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

Abstract

Autographa californicamultiple nucleopolyhedrovirus (AcMNPV) is in the familyBaculoviridae, genusAlphabaculovirus AcMNPVme53is a highly conserved immediate early gene in all lepidopteran baculoviruses that have been sequenced and is transcribed up to late times postinfection. Althoughme53is 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 occlusion-derived 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.

Importance: Baculovirusme53is 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.

FIG 1

Bioinformatic analysis of ME53s from group I and II alphabaculoviruses and betabaculoviruses. (A) Neighbor-joining phylogenetic analysis of ME53 protein sequences. The bootstrap scores of the nodes are shown. Clades 1, 2, and 3 are indicated by brackets. (B) Schematics of ME53 conservation relative to AcMNPV ME53 within each group of viruses showing three highly conserved amino acid regions, 111 to 138, 225 to 300, and 379 to 400.

FIG 2

Analysis of amino acid sequence conservation of ME53s from group I alphabaculoviruses. Shown are amino acid sequence alignments of the ME53 N terminus (A) and C terminus (B). The asterisks indicate 100% identical residues, the colons indicate conserved substitutions, and the periods indicate semiconserved substitutions. The sequences within the boxes represent the predicted alpha helix of aa 113 to 136 (A) and putative zinc finger domain of aa 379 to 399 (B). The numbered amino acids are the 100% conserved residues mentioned in the text.

FIG 3

Schematics of ME53 bacmid constructions. (A) Cloning strategy for HA-tagged ME53 peptides and internal deletions using the AcMNPV me53 knockout bacmid as the backbone. An HA epitope tag (open squares) was fused to the C terminus of ME53 (solid rectangles) to allow intracellular localization of ME53 by immunofluorescence microscopy. (B) Strategy for GFP-fused ME53 truncations and internal deletions using the AcMNPV me53 knockout bacmid as the backbone. A GFP tag (gray rectangles) was fused to the C terminus of ME53 (black rectangles) to follow ME53 localization by fluorescence microscopy. The white rectangles represent internal deletions.

FIG 4

Intracellular localization of HA-tagged ME53 peptides and internal deletions following transfection with bacmid DNA at 18 and 48 hpt. Cells were fixed and stained with mouse anti-HA primary antibody (1:20) and Alexa Fluor 594 goat anti-mouse secondary antibody (1:100). Forty transfected cells showing red fluorescence were counted, and the percentages of cells showing nuclear localization are indicated. Peptide aa 83 to 152 localized largely to the nucleus, while ME53 lacking aa 83 to 152 remained cytoplasmic. The monolayer column is at a lower magnification to show several cells in the same view. Scale bar is 2 µm.

FIG 5

Intracellular localization of plasmid-only expressed GFP fused to ME53 (ME53:GFP alone), bacmid DNA-expressed full-length ME53 fused to GFP (ME53:GFP), and ME53 lacking aa 250 to 449 at the C terminus fused to GFP (ME53_Δ250–449:GFP) at 48 hpt. Cells were fixed and examined under a Leica SP5 CLSM using a 63× dipping lens. Forty transfected cells with green fluorescence were counted, and the percentages of cells showing nuclear localization are indicated. Transient expression of ME53:GFP in the absence of virus infection showed only cytoplasmic localization, while truncation of the C terminus of ME53 did not abolish its nuclear translocation.

FIG 6

Intracellular localization of GFP-fused ME53 N-terminal truncations with bacmid DNA at 48 hpt. Cells were fixed and examined under a Leica SP5 CLSM using a 63× dipping lens. Forty transfected cells with green fluorescence were counted, and the percentages of cells showing any nuclear localization are indicated. Nuclear localization started to be reduced for truncations of ME53 downstream of amino acid 108.

FIG 7

Intracellular localization of GFP-fused ME53 with internal deletions at the C terminus of the putative NTS at 48 hpt. Cells were fixed and examined under a Leica SP5 CLSM using a 63× dipping lens. Forty transfected cells with green fluorescence were counted, and the percentages of cells showing any nuclear localization are indicated. ME53 nuclear translocation was observed for deletions of residues downstream of amino acid 137.

FIG 8

Intracellular localization of transiently expressed GFP (GFP alone), bacmid-expressed ME53 fused to GFP (ME53:GFP), GFP only tagged with the NTS (aa 109 to 137) (NTS:GFP), and GFP fused to ME53 with the NTS (aa 113 to 139) deleted (ME53ΔNTS:GFP) at 48 hpt. Cells were fixed and examined under a Leica SP5 CLSM using a 63× dipping lens. Forty transfected cells with green fluorescence were counted, and the percentages of cells showing any nuclear localization are indicated. NTS:GFP localized mostly to the nucleus, while deletion of the NTS from ME53 abolished its nuclear localization.

FIG 9

Intracellular localization of GFP-fused ME53 site-directed mutations with bacmid DNA at 48 hpt. Cells were fixed and examined under a Leica SP5 CLSM using a 63× dipping lens. Forty transfected cells with green fluorescence were counted, and the percentages of cells showing nuclear localization are indicated. The highly conserved residues E121, R122, and K126 were mutated to alanine (A). None of the single-site mutations altered its nuclear translocation.

FIG 10

Virus titration of ME53 truncations and internal deletions at 7 days posttransfection. The horizontal dotted lines represent internally deleted regions. Supernatant with BVs was collected and used in endpoint dilution (10⁰ to 10⁻⁹) to determine the virus titer. The values are relative to 100% virus yield for the wild-type ME53 and are the averages of two determinations.