Nuclear Translocation Sequence and Region in Autographa californica Multiple Nucleopolyhedrovirus ME53 That Are Important for Optimal Baculovirus Production

Abstract

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is in the family Baculoviridae, genus Alphabaculovirus. AcMNPV me53 is a highly conserved immediate early gene in all lepidopteran baculoviruses that have been sequenced and is transcribed up to late times postinfection. Although me53 is not essential for viral DNA synthesis, infectious budded virus (BV) production is greatly attenuated when it is deleted. ME53 associates with the nucleocapsid on both budded virus and occlusionderived virus, but not with the virus envelope. ME53 colocalizes in plasma membrane foci with the envelope glycoprotein GP64 in a GP64-dependent manner. ME53 localizes in the cytoplasm early postinfection, and despite the lack of a reported nuclear localization signal (NLS), ME53 translocates to the nucleus at late times postinfection. 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 hemagglutinin (HA) or green fluorescent protein (GFP) tags were constructed. Intracellular-localization studies identified residues within amino acids 109 to 137 at the N terminus of ME53 that acted as the nuclear translocation sequence (NTS), facilitating its nuclear transport at late times postinfection. The first 100 N-terminal amino acids and the last 50 C-terminal amino acids of ME53 are dispensable for high levels of budded virus production. The region within amino acids 101 to 398, which also contains the NTS, is critical for optimal levels of budded virus production. IMPORTANCEBaculovirus me53 is a conserved immediate early gene found in all sequenced lepidopteran alpha-and betabaculoviruses. We first identified residues within amino acids 109 to 137 at the N terminus that act as the ME53 nuclear translocation sequence (NTS) to facilitate its nuclear translocation and defined an internal region within amino acids 101 to 398, which includes the NTS, as being necessary for optimal budded virus production. Altogether, these results indicate a previously unidentified nuclear role that ME53 plays in virus replication. The Baculoviridae are a family of insect DNA viruses that infect insects, mostly from the order Lepidoptera but also from the orders Diptera and Hymenoptera. Baculoviruses are characterized by a circular double-stranded DNA genome ranging from 80 to 180 kb in size, packaged within a rod-shaped capsid and enclosed by a lipid envelope (1).The viral DNA genome is uncoated into the nucleus, followed by virus gene transcription, DNA replication, and eventually nucleocapsid assembly in the nucleus prior to budding from the cells or occlusion into polyhedra (2, 3). Baculovirus gene expression and regulation follow a temporal cascade. Immediate early genes are transcribed first within 30 min postinfection, followed by transactivation of viral early genes (4). The early gene products then allow viral DNA replication and late/very late gene transcription (5, 6). Late and very late gene transcription is ...

Molecular Mechanisms of Nuclear Import of Baculovirus Proteins

“…An illustrative example is the interaction between the Ran-binding protein 5 (RanBP5) of the influenza A virus (IAV) and the PB1-PA dimer in the host [35]. In baculovirus, certain nuclear proteins lacking functional NLS exhibit a sequence serving as a pivotal motif for nuclear translocation.For instance, Liu et al identified amino acid residues 109 to 137 at the N-terminal of ME53, the nuclear protein of AcMNPV, as a potential binding site for other viral or host proteins, including VP39 and GP64 [37].…”

Mechanisms Mediating Nuclear Trafficking Involved in Viral Propagation by DNA Viruses

“…Pan et al reported a novel NLS in the baculovirus late expression factor 11, and further research revealed that the NLS of baculovirus LEF-11 is important for viral DNA replication [25,88]. It has been reported that the cNLSs of baculovirus IE-1 and ME53 are critical for optimal levels of budded virus production [27,92]. Moreover, Yu and colleagues identified a bipartite NLS of KRK (residues 83 to 85) and KKVK (residues 120 to 123) that is essential for the final destination of E1 in the nucleus to exert its function during viral replication [93].…”