Autographa californica multiple nucleopolyhedrovirus me53 (ac140) is a nonessential gene required for efficient budded-virus production

Abstract

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. We generated an me53-null bacmid (AcDeltame53GFP), as well as a repair virus (AcRepME53:HA-GFP) carrying me53 with a C-terminal hemagglutinin (HA) tag, under the control of its native early and late promoter elements. Sf9 and BTI-Tn-5b1 cells transfected with AcDeltame53GFP resulted in a 3-log reduction in budded-virus (BV) production compared to both the parental Autographa californica multiple nucleopolyhedrosis virus and the repair bacmids, demonstrating that although me53 is not essential for replication, replication is compromised in its absence. Our data also suggest that me53 does not affect DNA replication. Cell fractionation showed that ME53 is found in both the nucleus and the cytoplasm as early as 6 h postinfection. Deletion of the early transcriptional start site resulted in a 10- to 360-fold reduction of BV yield; however, deletion of the late promoter (ATAAG) resulted in a 160- to 1,000-fold reduction, suggesting that, in the context of BV production, ME53 is required both early and late in the infection cycle. Additional Western blot analysis of purified virions from the repair virus revealed that ME53:HA is associated with both BV and occlusion-derived virions. Together, these results indicate that me53, although not essential for viral replication, is required for efficient BV production.

FIG. 1.

Schematic representation of the me53 and TN7 insertion loci for AcGFP, AcΔme53GFP, and AcRepME53:HA-GFP. All GFP viruses contain an eGFP reporter and polyhedrin under the control of its native promoter at the Tn7 site. AcΔme53GFP, me53 was replaced with cat under the control of a prokaryotic promoter. AcRepME53:HA-GFP carries me53 with a C-terminal HA tag under the control of its native early and late promoter at the Tn7 site. Promoter sites for p74, me53, ie0, and Opie1 are indicated by small arrows (E, early; L, late).

FIG. 2.

Cytopathology and GFP fluorescence of AcGFP, AcRepME53:HA-GFP, and AcΔme53GFP in Sf9 and BTI-Tn-5b1 cells. (A) Fluorescence images of monolayers of AcGFP, AcRepME53:HA-GFP, and AcΔme53GFP transfected cells at 48 and 96 h p.t. as indicated. (B) Representative viral plaques from AcGFP, AcRepME53:HA-GFP and AcΔme53GFP transfected Sf9 and BTI-Tn-5b1 monolayers at 120 h p.t. The inset shows the means and standard deviations of plaque sizes from 20 well- isolated plaques from each bacmid.

FIG. 3.

Virus growth curves of AcGFP and AcΔme53GFP in Sf9 (A) or BTI-Tn-5b1 cells (B) and viral DNA accumulation (C and D). Monolayers were transfected with either AcGFP or AcΔme53GFP bacmid, and supernatant samples were collected at the indicated time points. Note that time zero is when the transfection supernatant is removed and replaced with fresh medium. Sf9 monolayers were transfected with 2 μg of either AcGFP or AcΔme53GFP, and the fold increase in intracellular DNA (C) or total DNA (intracellular and budded virus) (D) was determined at the times noted. For panels C and D, the fold increase was calculated by dividing by viral DNA copy number at time zero. The data represent the averages of three independent experiments.

FIG. 4.

Western immunoblot of infected cells or virus particles. Sf9 and BTI-Tn-5b1 cells were infected with AcRepME53:HA-GFP. (A) Cells were collected and fractionated into cytoplasmic and nuclear fractions at various times p.i. as indicated. Blots were probed with anti-HA, anti-IE1, or anti-gp64 antibodies. (B) AcRepME53:HA-GFP BVs (HABV) and ODVs (HAODV) were purified and probed with anti-HA to detect HA-tagged ME53. Parental AcMNPV virus (AcBV and AcODV) was included as a control to ensure there was no cross-reactivity with the antibodies. Electron microscopy was used to confirm the isolation of intact BVs.

FIG. 5.

(A) Nucleotide sequence of the me53 upstream region for AcGFP, AcΔEΔLme53, AcΔLme53, and AcΔEme53. The early promoter is italicized and underlined, the late promoter is boldface and underlined, and the TATA element is underlined. Crossed-out nucleotides indicate those deleted in the constructs. (B) Titers of AcΔEme53, AcΔLme53, AcΔEΔLme53, or AcGFP bacmid transfected monolayers at 5 days p.t. of Sf9 and BTI-Tn-5b1 cells. The data represent the averages of two independent experiments. (C) Northern blot analysis of me53 transcripts in cells transfected with AcRepME53:HA-GFP, AcΔEΔLme53, AcΔLme53, and AcΔEme53 at 24 and 48 h p.t. Ribosomal bands are shown below to indicate relative RNA loading.