Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is in the family Baculoviridae, genus Alphabaculovirus. AcMNPV me53 is one of the conserved immediate early genes expressed immediately following AcMNPV infection in all sequenced lepidopteran baculoviruses. Me53 is transcribed both early and late post-infection. Although me53 is not essential for viral DNA synthesis, it greatly attenuates infectious budded virus (BV) production when deleted. ME53 is associated with the nucleocapsid on both budded virus and occlusion-derived virus, but not with the envelope. ME53 co-localizes in plasma membrane foci with viral envelope glycoprotein GP64. Despite lack of a nuclear localization signal (NLS), ME53 localizes in the cytoplasm early post-infection, and translocates to the nucleus late post-infection. To map determinants of ME53 that facilitate its nuclear translocation, recombinant AcMNPV bacmids containing a series of ME53 truncations, internal deletions and peptides fused with HA or GFP tags were constructed. Intracellular localization identified that residues within amino acids 109 to 137 act as the nuclear translocation sequence (NTS) to facilitate ME53 nuclear transport. Since ME53 translocates to the nucleus, and has a conserved zinc-finger domain which is usually considered to be related to transcriptional regulation, a possible nuclear function of ME53 was also investigated. The transcript levels of select viral genes were quantified by qRT-PCR in both wildtype bacmid and me53 knockout bacmid transfected cells. The presence of me53 increased the transcription of viral immediate early genes, genes encoding viral RNA polymerase subunits, and genes required for virus production. The most increased genes are late genes essential for viral nucleocapsid assembly and egress, suggesting that ME53 may function as a transcription factor to regulate viral gene expression. Furthermore, since ME53 translocates to the nucleus and co-localizes with viral major envelope protein GP64 at the plasma membrane, this study investigated the possible chaperone proteins ME53 recruits to facilitate its translocations. The HA or V5 epitope tag was fused to ME53 to help identify its binding partners in immunoprecipitation (IP) assays. Viral major envelope protein GP64 and major capsid protein VP39 were detected as ME53 binding partners that might facilitate its translocation to either the plasma membrane or the nucleus.