Doctor of Philosophy, The Ohio State University, 2012, Biochemistry Program, Ohio State

The primer for reverse transcription in human immunodeficiency virus type 1 (HIV-1), human tRNALys 3, is selectively packaged into the virion along with tRNALys 1, 2. Human lysyl-tRNA synthetase (LysRS), the only cellular factor known to interact specifically with all three tRNALys isoacceptors, is also selectively packaged into HIV-1. Previous work has defined a tRNALys packaging complex that includes the tRNALys isoacceptors, LysRS, Gag, GagPol, and viral RNA. Numerous studies support the hypothesis that during tRNALys packaging, a Gag/GagPol complex interacts with a tRNALys/LysRS complex, with the capsid (CA) domain of Gag interacting specifically with LysRS, and GagPol interacting with both Gag and tRNALys. In this work, we have identified critical residues along one face of the dimerization helix 7 (H7) of LysRS involved in packaging of LysRS into virions. Mutation of these residues affects binding to Gag in vitro, as well as the oligomerization state and aminoacylation activity of the synthetase. Taken together with previous work, these data support the conclusion that the LysRS H7–CA interaction interface represents a novel antiviral target. With this target in mind, a support-bound cyclic peptide (CP) library containing randomized amino acid sequences and different ring sizes was synthesized and screened against CA and the monomeric form of the CA C-terminal domain (WM-CA CTD). Out of 3 x 105 CPs screened, 21 hits were obtained and 6 CPs were chosen for detailed in vitro analysis. Two CPs, CP2 and CP4 showed strong binding (Kd ~ 500 nM) to both CA and WM-CA CTD in vitro. Scrambled variants of CP2 and CP4 and point mutants at each of the randomized positions eliminated binding, suggesting a sequence-specific mode of interaction. CP2 and CP4 also inhibited LysRS/CA interaction in vitro with an IC50 value of ~ 1 µM. Furthermore, nuclear magnetic resonance (NMR), mutational studies along with in silico analysis revealed that CP2 and CP4 bind to a site proximal (open full item for complete abstract)

Committee: Karin Musier-Forsyth (Advisor); Michael Ibba (Committee Member); Jennifer Ottesen (Committee Member); Christopher M Hadad (Committee Member) Subjects: Biochemistry; Biophysics; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2005, Veterinary Preventive Medicine

Infectious bursal disease virus (IBDV) causes an acute and contagious disease in young chickens from 3-6 weeks of age. Two serotypes of the virus are recognized of which serotype 1 viruses are pathogenic to chickens and are classified into classic, variant, and serotype 2 viruses are nonpathogenic. The disease is controlled by vaccination. In the first part of the study interactions between a mild and a pathogenic strain of IBDV in specific pathogen free (SPF) chickens were studied. Chickens were inoculated with the Bursine-2 vaccine followed by the pathogenic STC strain at various time intervals. Persistence of virus strains was monitored by the reverse transcriptase polymerase chain/restriction fragment length polymorphism (RT-PCR/RFLP), bursa/body weight ratios and histopathological lesion scores. The mild strain interfered with the replication of the pathogenic strain.Currently available ELISA kits were evaluated for their ability to detect antibodies elicited by serotype 1 and serotype 2 viruses. Virus neutralization (VN) test differentiates between antibodies elicited by the two serotypes as well as subtypes of serotype 1 viruses. SPF chickens were inoculated with either serotype 1 STC or serotype 2 OH strain. Sera from these chickens and naturally exposed chickens were tested by five commercial ELISA kits and the VN. The ELISA kits detected antibodies to both serotypes of the virus. Therefore, while determining the antibody profiles of the flocks, the presence of serotype 2 antibodies should be taken into account. Simple and quick diagnostic assays are needed for developing control strategies against IBDV. A differential RT-PCR assay was developed. Two primer sets were designed. Primer set one targeted the segment A of the virus and specifically amplified serotype 2 strains. Primer set 2 targeted the segment B of the virus and amplified the vv strains. These primer sets were validated with 26 different strains maintained in our laboratory. The primer set 2 wa (open full item for complete abstract)

Committee: Yehia Saif (Advisor) Subjects: Biology, Microbiology