Baculovirus F-Box Protein LEF-7 Modifies the Host DNA Damage Response To Enhance Virus Multiplication

Abstract

The DNA damage response (DDR) of a host organism represents an effective antiviral defense that is frequently manipulated and exploited by viruses to promote multiplication. We report here that the large DNA baculoviruses, which require host DDR activation for optimal replication, encode a conserved replication factor, LEF-7, that manipulates the DDR via a novel mechanism. LEF-7 suppresses DDR-induced accumulation of phosphorylated host histone variant H2AX (γ-H2AX), a critical regulator of the DDR. LEF-7 was necessary and sufficient to block γ-H2AX accumulation caused by baculovirus infection or DNA damage induced by means of pharmacological agents. Deletion of LEF-7 from the baculovirus genome allowed γ-H2AX accumulation during virus DNA synthesis and impaired both very late viral gene expression and production of infectious progeny. Thus, LEF-7 is essential for efficient baculovirus replication. We determined that LEF-7 is a nuclear F-box protein that interacts with host S-phase kinase-associated protein 1 (SKP1), suggesting that LEF-7 acts as a substrate recognition component of SKP1/Cullin/F-box (SCF) complexes for targeted protein polyubiquitination. Site-directed mutagenesis demonstrated that LEF-7's N-terminal F-box is necessary for γ-H2AX repression and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replication events. We concluded that LEF-7 expedites virus replication most likely by selective manipulation of one or more host factors regulating the DDR, including γ-H2AX. Thus, our findings indicate that baculoviruses utilize a unique strategy among viruses for hijacking the host DDR by using a newly recognized F-box protein.

INTRODUCTION

Viruses modify the environment of their host cell through diverse mechanisms that collectively impair cellular function, promote virus propagation, and induce pathogenesis. Unsurprisingly, host cells possess intrinsic pathways, including the DNA damage response (DDR), to combat virus infection. The DDR is capable of detecting incoming or replicating viral DNA (vDNA) and activating potent antiviral defenses, including apoptosis (reviewed in reference 1). Nonetheless, certain DNA viruses, such as the mammalian herpesviruses and insect baculoviruses, require the host DDR for efficient multiplication (2–7). Therefore, these viruses frequently activate the DDR but alter this response to ablate its antiviral effects and exploit its proviral functions (1, 8). To this end, DNA viruses encode factors that modify or degrade key DDR components. By disrupting canonical DDR signaling, these viral factors expedite virus multiplication, contribute to cellular transformation, and promote pathogenesis (reviewed in references 1 and 9). Thus, the interaction of viral proteins and host DDR factors constitutes a critical virus-host interface with direct implications for human disease.

Following detection of virus invasion, DDR signaling commences with the activation of phosphatidylinositol 3-kinase-like kinases, including ataxia telangiectasia-mutated kinase (ATM) and ATM- and Rad3-related kinase (ATR) (reviewed in references 10 and 11). Subsequently, ATM and ATR phosphorylate an array of proteins that lead to cell cycle arrest, DNA repair, or apoptosis. The chromatin-associated histone variant H2AX is rapidly phosphorylated by ATM at or near DNA breaks (reviewed in reference 12). As such, phosphorylated H2AX (γ-H2AX) marks the site of DNA damage and subsequently functions to recruit additional host factors that mediate DNA repair and amplify DDR signaling. Ablation of H2AX or loss of its phospho-acceptor residue abolishes repair factor recruitment and contributes to chromosomal abnormalities (13–15). Thus, γ-H2AX plays a central role in regulating and amplifying the DDR.

Most DNA viruses that activate the host DDR also trigger γ-H2AX accumulation. In contrast, the prototype baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) represses γ-H2AX during infection of permissive insect hosts (3). Replication of the circular DNA genome (134 kb) of AcMNPV activates the DDR, which provides activities essential for efficient virus multiplication (2, 3). As is the case for most DNA viruses, the baculovirus replication events that initiate the DDR are unknown, but it is expected that rolling-circle- and recombination-related intermediates (16) that resemble damaged DNA are involved. Despite required participation of the host DDR, γ-H2AX is suppressed during AcMNPV DNA replication (3). Furthermore, AcMNPV is capable of repressing γ-H2AX triggered by DNA damage-inducing pharmacological agents. These findings suggested that baculoviruses carry one or more genes that alter H2AX phosphorylation, presumably to neutralize antiviral DDR activities and facilitate vDNA replication.

By virtue of its crucial position in regulating the DDR, γ-H2AX represents an attractive target for viral manipulation. Here, we report that the baculovirus replicative factor LEF-7 modulates the host DDR by repressing γ-H2AX. We discovered that LEF-7 is a nuclear F-box protein that is also essential for efficient AcMNPV multiplication, extending previous findings on LEF-7 stimulation of vDNA replication and recombination (17–20). The F-box domain was required for LEF-7's enhancement of virus replication, γ-H2AX repression, and interaction with host S-phase kinase-associated protein 1 (SKP1), which is a component of the SKP1/Cullin/F-box (SCF) complex that mediates selective protein ubiquitination. Collectively, our findings establish LEF-7 as an F-box protein that represses γ-H2AX to promote virus multiplication by using a novel strategy for manipulating host DDR components.

MATERIALS AND METHODS

Cells and virus.

Spodoptera frugiperda IPLB-SF21 (SF21) cells (21) were maintained at 27°C in TC100 medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone). SF21 cells stably expressing GFP^HA-SfH2AX were described previously (3). RFP^HA-LEF-7-expressing SF21 cells were selected by neomycin resistance (22) and cloned by serial dilution of red fluorescent protein (RFP) fluorescent cells.

AcMNPV recombinants were bacmid derived. The wild-type bacmid AcBacWT (vWT) was generated by Tn7-mediated transposition (23) of the polh gene from vector pFAcT (24) into the polyhedrin (polh) locus of bMON14272. To generate the lef-7 knockout AcBacΔLEF-7 (vΔLEF-7), lef-7 of bMON14272 was replaced with a Zeocin resistance cassette by using plasmid pAcLEF-7(fr)-Zeo and the λ phage Red recombinase system (25, 26); the polh gene was then inserted into AcBacΔLEF-7 by Tn7-mediated transposition. The lef-7⁺ rescue bacmid AcBacLEF-7^HA (vLEF-7^HA) was generated by transposition using AcBacΔLEF-7 and the transfer vector pFAcT/ie-1^prm/^HALEF-7. All viruses were produced by bacmid transfection of Trichoplusia ni BTI-TN5B1-4 cells (27) and plaque purified. Virus genotypes were verified by analysis of PCR-amplified genomic segments. For infection, extracellular budded virus (BV) was added to cell monolayers using the indicated multiplicity of infection (MOI) measured in PFU per cell. BV yields were measured by using RFP^HA-LEF-7-expressing SF21 cells and 50% tissue culture infective dose (TCID₅₀) endpoint dilution (28).

Plasmids.

pGFP^HA-SfH2AX and pHSEpiHis expression plasmids for AcMNPV replication factors (kindly provided by L. Passarelli, Kansas State University) were described previously (3, 29). LEF-7 residues 15 to 52 were replaced with Gly-Ser and residues Leu15-Pro16 were replaced with Ala-Ala by site-directed mutagenesis to generate expression plasmids pHSEpiHis/LEF-7(ΔF-box) and pHSEpiHis/LEF-7(LP/AA), respectively. LEF-7 sequences were fused to those at the C terminus of hemagglutinin (HA) epitope-tagged red fluorescent protein (RFP) and placed under the control of the AcMNPV immediate early ie-1 promoter to generate expression plasmid pRFP^HA-LEF-7. The lef-7 knockout vector pAcLEF-7(fr)-Zeo was produced by replacing the flanking regions of ie-1 within plasmid pAcIE1(fr)-Zeo (26) (kindly provided by D. Theilmann, University of British Columbia) with the 5′ and 3′ noncoding flanking regions of lef-7, which were PCR amplified from AcMNPV bacmid bMON14272 (Invitrogen). LEF-7 transposition vector pFAcT/ie-1^prm/^HALEF-7 was generated by inserting N-terminally HA-tagged ^HAlef-7 under the control of the ie-1 promoter into transfer vector pFAcT (24) (provided by D. Theilmann). The reporter plasmid pPolh^prm/lacZ/hr5, used to measure AcMNPV very late gene expression, carries the lacZ gene under the control of the very late polh promoter (polh prm), which is cis linked to the AcMNPV hr5 enhancer inserted immediately downstream; the RNA start site lies within the very late baculovirus transcription motif ATAAG.

Transfections.

SF21 cells were transfected with expression plasmids as described previously (30). When indicated, they were treated for 1 h either with the topoisomerase II inhibitor etoposide (Calbiochem) at a final concentration of 100 μM or with dimethyl sulfoxide (DMSO) vehicle. Intracellular β-galactosidase activity in reporter pPolh^prm/lacZ/hr5-transfected cells was measured at 48 h after infection (Applied Biosystems) and is reported as the average value ± standard deviation (SD) determined from triplicate experiments.

Antisera and IP.

Standard immunoblot analyses used the following antisera: monoclonal anti-HA (Covance), anti-actin (BD Biosciences), anti-T7 (Novagen), polyclonal anti-γ-H2A.X pS139 (ab11174; Abcam), or anti-IE1 (31). Signals were developed with alkaline phosphatase-conjugated goat anti-rabbit or goat anti-mouse immunoglobulin G (Jackson ImmunoResearch Laboratories, Inc.) and the CDP-Star chemiluminescence detection system (Roche). For immunoprecipitations (IP), clarified supernatant of SF21 cells lysed in IP buffer (10 mM sodium phosphate at pH 7.2, 150 mM NaCl, 50 mM NaF, 2 mM EDTA, 0.5% NP-40, 1× protease inhibitors [Roche]) was mixed with anti-T7-cross-linked agarose beads (Novagen) for 4 h at 4°C. After washing with IP buffer, protein was eluted in electrophoresis sample buffer and subjected to immunoblot analysis. Films were scanned at 300 dpi using an Epson Expression 1680 scanner and prepared using Adobe Photoshop.

Immunofluorescence and microscopy.

SF21 cells on glass coverslips were fixed with 4% formaldehyde in TC100, treated for 15 min with 0.1% Triton X-100 in phosphate-buffered saline (pH 7.1) (PBS), washed with PBS, and blocked with 2% bovine serum albumin (BSA) in PBS. After incubation with polyclonal anti-IE1 and/or monoclonal anti-HA, the coverslips were washed and stained with Alexa Fluor 594-conjugated goat anti-rabbit IgG and/or Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes) in 2% BSA–PBS. After a PBS wash, coverslips were stained with 4′,6-diamidine-2-phenylindole dihydrochloride (DAPI) (Roche) and mounted with Fluoromount-G (Southern Biotech). Fluorescence images were collected with a Nikon Eclipse TE2000-U confocal microscope. SF21 cells with occluded virus were photographed at 48 h after infection by using a phase-contrast microscope (Axiovert 135TV; Zeiss) equipped with a Microfire camera (Optronics) and Picture Frame software (Optronics). All images were representative and were prepared using Adobe Photoshop.

Sequence identification and alignments.

The gene (sfskp1) encoding Spodoptera frugiperda S-phase kinase-associated protein 1 (SKP1) was identified by a BLAST search of an S. frugiperda cDNA database (32) by using sequences of human skp1 (GenBank accession number NM_170679.2) as the query. After PCR amplification from a λ phage cDNA library of SF21 poly(A)⁺ RNA (30), sfskp1 was inserted into the ie-1 promoter expression vector pIE1^prm/hr5/PA (22) to generate pIE1^prm/hr5/^T7SfSKP1/PA; sequences encoding an N-terminal T7-epitope tag were included.

Nucleotide sequence accession numbers.

The nucleotide sequence of sfskp1 was submitted to GenBank under accession number KC812498. Baculovirus lef-7s were compared by using the ClustalW2 multiple-sequence alignment program (33) (www.ebi.ac.uk/Tools/msa/clustalw2); virus abbreviations are those listed by the International Committee of Taxonomy of Viruses (www.ictvdb.org), including AcMNPV (GenBank accession AAA66755), BmNPV (AAC63791), CfMNPV (AAP29897), OpMNPV (AAC59122), AgMNPV (AAS83209), and HaGV (ABY47823). Sequences of human SKP1 (AAC50241) and human SKP2 (AAK31593) were also compared.

RESULTS

Baculovirus LEF-7 represses γ-H2AX.

To define the molecular mechanisms by which baculoviruses repress γ-H2AX and how this DDR deregulation contributes to virus multiplication, we sought to identify the virus-encoded factor(s) responsible. Because inhibition of γ-H2AX coincides with AcMNPV DNA replication and involves viral factors produced before vDNA synthesis (3), we first screened known early viral replicative genes. To this end, we developed an assay by which AcMNPV factors expressed from transfected plasmids were tested for the capacity to inhibit γ-H2AX triggered by etoposide, a potent DNA-damaging agent in Spodoptera cells (2, 3). Levels of γ-H2AX were monitored by using a Spodoptera H2AX (SfH2AX)-GFP fusion protein (Fig. 1A), which is phosphorylated within an ATM/ATR consensus motif (Ser135) in response to DNA damage (3). Of the screened virus replicative factors, including primase (LEF-1), primase-associated factor (LEF-2), single-stranded DNA (ssDNA)-binding protein (LEF-3), helicase (P143), DNA polymerase (DNA^pol), and replication factor LEF-7 of unknown function, only LEF-7 reduced accumulation of etoposide-induced γ-H2AX to background levels (Fig. 1B, lane 7). Each replicative factor was readily detected in transfected cells and had no effect on the total level of SfH2AX fusion protein (Fig. 1B, lanes 1 to 8). We concluded that LEF-7 was the candidate responsible for γ-H2AX suppression.

Fig 1.

Baculovirus LEF-7 represses γ-H2AX. (A) GFP^HA-SfH2AX fusion protein. Spodoptera frugiperda H2AX (SfH2AX) was fused to the C terminus of GFP^HA and used as an indicator for γ-H2AX. SfH2AX (138 residues) possesses a consensus ATM/ATR phosphorylation site (SQ), which includes the required residue Ser135 (3). (B) Etoposide-induced γ-H2AX. SF21 cells were transfected with a plasmid encoding GFP^HA-SfH2AX together with a plasmid vector or plasmids encoding the indicated baculovirus late expression factors (LEFs). Following transfection, cells were treated with DMSO vehicle (−) or 100 μM etoposide (+), and whole-cell lysates were prepared and subjected to immunoblot analysis using phospho-specific anti-H2AX (top), anti-HA (middle), or anti-actin (bottom) to verify protein loading. Due to various levels of expression, HA-tagged viral proteins appear at different intensities (lanes 3 to 8). SfH2AX-P denotes the phosphorylated form of GFP^HA-SfH2AX; SfH2AX denotes total GFP^HA-SfH2AX. (C) Baculovirus mutants. The LEF-7 deletion mutant (vΔLEF-7) possesses a Zeocin resistance (zeo^R) cassette in place of lef-7 (open reading frame nucleotides 3 to 582); all other sequences are identical to those of wild-type AcMNPV (vWT). HA-tagged lef-7 under the control of the immediate early promoter (open box) of the AcMNPV ie-1 gene was inserted adjacent to the polyhedrin (polh) gene for lef-7 rescue virus, vLEF-7^HA. (D) Virus-induced γ-H2AX. SF21 cells stably expressing GFP^HA-SfH2AX were mock infected (mi) or inoculated with the indicated viruses (MOI, 5). Whole-cell lysates were prepared at the indicated times (hours) after infection and subjected to immunoblot analysis by using phospho-specific anti-H2AX (top), anti-HA (middle top and middle), anti-IE1 (middle bottom), or anti-actin (bottom).

To determine whether LEF-7 represses γ-H2AX in the context of the DDR triggered by infection, we generated an AcMNPV LEF-7 deletion mutant (vΔLEF-7) in which the lef-7 gene was replaced with a selectable drug resistance marker (Fig. 1C). We then constructed a rescue mutant (vLEF-7^HA) in which HA-tagged lef-7 was inserted under the control of a strong immediate early AcMNPV promoter within the vΔLEF-7 genome. As expected, wild-type lef-7⁺ AcMNPV suppressed accumulation of γ-H2AX between 3 and 24 after infection (Fig. 1D, lanes 3 to 6). Conversely, γ-H2AX was readily detected from 6 to 24 h after infection with lef-7 deletion mutant vΔLEF-7 (lanes 7 to 10). When lef-7^HA was reinserted into the genome, the rescued mutant vLEF-7^HA once again reduced γ-H2AX accumulation to background levels (lanes 11 to 14); LEF-7^HA was detected as early as 3 h and accumulated through 24 h after infection with this rescued lef-7⁺ mutant. Each of the viruses tested initiated infection properly as indicated by the uniform early appearance of immediate early protein IE1 (Fig. 1D). Moreover, the normal timing of IE1 suggested that LEF-7 contributes minimally to early AcMNPV transcriptional events during infection. Altogether, these findings conclusively demonstrated that (i) AcMNPV triggers the host DDR, which induces γ-H2AX accumulation, and (ii) LEF-7 alters virus-induced DDR signaling as indicated by repression of γ-H2AX during the peak of vDNA replication. Surprisingly, γ-H2AX reappeared, albeit at reproducibly reduced levels, by 24 h after infection, when vDNA synthesis declines markedly and virus assembly begins. Because intracellular LEF-7^HA was still present (Fig. 1D, lanes 11 to 14), this finding raises the interesting possibility that LEF-7 is inactivated late in infection or that a LEF-7-insensitive kinase of viral or host origin becomes active then.

LEF-7 stimulates baculovirus multiplication.

Because DNA viruses often manipulate the host DNA damage response to facilitate replication (reviewed in reference 1), we hypothesized that LEF-7 repression of γ-H2AX facilitates AcMNPV multiplication. To test this prediction, we first assessed LEF-7's effect on virus production. Compared to wild-type lef-7⁺ AcMNPV, BV yields of vΔLEF-7 were reduced 125- and 65-fold at 24 and 48 h after infection, respectively (Fig. 2A). Reinsertion of lef-7^HA into the viral genome restored BV yields, as vLEF-7^HA produced >300-fold more BV than vΔLEF-7 by 48 h. The positive influence of LEF-7 on yields of infectious virus suggested that virus gene expression, vDNA replication, or both were affected. We tested this possibility by quantifying very late gene expression using a virus-dependent reporter assay in which the AcMNPV polyhedrin gene (polh) promoter directs lacZ expression; polyhedrin is the major component of occluded virus, the final morphological unit of infectivity. Transcription from the polh promoter requires vDNA replication (reviewed in references 16, 34, and 35). As expected, the polh reporter was highly responsive to infection with wild-type lef-7⁺ AcMNPV (Fig. 2B). In contrast, polh promoter activity was reduced by 95% upon infection with lef-7-deficient vΔLEF-7. Promoter activity was restored to greater-than-wild-type levels by expression of lef-7^HA in vLEF-7^HA. Consistent with these findings and previous studies (18), nuclear accumulation of occluded virus was dramatically reduced in vΔLEF-7-infected cells but was restored to wild-type levels in vLEF-7^HA-infected cells (Fig. 2C). We concluded that LEF-7 is required for optimal expression of very late viral genes, which suggested a direct role in virus DNA replication. Thus, our findings are consistent with previous reports that LEF-7 stimulates virus DNA replication (18–20).

Fig 2.

LEF-7 stimulates AcMNPV multiplication. (A) Budded virus. Extracellular BV was collected from SF21 cells at the designated times after infection with the indicated viruses (MOI, 0.5), quantified by TCID₅₀, and reported as the average virus concentration (PFU/ml) ± SD from triplicate experiments. (B) Very late gene expression. SF21 cells were transfected with a reporter plasmid carrying lacZ under the control of the very late promoter of the baculovirus polyhedrin gene and mock infected (mi) or inoculated with the indicated viruses (MOI, 5) 24 h later. Intracellular β-galactosidase was measured at 48 h after infection and is reported as the average ± SD of relative light units (RLU) from triplicate experiments. (C) Occluded virus (OV). SF21 cells were photographed (magnification, ×200) at 48 h after mock infection (mi) or inoculation with the indicated viruses (MOI, 5). Arrows denote nuclear OV particles. (D) LEF-7 localization. SF21 cells transfected with a plasmid encoding wild-type LEF-7^HA were mock infected (mi) or inoculated (MOI, 5) with wild-type AcMNPV (vWT). Cells were fixed at the indicated times (hours) after infection, stained with anti-HA, anti-IE1, and DAPI, and then viewed by fluorescence confocal microscopy; representative cells are shown. (E) Localization of mutated LEF-7. Fluorescence confocal microscopy was used to view anti-HA-stained SF21 cells transfected 24 h earlier with expression plasmids encoding HA-tagged wild-type (wt), F-box-deleted (ΔF-box), or F-box-mutated (LP/AA) LEF-7. Bars, 5 μm.

As predicted from its role in virus replication and γ-H2AX repression, LEF-7 localized to the nucleus as indicated by immunofluorescence confocal microscopy (Fig. 2D). A putative nuclear localization signal is positioned near the N terminus of LEF-7 (Fig. 3A) (36). Accumulation of LEF-7 in nuclei of plasmid-transfected SF21 cells (Fig. 2D, panels i to iv) indicated that nuclear import was independent of DNA damage or infection. LEF-7 was also concentrated in the nucleus at all times after infection (Fig. 2D, panels v to xvi). AcMNPV DNA replication centers, visualized as discrete nuclear foci containing the essential replicative factor IE1 (37, 38), grew in size to encompass a large portion of the nucleus by 24 h after infection (Fig. 2D, panels vii, xi, and xv). LEF-7 overlapped these replication centers (Fig. 2D, panels vi to xiv). Although host chromatin containing SfH2AX is excluded to the margin of the nucleus during expansion of the replication centers later in infection (3), there was no obvious enhancement of LEF-7 accumulation here (Fig. 2D, panel xiv).

Fig 3.

LEF-7 is an F-box protein. (A) Schematic. AcMNPV LEF-7 (226 residues) possesses a 38-residue domain (residues 15 to 52) with F-box sequence similarity and an adjacent hydrophobic region (residues 56 to 138) resembling the Leu-rich domains of other F-box proteins. A putative nuclear localization signal (NLS) is positioned next to the F-box (residues 6 to 14, KRPRAKRIR); a Cys-rich motif (residues 167 to 188) was noted previously (36). (B) The SCF complex. The F-box protein associates with the cullin protein scaffold by binding the adapter SKP1. Cullin simultaneously recruits an E2 ubiquitin-conjugating enzyme by interacting with RBX1. The E2 enzyme transfers ubiquitin (Ub) to the proximal substrate target, which is bound specifically by the F-box protein. (C) Alignments. F-box residues of AcMNPV LEF-7 are compared with those of baculoviruses and human SKP2. Diamonds denote residues that are similar in ≥90% of identified LEF-7s; Leu15 and Pro16 (bold) are invariant. An F-box consensus sequence (39) is shown; LEF-7 similarities are shaded, and residues conserved in ≥40% of the 234 F-boxes used to generate the consensus are in bold. F-box α-helices of SKP2 (40) are depicted along with those residues that contact human SKP1 (underlined). (D) Spodoptera SKP1. The amino acid sequence (162 residues) of SfSKP1 is compared to that of human SKP1; similar residues are shaded. Residues of human SKP1 that contact human F-box protein SKP2 (40) are underlined. (E) LEF-7 mutations. F-box-deleted LEF-7 (ΔF-box) that lacks residues 15 to 52 and F-box-mutated LEF-7 (LP/AA) that bears alanine substitutions of Leu15 and Pro16 are compared to wild-type (wt) LEF-7; the N-terminal HA tag is indicated. (F) Immunoprecipitations. Lysates of SF21 cells transfected 24 h earlier with plasmids encoding the indicated HA-tagged LEF-7s with or without plasmid encoding T7-tagged SfSKP1 were immunoprecipitated with anti-T7 serum. Immunoblots of the precipitate (IP) and lysate using anti-T7 and anti-HA sera are shown.

LEF-7 is a viral F-box protein.

To define the molecular mechanism of LEF-7 function, we searched for recognizable motifs within its primary sequence. This effort revealed a predicted F-box domain spanning LEF-7 residues 15 to 52 (Fig. 3A). F-box proteins function as the substrate recognition component of SKP1/Cullin/F-box (SCF) ubiquitin ligase complexes (reviewed in reference 39). These F-box proteins recruit their selected substrate(s) to the SCF complex for polyubiquitination (Fig. 3B), which often mediates substrate degradation. The F-box domain itself interacts with the adaptor protein SKP1, which in turn binds Cullin and associated E2 ubiquitin-conjugating enzymes. Alignment of known F-box domains (40) with that of AcMNPV LEF-7 (Fig. 3C) revealed conserved residues critical for interaction with SKP1, including a Leu-Pro pair (Leu15-Pro16) present in all baculovirus homologs of LEF-7. This Leu-Pro pair is also conserved among mammalian F-box proteins and is also required for SKP1 association (39, 41, 42).

To assess LEF-7 participation in an SCF complex, we first investigated LEF-7/SKP1 interactions. To this end, we cloned host Spodoptera SKP1 (SfSKP1) from a cDNA expression library. We discovered that SfSKP1 is 90% similar to human SKP1 (Fig. 3D) and possesses conserved residues required for F-box interactions (40). When wild-type LEF-7^HA (Fig. 3E) was expressed in plasmid-transfected SF21 cells along with T7-tagged SfSKP1, both proteins formed an immunoprecipitable complex (Fig. 3F, lane 2). Conversely, SfSKP1 failed to interact with F-box-deleted LEF-7 (Fig. 3F, lane 3), which lacks residues 15 to 52 (Fig. 3E). Because the highly conserved Leu-Pro pair within mammalian F-boxes is critical to SKP1 interaction (41, 42), we replaced LEF-7's corresponding residues with Ala (Fig. 3E). LP/AA-mutated LEF-7 also failed to immunoprecipitate with SfSKP1 (Fig. 3F, lane 4). These findings indicated that the LEF-7 F-box is required for interaction with SfSKP1. We concluded that LEF-7 is an F-box protein that associates with host SKP1, most likely in the context of a Spodoptera SCF complex.

The F-box is required for LEF-7 repression of γ-H2AX and virus replication.

To define the biochemical role of the F-box, we tested the effect of F-box disruptions on LEF-7's ability to repress DNA damage-induced γ-H2AX in plasmid-transfected SF21 cells. As expected, wild-type LEF-7 reduced etoposide-induced γ-H2AX to background levels (Fig. 4A, compare lane 1 with lanes 3 to 5). However, neither F-box-deleted nor LP/AA-mutated LEF-7 repressed γ-H2AX when expressed at comparable levels (lanes 6 to 11). Furthermore, higher levels of either mutated protein conferred only partial suppression of etoposide-induced γ-H2AX. Similarly, disruption of the F-box abolished LEF-7's capacity to repress virus-induced γ-H2AX (Fig. 4B). In contrast to wild-type LEF-7, neither F-box-deleted nor LP/AA-mutated proteins fully suppressed γ-H2AX in SF21 cells infected with lef-7-deficient virus (vΔLEF-7) (Fig. 4B, lanes 6 to 11). Loss of activity was not due to altered protein localization, as both F-box-deleted and LP/AA-mutated LEF-7 accumulated within the nuclei of plasmid-transfected cells (Fig. 2E). We concluded that loss of SKP1 interaction was sufficient to diminish or abolish LEF-7 repression of γ-H2AX triggered by diverse stimuli.

Fig 4.

The F-box is required for LEF-7 function. (A) Etoposide treatment. SF21 cells transfected 24 h earlier with a plasmid encoding GFP^HA-SfH2AX together with a plasmid vector or increasing amounts of plasmid encoding HA-tagged wild-type (wt), F-box-deleted (ΔF-box), or L15/P16-substituted (LP/AA) LEF-7 were treated for 1 h with DMSO vehicle (−) or 100 μM etoposide (+). Whole-cell lysates were subjected to immunoblotting by using phospho-specific anti-H2AX (top), anti-HA (middle), or anti-actin (bottom). (B) vΔLEF-7 infection. SF21 cells transfected as described for panel A were mock infected (−) or inoculated (+) with AcMNPV mutant vΔLEF-7 (MOI, 5), lysed after 12 h, and subjected to immunoblotting as indicated. (C) Very late gene expression. SF21 cells were transfected with very late gene reporter pPolh^prm/lacZ/hr5 together with plasmid vector or with increasing amounts of plasmid encoding the indicated LEF-7s and inoculated (MOI, 5) with vΔLEF-7. Intracellular β-galactosidase was measured at 48 h after infection and reported as the average ± SD of relative light units (RLU) from triplicate experiments. Intracellular LEF-7^HA was monitored by immunoblotting (bottom).

To determine the role of the LEF-7 F-box in AcMNPV replication, we measured the impact of F-box disruption on LEF-7 replicative activity using our very late gene (polh) reporter. In plasmid-transfected cells infected with lef-7-deficient vΔLEF-7, both F-box-deleted and LP/AA-mutated LEF-7 exhibited a greatly reduced capacity to promote late gene expression compared to wild-type LEF-7 (Fig. 4C). Interestingly, increasing levels of wild-type LEF-7 conferred only minimal increases in promoter activity, suggesting that LEF-7's replicative activity is saturable. In contrast, F-box-disrupted LEF-7 proteins failed to recapitulate wild-type levels of reporter activation even at high levels of expression. We concluded that LEF-7 promotes virus replication in an F-box-dependent manner. Taken together, our findings suggest a mechanistic link between LEF-7-mediated inhibition of γ-H2AX and stimulation of baculovirus multiplication. Moreover, our study suggests that LEF-7 acts as the substrate recognition component of an SKP1/Cullin/F-box (SCF) complex for targeted polyubiquitination of a DDR component affecting γ-H2AX regulation.

DISCUSSION

We report here that baculovirus LEF-7 is a novel modulator of the host DNA damage response (DDR). LEF-7 was necessary and sufficient to suppress phosphorylation of the histone variant H2AX following either baculovirus infection or pharmacologically induced DNA damage (Fig. 5). Because γ-H2AX is a key regulator of the DDR, baculoviruses target a central step in the DDR for modification. LEF-7-mediated repression of γ-H2AX correlated with enhanced virus replication and increased virus yields. Thus, this conserved baculovirus protein manipulates the host DDR to promote virus multiplication (Fig. 5). Furthermore, our finding that LEF-7 is an F-box protein that interacts with a component of SKP1/Cullin/F-box (SCF) complexes suggests that LEF-7 uses targeted polyubiquitination for altering or inactivating the function of a DDR regulator.

Fig 5.

Model for baculovirus LEF-7 modulation of the host DDR. Following genotoxic stress-induced DNA damage (left), the DNA damage response (DDR) activates ATM, which phosphorylates host histone variant H2AX and other DDR factors that recruit DNA repair components to sites of host chromatin (11, 12). Likewise, the initiation of baculovirus DNA replication (right) activates ATM, which expedites virus genome production, presumably by mobilizing DDR components for viral purposes (2, 3). So as to prevent nonproductive sequestration of DDR repair components on host chromatin, virus-expressed LEF-7 represses ATM-mediated accumulation of γ-H2AX, thereby liberating host DDR factors for vDNA replication and packaging. In the context of DNA damage inflicted by genotoxic stress (left), LEF-7 is sufficient to downregulate γ-H2AX and likely hinders or limits cellular DNA repair.

Viral regulation of H2AX phosphorylation.

It is well established that diverse viruses, including the human herpesviruses, papillomaviruses, HIV, and adenoviruses, activate the host DDR and in the process trigger γ-H2AX accumulation (1, 8, 12). The DNA baculoviruses also engage the host DDR as a means to facilitate vDNA replication and stimulate multiplication (2, 3). Because baculovirus-induced DDR signaling involves ATM activation, which is required for virus replication, it was unexpected that γ-H2AX was suppressed in infected cells (3). We discovered that baculovirus replicative factor LEF-7 is responsible for this repression of γ-H2AX. Our findings establish LEF-7 as one of the first known suppressors of virus-induced γ-H2AX.

LEF-7 homologs are found in all group I alphabaculoviruses, several viruses within group II, and at least two betabaculoviruses in a survey of available genomic sequences (16). This conservation suggests an influential function for LEF-7 during multiplication. Indeed, LEF-7 enhances baculovirus DNA replication and gene expression, as indicated by using virus deletion mutants and complementation assays (Fig. 2 and 4) (18, 19). Thus, LEF-7's alteration of host DDR signaling through γ-H2AX correlates with its enhancement of viral replicative activity. How LEF-7's suppression of γ-H2AX generates a cellular environment better suited for vDNA replication while maintaining activity of virus-required DDR kinases is unclear. Due to the complexity of DDR pathways, there are multiple possibilities.

A model (Fig. 5) in which LEF-7 liberates virus-activated DDR factors, including kinases and DNA repair factors, to facilitate vDNA replication and recombination is consistent with our data. Upon virus-mediated induction, the host DDR activates ATM, which phosphorylates H2AX. Normally, γ-H2AX recruits these DDR factors to host chromatin that is either damaged or reorganized (12). However, LEF-7 represses γ-H2AX during peak vDNA replication, most likely to divert required DDR components away from host chromatin toward vDNA replication centers. It is relevant that during infection, H2AX (as GFP^HA-SfH2AX) is forced to the nuclear margin along with other host histones and away from AcMNPV replication centers (3, 37). This redistribution suggests that H2AX is associated with host chromatin, not replicating vDNA. How the liberated host DDR components are recruited to viral replication centers is unknown, but we predict that specific baculovirus replicative proteins are involved. A virus-mediated mechanism that diverts host factors for vDNA replication would be especially advantageous for viruses evolved for high-level multiplication. Indeed, baculoviruses are unequivocally among the most robust and productive of DNA viruses, where vDNA can constitute more than half of the total DNA of an infected cell (16, 35).

DDR modification via the F-box of LEF-7.

Our study revealed that LEF-7 possesses an F-box domain (Fig. 3A), which is highly conserved and required for LEF-7 modulation of the host DDR. F-box proteins are components of SCF ubiquitin ligases that mediate recruitment of specific substrates for ubiquitination (Fig. 3B). For example, the human F-box protein SKP2 interacts with the SCF adaptor SKP1, which tethers SKP2 to the SCF complex for ubiquitination of SKP2 substrates, including multiple cell cycle control factors (reviewed in references 43 and 44). We determined here that the LEF-7 F-box is required for interaction with the Spodoptera SKP1 homolog, SfSKP1 (Fig. 3). Mutations that disrupted LEF-7 interactions with SfSKP1 also abolished or hindered LEF-7 repression of γ-H2AX and enhancement of AcMNPV multiplication (Fig. 4). Thus, LEF-7 presumably acts as the substrate recognition component of an SCF complex for targeted polyubiquitination of a DDR component (see below) that affects γ-H2AX regulation. Mammalian F-box proteins also possess a characteristically rigid domain positioned carboxy terminal to the F-box that interacts with the intended substrate protein; Skp2 contains a Leu-rich substrate-binding domain (44). Like SKP2, LEF-7 possesses a Leu-rich domain of repetitive hydrophobic residues (Fig. 3A), which is located adjacent to its F-box domain.

Possible DDR targets of LEF-7.

Regulation of protein function by ubiquitination has emerged as a common strategy for viral manipulation of host pathways. By altering ubiquitination of key DDR components, viruses can target these factors for either proteasomal degradation or modification of their activity (45). Our finding that LEF-7 is necessary and sufficient to suppress γ-H2AX induced by infection and by pharmacological DNA damage suggests that LEF-7 regulates a central component in the DDR. Because intracellular H2AX levels were unaffected (Fig. 2 and 4), it is unlikely that LEF-7 degrades γ-H2AX. Due to lack of sequence similarity, it is also unlikely that LEF-7 has phosphatase activity. Rather, LEF-7 may affect those factors, including kinases, which contribute to H2AX phosphorylation. The interferon regulatory factor vIRF-1 of Kaposi's sarcoma-associated herpesvirus can bind and inhibit ATM (6). Whereas vIRF-1 blocks DNA damage-induced γ-H2AX, its role during infection is unclear. If LEF-7 targets a kinase, it must be selective, since AcMNPV requires DDR kinases for optimal replication (2, 3). Alternatively, LEF-7 may mediate nondegradative ubiquitination of a DDR factor that is indirectly responsible for γ-H2AX. For example, F-box protein SKP2 promotes K63-linked polyubiquitination of NBS1 (46), a component of the host Mre11-Rad50-Nbs1 (MRN) complex. This modification of the MRN complex is necessary to recruit and activate ATM at the site of a double-strand DNA (dsDNA) break.

By virtue of its ability to coordinate DNA repair and to amplify DDR signaling, γ-H2AX is an advantageous target for viral manipulation of the DDR. Hence, DNA viruses have evolved diverse mechanisms that alter H2AX. Murine gammaherpesvirus 68 and Epstein-Barr virus encode kinases that phosphorylate H2AX (47). The E3 ubiquitin ligase ICP0 of herpes simplex virus 1 triggers degradation of the cellular ubiquitin ligases RNF8 and RNF168 to block ubiquitination of H2AX and prevent recruitment of inhibitory DDR factors to incoming viral DNA (48). Human papillomavirus triggers the host DDR and activates ATM and γ-H2AX through a mechanism involving viral protein E7, which interacts with ATM (49). We now report that baculoviruses have evolved a distinct strategy for regulating H2AX that involves the activity of the viral F-box protein LEF-7, specifically for repressing H2AX phosphorylation. Additional studies should reveal the targets of LEF-7 and the molecular mechanism whereby DNA viruses like the baculoviruses manipulate the host DDR to facilitate their multiplication.