucleocapsid (N) protein that encapsidates the single stranded RNA genome. Common to paramyxovirus N proteins is a C-terminal tail (Ntail), which contributes to docking of the polymerase complex to the genome through defined interaction domains. The central Ntail section is structurally disordered and thought to provide spatial flexibility required for productive interaction of the polymerase with the encapsidated viral genome, but its mechanistic role and relevance for successful virus replication is untested. Focusing initially on members of the morbillivirus genus, a series of Measles virus (MeV) and Canine distemper virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah virus (NiV), a member of the henipavirus genus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbillivirus disease. The recombinant viruses displayed different stages of attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated was genetically stable after extensive ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral attenuation coincided with stepwise-altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual virus attenuation for next-generation paramyxovirus vaccine design.

Available for download on Saturday, April 20, 2019

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