Autographa californica multiple nucleopolyhedrovirus DNA polymerase C terminus is required for nuclear localization and viral DNA replication

Abstract

The DNA polymerase (DNApol) of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for viral DNA replication. The DNApol exonuclease and polymerase domains are highly conserved and are considered functional in DNA replication. However, the role of the DNApol C terminus has not yet been characterized. To identify whether only the exonuclease and polymerase domains are sufficient for viral DNA replication, several DNApol C-terminal truncations were cloned into a dnapol-null AcMNPV bacmid with a green fluorescent protein (GFP) reporter. Surprisingly, most of the truncation constructs, despite containing both exonuclease and polymerase domains, could not rescue viral DNA replication and viral production in bacmid-transfected Sf21 cells. Moreover, GFP fusions of these same truncations failed to localize to the nucleus. Truncation of the C-terminal amino acids 950 to 984 showed nuclear localization but allowed for only limited and delayed viral spread. The C terminus contains a typical bipartite nuclear localization signal (NLS) motif at residues 804 to 827 and a monopartite NLS motif at residues 939 to 948. Each NLS, as a GFP fusion peptide, localized to the nucleus, but both NLSs were required for nuclear localization of DNApol. Alanine substitutions in a highly conserved baculovirus DNApol sequence at AcMNPV DNApol amino acids 972 to 981 demonstrated its importance for virus production and DNA replication. Collectively, the data indicated that the C terminus of AcMNPV DNApol contains two NLSs and a conserved motif, all of which are required for nuclear localization of DNApol, viral DNA synthesis, and virus production.

Importance: The baculovirus DNA polymerase (DNApol) is a highly specific polymerase that allows viral DNA synthesis and hence virus replication in infected insect cells. We demonstrated that the exonuclease and polymerase domains of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) alone are insufficient for viral DNA synthesis and virus replication. Rather, we identified three features, including two nuclear localization signals and a highly conserved 10-amino-acid sequence in the AcMNPV DNApol C terminus, all three of which are important for both nuclear localization of DNApol and for DNApol activity, as measured by viral DNA synthesis and virus replication.

FIG 1

DNApol truncations and repair virus bacmids. (A) Schematic diagram of AcMNPV DNApol and of the C-terminal DNApol truncations in bacmids Bac-GFP-Pol800 to Bac-GFP-Pol949 and WT repair viruses (WT^rep). Conserved exonuclease domains are labeled Exo I to Exo III, with corresponding DNApol amino acid numbers above the label and polymerase domains labeled I to VII with corresponding amino acid numbers below the label. C-terminal DNApol truncations are numbered according to the last residue of the WT DNApol present in the mutant. (B) Schematic of cassettes with truncated dnapol fragments (Pol800, Pol831, Pol841, Pol878, and Pol949), egfp as a reporter under the Opie1 promoter, and the adjoining polyhedrin (polh) gene under the polh promoter. The cassettes were inserted in the original polyhedrin locus of the DNApol KO, Ac^KO bacmid by Tn7-mediated transposition. The WT and truncated DNApol constructs were expressed by the dnapol native promoter and SV40 poly(A). (C) Schematic of truncated fragments (Pol800, Pol831, Pol841, Pol878, and Pol949) with GFP fusions following insertion into a pBluescript II(+) vector. The truncated DNApol constructs were expressed by the Acie-1 promoter and SV40 poly(A).

FIG 2

GFP fluorescence and virus production analysis of C-terminal-truncated DNApol:GFP fusion bacmids and WT^rep and Ac^KOGFP bacmid in Sf21 cells. (A) GFP fluorescence of C-terminal truncation bacmids and WT^rep and Ac^KOGFP bacmids in Sf21 cells. Fluorescence images of monolayers of cells transfected with WT^rep, Ac^KOGFP, Bac-Pol800:GFP, Bac-Pol831:GFP, Bac-Pol841:GFP, Bac-Pol878:GFP, Bac-Pol949:GFP, and Bac-Pol984:GFP a 24, 96, and 144 hpt (top, middle, and bottom rows, respectively). Ac^KOGFP was used as a DNApol negative control, and WT^rep was used as a positive control. All images were taken at the same magnification. (B) Light microscopy images of monolayers of cells transfected withWT^rep, Ac^KOGFP, Bac-Pol800:GFP, Bac-Pol831:GFP, Bac-Pol841:GFP, Bac-Pol878:GFP, Bac-Pol949:GFP, and Bac-Pol984:GFP at 144 hpt. (C) Monolayers were transfected with 2 μg of the indicated bacmid DNAs, Ac^KOGFP, or WT^rep bacmid. The production of infectious BV was determined in TCID₅₀ endpoint dilution assays. (D) AcMNPV DNApol accumulation kinetics of the WT^rep, Ac^KOGFP, Bac-Pol800:GFP, Bac-Pol831:GFP, Bac-Pol841:GFP, Bac-Pol878:GFP, Bac-Pol949:GFP, and Bac-Pol984:GFP in independently transfected Sf21 cells. Total intracellular DNA was extracted from transfected cells at 0 hpt and 24 hpt, and AcMNPV DNA copy numbers were determined by qPCR. Bars in the panels represent standard deviations determined from three independent replicates.

FIG 3

Nuclear localization of C-terminal-truncated DNApol via pBlue transient-expression plasmids. Fluorescence localization of DNApol-GFP fusion proteins was performed by using transient-expression plasmids following transfection of Sf21 cells at 24 hpt. Nuclei were stained with DAPI, and cells were visualized by confocal microscopy. Cells were transfected by pBlue-Pol800:GFP (Pol800, A1 to A5), pBlue-Pol831:GFP (Pol831, B1 to B5), pBlue-Pol841:GFP (Pol841, C1 to C5), pBlue-Pol878:GFP (Pol878, D1 to D5), pBlue-Pol949:GFP (Pol949, E1 to E5), and pBlue-Pol984:GFP (Pol984, F1 to F5). Photographs were taken via light microscopy (A1 to F1), GFP green fluorescence (A2 to F2), and blue DAPI fluorescence (A3 to F3). Merged green and blue fluorescence images (A4 to F4) are in the column labeled G+D. Light microscopy and green and blue fluorescence images were also merged (A5 to F5) in the column labeled L+G+D.

FIG 4

Schematic of AcMNPV C-terminal DNApol and conserved sequences. (A) Schematic of AcMNPV DNApol with expanded predicted NLS motifs and a conserved 10-aa C-terminal sequence. A 16-aa sequence within a 24-amino-acid stretch from D804 to T827 closely matches the consensus classic bipartite NLS sequence ([K/R]₂[X]_10–12[K/R]_>3-5) (33, 47); the second motif within a 10-amino-acid fragment at positions C939 to D948 corresponds to the classical monopartite consensus sequence (K[K/R]X[K/R]) (33). (B) Sequence alignment of C-terminal DNApols in 11 group I alphabaculoviruses. Sequence alignment was performed using ClustalX and was edited by using GeneDoc software; the black shading represents 100% conservation in aa 972 to 981. AgMNPV, Anticarsia gemmatalis MNPV; AnpeNPV, Antheraea pernyi NPV; BmNPV, Bombyx mori NPV; CfDEFNPV, Choristoneura fumiferana DEF NPV; CfMNPV, Choristoneura fumiferana NPV; EppoNPV, Epiphyas postvittana NPV; HcNPV, Hyphantria cunea NPV; MaviMNPV, Maruca vitrata MNPV; OpMNPV, Orgyia pseudotsugata MNPV; RoMNPV, Rachiplusia ou MNPV.

FIG 5

Functional analysis of putative NLSs in the DNApol C terminus performed with pBlue transient-expression plasmids. Localization of the indicated DNApol-GFP fusion peptides was performed by using transient-expression plasmids in transfected Sf21 cells at 24 hpt. Nuclei were stained with DAPI and visualized via confocal microscopy. Cells were transfected with egfp-only plasmid (A1 to A5) or with EGFP-tagged DNApol plasmids pBlue-egfp:Pol804-827 (Pol804-827; B1 to B5), egfp:Pol939–948 (Pol939-948; C1 to C5), or with EGFP-tagged DNApol deletions pBlue-PolΔ804-827:GFP (PolΔ804-827; D1 to D5), pBlue-PolΔ939-948:GFP (PolΔ939-948; E1 to E5). The constructs were expressed by an Acie-1 promoter and SV40 poly(A). Photographs were taken at 24 hpt by using light microscopy (A1 to E1), GFP fluorescence (A2 to E2), or blue DAPI staining (A3 to F3). Green and blue fluorescence images were merged (A4 to E4) in the column labeled G+D, and light and green and blue fluorescence images were merged (A5 to E5) in the column labeled L+G+D. All images are at the same magnification. Compare with Fig. 3F for the full-length pBlue-Pol984:GFP as a positive control.

FIG 6

GFP fluorescence and virus production analysis of bacmids of alanine-replaced aa 972 to 981 as well as Ac^KOGFP and WT^rep bacmids in Sf21 cells. (A) Fluorescence and light microscopy images of monolayers of cells transfected with Ac^KOGFP, Bac-GFP-Pol10A, and WT^rep at 24, 96, and 144 hpt. Ac^KOGFP was used as a negative control, and WT^rep was a positive control. (B) Monolayers were transfected with 2 μg of the bacmid DNAs. Infectious BV titers were determined in TCID₅₀ endpoint dilution assays. (C) Viral DNA accumulation kinetics following independent transfections of Sf21 cells with bacmids Bac-GFP-Pol10A, Ac^KOGFP, or WT^rep. Total intracellular DNA was extracted at 0 and 24 hpt, and AcMNPV DNA copy numbers were determined by qPCR of Acie1. Bars in the panels represent standard deviations determined from three independent replicates.

FIG 7

GFP fluorescence and viral production analysis of DNApol C-terminal substitution bacmid, WT^rep, and Ac^KOGFP bacmid in Sf21 cells. (A) Schematic of AcMNPV C-terminal DNApol (amino acids 801 to 984) replaced with that of CfMNPV (amino acids 798 to 990). (B) GFP fluorescence images of monolayers of cells transfected with bacmid Ac^KOGFP, Bac-GFP-AcPol800:CfPol798–990, or WT^rep at 24, 72, and 120 hpt. (C) Monolayers were transfected with 2 μg of Ac^KOGFP, Bac-GFP-AcPol800:CfPol798-990, or WT^rep bacmid DNA, and BV production was determined in a TCID₅₀ assay.