Identification of a domain of the baculovirus Autographa californica multiple nucleopolyhedrovirus single-strand DNA-binding protein LEF-3 essential for viral DNA replication

Abstract

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) lef-3 is one of nine genes required for viral DNA replication in transient assays. LEF-3 is predicted to contain several domains related to its functions, including nuclear localization, single-strand DNA binding, oligomerization, interaction with P143 helicase, and interaction with a viral alkaline nuclease. To investigate the essential nature of LEF-3 and the roles it may play during baculovirus DNA replication, a lef-3 null bacmid (bKO-lef3) was constructed in Escherichia coli and characterized in Sf21 cells. The results showed that AcMNPV lef-3 is essential for DNA replication, budded virus production, and late gene expression in vivo. Cells transfected with the lef-3 knockout bacmid produced low levels of early proteins (P143, DNA polymerase, and early GP64) and no late proteins (P47, VP39, or late GP64). To investigate the functional role of domains within the LEF-3 open reading frame in the presence of the whole viral genome, plasmids expressing various LEF-3 truncations were transfected into Sf21 cells together with bKO-lef3 DNA. The results showed that expression of AcMNPV LEF-3 amino acids 1 to 125 was sufficient to stimulate viral DNA replication and to support late gene expression. Expression of Choristoneura fumiferana MNPV lef-3 did not rescue any LEF-3 functions. The construction of a LEF-3 amino acid 1 to 125 rescue bacmid revealed that this region of LEF-3, when expressed in the presence of the rest of the viral genome, stimulated viral DNA replication and late and very late protein expression, as well as budded virus production.

FIG. 1.

Construction of lef-3 knockout and rescue AcMNPV bacmids. (A) Schematic drawing of construction of a lef-3 knockout bacmid (bKO-lef3-cat) from bMON14272. The relative location and orientations of orf66, orf68, and orf69 in the lef-3 locus of bMON14272 (AcMNPV bacmid) are indicated. The cat gene flanked by the FLP recognition target was amplified by PCR using primers C-25716 and C-25717, and the product was electroporated into DH10Bac-pKD46 cells to replace the lef-3 by homologous recombination (bKO-lef3-cat). The location and orientation of primers used to confirm the constructs are also shown. (B) Schematic diagram of construction of polyhedrin-positive bacmid bKO-lef3 and polyhedrin (polh)-positive rescue bacmids bKO-lef3-Ac and bKO-lef3-Cf from bKO-lef3-cat. The names and locations of primers used in the donor plasmid construction and the location of the selection antibiotic (Gen) are shown for each construct. (C) Results of PCR confirmation of bacmid sequence modifications. Purified bacmid DNA from bAcP (lane 1), bKO-lef3 (lane 2), bKO-lef3-Ac (lane 3), and bKO-lef3-Cf (lane 4) were used as templates with primer pairs specific for AcMNPV lef-3 (Ac-lef3; C-23321and C-22915) or the cat gene in the appropriate locations (cat-lef3; C-25459 and C-24244) or CfMNPV lef-3 (Cf-lef3; C-6721 and C-24244). Primers C-23321 and C-22915 were used to confirm the loss of the lef-3 coding region. Primers C-25459 and C-24244 were used to confirm the recombination junction of flanking regions.

FIG. 2.

Viral DNA replication in bacmid-transfected Sf21 cells. Total intracellular DNA from Sf21 cells transfected with bAcP, bKO-lef3, bKO-lef3-Ac, or bKO-lef3-Cf was isolated at 24 and 48 h posttransfection. The DNA was digested with DpnI for 16 h and analyzed by real-time PCR using host genome- and virus-specific primer pairs. The relative viral genome copy number per cell was calculated for each bacmid transfection. The results are expressed as the number of viral genome equivalents per cell and are displayed as averages of samples analyzed in triplicate from transfections performed in duplicate. Error bars indicate standard deviations.

FIG. 3.

Budded virus production in bacmid-transfected and infected cells. (A) Sf21 cells were transfected with bAcP, bKO-lef3, bKO-lef3-Ac, or bKO-lef3-Cf bacmid DNA. Cell culture supernatants were harvested at 12, 24, 36, 48, 72, and 96 h posttransfection and titrated by TCID₅₀ endpoint dilution assays for the presence of infectious budded virus. The results represent the average titers derived from three independent assays. Error bars represent standard deviations. (B) Sf21 cells were infected (multiplicity of infection of 1) with supernatants harvested at 96 h posttransfection from cells transfected with bAcP, bKO-lef3, bKO-lef3-Ac, or bKO-lef3-Cf bacmid DNA. Cell culture supernatants from these infections were harvested and titrated by TCID₅₀ assays for the presence of infectious budded virus. The results represent the average titers derived from three independent assays. Error bars represent standard deviations.

FIG. 4.

Viral protein expression of bKO-lef3-transfected cells in the absence or presence of various truncations of LEF-3. (A) Sf21 cells were transfected with bacmid bAcP (WT; lane 1), AcMNPV lef-3 rescue bacmid (Ac-R; lane 2), or AcMNPV lef-3 knockout bacmid (KO; lane 3). Transfected cells were harvested at 48 h posttransfection and analyzed by immunoblotting for the expression of early and late viral proteins. Antibodies used as probes on the immunoblots were directed against the specific proteins indicated on the right. (B) The AcMNPV lef-3 knockout bacmid bAcKO-lef3 was transfected alone (lane 1) or cotransfected with plasmids expressing the full-length LEF-3 (lane 2), LEF-3 amino acids (aa) 2 to 189 (lane 3), LEF-3 aa 2 to 125 (lane 4), LEF-3 aa 2 to 83 (lane 5), or LEF-3 aa 190 to 385 (lane 5). At 48 h posttransfection, cell extracts were prepared and analyzed by immunoblotting for the expression of early and late viral proteins. Antibodies used as probes were directed against the specific proteins indicated on the right. The lower panel shows the results of probing the immunoblot with antibody directed against the HA tag fused to the various truncated LEF-3 proteins. The results of probing mock-transfected cells are also shown (lane 7).

FIG. 5.

Transient viral DNA replication assays. (A) Sf21 cells were transfected with a collection of plasmids, which together expressed the AcMNPV genes necessary for plasmid DNA replication (ie-1, dnapol, lef-1, lef-2, p35, pe38, and ie-2) in the absence of lef-3 (lane 1) or in the presence of plasmids expressing the full-length LEF-3 (lanes 2 and 8), LEF-3 amino acids (aa) 2 to 189 (lanes 3 and 9), LEF-3 aa 2 to 125 (lanes 4 and 10), LEF-3 aa 2 to 83 (lanes 5 and 11), or LEF-3 aa 190 to 385 (lanes 6 and 12). Following incubation for 48 h, total intracellular DNA was prepared and digested with EcoRI to linearize the plasmids or with EcoRI and DpnI to detect replicated plasmid DNA. Restricted DNA was prepared by Southern blotting, and replicated DNA was detected with a probe prepared from the viral DNA polymerase gene (dnapol). (B) Sf21 cells were transfected with rescue bacmid bKO-lef3-Ac (lane 1), bKO-lef3 (lane 2), or bKO-lef3 cotransfected with plasmids expressing the full-length LEF-3 (lane 3), LEF-3 aa 2 to 189 (lane 4), LEF-3 aa 2 to 125 (lane 5), LEF-3 aa 2 to 83 (lane 6), or LEF-3 aa 190 to 385 (lane 7). A control transfected with only herring sperm DNA was included (lane 8). At 48 h, total intracellular DNA was prepared and probed as described above. DpnI-resistant DNA was detected with a probed prepared from the viral DNA polymerase gene (dnapol). (C) The same DNA samples shown in panel B were digested with DpnI for 16 h and analyzed by real-time PCR using host genome- and virus-specific primer pairs. The relative viral copy number per cell was calculated for each bacmid transfection. The results are expressed on a log₁₀ scale as the number of viral genome equivalents per cell and are displayed as averages of samples analyzed in triplicate. Error bars indicate standard deviations.

FIG. 6.

Construction and characterization of bKO-lef3-125. (A) Schematic diagram describing the construction of the LEF-3 amino acid 1 to 125 rescue bacmid, bKO-lef3-125. The location and orientation of primers used in donor plasmid constructions and the diagnostic PCR are shown as arrows with names. (B) Viral DNA replication in bacmid-transfected Sf21 cells. Total intracellular DNA was isolated from Sf21 cells transfected with bAcP, bKO-lef3-Ac, bKO-lef3-125, or bKO-lef3-Cf at various times posttransfection. The DNA was digested with DpnI for 16 h and analyzed by real-time PCR. The relative viral copy number per cell was calculated for each bacmid transfection. The results are expressed as the number of viral genome equivalents per cell and are displayed as averages of samples analyzed in triplicate from transfections performed in duplicate. Error bars indicate standard deviations. (C) Budded virus growth curves in Sf21 cells transfected with bAcP or bKO-lef3-125 DNA. Supernatants were harvested at various times posttransfection and titrated for the presence of infectious virus in TCID₅₀ endpoint dilution assays. (D) The virus supernatants harvested at 96 h from bacmid transfection mixtures were used to infect Sf21 cells (multiplicity of infection, 0.1). At various times after infection, cell culture supernatants were harvested and titer were determined in TCID₅₀ assays. Virus titers represent the averages derived from three independent assays. Error bars represent standard deviations.