| Me53 is a highly conserved baculovirus gene found in all lepidopteran baculoviruses that have been fully sequenced to date. The putative ME53 protein contains a zinc finger domain and has been previously described as a major early transcript. A me53-null bacmid (AcDeltame53GFP) as well as a repair virus (AcRepME53:HA-GFP) carrying me53 with a C-terminal HA tag, under the control of its native early and late promoter elements. Cells transfected with AcDeltame53GFP resulted in a 3 log reduction in budded virus (BV) production compared to both the parental and repair bacmids, demonstrating that although me53 was not essential, viral replication was compromised in its absence. Our data also suggested that me53 did not affect DNA replication. Deletion of the early transcriptional start site resulted in a 10-360 fold reduction of BV yield; however, deletion of the late promoter resulted in a 160-1,000 fold reduction in BV yield, suggesting that ME53 was required both early and late in the infection cycle. Western blot analysis of purified virions from the repair virus revealed that ME53:HA was associated with both BV and occlusion derived virions. In localization studies, ME53:GFP adopted a primarily cytoplasmic distribution at early times post-infection, and a primarily nuclear distribution at late times post-infection. Additionally, at late times ME53:GFP formed distinct foci at the cell periphery. These foci co-localized with the major envelope fusion protein GP64, and frequently with VP39 capsid protein, suggesting these regions may represent viral budding sites. Deletion of vp39 did not influence the distribution of ME53:GFP, however deletion of gp64 abolished ME53:GFP foci at the cell periphery, implying an association between ME53 and GP64. Despite the association of ME53 and GP64, ME53 also associated with the nucleocapsid based on budded virus fractionation. Together this suggests that ME53 may be providing a scaffold that bridges the viral envelope and nucleocapsid. Furthermore, truncation studies revealed that domains within both the amino- and carboxy- halves of ME53 contributed to efficient production and accumulation of budded virus at the cell periphery, while nuclear localization at late times was dependent on residues within the amino half of the protein. |
