HCF-1 encoded by baculovirus AcMNPV is required for productive nucleopolyhedrovirus infection of non-permissive Tn368 cells

Baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replicates in both Spodoptera frugiperda Sf21 and Trichoplusia ni Tn368 cells, whereas AcMNPV defective in hcf-1 (host cell-factor 1) gene productively infects only Sf21 cells, indicating that HCF-1 is indispensable for the AcMNPV productive infection of Tn368 cells. Here, we demonstrated that HCF-1 protein transiently expressed in Tn368 cells promotes the DNA synthesis of Hyphantria cunea MNPV ﻿(HycuMNPV), Orygia pseudotsugata MNPV and Bombyx mori NPV, which are normally unable to replicate in Tn368 cells. We also demonstrated that a recombinant HycuMNPV harboring the hcf-1 gene successfully replicates in Tn368 cells, generating substantial yields of progeny viruses and polyhedra. These results indicate that HCF-1 encoded by AcMNPV is an essential viral factor for productive NPV infection of Tn368 cells. Taken together with the previous findings on HRF-1 (host range factor 1), the present results provide strong evidence that viral genes acquired through horizontal gene transfer play an important role in baculovirus evolution, serving to expand the host range of baculoviruses.

hcf-1 promoter and this repression activity of HCF-1 is required for the efficient expression of viral late genes and production of polyhedra in Tn368 cells 18 . However, the functional role of HCF-1 protein in AcMNPV-infected Tn368 cells has not been conclusively determined.
In the present study, we demonstrated that transiently expressed HCF-1 protein promotes the productive infection of non-permissive Tn368 cells by Hyphantria cunea MNPV (HycuMNPV). Recombinant HycuMNPV harboring the hcf-1 gene also successfully replicated in Tn368 cells, indicating that hcf-1 gene embedded in the HycuMNPV genome functions effectively for HycuMNPV productive infection of Tn368 cells. In contrast, Orgyia pseudotsugata MNPV (OpMNPV) and Bombyx mori NPV (BmNPV) were capable of viral DNA replication in HCF-1-expressing Tn368 cells, but not the synthesis of viral structural or polyhedral proteins. Taken together, these results indicate that HCF-1 protein is an essential viral factor for the productive NPV infection of Tn368 cells, but is not sufficient to promote viral protein synthesis of certain NPVs in infected Tn368 cells.

Results
HCF-1 promotes viral DNA and viral protein production of certain NPVs in non-permissive Tn368 cells. To determine whether HCF-1 promotes the productive infection of NPVs other than AcMNPV in Tn368 cells, transfection-infection experiments were performed using four different NPVs that are non-permissive in T. ni cells. Tn368 cells were first transfected with plasmids pFBD/hcf-1 and pFBD/luc, which express HCF-1 and luciferase proteins, respectively, under transcriptional control of the Drosophila melanogaster heat shock protein 70 (HSP70) gene promoter (Fig. 1a). At 24 h post-transfection, Tn368 cells were heat-shocked at 42 °C for 30 min and incubated for 6 h at 28 °C. The cells were then infected with HycuMNPV, OpMNPV, BmNPV and Lymantria dispar MNPV (LdMNPV), and examined for viral DNA, viral proteins, progeny budded viruses (BVs) and polyhedra. Microscopic examination at 72 h post-infection showed that polyhedra were only produced in a small number of HycuMNPV-infected Tn368 cells (Fig. 1b). However, progeny BV production by any of the examined NPVs was not detected (Fig. 1c). Although BV production was not observed, viral DNA synthesis was promoted by HCF-1 protein in Tn368 cells infected with HycuMNPV, OpMNPV and BmNPV (Fig. 1d). Notably, the production of major capsid protein VP39 and polyhedrin (matrix protein of polyhedra) were observed clearly only in HycuMNPV-infected Tn368 cells (Fig. 1e). In luciferase-expressing Tn368 cells, no significant increases in viral DNA, VP39 protein, polyhedrin, progeny BVs or polyhedra were detected following infection with any of the examined NPVs ( Fig. 1b-e). Immunoblot analysis showed that substantial amounts of luciferase and HCF-1 proteins were expressed in the plasmid-transfected and virus-infected Tn368 cells (Fig. 1f).

Construction and characterization of HycuMNPV bacmid (HycuBac) in SpIm cells. The results
of the transfection-infection experiments presented in Fig. 1 suggested that HCF-1 protein promotes productive HycuMNPV replication in non-permissive Tn368 cells. To confirm this speculation, we constructed and characterized a HycuMNPV bacmid (HycuBac; Fig. 2a) in SpIm cells, which are permissive for HycuMNPV. HycuBac DNA or a plasmid that expresses EGFP protein was transfected into SpIm cells. At 72 h post-transfection, a number of polyhedra were found only in HycuBac DNA-transfected SpIm cells (Fig. 2b). Progeny BVs (vHycuBac) from HycuBac DNA-transfected SpIm cells and those of parental HycuMNPV were used to infect SpIm cells to compare the kinetics of polyhedra production, progeny BV yield and polyhedrin expression. All measured kinetic parameters of vHycuBac were comparable to those of parental HycuMNPV in infected SpIm cells up to 72 h post-infection ( Fig. 2c-e).
Construction and characterization of recombinant HycuBac harboring hcf-1 or the luciferase gene in SpIm cells. As HycuBac was shown to produce progeny BVs in SpIm cells with biological activity comparable to those of parental HycuMNPV, we constructed recombinant HycuBac harboring hcf-1 (HycuBac/ hcf-1) or the luciferase gene (HycuBac/luc) (Fig. 3a) to determine whether HCF-1 protein promoted HycuMNPV replication in Tn368 cells. The two types of progeny BVs, vHycuBac/hcf-1 and vHycuBac/luc, were obtained from SpIm cells transfected with HycuBac/hcf-1 and HycuBac/luc, respectively, and were then used to infect SpIm cells. No significant kinetic differences were observed between vHycuBac/hcf-1 and vHycuBac/luc with respect to polyhedra production, BV yield, and syntheses of envelope fusion protein GP64, major capsid protein VP39 and polyhedrin until 72 h post-infection ( Fig. 3b-d). The luciferase and HCF-1 proteins were expressed at high levels in SpIm cells following infection with vHycuBac/hcf-1 and vHycuBac/luc, respectively (Fig. 3e).
Recombinant HycuMNPV harboring hcf-1 (vHycuBac/hcf-1) replicates in Tn368 cells. As no significant differences in the biological properties of vHycuBac/hcf-1, vHycuBac/luc and vHycuBac in SpIm cells were detected (Fig. 3), Tn368 cells were infected with vHycuBac/hcf-1 to determine whether HCF-1 protein expressed from the HycuMNPV genome promoted HycuMNPV replication in Tn368 cells. Microscopic examination showed that polyhedra were first produced at 48 h post-infection and that the number of cells with polyhedra had markedly increased by 72 h post-infection (Fig. 4a). BV production in vHycuBac/hcf-1-infected Tn368 cells increased continuously from 48 to 72 h post-infection (Fig. 4b) and the amount of viral DNA increased strikingly at 24 h post-infection (Fig. 4c). Envelope fusion protein GP64 accumulated substantially in vHycuBac/hcf-1-infected Tn368 cells from 24 h post-infection onwards (Fig. 4d) and VP39 and polyhedrin proteins were clearly detected at 48 h post-infection and remained detectable at 72 h post-infection in vHycuBac/hcf-1-infected Tn368 cells (Fig. 4d). In contrast, no significant production of polyhedra, BVs, viral DNA, VP39 protein or polyhedrin was observed in vHycuBac/luc-infected Tn368 cells (Fig. 4a-d). The GP64 band that was detected temporarily at 24 h post-infection in vHycuBac/luc-infected Tn368 cells (Fig. 4d) represents GP64 protein expressed through the gp64 early promoter, which is occasionally observed upon infection with group I alphabaculoviruses even when inoculated viruses are unable to replicate productively. Immunoblot analysis showed that the luciferase and HCF-1 proteins were expressed at high levels from 24 to 72 h post-infection (Fig. 4e). Polypeptides from infected Tn368 cells were resolved on 12% SDS-polyacrylamide gels and transferred onto Immobilon-P (for VP39 protein) or nitrocellulose membranes (for polyhedrin). VP39 protein was probed using anti-BmNPV/AcMNPV VP39 polyclonal antibody and visualized with ECL Select Western Blotting Detection Reagent. Polyhedrin protein was probed with anti-BmNPV polyhedrin polyclonal antibody and visualized Scientific RepoRts | 7: 3807 | DOI:10.1038/s41598-017-03710-z

Discussion
The hcf-1 gene is required for the efficient replication of AcMNPV in Tn368 cells, but is dispensable for the productive infection of Sf21 cells 14,15 , indicating that the HCF-1 functions specifically in Tn368 cells to expand the host range of AcMNPV. In the present study, we examined whether HCF-1 also promotes the replication of HycuMNPV, OpMNPV, BmNPV and LdMNPV in non-permissible Tn368 cells, and found that HycuMNPV harboring the hcf-1 gene replicates in Tn368 cells, producing substantial amounts of BVs and polyhedra. These findings demonstrate that expansion of the AcMNPV host range to Tn368 cells by hcf-1 can also be extended to other NPVs, suggesting that HCF-1 is an essential viral factor for productive NPV infection of Tn368 cells.
The hcf-1 gene of AcMNPV is one of the lef (late expression factor) genes, which is essential for viral DNA replication and expression of late genes in transient assays in Tn368, but not Sf21, cells 14 . In AcMNPV-infected Tn368 cells, the HCF-1 protein is synthesized early during infection and localizes in punctuate nuclear structures 17,18 . Recombinant AcMNPV defective in functional hcf-1 gene replicates normally in Sf21 cells, but exhibits various mutant phenotypes in Tn368 cells, including defective viral DNA replication and late gene transcription, and complete cessation of cellular and viral protein syntheses 15 . These results collectively imply that the HCF-1 protein is involved in viral DNA replication and late gene expression, as well as cellular protein synthesis, in AcMNPV-infected Tn368 cells. However, the present results also showed that although OpMNPV and BmNPV DNAs were synthesized successfully in HCF-1-expressing Tn368 cells, major capsid protein VP39 and polyhedrin were not detected, and neither BVs nor polyhedra were produced. Quantitative qRT-PCR analysis revealed that transcripts from the vp39 genes of HycuMNPV and OpMNPV, but not those of BmNPV or LdMNPV, increased markedly at 48 h post-infection in HCF-1-expressing Tn368 cells (Supplementary Figure S1). As no vp39 gene transcripts were detected in Tn368 cells infected with BmNPV or LdMNPV, the hcf-1 gene appears to promote viral late gene transcription in an NPV species-specific manner in Tn368 cells. In addition, because the OpMNPV vp39 gene was transcribed at high levels in HCF-1-expressing Tn368 cells, recombinant OpMNPV harboring hcf-1 may also be capable of replicating productively in non-permissive Tn368 cells.
A similar cell-line specific viral factor to HCF-1, designated as HRF-1, was identified in LdMNPV and functions specifically in Ld652Y cells derived from L. dispar 13,16 . The hrf-1 gene was shown to rescue the global translation arrest of Ld652Y cells caused by AcMNPV infection and promotes productive AcMNPV replication in Ld652Y cells 13,16 . We previously demonstrated that HRF-1 promotes the replication of BmNPV, HycuMNPV, and Spodoptera exigua MNPV in non-permissive Ld652Y cells, indicating that the HRF-1 protein also functions to expand the host range of various NPVs to non-permissive Ld652Y cells 19,20 .
The hcf-1 and hrf-1 genes are encoded by only a few members of the family Baculoviridae. Evidence suggests that the hrf-1 gene was acquired quite recently by LdMNPV through horizontal gene transfer 21 , and that homologs of HRF-1 with low amino acid sequence identity are found exclusively in OpMNPV 13,22 , which productively infects Ld652Y cells 23 , and Dasychira pudibunda NPV, which is closely related to OpMNPV 24 . Homologs of hcf-1 have been identified in only four NPV species: three close relatives of AcMNPV (Rachiplusia ou MNPV, Plutella xylostella NPV and Thysanoplusia orichalcea MNPV), and Clanis bilineata NPV, which is more distantly related to AcMNPV 8, 25-27 , suggesting that the hcf-1 gene of AcMNPV was also acquired by a gene acquisition event. Notably, despite marked differences in structure and mode of action in virus-infected cells, HCF-1 and HRF-1 proteins have convergent functional roles in the host range expansion of NPVs to non-permissive cell lines derived from T. ni and L. dispar, respectively. Taken together with previous findings for the hrf-1 gene 19,20 , the present study indicates that several NPVs contain genes that are acquired by gene acquisition events and contribute to evolution of baculoviruses in terms of the host range expansion. 28 were maintained at 28 °C in TC100 medium (AppliChem) supplemented with 0.26% tryptose broth (Sigma) and 10% fetal bovine serum (FBS). FRI-SpIm (SpIm) cells derived from Spilosoma imparilis 29 were grown at 28 °C in MM medium 30 supplemented with 3% FBS. The following clonal isolates of four NPVs were used in these experiments: BmNPV N9 (BmNPV) from the silkworm B. mori 31 , HycuMNPV N9 (HycuMNPV) from the fall webworm H. cunea 32 , LdMNPV A21-MPV (LdMNPV) from the gypsy moth L. dispar 33 , and OpMNPV from the Douglass-fir tussock moth, O. pseudotsugata 34 .

Cells, viruses and virus infection. Tn368 cells derived from Trichoplusia ni
Tn368 and SpIm cells were infected with viruses at varying multiplicity of infection (MOI) during a 60-min incubation at room temperature, as described previously 35 . Infected Tn368 and SpIm cells were washed three times with TC100 and MM media, respectively, and used for analyses at the indicated time points. The time zero of infection was defined as the time when the viral inoculum was removed from the cell culture.
Plaque assay. Plaque assays were performed using SpIm cells for HycuMNPV, BM-N cells for BmNPV and Ld652Y cells for OpMNPV and LdMNPV, as described previously 35 . Briefly, monolayer cultures of these cells (0.5 × 10 6 cells) were prepared in 35 mm culture dishes (Falcon 3001) and inoculated with 200 μl of the virus suspensions to be titrated. After a 60 min adsorption period, the viral inoculum was replaced with 3 ml of 0.75% SeaPlaque agarose (FMC) in the medium appropriate for each cell line. The cultures were incubated at 28 °C for 5-6 days and formed plaques were counted under a microscope.