Analysis of an Autographa californica multicapsid nucleopolyhedrovirus lef-6-null virus: LEF-6 is not essential for viral replication but appears to accelerate late gene transcription

Abstract

The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-6 gene was previously shown to be necessary for optimal transcription from an AcMNPV late promoter in transient late expression assays. In the present study, we examined the expression and cellular localization of lef-6 during the AcMNPV infection cycle and generated a lef-6-null virus for studies of the role of lef-6 in the infection cycle. Transcription of lef-6 was detected from 4 to 48 h postinfection, and the LEF-6 protein was identified in dense regions of infected cell nuclei, a finding consistent with its potential role as a late transcription factor. To examine lef-6 in the context of the AcMNPV infection cycle, we deleted the lef-6 gene from an AcMNPV genome propagated as an infectious BACmid in Escherichia coli. Unexpectedly, the resulting AcMNPV lef-6-null BACmid (vAc(lef6KO)) was able to propagate in cell culture, although virus yields were substantially reduced. Thus, the lef-6 gene is not essential for viral replication in Sf9 cells. Two "repair" AcMNPV BACmids (vAc(lef6KO-REP-P) and vAc(lef6KO-REP-ie1P)) were generated by transposition of the lef-6 gene into the polyhedrin locus of the vAc(lef6KO) BACmid. Virus yields from the two repair viruses were similar to those from wild-type AcMNPV or a control (BACmid-derived) virus. The lef-6-null BACmid (vAc(lef6KO)) was further examined to determine whether the deletion of lef-6 affected DNA replication or late gene transcription in the context of an infection. The lef-6 deletion did not appear to affect viral DNA replication. Using Northern blot analysis, we found that although early transcription was apparently unaffected, both late and very late transcription were delayed in cells infected with the lef-6-null BACmid. This phenotype was rescued in viruses containing the lef-6 gene reinserted into the polyhedrin locus. Thus, the lef-6 gene was not essential for either viral DNA replication or late gene transcription, but the absence of lef-6 resulted in a substantial delay in the onset of late transcription. Therefore, lef-6 appears to accelerate the infection cycle of AcMNPV.

FIG.1.

Transcription and localization of lef-6 in AcMNPV-infected Sf9 cells. (A) A schematic representation of the AcMNPV lef-6 locus shows the relative positions and orientations of the iap1, lef-6, and orf29 ORFs. Primers used for mapping the lef-6 transcripts by 5′ and 3′ RACE analyses are shown as short arrows below the lef-6 ORF (GSP1, GSP2, and 3′ RACE), and the position of the cRNA probe used for Northern blot analysis is shown as a long arrow (Probe: 343 nt). Relative positions of lef-6 and iap1 RNAs are indicated as dashed lines above the ORFs. (B) Sequences derived from 5′ and 3′ RACE analyses are shown below AcMNPV genomic sequences (AcMNPV). A large arrow (labeled 23,397) indicates the position of the 5′ end mapped by 5′ RACE, and single underlined nucleotides represent 5′ ends mapped in a previous study (34). (C) Northern blot analysis of lef-6 transcription in AcMNPV-infected Sf9 cells. Numbers above each lane indicate the time (hours) p.i. when RNAs were isolated (Sf9, mock-infected Sf9 cells). The sizes (kb) of le-6 and iap1 RNAs are indicated on the right. (D to G) Localization of LEF-6 in AcMNPV-infected Sf9 cells. LEF-6 was localized by immunofluorescent staining of AcMNPV-infected Sf9 cells by using an anti-LEF-6 antiserum as described in Materials and Methods. Left panels show phase-contrast images of uninfected Sf9 cells (D) or AcMNPV-infected Sf9 cells (F) at 18 h p.i. Panels on the right show epifluorescence micrographs of uninfected (E) or AcMNPV-infected (G) Sf9 cells, and arrowheads indicate dense areas of infected cell nuclei with intense staining (in panel G).

FIG. 2.

Construction of a lef-6-null virus. (A) The strategy for construction of a lef-6-null BACmid containing a deletion of the AcMNPV lef-6 gene is shown in the diagram. The structure of the lef-6 locus is shown above that of the linear DNA fragment excised from transfer vector (pBluIAPGUSorf29CAT) and used for recombination in E. coli. In pBluIAPGUSorf29CAT, the lef-6 ORF was excised and replaced with a p6.9 promoter-driven GUS gene plus a chloramphenicol resistance gene cassette (cat). The structure of the resulting lef-6 locus in BACmid vAc^lef6KO is shown in the lower portion of the diagram. The positions of primers used for PCR analysis of the resulting AcMNPV BACmid (vAc^lef6KO) are indicated by small arrows, and the locations and sizes of predicted PCR products are indicated by brackets below the diagrams. (B) The results of PCR analysis of the lef-6 locus of the lef-6-null BACmid (vAc^lef6KO) are shown on an ethidium bromide-stained agarose gel. Templates (vAc^lef6KO and wild-type AcMNPV) and primer pairs (A+B, C+D, and E+F) are shown above the lanes, and the sizes of PCR amplification products (in kilobase pairs) are indicated on the right and left. M, marker DNAs. Primer pairs (A+B, C+D, and E+F) correspond to those shown in panel A. (C) Southern blot analysis was used to confirm the absence of lef-6 in the lef-6-null BACmid (vAc^lef6KO). For hybridization analysis, a lef-6-specific DNA probe was hybridized to XhoI-digested virus or the BACmid DNAs indicated above each lane.

FIG. 3.

Construction and analysis of lef6 repair viruses. Two lef-6 gene constructs were inserted into the polyhedrin locus of the lef-6-null BACmid (vAc^lef6KO) to generate repair BACmids vAc^lef6KO-REP-P and vAc^{lef6KO-REP-ie1P}. The structures of the inserted lef-6 constructs are shown, and the promoter (wild-type lef-6 or wild-type ie-1) used to drive lef-6 expression is indicated along with the gentamicin resistance gene (Gm^r), and transposon Tn7 attachment sites (Tn7 att). The relative locations of PCR primers used for analysis of the polyhedrin locus are indicated below each construct (indicated by A and B), and the relative sizes of the predicted PCR amplification products are indicated below with brackets. The results of PCR analysis are shown on an ethidium bromide-stained agarose gel below. Template DNAs are indicated on the right of the gel, and the sizes of the PCR products (in kilobase pairs) are indicated on the left.

FIG. 4.

Analysis of viral replication by a lef-6-null virus. (A) A transfection-infection assay was used to examine lef-6-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. (B) The results of GUS staining of infected cells are shown in the panels on the right. (C) Virus growth curves were generated to measure virus production from Sf9 cells infected with viruses derived from each of the BACmid constructs or wild-type AcMNPV. Infectious budded virions were prepared from the BACmids vAc^lef6KO, vAc^lef6KO-REP-P, vAc^{lef6KO-REP-ie1P}, or vAc^64−/+GUS or from wild-type AcMNPV. Infections were performed in triplicate at an MOI of 5, and supernatants were collected and assayed for production of infectious virus by TCID₅₀.

FIG. 5.

Analysis of viral DNA replication in Sf9 cells infected with lef-6-null and control viruses. (A) Sf9 cells were infected with viruses derived from either wild-type AcMNPV, control BACmid (vAc^64−/+GUS), lef-6-null BACmid (vAc^lef6KO), or lef-6 repair BACmids (vAc^lef6KO-REP-P or vAc^{lef6KO-REP-ie1P}), and total cellular DNAs were isolated at various times posttransfection (6 to 96 h) and examined by Southern dot blot hybridization. Total AcMNPV DNA was labeled with [³²P]dATP as a hybridization probe. A standard curve of AcMNPV DNA is shown on the right (10 to 400 ng of AcMNPV DNA). (B) Quantitative analysis of viral DNA replication by Southern dot blot analysis. Three replicates of each virus infection and time point were examined as in panel A, and DNA was quantified by phosphorimager analysis. Bars represent the average of three dot blot samples, and error bars represent the standard deviation.

FIG. 6.

Analysis of viral early, late, and very late transcripts in lef-6-null and control virus-infected Sf9 cells. Each panel represents Northern blot analysis of early, late, or very late RNAs from cells infected with lef-6-null (vAc^lef6KO) or control (vAc^lef6KO-REP-P, vAc^{lef6KO-REP-ie1P}, or vAc^64−/+GUS) viruses. Sf9 cells were infected at an MOI of 5. At various times p.i. (12, 18, 24, 48, or 72 h), total RNAs were isolated and used for Northern blot analysis with early (ie-1), late (p6.9, orf133, and vp39), or very late (p10) gene-specific probes. Viruses used for infections 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. For comparison, RNAs isolated from wild-type AcMNPV-infected Sf9 cells at 18, 24, and 48 h p.i. are shown on the right. M, mock-infected cells. (Note that the 24-h-p.i. time point for vAc^{lef6KO-REP-ie1P} is missing RNA samples on blots for p6.9, vp39, and p10. The sample name is therefore indicated in parentheses above.)

FIG. 7.

Late p6.9 promoter-reporter analysis in lef-6-null and control virus-infected cells. Temporal expression of a GUS reporter gene under the control of a p6.9 late promoter was measured from extracts of Sf9 cells infected with either the lef-6-null (vAc^lef6KO) or repair (vAc^lef6KO-REP-P and vAc^{lef6KO-REP-ie1P}) viruses. GUS activity was measured as described in Materials and Methods. Times p.i. are indicated below the x axis. Error bars represent the standard deviations from three independent infections.