Essential function of VCP/p97 in infection cycle of the nucleopolyhedrovirus AcMNPV in Spodoptera frugiperda Sf9 cells

Yulia V. Lyupinaa, Pavel A. Erokhova, Oksana I. Kravchuka, Alexander D. Finoshina, Svetlana B. Abaturovaa, Olga V. Orlovab, Svetlana N. Beljelarskayab, Margarita V. Kostyuchenkoc, Victor S. Mikhailova,⁎


The protein VCP/p97 (also named CDC48 and TER94) belongs to a type II subfamily of the AAA+ATPases and controls cellular proteostasis by acting upstream of proteasomes in the ubiquitin-proteasome protein degrada- tion pathway. The function of VCP/p97 in the baculovirus infection cycle in insect cells remains unknown. Here, we identified VCP/p97 in the fall armyworm Spodoptera frugiperda (Sf9) cells and analyzed the replication of the Autographa californica multiple nucleopolyhedrovirus, AcMNPV, in Sf9 cells in which the VCP/p97 function was inhibited. The specific allosteric inhibitor of the VCP/p97 ATPase activity, NMS-873, did not deplete VCP/p97 in infected cells but caused a dose-dependent inhibition of viral DNA synthesis and efficiently suppressed ex- pression of viral proteins and production of budded virions. NMS-873 caused accumulation of ubiquitinated proteins in a manner similar to the inhibitor of proteasome activity, Bortezomib. This suggests the essential function of VCP/p97 in the baculovirus infection cycle might be associated, at least in part, with the ubiquitin- proteasome system.

Keywords: VCP/p97 TER94
Baculovirus AcMNPV
Spodoptera frugiperda

1. Introduction

Baculoviruses contain circular, double-stranded DNA genomes of 80 to 180 kb and infect insects of the orders Lepidoptera, Hymenoptera, and Diptera. In infected species they produce two types of mature viruses, budded virions (BV) required for systemic infection of insect tissues and occlusion derived viruses (ODV) for transmission in insect populations (for review see (Rohrmann, 2013)). Baculoviruses cause stress in infected cells as demonstrated by the induction of the apoptotic pathway (Clem, 2007; Clem et al., 1991; Schultz and Friesen, 2009), signal kinases (Chen et al., 2009; Katsuma et al., 2007; Schultz and Friesen, 2009; Xiao et al., 2009), the DNA damage response (Huang et al., 2011; Mitchell and Friesen, 2012), oxidative stress (Micheal and Subramanyam, 2013; Wang et al., 2001), and the heat shock response (Breitenbach and Popham, 2013; Iwanaga et al., 2014; Lyupina et al., 2010, 2011; Tung et al., 2016). Although, baculoviruses encode most of factors required for replication in cell nuclei, they utilize host cell systems to maintain proteostasis. The conserved VCP protein, also named p97, CDC48, or TER (Transitional Endoplasmic Reticulum) ATPase, plays an important role in control of the cellular proteome.
VCP/p97 is a member of a type II subfamily of the AAA+ATPase family (extended family of ATPases associated with various cellular activities) and contains two tandem ATPase domains and forms a double ring- shaped homohexamer structure inside cells (for review see (Hanzelmann and Schindelin, 2017; Ripstein et al., 2017; van den Boom and Meyer, 2017)). VCP/p97 acts as an unfoldase/segregase and dis- assembles protein aggregates in an ubiquitin-dependent manner by substrate threading through the central channel of the p97 hexamer (Bodnar and Rapoport, 2017b). Recent data suggest an involvement of VCP/p97 in the infection cycle of several DNA and RNA viruses (Arita et al., 2012; Lin et al., 2017; Panda et al., 2013; Phongphaew et al., 2017; Wang et al., 2017; Wong et al., 2015; Wu et al., 2016; Yi and Yuan, 2017). VCP/p97 is a key member of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. It pulls poly- ubiquitinated, misfolded proteins out of the ER and transfers them to the proteasome (for review see (Bodnar and Rapoport, 2017a; Brodsky, 2012; Locke et al., 2014; Stein et al., 2014; van den Boom and Meyer, 2017)). Induction of the GRP78 homolog by baculoviruses infection suggests that it activates ERAD in infected cells (Lyupina et al., 2011). The functional association of VCP/p97 with proteasomes is conserved in evolution. In Archaea, the VCP/p97 homolog forms a stable complex with the 20S proteasome (Barthelme et al., 2014; Barthelme and Sauer, 2012). The essential role of the host cell proteasomes and ubiquitin- proteasome system (UPS) in the baculovirus replication cycle was shown in several laboratories (Katsuma et al., 2011; Lyupina et al., 2013; Xue et al., 2012). In order to control UPS, baculoviruses encode ubiquitin (Guarino et al., 1995; Ma et al., 2015) and members of the RING-family of ubiquitin ligases (Imai et al., 2003; Katsuma et al., 2008) which were shown recently to participate in the maturation of viral nucleocapsids and their egress from cells (Biswas et al., 2018). All these data suggest that VCP/p97 functions upstream of proteasomes in the UPS and might play an important role in the infection cycle of baculoviruses. In this report, this prediction was confirmed in experi- ments with NMS-873, the most potent and specific inhibitor of the VCP/ p97 ATPase activity identified to date.

2. Materials and methods

2.1. Cells and reagents

Spodoptera frugiperda Sf9 cells were cultured in SF-900 II SFM media (Invitrogen) supplemented with 10% fetal bovine serum (FBS) in the flasks at 27 °C. The cells were infected with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV, the family Baculoviridae) at the MOI of 10. NMS-873 and Bortezomib (PS-341) from Sigma were dissolved in DMSO. The following antibodies (Abs) were used: poly- clonal Ab to human VCP protein from Cell Signalling Technology (2648S); polyclonal Ab to BmNPV DBP (Okano et al., 1999); mouse mAb (FK2) to mono- and polyubiquitinated conjugates from Enzo Life Sciences (BML-PW8810; mouse mAb to beta-actin from Santa Cruz Biotechnology. Peroxidase-conjugated anti-rabbit IgG and ECL reagents were purchased from GE Healthcare Life Sciences.

2.2. Real-time PCR

Measurement of viral DNA content in AcMNPV-infected cells by real-time PCR (RT-PCR) was carried out by method of Rosinski et al. (Rosinski et al., 2002) as described (Lyupina et al., 2010) by using Syntol R-442 Reagent Kit (www.sintol.ru) and the Applied Biosystems 7500 Real-Time PCR System. DNA was purified from AcMNPV-infected cells by using the Genomic DNA Purification Kit K0512 (Thermo Scientific). The forward primer was 5′-ATTAGCGTGGCGTGCTTTTAC-3′. The reverse primer was 5′-GGGTCAGGCTCCTCTTTGC-3′. Viral DNA purified from budded virions was used as a standard.

2.3. Immunohistochemistry and confocal microscopy

Virus- or mock-infected Sf9 cells were fixed for 15 min with 4% paraformaldehyde in phosphate-buffered saline (PBS), washed three times with PBS, and permeabilized for 2 min in cold acetone (−20 °C). The cells were rehydrated with PBS, treated with 1% SDS in PBS for 5 min at room temperature, washed three times with PBS for 5 min each, blocked with 5% FBS and 0.3% Triton X100 in PBS for 1 h, and then subjected to antibody treatments. Antigen localization was de- termined by incubation of the cells with mouse mAb (FK2) to mono- and polyubiquitinated conjugates (1:1000 dilution with 5% FBS and 0.3% Triton X100 in PBS) overnight at room temperature. After in- cubation with the primary antibody, cells were washed four times (5 min per wash) with PBS and then treated with the secondary anti- body, Alexa 594-conjugated Donkey anti-mouse IgG (1:700 dilution, Invitrogen, USA) for 2 h. After four washes with PBS (10 min per wash), the slides were mounted with the Mowiol and analyzed under confocal microscope Leica SPE equipped with an Ar-Kr laser at the Core Facility on Cell Technologies and Optical Research Methods in Developmental Biology of IDB RAS. To ensure equal illumination for all treatments, the same intensity and filter settings were used throughout. Images were recorded at a resolution of 1024 × 1024 pixels and processed with the Leica LCS software. Control experiments were performed by omitting primary or secondary antibodies.

2.4. Determination of proteasome activity

Determination of proteasome activity in cell extract was performed as described (Lyupina et al., 2013). Cells for analysis were washed with PBS buffer and collected by centrifugation. Samples of 106 cells were allowed to swell for 30 min at 4 °C in 30 μl of a hypotonic buffer con- taining 5 mM Tris−HCl, pH 8.0, 2 mM EDTA, and 1 mM DTT, vortexed and disrupted by freezing (−80 °C)-thawing. The extracts were clarified by centrifugation at 10,000×g for 30 min. The chymotrypsin-like proteasome activities was determined by hydrolysis of fluorogenic substrate Suc-LLVY-AMC. The activity was determined in portions of 0.5 to 10 μl from the extracts in final volume of 100 μl containing 30 mM Tris−HCl (pH 7.5), 5 mM MgCl2, 1 mM ATP, 1 mM DTT, and 30 μM Suc-LLVY-AMC. Inhibitors were added to the reaction mixtures on ice. The reactions were carried out at 37°C for 20 min and termi- nated by the addition of 1% SDS and chilling. The digestion product was detected by using fluorimeter VersaFluor (Bio-Rad) with the ex- citation wavelength of 380 nm and the emission wavelength of 440 nm. The proteasome-independent activity was determined in the presence of 3 μM MG-132 (less than 10% in this report) and subtracted from the values obtained in the absence of MG-132.

2.5. Other methods

Electrophoresis in a native polyacrylamide gel followed by detection of proteasome activity in the gel was performed as described earlier (Lyupina et al., 2013). Two-dimensional PAGE was carried out by the method of O’Farrell (O’Farrell et al., 1977) as previously described (Lyupina et al., 2013). Mass spectra of the tryptic peptides of Sf9 proteins were obtained by using the matrix-assisted laser desorption/io- nization (MALDI) time-of-flight mass (TOF) spectrometer as described (Lyupina et al., 2016, 2011). Identification of Sf9 proteins was per- formed by using Mascot software (www.matrixscience.com) in the NCBI database taking into account possible oxidation of methionines and modification of cysteines by acrylamide. SDS-polyacrylamide gel electrophoresis (PAGE) was performed according to Laemmli (Laemmli, 1970). Polyhedrin was visualized by Coomassie staining. For Western blotting, proteins were transferred on Hybond-ECL membrane (Amer- sham) and probed with respective primary antibody. BV titer was de- termined by the endpoint dilution assay (Reed and Muench, 1938) as described (Langfield et al., 2011). Viability of Sf9 cells was examined by the trypan blue exclusion as described In Growth and Maintenance of Insect Cell Lines Version K July 12, 2002 25-0127 (www.invitrogen. [email protected]: http://wolfson.huji.ac.il/ expression/insect/insect_man.pdf).

3. Results

3.1. Identification of S. frugiperda VCP/p97

The VCP/p97 protein was identified in S. frugiperda cells by mass spectrometry after fractionation of the Sf9 cell extracts in poly- acrylamide gels by 2D electrophoresis (Fig. 1A) and by a native elec- trophoresis (Fig. 1B). A Mascot search using the NCBI database con- firmed the protein homology to insect TER94 ATPases with the best score to the Helicoverpa armigera homolog (XP_021183018.1) (E value of 4.1e-16). Blast searches in the database for the lepidopteran genus Spodoptera, spodobase (http://bioweb.ensam.inra.fr/spodobase/) re- vealed several mRNAs encoded by the vcp/p97 gene. A complete se- quence of S. frugiperda VCP/p97 was constructed from three partial overlapped mRNAs coding the N-terminus (Sf9LR126164-5-1-C1), the middle fragment (Sf9LR817849-5-1-C1) and the C-terminusThe S. frugiperda VCP/p97 (Sf VCP/p97) contains 806 amino acids and has the theoretical pI/Mw values of 5.30 / 89,101.41. Fragments verified by mass spectrometry covered more than 80% of the predicted amino acids and confirmed the intact N-terminus and C-terminus in the protein isolated from Sf9 cells. The Sf VCP/p97 includes all specific motifs of the AAA family, CDC48 subfamily ATPases, namely the N-terminal domain (aa 25–97), ATP binding site (aa 479–646), arginine finger (aa 634), Walker A motif GxxxxGK[S/T] (aa 517–524) and Walker B motif hhhh[D/E], where h is a hydrophobic residue (aa 572–577). Sequence alignment confirmed a high homology of Sf VCP/ p97 to animal TER ATPases. The identity level was 97.9% with the lepidopteran homolog (B. mori, NP_001037003.1) and 83.7% with the human homolog (P55072-1). In native gels (Fig. 1B), Sf VCP/p97 mi- grated ahead the thyroglobulin standard (670 kDa) and the 20S pro- teasome cluster (calculated mass of approximately 700 kDa) and behind a soluble V1 subdomain of the vacuolar-type proton ATPase (V-ATPase) (confirmed by presence of homologues to the V-ATPase subunit A of Helicoverpa armigera, XP_021181049.1 (E-value of 1e-06) and to subunit B of Heliothis virescens, P31410.1 (E-value of 0.055)). A hexamer of alternating A and B subunits comprises a catalytic core of the V1 do- main. The migration pattern of VCP/p97 in native gels suggested that it forms in Sf9 cells a homohexamer complex (cal. mass of 535 kDa) as was shown for other eukaryotic CDC48-type ATPases (Bodnar and Rapoport, 2017a; Hanzelmann and Schindelin, 2017; Ripstein et al., 2017; van den Boom and Meyer, 2017).

3.2. Effect of NMS-873 on Sf9 cells

The compound NMS-873, a specific allosteric inhibitor of the ATPase activity of VCP/p97 proteins (Magnaghi et al., 2013), produced a cytostatic effect on Sf9 cells at concentrations of 3 and 10 μM (Fig. 3A). The inhibitor showed a negligible toxicity to Sf9 cells at 3-μM concentration while decreased numbers of viable cells to approximately 70% after 48 h incubation with 10 μM NMS-873 (Fig. 3B). Considering the possible function of VCP/p97 on upstream proteasomes in the ubiquitin-proteasome pathway (UPP), we compared the NMS-873 ef- fects with that of Bortezomib, a potent inhibitor of proteasome activity.
The cytostatic effect and cytotoxicity of 10 μM Bortezomib were com- parable with NMS-873 (Fig. 3A,B, bottom panels). Bortezomib showed a steady cytotoxic effect and killed most of cells in four days, whereas Sf9 cells incubated with NMS-873 resumed proliferation after two days probably due to metabolic neutralization of the inhibitor. To minimize indirect effects, the incubation of cells with inhibitors did not exceed 48 h. These experiments with Sf9 cells confirmed that both, NMS-873 and Bortezomib, are inhibitors of essential metabolic pathways in insect cells.

3.3. Effect of NMS-873 on AcMNPV DNA replication in Sf9 cells

In order to elucidate the VCP/p97 function in the baculovirus re- plication cycle, we examined the effect of NMS-873 on viral DNA am- plification. The inhibitor was added after the standard infection of 1 h, the period sufficient for the virus endocytosis, fusion with acidic en- dosomes, release into the cytoplasm and internalization into the nu- cleus (Wen et al., 2017). Further incubation was carried out in the presence of NMS-873 at concentrations of 3 and 10 μM. The NMS-873 addition produced a dose-dependent and robust inhibition of viral DNA synthesis that was monitored by a real-time PCR (Fig. 4). The amount of progeny viral DNA at 24 hpi was decreased by approximately two or- ders of magnitude under incubation in the presence of 10 μM NMS-873.
A comparable inhibition of viral DNA synthesis was observed in the presence of 10 μM Bortezomib (data not shown) that was in agreement with published data showing a strong inhibitory effect of the protea- some inhibitors on baculovirus DNA replication (Katsuma et al., 2011; Lyupina et al., 2013; Xue et al., 2012).

3.4. Effect of NMS-873 on protein synthesis in Sf9 cells

In agreement with efficient inhibition of viral DNA replication, NMS-873 produced a strong and dose-dependent inhibitory effect on synthesis of viral proteins. Production of viral protein DBP (determined by Western blotting) was markedly suppressed by NMS-873 at con- centration of 3 μM (Fig. 5A) and almost blocked at concentration of 10μM (Fig. 5B). The very-late viral protein polyhedrin (determined by staining in gel) was not detected at 48 hpi in the infected cells incubated in the presence of both concentrations of NMS-873. The inhibition of viral protein synthesis with 10 μM NMS-873 was similar to that produced by 10 μM Bortezomib (Fig. 5B). This result corresponded to robust inhibitory effect of both compounds on the amplification of viral DNA, which serves as a template for viral mRNAs in the infection cycle. The amount of VCP/p97 in AcMNPV-infected Sf9 cells was monitored by Western blot analysis using commercial polyclonal antibody (Fig. 5B). Neither infection with AcMNPV nor incubation with NMS-873 and Bortezomib caused depletion of VCP/p97 in infected cells.
The amount of the ubiquitinated proteins in cells was estimated as described previously by measuring overall immunoreaction with mouse mAb (FK2) on each lane of Western blots normalized to the amount of cellular protein loaded onto the lane (Lyupina et al., 2013). As ex- pected, Bortezomib caused a prominent increase in the amount of the ubiquitinated proteins in infected cells due to inhibition of proteasome activity (Fig. 5B). The accumulation of proteins modified by ubiquitin in the presence of proteasome inhibitors was described previously in baculovirus-infected B. mori and Sf9 cells (Katsuma et al., 2011; Lyupina et al., 2013). Interestingly, NMS-873 also caused accumulation of ubiquitinated proteins at least for first 24 h of infection. The content of the ubiquitinated proteins in AcMNPV-infected Sf9 cells was de- termined also by fluorescent confocal microscopy (Fig. 6A,B). The fluorescence signal of the ubiquitin conjugates in cells was measured by using ImageJ software in the mock-infected cells, and AcMNPV-infected cells incubated for 48 hpi in the presence of NMS-873 and Bortezomib. The virus infection caused a significant increase in the amount of the ubiquitin conjugates. Additional accumulation of the ubiquitin con- jugates was observed under incubation in the presence of NMS-873 and Bortezomib. All these data correspond well to a proposed function of VCP/p97 in the ubiquitin-proteasome pathway. To exclude a direct inhibitory effect of NMS-873 on proteasomes, we performed a control experiment with extracts from Sf9 cells using a standard assay for proteasome activity with fluorogenic peptides. Bortezomib produced a dose-dependent inhibition of the proteasome activity in the extracts, whereas NMS-873 did not inhibit the activity (Fig. 7A). When these reaction mixtures were analyzed by native gel electrophoresis followed by recovery of the proteasome activity in situ, the probes with Borte- zomib showed trace activities indicating a steady inhibitory effect of this compound (Fig. 7B). The probes incubated with NMS-873 showed the standard pattern of the proteasome activities. The results argued against a direct action of NMS-873 on proteasomes in Sf9 cells.

3.5. Effect of NMS-873 on BV production

The efficient inhibition of the viral DNA amplification and viral genes expression by NMS-873 and Bortezomib should prevent produc- tion of mature BV virions. It was shown previously, that the sensitivity of BV production to proteasome inhibitors declines in the course of infection (Katsuma et al., 2011; Xue et al., 2012). In experiments with VER-155008, we observed that suppression of the ATPase of HSP70 s produces a strong inhibitory effect on BV production only when VER- 155008 was added early in infection (Lyupina et al., 2014). Therefore in subsequent experiments, we compared BV production when NMS- 873 was applied to the infected cells at different times in infection, 0 hpi (just after infection), 6 hpi (initiation of viral DNA replication), and 16 hpi (mid-phase in replication). The inhibitory effect of 10 μM NMS-873 on BV production was gradually decreased in the course of infection, however it remained prominent (Fig. 8A). Similar result was obtained with 10 μM Bortezomib, although the inhibitory effect of this compound was lower than in the case of NMS-873 (Fig. 8B).

4. Discussion

The AAA+ATPase VCP/p97 was found in Sf9 cells in amounts comparable with members of the HSP/HSC70 family that are among the most abundant proteins in animal cells (Fig. 1A). When isolated from cells, VCP/p97 was associated into homohexamer complexes (Fig. 1B). Blast searches confirmed a high sequence homology of the Sf9 protein to VCP/p97/TER94 from other animals that was consistent with the conserved nature of the AAA+ATPases (Barthelme and Sauer, 2016; Erzberger and Berger, 2006). When this report was in the re- viewing process, S. frugiperda VCP/p97 sequence has been submitted to the UniProt database by another research group (accession number A0A2H1V392). The loaded sequence was identical to that shown in Fig. 2.
RNA interference (RNAi) and chemical compounds are commonly used in functional analysis of cellular proteins. Inefficient RNAi was reported in many lepidopteran insects (Terenius et al., 2011) that are hosts for baculoviruses. Double-stranded RNAs enter Spodoptera fru- giperda Sf9 cells but are accumulated in the acidic bodies, and not available for RNA interference (Yoon et al., 2017). In addition, bacu- loviruses strongly inhibit the host RNA interference antiviral response (Mehrabadi et al., 2015). All these data suggest that the chemical in- hibitors may serve as a preferred tool to study function of host proteins in baculovirus infection. In this report, we analyzed role of VCP/97 in AcMNPV infection cycle in Sf9 cells by using the compound NMS-873, that is specific allosteric inhibitor of the ATPase activity of VCP/p97 (Magnaghi et al., 2013).
NMS-873 produced cytostatic and cytotoxic effects on Sf9 cells (Fig. 3A,B). Similar effects were produced by the inhibitor of the pro- teasome activity, Bortezomib. Addition of NMS-873 did not deplete VCP/p97 in infected cells but caused a dose-dependent inhibition of viral DNA replication and suppressed expression of viral proteins (Figs. 4 and 5). It is important that the inhibitory effect on viral infection cycle was observed in the presence of 3 μM NMS-873, the concentration that showed a negligible toxicity to Sf9 cells. These data confirmed that the ATPase activity of VCP/p97 is essential for pro- ductive infection of insect cells with baculoviruses. Interestingly, the VCP/p97 inhibition resulted in accumulation of ubiquitinated proteins for 24 hpi as was observed also in the presence of Bortezomib (Figs. 5 and 6). Accumulation of the ubiquitinated proteins in cells under pro- teasome inhibition is a general phenomenon that was observed also in baculovirus-infected B. mori and S. frugiperda cells (Katsuma et al., 2011; Lyupina et al., 2013). Control experiments excluded a direct in- hibitory effect of NMS-873 on proteasome activity (Fig. 7). Experiments with NMS-873 confirmed the involvement of VCP/p97 in the ubiquitin- dependent degradation pathway in infected cells. The essential role of VCP/p97 in the baculovirus infection cycle might be connected, at least in part, to processing of the ubiquitinated proteins toward proteasomes for proteolysis. This proposal is consistent with VCP/p97 functioning upstream of proteasomes in the ubiquitin-proteasome system. It is known that baculoviruses cause proteotoxic stress in infected cells accompanied by induction of GRP78 and by accumulation of ubiquiti- nated proteins in aggresomes (Guo et al., 2015; Lyupina et al., 2013, 2011). By controlling proteostasis in infected cells, VCP/p97 cells might alleviate detrimental complications of the proteotoxic stress.
The release of mature BV virions was markedly suppressed under inhibition of the VCP/p97 activity by NMS-873 added at different stages in infection, 0 hpi (after infection), 6 hpi (initiation of viral DNA replication), and 16 hpi (mid-phase in replication) (Fig. 8A). The sen- sitivity of BV production to NMS-873 decreased when the inhibitor was added at 16 hpi instead of 0 hpi or 6 hpi. Similar but slightly lower inhibitory effects on BV production was observed under inhibition of the proteasome activity by Bortezomib (Fig. 8B). This was in agreement with the published data showing the decrease in inhibitory effect of Bortezomib in the course of infection (Katsuma et al., 2011; Xue et al., 2012). The lower inhibitory effect of Bortezomib in comparison to that of NMS-873 suggested indirectly that some other functions of VCP/p97 besides processing of ubiquitinated proteins to proteasomes might be important for BV production. The maturation and release of BV does not require ATPase activity of heat shock proteins HSP/HSC70 after the mid-replication stage (Lyupina et al., 2014). However, both compo- nents of UPS, VCP/97 and proteasomes, are involved in the virus in- fection cycle past the mid-replication stage.

5. Conclusions

The VCP/p97 ATPase is an abundant protein in Sf9 cells that is essential for productive infection cycle of AcMNPV. Experiments with specific inhibitor NMS-873 indicated that VCP/p97 is required for processing of the ubiquitinated proteins in infected cells.


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