GSBS Dissertations and Theses

Approval Date

5-5-2008

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Department of Molecular Genetics & Microbiology

Subjects

Newcastle disease virus; Virulence; Viral Fusion Protein; Viral Proteins; HN Protein; Interferons; Academic Dissertations

Abstract

Newcastle disease virus (NDV) is a member of the genus Avulavirus of the Paramyxoviridae family of enveloped negative-stranded RNA viruses. The virus causes respiratory, neurological, or enteric disease in many species of birds, resulting in significant losses to the poultry industry worldwide. Strains of the virus are classified into three pathotypes based on the severity of disease in chickens. Avirulent strains that produce mild or asymptomatic infections are termed lentogenic, whereas virulent strains are termed velogenic. Strains of intermediate virulence are termed mesogenic.

The envelope of NDV virions contains two types of glycoproteins, the hemagglutinin-neuraminidase (HN) and fusion (F) proteins. HN mediates three functions: 1) virus attachment to sialic acid-containing receptors; 2) neuraminidase activity that cleaves sialic acid from progeny virions to prevent self-aggregation; and, 3) complementation of the F protein in the promotion of fusion.

Though it is widely accepted that cleavage of a fusion protein precursor is the primary determinant of NDV virulence, it is not the sole determinant. At least two other proteins, HN and the V protein, contribute to virulence. The V protein possesses interferon (IFN) antagonistic activity. The long-range goal of these studies is to understand the roles of HN and V in the differential virulence patterns exhibited by members of the NDV serotype.

The first aim is to compare the IFN antagonistic activity of the V protein from a lentogenic and a mesogenic strain of the virus. The results of this study demonstrate that the V protein of the mesogenic strain Beaudette C (BC) exhibits greater IFN antagonistic activity than that of the lentogenic strain La Sota. Hence, the IFN antagonistic activities of the two V proteins correlate with their known virulence properties.

Comparison of the C-terminal regions of La Sota and BC V proteins revealed four amino acid differences. The results demonstrate that the IFN antagonistic activity of La Sota V increases when any one of these residues is mutated to the corresponding residue in BC V. Conversely, the IFN antagonistic activity of BC V decreases when any one of these four residues is mutated to the corresponding residue in La Sota V. However, no single residue accounts for the difference in IFN antagonistic activity between the two V proteins. Also, analysis of La Sota V and BC V proteins with multiple mutations in these positions revealed that the four residues are collectively responsible for the difference in the IFN antagonistic activity of the two V proteins. Finally, characterization of chimeric La Sota/BC V proteins showed that the N-terminal region also contributes to the IFN antagonistic activity of V.

Contrary to an earlier report, results described here demonstrate that the NDV V protein does not target STAT1 for degradation. However, both La Sota and BC V proteins target interferon regulatory factor (IRF)-7 for degradation and promote the conversion of full-length IRF-7 to a lower molecular weight form (IRF-7*). This is the first demonstration that IRF-7 is targeted by a paramyxovirus V protein. The amount of IRF-7* decreases in a dose-dependent manner in the presence of a proteasome inhibitor, suggesting that IRF-7* is a degradation product of IRF-7. Furthermore, the BC V protein promotes complete conversion of IRF-7 to IRF7*, whereas the La Sota V protein does so less efficiently. Again, this is consistent with the difference in IFN antagonistic activity of the two V proteins, and in turn, with their virulence.

The second aim is to characterize an HN-specific monoclonal antibody called AVS-I. A previous study suggested that AVS-I recognizes an epitope that is conserved in lentogenic strains and raises the possibility that this epitope may colocalize with a determinant of virulence in HN. To further characterize antibody AVS-I and the epitope it recognizes, we (i) determined its specificity for several additional strains of the virus, (ii) mapped its binding to HN in competition with our own antibodies, (iii) determined its functional inhibition profile, and (iv) isolated and sequenced an AVS-I escape mutant. The results demonstrate that AVS-I binds to a conformational epitope at the carboxy terminus of HN. This suggests that this region of HN may define a determinant of virulence. However, it was also shown that AVS-I, which was previously thought to be specific for avirulent strains of NDV, actually recognizes individual mesogenic and velogenic strains.

In conclusion, the data presented in this dissertation contributes to a greater understanding of the molecular basis for NDV virulence and may aid in development of antiviral strategies and generation of recombinant NDVs suitable for use in cancer and gene therapy.

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