Analysis of an Autographa californica nucleopolyhedrovirus lef-11 knockout: LEF-11 is essential for viral DNA replication

Abstract

The Autographa californica nucleopolyhedrovirus (AcMNPV) lef-11 gene was previously identified by transient late expression assays as a gene important for viral late gene expression. The lef-11 gene was not previously identified as necessary for DNA replication in transient origin-dependent plasmid DNA replication assays. To examine the role of lef-11 in the context of the infection cycle, we generated a deletion of the lef-11 gene by recombination in an AcMNPV genome propagated as a BACmid in Escherichia coli. The resulting AcMNPV lef-11-null BACmid (vAc(lef11KO)) was unable to propagate in cell culture, although a "repair" AcMNPV BACmid (vAc(lef11KO-REP)), which was generated by transposition of the lef-11 gene into the polyhedrin locus of the vAc(lef11KO) BACmid, was able to replicate in a manner similar to wild-type or control AcMNPV viruses. Thus, the lef-11 gene is essential for viral replication in Sf9 cells. The vAc(lef11KO) BACmid was examined to determine if the defect in viral replication resulted from a defect in DNA replication or from a defect in late transcription. The lef-11-null BACmid and control BACmids were transfected into Sf9 cells, and viral DNA replication was monitored. The viral DNA genome of the lef-11-null BACmid (vAc(lef11KO)) was not amplified, whereas replication and amplification of the genomes of the repair BACmid (vAc(lef11KO-REP)), wild-type AcMNPV, and a nonpropagating gp64-null control BACmid (vAc(GUSgp64KO)) were readily detected. Northern blot analysis of transcripts from selected early, late, and very late genes showed that late and very late transcription was absent in cells transfected with the lef-11-null BACmid. Thus, in contrast to prior studies using transient replication and late expression assays, studies of a lef-11-null BACmid indicate that LEF-11 is required for viral DNA replication during the infection cycle.

FIG. 1.

Strategy for construction of a lef-11-null BACmid containing a deletion of the AcMNPV lef-11 gene and rescue by reinsertion of the wild-type (wt) lef-11 gene. (A) Relative locations and orientations of overlapping ORFs in the lef-11 locus of AcMNPV. The relative locations of lef-11 and pp31 transcripts are indicated by dashed lines. (B) Organization of the transfer vector DNA used to generate the lef-11 knockout BACmid by recombination in E. coli. A linear DNA fragment containing a poly(A) site, a p6.9 promoter-driven GUS gene, a chloramphenicol resistance gene cassette (CAT), and an ie1 promoter, are flanked by 1,034- and 1,026-bp regions from the orf38 and pp31 genes, as indicated. The linear DNA fragment was excised from plasmid pGEM72(f+)polyAie1Ppp31GUSorf38CAT, as described in Materials and Methods, and cotransfected with BACmid bMON14272 into E. coli strain BJ5183. (C) The organization of the lef-11-null BACmid (vAc^lef11KO) is shown. vAc^lef11KO was derived from BACmid bMON14272 and contains a chloramphenicol resistance gene cassette and a p6.9 promoter-driven GUS reporter gene in the lef-11 locus. The majority of the lef-11 ORF was removed. (D) Structure of the lef-11-null repair BACmid (vAc^lef11KO-REP). vAc^lef11KO-REP was derived from BACmid vAc^lef11KO by insertion of the wild-type lef-11 gene (under the control of the lef-11 promoter) into the polyhedrin locus by transposition with plasmid pFastBAClef11-REP.

FIG.2.

Confirmation of BACmid constructs vAc^lef11KO and vAc^lef11KO-REP by PCR analysis. (A) The strategy for PCR analysis of the lef-11 locus in BACmid vAc^lef11KO is indicated by the positions of primer pairs (arrows and brackets). The top diagram shows the structure of the wild-type (wt) lef-11 locus, and the lower diagram shows the structure of the BACmid vAc^lef11KO. To confirm the insertion of the polyA-GUS-CAT-ie1 cassette in BACmid vAc^lef11KO, primer pairs A+B and C+D were used to examine the recombination junctions by PCR analysis. For each primer pair, one primer corresponded to sequences within the inserted sequence (the GUS or CAT ORF), and the second primer was from the baculovirus genome, just outside the homologous flanking sequences used for recombination. Primer pair E+F was designed to amplify a fragment from within the lef-11 ORF and was used to confirm the absence of the lef-11 ORF in vAc^lef11KO. The sizes of expected PCR amplification products are shown below each primer pair on the diagram, and the panels below show the agarose gel electrophoresis results of each PCR, with the sizes of PCR products indicated beside an arrowhead. Primer pairs used for each PCR analysis are indicated below each panel and template DNAs are indicated above the panels. M, DNA size markers. (B) Analysis of the polyhedrin locus in BACmid vAc^lef11KO-REP. PCR analysis was used to confirm the insertion of a cassette containing the lef-11 ORF under the control of the wild-type lef-11 promoter from plasmid pFastBAClef11-REP, into BACmid vAc^lef11KO. The relative location of the primer pair used to confirm the insertion of the lef-11 gene is shown below the diagram of the resulting BACmid (vAc^lef11KO-REP). The panel below shows an ethidium bromide-stained agarose gel with the expected 1.47-kbp DNA product of PCR amplification from vAc^lef11KO-REP (lane 1). A similar PCR amplification from a negative control (wild-type AcMNPV) is also shown (lane 2).

FIG. 3.

Analysis of viral replication by a lef-11-null BACmid. (A) A transfection-infection assay was used to examine lef-11-null BACmids for viral replication in Sf9 cells. BACmid DNAs from the indicated constructs were used to transfect Sf9 cells, and cells were incubated for 5 days. Supernatants from transfected cells were transferred to a second group of Sf9 cells, which were subsequently incubated for 3 days and then stained for GUS expression from the p6.9 late promoter-GUS reporter. The results of GUS staining are shown in the three lower panels. (B) Virus growth curves were determined from either transfected cells or infected cells. For transfections, Sf9 cells were transfected in triplicate with either vAc^lef11KO or vAc^64−/+GUS, and then supernatants were removed at the indicated times posttransfection, and the TCID₅₀ in Sf9 cells was determined. For infections, infectious budded virions were prepared from wild-type AcMNPV or BACmids vAc^lef11KO-REP or vAc^64−/+GUS. Infections were performed at an MOI of 5 in triplicate, and supernatants were collected and assayed for production of infectious virus by TCID₅₀.

FIG. 4.

Complementation of a lef-11-null BACmid by a stable cell line expressing LEF-11. The diagram shows the strategy used to examine a LEF-11-expressing cell line (Sf9^lef11) for complementation of the lef-11-null BACmid (vAc^lef11KO). GUS activity results are indicated above the wells (GUS+ or GUS−). Quantitative measurements from triplicate transfection-infections are shown for the infected wells (A, B, and C) in the graph on the right.

FIG. 5.

Analysis of AcMNPV DNA replication in Sf9 cells transfected with lef-11-null and control BACmids. (A) Sf9 cells were transfected with either wild-type (Wt) AcMNPV, control BACmid (vAc^64−/+GUS), lef-11 repair BACmid (vAc^lef11KO-REP), gp64-null control BACmid (vAc^GUSgp64KO), or lef-11-null BACmid (vAc^lef11KO) DNA (lanes 1 to 5, respectively), and total cellular DNAs were isolated at various times (0 to 96 h) posttransfection. Viral DNA replication was detected by Southern dot blot hybridization with total AcMNPV DNA as a ³²P-labeled hybridization probe. Cells transfected with the lef-11-null BACmid (vAc^lef11KO) were also examined after an extended period (right lane, 5 to 10 days). A standard curve of AcMNPV DNA is shown below (10 to 200 ng of DNA). (B) Quantitative analysis of BACmid DNA replication by Southern dot blot analysis. Three replicates of each virus and time point were examined as shown in panel A, and DNA was measured by PhosphorImager analysis. Bars represent the average of three dot blot samples, and error bars represent standard deviation. (C) Sf9 cells transfected with each of the indicated DNAs (wild-type AcMNPV, vAc^64−/+GUS, vAc^lef11KO-REP, vAc^GUSgp64KO, or vAc^lef11KO) were stained for GUS activity at 4 days posttransfection.

FIG. 6.

Northern blot analysis of early, late, and very late transcripts from cells transfected with lef-11-null or control BACmids. Sf9 cells were transfected with either the lef-11-null BACmid (vAc^lef11KO), a control BACmid (vAc^64−/+GUS), or the lef-11 repair BACmid (vAc^lef11KO-REP). At various times (12, 18, 48, or 72 h) posttransfection, total RNAs were isolated and used for Northern blot analysis with early (ie1), late (p6.9, p24, and gp16), or very late (p10) gene-specific probes. BACmids used for transfections are indicated at the top of the lanes, and gene-specific probes are indicated on the left. The sizes of expected RNAs from each gene-specific probe are indicated in kilobases on the right.