Investigating the Function of Vaccinia Virus Protein A33 during Morphogenesis, Egress, and Infectivity

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2011.Vaccinia virus, the prototypical member of the orthopoxviruses, was used as a live attenuated vaccine for the eradication of smallpox. It is an enveloped DNA virus with a large genome of about 200 kbp that is predicated to encode approximately 200 functional open reading frames. During vaccinia virus infection, two morphologically and antigenically distinct forms of infectious virions are produced: intracellular mature virions (IMV) and extracellular virions (EV). The first form of infectious virions produced within the viral factory is IMV. Due to the intracellular envelopment that occurs at the trans-Golgi network or early endosome, a small subset of IMV obtain an additional double membrane envelope and become intracellular enveloped virions (IEV). IEV are transported to the cell surface along microtubules and released from the cytoplasm by fusion of their outermost membrane with the plasma membrane. Virions that are retained on the cell surface are called cell-associated enveloped virion (CEV) and those released from the cell surface are called extracellular enveloped virion (EEV). CEV and EEV are collectively termed EV, which are responsible for severe systemic infection. Although eight proteins encoded by the virus, A33, A34, A36, A56, B5, F12, F13, and K2, are known to be specific for IEV/EV, understanding of the molecular mechanism of intracellular envelopment is far from complete. The major goal of this thesis study is to elucidate the function of A33 during viral morphogenesis, egress, and/or infectivity. A recombinant virus that has A33R deleted (v∆A33R) has been shown to produce small plaques due to the inability of the virus to induce actin tails. Thus, A33 is known to be required for efficient cell-to-cell spread. However, its exact role during infection remains to be determined. To study poxvirus morphogenesis in the absence of A33, a recombinant virus that has A33R deleted and expresses B5R-GFP in place of the normal B5R (vB5R-GFP/∆A33R) was generated. vB5R-GFP/∆A33R makes plaques that are smaller in size to those made by v∆A33R, indicating additional defects due to the fusion of GFP to the cytoplasmic tail of B5 in the absence of A33. Compared to v∆A33R, vB5R-GFP/∆A33R produces less infectious EV, which accounts for the smaller plaque phenotype. Additionally, we have found that A33R-deficient viruses are not fully infectious due to their inability to bind target cells efficiently. Further analysis of purified EEV demonstrates that A33 is required for the incorporation of B5-GFP into progeny EEV while the incorporation of B5 is independent of A33, suggesting there exists two pathways for the incorporation of B5 into EV envelope, A33-dependent and A33-independent. Based on our previous study, we have hypothesized that the A33-dependent mechanism for the incorporation of B5 into EV envelope requires the A33-B5 interaction. Using co-immunoprecipitation, we confirmed that A33 and B5-GFP interacted and found that the interaction was through their lumenal domains. A33 is a multifunctional protein and we wanted to study the function of the A33-B5 interaction during infection. Therefore, we generated seven A33 charge-to-alanine mutants, in which charge residues in clusters in the lumenal domain were mutated to alanines. Using co-IP, we have found that all of the mutants still interact with B5-GFP. Interestingly, we isolated A33 charge-to-alanine mutants that had a stronger interaction with B5. Characterization of the recombinant viruses expressing A33R that contains the charge-to-alanine mutations reveals that the mutants with the stronger interaction with B5 have a defective plaque phenotype. Furthermore, we have found that the aberrant interaction between A33 and B5-GFP on the surface of EEV reduces the binding ability of EEV. A33 has been shown to migrate as a high molecular weight species on SDS-PAGE under non-reducing conditions, indicating that it is present as an intermolecular disulfide-bonded homodimer or heteromultimer during infection. We demonstrate that A33 forms an intermolecular disulfide-bonded homodimer through the cysteine at amino acid 62. Surprisingly, we have found that A33 is still capable of forming a homodimer even in the absence of intermolecular disulfide bond formation and that intermolecular disulfide bonding is not required for its function during infection. In conclusion, this body of work has uncovered previously unidentified function of A33 in target cell binding. We have revealed two mechanisms for the dimerization of A33, demonstrating the importance of homodimerization for its function during infection. Characterization of vB5R-GFP/∆A33R has provided evidence that there exists two pathways for the incorporation of B5 into the envelope of EV. Furthermore, a regulatory mechanism appears to be required for the incorporation of B5 via the A33-dependent pathway to produce EV that are infectious