Date on Master's Thesis/Doctoral Dissertation


Document Type

Doctoral Dissertation

Degree Name

Ph. D.


Microbiology and Immunology

Degree Program

Microbiology and Immunology, PhD

Committee Chair

Sokoloski, Kevin

Committee Co-Chair (if applicable)

Casella, Carolyn

Committee Member

Casella, Carolyn

Committee Member

Chung, Donghoon

Committee Member

Klinge, Carolyn

Committee Member

Lukashevich, Igor

Author's Keywords

alphavirus; hnRNP I; tethering


Old World alphaviruses cause significant outbreaks of illness and debilitating multi-joint arthritis for prolonged periods. Currently, there are no FDA approved vaccines or antiviral therapies; and thus, there is a critical need to identify and characterize the molecular biology of alphaviruses. Alphaviruses rely on the host cell machinery to complete the viral lifecycle and are dependent on interactions with host RNA binding proteins. Accordingly, several host heterogenous nuclear ribonucleoprotein proteins (hnRNPs) have been found to bind to the Sindbis virus (SINV) RNAs. Disrupting the interaction sites in the viral RNAs of these RNA:Protein interactions results in decreased viral titers in tissue culture models of infection. Nonetheless, whether the observed phenotypes were due to loss of hnRNP binding, or the incorporation of polymorphisms into the primary nucleotide sequence of SINV remained unknown. To determine if the loss of hnRNP binding was the primary cause of attenuation, or if the disruption of the RNA sequence itself was responsible for the observed phenotypes, we utilized an innovative protein tethering approach to vi restore the binding of a candidate hnRNP protein in the absence of the native interaction site. Specifically, we reconstituted the hnRNP I interaction with the viral RNA by replacing the native interaction site with the 20nt Bovine Immunodeficiency virus Transactivation RNA Response element (BIV-TAR). Importantly, the BIV-TAR element will bind with high specificity to proteins tagged with a TAT peptide. Reestablishment of the hnRNP I:vRNA interaction via the BIV-TAR / TAT tethering approach restored the phenotype to wild-type like levels. As the reconstitution of the hnRNP I interaction in the absence of the native interaction site repaired the mutant phenotype we can conclude that hnRNP I binding, and not primary sequence, is responsible for the observed mutant phenotype following the loss of the native interaction site. Further examinations of the mutant phenotype revealed that the increased structural protein expression observed following the loss of hnRNP I binding led to an apparent overwhelming of the host glycosylation machinery which in turn caused poor viral particle function as manifested by decreased specific infectivity.

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