UBC Theses and Dissertations
The analysis of Autographa Californica multiple Nucleopolyhedrovirus EXONO (ORF141) function and its role in virus budding Fang, Minggang
Baculoviruses have a biphasic replication cycle producing two types of virions, budded virus (BV) and occlusion derived virus (ODV) which are required for the systemic spread or oral infection with the insect host respectively. Little is known about the events of the BV pathway and the mechanism by which nucleocapsids are selected and directed from the nucleus to plasma membrane to form BV. The Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) gene exon0 (orf141) is known to be required for the efficient production of BV and in this study the function and mechanism by which EXON0 affects BV production was investigated. Confocal microscopic analysis showed that EXON0 localized in the nucleus in the ring zone of virogenic stroma where nucleocapsids are assembled. In addition EXON0 also concentrated in the cytoplasm at the plasma membrane. Analysis of virions revealed that EXON0 copurified with nucleocapsid fractions of both BV and ODV. In support of this yeast 2-hybrid screening, co-immunoprecipitation, and confocal microscopy revealed that EXON0 interacted with the known nucleocapsid proteins FP25 and BV/ODV-C42. Transmission electron microscopy showed that deletion of exon0 results in nucleocapsids being unable to efficiently egress from the nucleus to the cytoplasm. Cellular protein interaction analyzed by tandem affinity purification and co-immunoprecipitation showed that beta-tubulin co-purified with EXON0. Immunofluorescence also showed that EXON0 and microtubules co-localized during virus infection. The microtubule inhibitors colchicine and nocodazole affected the localization of EXON0 and significantly reduced BV production. These data support the conclusion that egress of AcMNPV nucleocapsids is facilitated by interaction of EXON0 with beta-tubulin and microtubules. Deletion and point mutation analysis mapped domains of EXON0 required for efficient budding, dimer formation and association with FP25, BV/ODV-C42 and beta-tubulin. The Leucine zipper domain was required for dimer formation, beta-tubulin and BV/ODV-C42 interaction and also reduced interaction with FP25. Multiple domains were also shown to affect BV production. This study provides a detailed analysis of EXON0 which is one of the first baculovirus genes shown to be specific for the BV pathway. The results extend our understanding of the BV pathway which is a major determinant of baculovirus pathogenesis.
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