Investigations into the mechanisms involved in baculovirus nucleocapsid egress

The mechanism of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) nucleocapsid egress from the nucleus to the plasma membrane leading to the formation of budded virus (BV) is not known. AC141 is a nucleocapsid protein and has been shown to be associated with β-tubulin. We hypothesized that nucleocapsid proteins associate with the lepidopteran microtubule and kinesin-1 during egress. Kinesin-1 is a motor protein that moves along the microtubules and carries cargo. Experiments showed that nucleocapsid proteins associate with kinesin-1 during infection. Downregulation of kinesin-1 by siRNA results in reduced BV production. These studies support that AcMNPV utilizes kinesin-1 and microtubules for nucleocapsid transport and BV production. GP64 (integral membrane protein) and ME53 associate at the plasma membrane and are believed to be at budding foci of nucleocapsids. AC141 was shown to associate with the ME53-GP64 complex at the plasma membrane and potentially facilitates the budding of nucleocapsids. The interaction between AC141, GP64, ME53 may enhance the cellular relocation of ME53. AcMNPV-encoded viral ubiquitin (vUbi) and AC141 (a predicted E3 ubiquitin ligase) have been shown to be required for efficient BV production. We hypothesized that vUbi interacts with AC141 and this interaction is required for BV production. Deletion of both ac141 and vubi restricted the infection to a single cell. AC141 was ubiquitinated by either vUbi or cellular Ubi and this interaction was required for optimal BV production. Virion fractionation showed that a nucleocapsid protein of 100 kDa, potentially AC66, is specifically ubiquitinated with vUbi in BV and but not in occlusion derived virus (ODV). These data suggest ubiquitination of nucleocapsid protein acts as a signal that determines how a nucleocapsid is directed to become a BV or ODV. Polyubiquitin chains are formed by the internal lysines present within the ubiquitin and serve as a signal for various cellular pathways. Mutations of lysines to arginine showed that vUbi is involved in cellular processes mediated by K6 and K27 polyubiquitin chains. The collective results of this study provide significant new data on the role of viral and host proteins and the mechanism by which baculovirus nucleocapsids egress from the nucleus to form BV.Land and Food Systems, Faculty ofGraduat