- Email: [email protected]
HSC70 chaperones
G Model
ARTICLE IN PRESS
VIRUS 96375 1–5
Virus Research xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Short communication
1
Egress of budded virions of Autographa californica nucleopolyhedrovirus does not require activity of Spodoptera frugiperda HSP/HSC70 chaperones
2
3
4
5
Q1
6
Yulia V. Lyupina a,1 , Olga V. Orlova b,1 , Svetlana B. Abaturova a , Svetlana N. Beljelarskaya b , Andrey N. Lavrov c , Victor S. Mikhailov a,∗ a
N.K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilova Str., Moscow 119334, Russia V.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova Str., Moscow 119334, Russia c M.V. Lomonosov Moscow State University, Faculty of Biology, 1-12 Leninskie Gory, Moscow 119991, Russia
7
b
8 9 10
a r t i c l e
11 26
i n f o
a b s t r a c t
12
Article history: Received 28 June 2014 Received in revised form 4 August 2014 Accepted 5 August 2014 Available online xxx
13 14 15 16 17 18
Keywords: Baculovirus AcMNPV DNA replication Heat shock proteins Budded virions Virus egress
19 20 21 22 23 24 25
The induction of heat shock proteins in baculovirus infected cells is well documented. However a role of these chaperones in infection cycle remains unknown. The observation that HSP70s are associated with virions of different baculoviruses reported by several researchers suggests that HSPs might be structural components of viruses or involved in virion assembly. These hypotheses were examined by using a novel inhibitor of the ATPase and chaperoning activity of HSP/HSC70s, VER-155008. When VER-155008 was added early in infection, the synthesis of viral proteins, genome replication and the production of budded virions (BV) were markedly inhibited indicating the dependence of virus reproduction on host chaperones. However, BV production was unaffected when VER-155008 was added in the mid-replication phase which is after accumulation of products required for completion of the viral DNA replication. These results suggest that the final stages in assembly of BV and their egress from cells do not depend on chaperone activity of host HSP/HSC70s. © 2014 Published by Elsevier B.V.
The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as a model for the study of a baculovirus infection cycle in insect cells. AcMNPV contains a 134-kbp double-stranded DNA genome that encodes approximately 150 proteins. Replication of viral DNA (vDNA) and the assembly of viral capsids occur in cell nuclei and yield two types of mature viruses, the budded virions (BV) and occlusion-derived virions (ODV) (for review see Rohrmann, 2013). Baculoviruses cause stress of infected cells that is 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 et al., 2013; Mitchell and Friesen, 2012), the oxidative (Wang et al., 2001) and proteotoxic (Lyupina et al., 2013, 2011) stress, and the heat shock response (Breitenbach and Popham, 2013; Iwanaga et al., 2014; Lyupina et al., 2010, 2011). Induction of HSP/HSC70s in infected cells is well-documented at both, the
27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
Q2
∗ Corresponding author. Tel.: +7 4991358847; fax: +7 499 1358012. E-mail address: [email protected] (V.S. Mikhailov). 1 These authors contributed equally to the report.
mRNA (Breitenbach and Popham, 2013; Breitenbach et al., 2011; Choi et al., 2012; Nguyen et al., 2013, 2012; Nobiron et al., 2003; Sagisaka et al., 2010; Salem et al., 2011; Xue et al., 2012) and protein level (Carinhas et al., 2011; Lyupina et al., 2010, 2011; Popham et al., 2010). Suppression of the heat shock response markedly decreases synthesis of vDNA and viral proteins and disturbs the progression of the infection cycle (Iwanaga et al., 2014; Lyupina et al., 2010). Major role in guarding the cellular proteome is played by chaperones of the HSP/HSC70 family that are ubiquitous proteins that assist folding of nascent proteins, support intracellular trafficking and regulate proteolysis. In Spodoptera frugiperda cells, AcMNPV infection increased expression of several members of the HSP/HSC70 family, one cognate protein HSC70(1) and three or four inducible HSP70s (Lyupina et al., 2011). HSC70(1) has sequence homology to GRP78, a factor that associates with the endoplasmic reticulum and regulates the unfolded protein response in cytoplasm. Therefore, this chaperone may protect infected cells from proteotoxicity caused by infection. The role of other HSP/HSC70s in the baculovirus infection cycle remains unknown. The observation that HSP70s are associated with some baculoviruses (Braconi et al., 2014; Hou et al., 2013; Iwanaga et al., 2014; Liu et al., 2008) suggests that these chaperones may be structural components of
http://dx.doi.org/10.1016/j.virusres.2014.08.002 0168-1702/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Lyupina, Y.V., et al., Egress of budded virions of Autographa californica nucleopolyhedrovirus does not require activity of Spodoptera frugiperda HSP/HSC70 chaperones. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.08.002
44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
G Model VIRUS 96375 1–5 2 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109
ARTICLE IN PRESS Y.V. Lyupina et al. / Virus Research xxx (2014) xxx–xxx
viruses or they may assist in the assembly of viral structures. In this report, we attempted to verify these hypotheses by using VER155008, a specific inhibitor of the ATPase and chaperoning activity of HSP70s and HSC70s (Massey et al., 2010). S. 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 AcMNPV at the MOI of 10. VER-155008 from Tocris Bioscience was dissolved in DMSO. The following antibodies (Abs) were used: rat mAb 7.10.3 to HSP/HSC70s of Drosophila melanogaster from Lindquist Lab.; polyclonal Ab to Human SQSTM1/p62 from Abcam (ab91526); polyclonal Ab to BmNPV DBP (Okano et al., 1999). Peroxidaseconjugated anti-rat IgG, anti-rabbit IgG, and ECL reagents were purchased from GE Healthcare Life Sciences. Viability of Sf9 cells was examined by the trypan blue exclusion. BV titer was determined by the endpoint dilution assay (Reed and Muench, 1938) as described (Langfield et al., 2011). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (1970). Polyhedrin was visualized by Coomassie staining. For Western blotting, proteins were transferred on Hybond-ECL membrane (Amersham) and probed with respective primary antibody. Measurement of viral DNA content in AcMNPV-infected cells by real-time PCR (RT-PCR) was carried out by method of Rosinski et al. (2002) as described (Lyupina et al., 2010). Two concentrations of VER-155008, low (20 M) and high (100 M), were tested in experiments with uninfected Sf9 cells. The inhibitor produced a dose-dependent cytostatic effect on proliferation of Sf9 cells and decreased the cell viability (Fig. 1). These data confirmed the cytotoxicity of VER-155008 for insect cells as was observed previously for human cell lines (Massey et al., 2010). Whereas the low concentration slightly affected the Sf9 cells, the 100-M concentration of VER-155008 efficiently blocked the proliferation and decreased the viability by approximately 20%. Addition of VER-155008 to AcMNPV-infected cells caused a dosedependent inhibition of vDNA replication (Fig. 2A). Despite potent cytotoxicity, the 100-M VER-155008 did not completely block synthesis of vDNA although it decreased the rate by approximately one-order of magnitude. Inhibition of the ATPase activity of HSP90 by 2.5 M of 17-AAG additionally suppressed the vDNA synthesis in infected cells incubated with 100 M of VER-155008 (Fig. 2B). This result confirmed the sensitivity of baculovirus replication to 17AAG that was observed in the earlier paper (Lyupina et al., 2011) and is in agreement with the specific affinity of VER-155008 for the active site of HSP70 in comparison to that of HSP90 reported by
Massey et al. (2010). In parallel with the assay of vDNA replication, the effect of VER-155008 on expression of selected host and viral proteins was analyzed (Fig. 2C). VER-155008 at 100-M inhibited the induction of HSP/HSP70s in infected cells that is typical for a heat-shock response (Lyupina et al., 2010) and markedly inhibited production of late DNA-binding protein (DBP) and the very late protein polyhedrin. The addition of 100-M VER-155008 to AcMNPV-infected cells at 0 hpi blocked production of BV at 24, 32 and 48 hpi (Fig. 3A). This result was expected considering that 100-M VER-155008 inhibited vDNA amplification (Fig. 2A) and suppressed viral products that was observed at 24 and 48 hpi in the cells used for titration of BV (Fig. 3B). When VER-155008 was added at 6 hpi, i.e. after the initiation of viral genome replication, a less efficient although high inhibition of BV and viral protein production was observed (Fig. 3A and B). The induction of host HSP/HSC70s was also incomplete similar to that observed when VER-155008 was added at 0 hpi (Fig. 3B). In the next experiment, VER-155008 was added at 24 hpi and BV production was determined at 24, 32 and 48 hpi (Fig. 3C). Synthesis of late viral proteins was nearly completed at 24 hpi as confirmed by the accumulation of DBP (Fig. 3D). However, induction of the very late proteins in the infection cycle was blocked by VER-155008 as shown for polyhedrin (Fig. 3D). Surprisingly, the addition of 100M VER-155008 at 24 hpi did not affect the release of BV at 32 and 48 hpi. Microscopic examination showed that the efficient egress of BV in the presence of VER-155008 was not caused by disintegration of infected cells in the presence of inhibitor. Although the morphology of the infected cells incubated with VER-155008 for 24 h was altered, most of these cells showed structural integrity and retained apparently intact cellular membranes (Fig. 3E). To examine the role of the host HSP/HSC70s in the egress of BV in more detail, we analyzed the dynamics of BV release from 20 to 32 hpi at 4-h intervals (Fig. 3F). In these experiments, 100-M VER-155008 was added at 16 hpi, at the mid-phase in the virus replication cycle. The dynamics of BV production was essentially the same in infected cells incubated in the presence or absence of VER-155008. The inhibitory effect of 100-M VER-155008 on the viral infection cycle was significant, when the inhibitor was added early in infection (0 or 6 hpi). These data confirmed that the early stages in the virus infection cycle directly or indirectly depend on the ATPase activity of cellular HSP/HSC70s. VER-155008 strongly inhibited the synthesis of viral proteins (Figs. 2 and 3). This allowed connecting the VER-induced inhibition with depletion of viral products required for the genome replication and BV assembly. However,
Fig. 1. Effect of VER-155008 on proliferation (A) and viability (B) of Sf9 cells. The quantity and percent of viable cells were determined in triplicate at 6, 24, and 48 h after the addition of VER-155008 (VER) to the media as indicated. Pairwise statistical comparisons to the DMSO group were performed using Student’s t test (*P < 0.05). The bars indicate the standard deviation.
Please cite this article in press as: Lyupina, Y.V., et al., Egress of budded virions of Autographa californica nucleopolyhedrovirus does not require activity of Spodoptera frugiperda HSP/HSC70 chaperones. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.08.002
110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153
G Model VIRUS 96375 1–5
ARTICLE IN PRESS Y.V. Lyupina et al. / Virus Research xxx (2014) xxx–xxx
3
Fig. 2. Effect of VER-155008 on viral DNA synthesis (A and B) and expression of the host and viral proteins (C). The Sf9 cells were infected with AcMNPV at a MOI of 10. VER-155008 (VER) was added to the media at 0 hpi as indicated. (A) The amount of viral DNA (vDNA) was determined by RT-PCR at 18, 24, and 30 hpi. Pairwise statistical comparisons to the DMSO group were performed using Student’s t test (*P < 0.05). (B) The amount of vDNA was determined at 24 hpi after the addition of VER-155008 (100 M) and 17-AAG (2.5 M) singly or in combination. Pairwise statistical comparisons of the VER group and the 17-AAG group to the DMSO group were performed using Student’s t test. The (VER + 17-AAG) group was compared to each of the VER and 17-AAG groups (*P < 0.05). The bars present the standard deviation for triplicate probes for each time point. (C) The amount of host HSP/HSC70s and viral DBP was determined by the Western blotting using respective polyclonal antibodies after fractionation of the cell extracts by SDS-8%PAGE. The regular and overexposed blots are shown for DBP. The polyhedrin band was visualized by the Coomassie staining on a parallel gel. The extracts were normalized according to their protein content. The host protein SQSTM1/p62 was used additionally as a standard in some experiments as shown in the bottom panel.
154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169
VER-155008 did not affect the BV production when added to infected cells at 16 or 24 hpi (Fig. 3) which is after the accumulation of products required for completion of the vDNA replication. This result strongly suggests that the final stages in maturation of BV and their egress from infected cells do not require the ATPase and chaperoning activity of cellular HSP/HSC70s. The association of HSP70s with different baculoviruses indicates that they may play a structural role in viral particles or may participate in the assembly of viruses and become trapped within the virions as they are assembled. The Bombyx mori HSC70-4 that is homolog of S. frugiperda HSC70(2) (Lyupina et al., 2011) was identified as a component of BV and ODV in BmNPV (Iwanaga et al., 2014; Liu et al., 2008). HSP70 was reported to be associated with BV of the Helicoverpa armigera NPV (HearNPV) together with about 100 other host proteins (Hou et al., 2013). This chaperone was also found among 21 host proteins associated with HearNPV ODV (Hou
et al., 2013). In addition, HSP70 was found in ODV but not BV of Anticarsia gemmatalis NPV (AgMNPV) (Braconi et al., 2014). However, HSP70s were not reported to be associated with AcMNPV BV particles (Wang et al., 2010). Whereas BV can easily trap envelope associated host proteins when they acquire their envelope during budding from cell; it is possible that ODV could acquire HSP70s in their envelope if it is derived nuclear membrane components (Braunagel et al., 2009). It is unlikely that baculoviruses use different mechanisms for the assembly of viral particles in different insect cell lines. The insensitivity of BV production to the inhibitor of chaperoning activity of HSP/HSC70s (VER-155008) added at the mid-phase in viral replication cycle reported here suggests strongly that the virus maturation may proceed without assistance of cellular HSP/HSC70s. It is known that baculoviruses are capable of the nonspecific incorporation of alien proteins in polyhedra (Xiang et al., 2011) or in viral particles as was shown previously for
Please cite this article in press as: Lyupina, Y.V., et al., Egress of budded virions of Autographa californica nucleopolyhedrovirus does not require activity of Spodoptera frugiperda HSP/HSC70 chaperones. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.08.002
170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185
G Model VIRUS 96375 1–5 4
ARTICLE IN PRESS Y.V. Lyupina et al. / Virus Research xxx (2014) xxx–xxx
Fig. 3. Effect of VER-155008 on the budded virions (BV) production. Sf9 cells were infected with AcMNPV at a MOI of 10, and 100 M of VER-155008 (VER) was added to the media at 0 hpi, 6 hpi (A and B), 24 hpi (C and D), or 16 hpi (F). The BV production was determined at the indicated time points. The bars present the standard deviation for triplicate probes for each time point. Panels B and D present expression patterns of selected proteins for the samples analyzed in panels A and C, respectively. (E) The infected cells were harvested for microscopy at 48 hpi after incubation in the absence or in the presence of 100 M VER-155008 added at 24 hpi. The images were taken at a magnification of 40. Scale bars: 20 m.
Please cite this article in press as: Lyupina, Y.V., et al., Egress of budded virions of Autographa californica nucleopolyhedrovirus does not require activity of Spodoptera frugiperda HSP/HSC70 chaperones. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.08.002
G Model VIRUS 96375 1–5
ARTICLE IN PRESS Y.V. Lyupina et al. / Virus Research xxx (2014) xxx–xxx
205
bacterial chloramphenicol acetyltransferase (CAT) (Carbonell and Miller, 1987). This property is widely used for vaccine production (for review see Madhan et al., 2010; Mena and Kamen, 2011). Only one host protein, cyclophilin A, which catalyzes the isomerization of peptide bonds, has been predicted to play a specific role in baculovirus assembly so far (Hou et al., 2013). The ubiquitous HSPs are the most abundant proteins in eukaryotic cell comprising up to 10% of total protein. Non-specific trapping of nuclear HSP70s during the assembly of viral particles looks as a possible source for contamination with host HSPs. The egress of BV from infected cells may also proceed without participation of HSP/HSC70s. Microtubules may play major role in BV trafficking to the plasma membrane (Fang et al., 2009), and actin-based machinery may also be involved in this process (Ohkawa et al., 2010). The budding from plasma membrane is presumably mediated by the endosomal sorting complex required for transport (ESCRT) machinery (Li and Blissard, 2012). The results obtained in this report suggest an essential function of host HSP/HSC70 chaperones in supporting expression of viral products but a minor role in assembling and processing of budded virions and their trafficking outside the cell.
206
Acknowledgement
186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204
We thank George Rohrmann for critical reading of the 208 manuscript. This research was supported by grants from the 209 Q3 Russian Foundation for Basic Research to V.S.M. (12-04-00085) and 210 to S.N.B. (14-04-00792). 207
211
212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258
References Braconi, C.T., Ardisson-Araujo, D.M., Leme, A.F., Oliveira, J.V., Pauletti, B.A., GarciaMaruniak, A., Ribeiro, B.M., Maruniak, J.E., Zanotto, P.M., 2014. Proteomic analyses of baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus budded and occluded virus. J. Gen. Virol. 95, 980–989. Braunagel, S.C., Cox, V., Summers, M.D., 2009. Baculovirus data suggest a common but multifaceted pathway for sorting proteins to the inner nuclear membrane. J. Virol. 83, 1280–1288. Breitenbach, J.E., Popham, H.J., 2013. Baculovirus replication induces the expression of heat shock proteins in vivo and in vitro. Arch. Virol. 158, 1517–1522. Breitenbach, J.E., Shelby, K.S., Popham, H.J., 2011. Baculovirus induced transcripts in hemocytes from the larvae of Heliothis virescens. Viruses 3, 2047–2064. Carbonell, L.F., Miller, L.K., 1987. Baculovirus interaction with nontarget organisms: a virus-borne reporter gene is not expressed in two mammalian cell lines. Appl. Environ. Microbiol. 53, 1412–1417. Carinhas, N., Robitaille, A.M., Moes, S., Carrondo, M.J., Jenoe, P., Oliveira, R., Alves, P.M., 2011. Quantitative proteomics of Spodoptera frugiperda cells during growth and baculovirus infection. PLoS ONE 6, e26444. Chen, G.Y., Shiah, H.C., Su, H.J., Chen, C.Y., Chuang, Y.J., Lo, W.H., Huang, J.L., Chuang, C.K., Hwang, S.M., Hu, Y.C., 2009. Baculovirus transduction of mesenchymal stem cells triggers the toll-like receptor 3 pathway. J. Virol. 83, 10548–10556. Choi, J.Y., Roh, J.Y., Wang, Y., Zhen, Z., Tao, X.Y., Lee, J.H., Liu, Q., Kim, J.S., Shin, S.W., Je, Y.H., 2012. Analysis of genes expression of Spodoptera exigua larvae upon AcMNPV infection. PLOS ONE 7, e42462. Clem, R.J., 2007. Baculoviruses and apoptosis: a diversity of genes and responses. Curr. Drug Targets 8, 1069–1074. Clem, R.J., Fechheimer, M., Miller, L.K., 1991. Prevention of apoptosis by a baculovirus gene during infection of insect cells. Science 254, 1388–1390. Fang, M., Nie, Y., Theilmann, D.A., 2009. AcMNPV EXON0 (AC141) which is required for the efficient egress of budded virus nucleocapsids interacts with betatubulin. Virology 385, 496–504. Hou, D., Zhang, L., Deng, F., Fang, W., Wang, R., Liu, X., Guo, L., Rayner, S., Chen, X., Wang, H., Hu, Z., 2013. Comparative proteomics reveal fundamental structural and functional differences between the two progeny phenotypes of a baculovirus. J. Virol. 87, 829–839. Huang, N., Wu, W., Yang, K., Passarelli, A.L., Rohrmann, G.F., Clem, R.J., 2011. Baculovirus infection induces a DNA damage response that is required for efficient viral replication. J. Virol. 85, 12547–12556. Iwanaga, M., Shibano, Y., Ohsawa, T., Fujita, T., Katsuma, S., Kawasaki, H., 2014. Involvement of HSC70-4 and other inducible HSPs in Bombyx mori nucleopolyhedrovirus infection. Virus Res. 179, 113–118. Katsuma, S., Mita, K., Shimada, T., 2007. ERK- and JNK-dependent signaling pathways contribute to Bombyx mori nucleopolyhedrovirus infection. J. Virol. 81, 13700–13709. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227, 680–685. Langfield, K.K., Walker, H.J., Gregory, L.C., Federspiel, M.J., 2011. Manufacture of measles viruses. In: Merten, O.-W., Al-Rubeai, M. (Eds.), Viral
5
Vectors for Gene Therapy. Methods and Protocols. Humana Press, New York/Dordrecht/Heidelberg/London, pp. 345–366. Li, Z., Blissard, G.W., 2012. Cellular VPS4 is required for efficient entry and egress of budded virions of Autographa californica multiple nucleopolyhedrovirus. J. Virol. 86, 459–472. Liu, X., Chen, K., Cai, K., Yao, Q., 2008. Determination of protein composition and host-derived proteins of Bombyx mori nucleopolyhedrovirus by 2-dimensional electrophoresis and mass spectrometry. Intervirology 51, 369–376. Lyupina, Y.V., Abaturova, S.B., Erokhov, P.A., Orlova, O.V., Beljelarskaya, S.N., Mikhailov, V.S., 2013. Proteotoxic stress induced by Autographa californica nucleopolyhedrovirus infection of Spodoptera frugiperda Sf9 cells. Virology 436, 49–58. Lyupina, Y.V., Dmitrieva, S.B., Timokhova, A.V., Beljelarskaya, S.N., Zatsepina, O.G., Evgen’ev, M.B., Mikhailov, V.S., 2010. An important role of the heat shock response in infected cells for replication of baculoviruses. Virology 406, 336–341. Lyupina, Y.V., Zatsepina, O.G., Timokhova, A.V., Orlova, O.V., Kostyuchenko, M.V., Beljelarskaya, S.N., Evgen’ev, M.B., Mikhailov, V.S., 2011. New insights into the induction of the heat shock proteins in baculovirus infected insect cells. Virology 421, 34–41. Madhan, S., Prabakaran, M., Kwang, J., 2010. Baculovirus as vaccine vectors. Curr. Gene Ther. 10, 201–213. Massey, A.J., Williamson, D.S., Browne, H., Murray, J.B., Dokurno, P., Shaw, T., Macias, A.T., Daniels, Z., Geoffroy, S., Dopson, M., Lavan, P., Matassova, N., Francis, G.L., Graham, C.J., Parsons, R., Wang, Y., Padfield, A., Comer, M., Drysdale, M.J., Wood, M., 2010. A novel, small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells. Cancer Chemother. Pharmacol. 66, 535–545. Mena, J.A., Kamen, A.A., 2011. Insect cell technology is a versatile and robust vaccine manufacturing platform. Expert Rev. Vaccines 10, 1063–1081. Mitchell, J.K., Byers, N.M., Friesen, P.D., 2013. Baculovirus F-box protein LEF-7 modifies the host DNA damage response to enhance virus multiplication. J. Virol. 87, 12592–12599. Mitchell, J.K., Friesen, P.D., 2012. Baculoviruses modulate a proapoptotic DNA damage response to promote virus multiplication. J. Virol. 86, 13542–13553. Nguyen, Q., Nielsen, L.K., Reid, S., 2013. Genome scale transcriptomics of baculovirus–insect interactions. Viruses 5, 2721–2747. Nguyen, Q., Palfreyman, R.W., Chan, L.C., Reid, S., Nielsen, L.K., 2012. Transcriptome sequencing of and microarray development for a Helicoverpa zea cell line to investigate in vitro insect cell–baculovirus interactions. PLOS ONE 7, e36324. Nobiron, I., O’Reilly, D.R., Olszewski, J.A., 2003. Autographa californica nucleopolyhedrovirus infection of Spodoptera frugiperda cells: a global analysis of host gene regulation during infection, using a differential display approach. J. Gen. Virol. 84, 3029–3039. Ohkawa, T., Volkman, L.E., Welch, M.D., 2010. Actin-based motility drives baculovirus transit to the nucleus and cell surface. J. Cell Biol. 190, 187–195. Okano, K., Mikhailov, V.S., Maeda, S., 1999. Colocalization of baculovirus IE-1 and two DNA-binding proteins DBP and LEF-3, to viral replication factories. J. Virol. 73, 110–119. Popham, H.J., Grasela, J.J., Goodman, C.L., McIntosh, A.H., 2010. Baculovirus infection influences host protein expression in two established insect cell lines. J. Insect Physiol. 56, 1237–1245. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493–497. Rohrmann, G.F., 2013. Baculovirus Molecular Biology, third ed. http://www.ncbi.nlm.nih.gov/pubmed/24479205 Rosinski, M., Reid, S., Nielsen, L.K., 2002. Kinetics of baculovirus replication and release using real-time quantitative polymerase chain reaction. Biotechnol. Bioeng. 77, 476–480. Sagisaka, A., Fujita, K., Nakamura, Y., Ishibashi, J., Noda, H., Imanishi, S., Mita, K., Yamakawa, M., Tanaka, H., 2010. Genome-wide analysis of host gene expression in the silkworm cells infected with Bombyx mori nucleopolyhedrovirus. Virus Res. 147, 166–175. Salem, T.Z., Zhang, F., Xie, Y., Thiem, S.M., 2011. Comprehensive analysis of host gene expression in Autographa californica nucleopolyhedrovirus-infected Spodoptera frugiperda cells. Virology 412, 167–178. Schultz, K.L., Friesen, P.D., 2009. Baculovirus DNA replication-specific expression factors trigger apoptosis and shutoff of host protein synthesis during infection. J. Virol. 83, 11123–11132. Wang, R., Deng, F., Hou, D., Zhao, Y., Guo, L., Wang, H., Hu, Z., 2010. Proteomics of the Autographa californica nucleopolyhedrovirus budded virions. J. Virol. 84, 7233–7242. Wang, Y., Oberley, L.W., Murhammer, D.W., 2001. Evidence of oxidative stress following the viral infection of two lepidopteran insect cell lines. Free Radic. Biol. Med. 31, 1448–1455. Xiang, X.W., Yang, R., Chen, L., Hu, X.L., Yu, S.F., Wu, X.F., 2011. Co-occlusion of foreign protein into polyhedra with BmNPV polyhedrin. Bing Du Xue Bao 27, 366–371. Xiao, W., Yang, Y., Weng, Q., Lin, T., Yuan, M., Yang, K., Pang, Y., 2009. The role of the PI3K-Akt signal transduction pathway in Autographa californica multiple nucleopolyhedrovirus infection of Spodoptera frugiperda cells. Virology 391, 83–89. Xue, J., Qiao, N., Zhang, W., Cheng, R.L., Zhang, X.Q., Bao, Y.Y., Xu, Y.P., Gu, L.Z., Han, J.D., Zhang, C.X., 2012. Dynamic interactions between Bombyx mori nucleopolyhedrovirus and its host cells revealed by transcriptome analysis. J. Virol. 86, 7345–7359.
Please cite this article in press as: Lyupina, Y.V., et al., Egress of budded virions of Autographa californica nucleopolyhedrovirus does not require activity of Spodoptera frugiperda HSP/HSC70 chaperones. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.08.002
259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343
ARTICLE IN PRESS
VIRUS 96375 1–5
Virus Research xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Short communication
1
Egress of budded virions of Autographa californica nucleopolyhedrovirus does not require activity of Spodoptera frugiperda HSP/HSC70 chaperones
2
3
4
5
Q1
6
Yulia V. Lyupina a,1 , Olga V. Orlova b,1 , Svetlana B. Abaturova a , Svetlana N. Beljelarskaya b , Andrey N. Lavrov c , Victor S. Mikhailov a,∗ a
N.K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilova Str., Moscow 119334, Russia V.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova Str., Moscow 119334, Russia c M.V. Lomonosov Moscow State University, Faculty of Biology, 1-12 Leninskie Gory, Moscow 119991, Russia
7
b
8 9 10
a r t i c l e
11 26
i n f o
a b s t r a c t
12
Article history: Received 28 June 2014 Received in revised form 4 August 2014 Accepted 5 August 2014 Available online xxx
13 14 15 16 17 18
Keywords: Baculovirus AcMNPV DNA replication Heat shock proteins Budded virions Virus egress
19 20 21 22 23 24 25
The induction of heat shock proteins in baculovirus infected cells is well documented. However a role of these chaperones in infection cycle remains unknown. The observation that HSP70s are associated with virions of different baculoviruses reported by several researchers suggests that HSPs might be structural components of viruses or involved in virion assembly. These hypotheses were examined by using a novel inhibitor of the ATPase and chaperoning activity of HSP/HSC70s, VER-155008. When VER-155008 was added early in infection, the synthesis of viral proteins, genome replication and the production of budded virions (BV) were markedly inhibited indicating the dependence of virus reproduction on host chaperones. However, BV production was unaffected when VER-155008 was added in the mid-replication phase which is after accumulation of products required for completion of the viral DNA replication. These results suggest that the final stages in assembly of BV and their egress from cells do not depend on chaperone activity of host HSP/HSC70s. © 2014 Published by Elsevier B.V.
The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as a model for the study of a baculovirus infection cycle in insect cells. AcMNPV contains a 134-kbp double-stranded DNA genome that encodes approximately 150 proteins. Replication of viral DNA (vDNA) and the assembly of viral capsids occur in cell nuclei and yield two types of mature viruses, the budded virions (BV) and occlusion-derived virions (ODV) (for review see Rohrmann, 2013). Baculoviruses cause stress of infected cells that is 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 et al., 2013; Mitchell and Friesen, 2012), the oxidative (Wang et al., 2001) and proteotoxic (Lyupina et al., 2013, 2011) stress, and the heat shock response (Breitenbach and Popham, 2013; Iwanaga et al., 2014; Lyupina et al., 2010, 2011). Induction of HSP/HSC70s in infected cells is well-documented at both, the
27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
Q2
∗ Corresponding author. Tel.: +7 4991358847; fax: +7 499 1358012. E-mail address: [email protected] (V.S. Mikhailov). 1 These authors contributed equally to the report.
mRNA (Breitenbach and Popham, 2013; Breitenbach et al., 2011; Choi et al., 2012; Nguyen et al., 2013, 2012; Nobiron et al., 2003; Sagisaka et al., 2010; Salem et al., 2011; Xue et al., 2012) and protein level (Carinhas et al., 2011; Lyupina et al., 2010, 2011; Popham et al., 2010). Suppression of the heat shock response markedly decreases synthesis of vDNA and viral proteins and disturbs the progression of the infection cycle (Iwanaga et al., 2014; Lyupina et al., 2010). Major role in guarding the cellular proteome is played by chaperones of the HSP/HSC70 family that are ubiquitous proteins that assist folding of nascent proteins, support intracellular trafficking and regulate proteolysis. In Spodoptera frugiperda cells, AcMNPV infection increased expression of several members of the HSP/HSC70 family, one cognate protein HSC70(1) and three or four inducible HSP70s (Lyupina et al., 2011). HSC70(1) has sequence homology to GRP78, a factor that associates with the endoplasmic reticulum and regulates the unfolded protein response in cytoplasm. Therefore, this chaperone may protect infected cells from proteotoxicity caused by infection. The role of other HSP/HSC70s in the baculovirus infection cycle remains unknown. The observation that HSP70s are associated with some baculoviruses (Braconi et al., 2014; Hou et al., 2013; Iwanaga et al., 2014; Liu et al., 2008) suggests that these chaperones may be structural components of
http://dx.doi.org/10.1016/j.virusres.2014.08.002 0168-1702/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Lyupina, Y.V., et al., Egress of budded virions of Autographa californica nucleopolyhedrovirus does not require activity of Spodoptera frugiperda HSP/HSC70 chaperones. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.08.002
44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
G Model VIRUS 96375 1–5 2 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109
ARTICLE IN PRESS Y.V. Lyupina et al. / Virus Research xxx (2014) xxx–xxx
viruses or they may assist in the assembly of viral structures. In this report, we attempted to verify these hypotheses by using VER155008, a specific inhibitor of the ATPase and chaperoning activity of HSP70s and HSC70s (Massey et al., 2010). S. 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 AcMNPV at the MOI of 10. VER-155008 from Tocris Bioscience was dissolved in DMSO. The following antibodies (Abs) were used: rat mAb 7.10.3 to HSP/HSC70s of Drosophila melanogaster from Lindquist Lab.; polyclonal Ab to Human SQSTM1/p62 from Abcam (ab91526); polyclonal Ab to BmNPV DBP (Okano et al., 1999). Peroxidaseconjugated anti-rat IgG, anti-rabbit IgG, and ECL reagents were purchased from GE Healthcare Life Sciences. Viability of Sf9 cells was examined by the trypan blue exclusion. BV titer was determined by the endpoint dilution assay (Reed and Muench, 1938) as described (Langfield et al., 2011). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (1970). Polyhedrin was visualized by Coomassie staining. For Western blotting, proteins were transferred on Hybond-ECL membrane (Amersham) and probed with respective primary antibody. Measurement of viral DNA content in AcMNPV-infected cells by real-time PCR (RT-PCR) was carried out by method of Rosinski et al. (2002) as described (Lyupina et al., 2010). Two concentrations of VER-155008, low (20 M) and high (100 M), were tested in experiments with uninfected Sf9 cells. The inhibitor produced a dose-dependent cytostatic effect on proliferation of Sf9 cells and decreased the cell viability (Fig. 1). These data confirmed the cytotoxicity of VER-155008 for insect cells as was observed previously for human cell lines (Massey et al., 2010). Whereas the low concentration slightly affected the Sf9 cells, the 100-M concentration of VER-155008 efficiently blocked the proliferation and decreased the viability by approximately 20%. Addition of VER-155008 to AcMNPV-infected cells caused a dosedependent inhibition of vDNA replication (Fig. 2A). Despite potent cytotoxicity, the 100-M VER-155008 did not completely block synthesis of vDNA although it decreased the rate by approximately one-order of magnitude. Inhibition of the ATPase activity of HSP90 by 2.5 M of 17-AAG additionally suppressed the vDNA synthesis in infected cells incubated with 100 M of VER-155008 (Fig. 2B). This result confirmed the sensitivity of baculovirus replication to 17AAG that was observed in the earlier paper (Lyupina et al., 2011) and is in agreement with the specific affinity of VER-155008 for the active site of HSP70 in comparison to that of HSP90 reported by
Massey et al. (2010). In parallel with the assay of vDNA replication, the effect of VER-155008 on expression of selected host and viral proteins was analyzed (Fig. 2C). VER-155008 at 100-M inhibited the induction of HSP/HSP70s in infected cells that is typical for a heat-shock response (Lyupina et al., 2010) and markedly inhibited production of late DNA-binding protein (DBP) and the very late protein polyhedrin. The addition of 100-M VER-155008 to AcMNPV-infected cells at 0 hpi blocked production of BV at 24, 32 and 48 hpi (Fig. 3A). This result was expected considering that 100-M VER-155008 inhibited vDNA amplification (Fig. 2A) and suppressed viral products that was observed at 24 and 48 hpi in the cells used for titration of BV (Fig. 3B). When VER-155008 was added at 6 hpi, i.e. after the initiation of viral genome replication, a less efficient although high inhibition of BV and viral protein production was observed (Fig. 3A and B). The induction of host HSP/HSC70s was also incomplete similar to that observed when VER-155008 was added at 0 hpi (Fig. 3B). In the next experiment, VER-155008 was added at 24 hpi and BV production was determined at 24, 32 and 48 hpi (Fig. 3C). Synthesis of late viral proteins was nearly completed at 24 hpi as confirmed by the accumulation of DBP (Fig. 3D). However, induction of the very late proteins in the infection cycle was blocked by VER-155008 as shown for polyhedrin (Fig. 3D). Surprisingly, the addition of 100M VER-155008 at 24 hpi did not affect the release of BV at 32 and 48 hpi. Microscopic examination showed that the efficient egress of BV in the presence of VER-155008 was not caused by disintegration of infected cells in the presence of inhibitor. Although the morphology of the infected cells incubated with VER-155008 for 24 h was altered, most of these cells showed structural integrity and retained apparently intact cellular membranes (Fig. 3E). To examine the role of the host HSP/HSC70s in the egress of BV in more detail, we analyzed the dynamics of BV release from 20 to 32 hpi at 4-h intervals (Fig. 3F). In these experiments, 100-M VER-155008 was added at 16 hpi, at the mid-phase in the virus replication cycle. The dynamics of BV production was essentially the same in infected cells incubated in the presence or absence of VER-155008. The inhibitory effect of 100-M VER-155008 on the viral infection cycle was significant, when the inhibitor was added early in infection (0 or 6 hpi). These data confirmed that the early stages in the virus infection cycle directly or indirectly depend on the ATPase activity of cellular HSP/HSC70s. VER-155008 strongly inhibited the synthesis of viral proteins (Figs. 2 and 3). This allowed connecting the VER-induced inhibition with depletion of viral products required for the genome replication and BV assembly. However,
Fig. 1. Effect of VER-155008 on proliferation (A) and viability (B) of Sf9 cells. The quantity and percent of viable cells were determined in triplicate at 6, 24, and 48 h after the addition of VER-155008 (VER) to the media as indicated. Pairwise statistical comparisons to the DMSO group were performed using Student’s t test (*P < 0.05). The bars indicate the standard deviation.
Please cite this article in press as: Lyupina, Y.V., et al., Egress of budded virions of Autographa californica nucleopolyhedrovirus does not require activity of Spodoptera frugiperda HSP/HSC70 chaperones. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.08.002
110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153
G Model VIRUS 96375 1–5
ARTICLE IN PRESS Y.V. Lyupina et al. / Virus Research xxx (2014) xxx–xxx
3
Fig. 2. Effect of VER-155008 on viral DNA synthesis (A and B) and expression of the host and viral proteins (C). The Sf9 cells were infected with AcMNPV at a MOI of 10. VER-155008 (VER) was added to the media at 0 hpi as indicated. (A) The amount of viral DNA (vDNA) was determined by RT-PCR at 18, 24, and 30 hpi. Pairwise statistical comparisons to the DMSO group were performed using Student’s t test (*P < 0.05). (B) The amount of vDNA was determined at 24 hpi after the addition of VER-155008 (100 M) and 17-AAG (2.5 M) singly or in combination. Pairwise statistical comparisons of the VER group and the 17-AAG group to the DMSO group were performed using Student’s t test. The (VER + 17-AAG) group was compared to each of the VER and 17-AAG groups (*P < 0.05). The bars present the standard deviation for triplicate probes for each time point. (C) The amount of host HSP/HSC70s and viral DBP was determined by the Western blotting using respective polyclonal antibodies after fractionation of the cell extracts by SDS-8%PAGE. The regular and overexposed blots are shown for DBP. The polyhedrin band was visualized by the Coomassie staining on a parallel gel. The extracts were normalized according to their protein content. The host protein SQSTM1/p62 was used additionally as a standard in some experiments as shown in the bottom panel.
154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169
VER-155008 did not affect the BV production when added to infected cells at 16 or 24 hpi (Fig. 3) which is after the accumulation of products required for completion of the vDNA replication. This result strongly suggests that the final stages in maturation of BV and their egress from infected cells do not require the ATPase and chaperoning activity of cellular HSP/HSC70s. The association of HSP70s with different baculoviruses indicates that they may play a structural role in viral particles or may participate in the assembly of viruses and become trapped within the virions as they are assembled. The Bombyx mori HSC70-4 that is homolog of S. frugiperda HSC70(2) (Lyupina et al., 2011) was identified as a component of BV and ODV in BmNPV (Iwanaga et al., 2014; Liu et al., 2008). HSP70 was reported to be associated with BV of the Helicoverpa armigera NPV (HearNPV) together with about 100 other host proteins (Hou et al., 2013). This chaperone was also found among 21 host proteins associated with HearNPV ODV (Hou
et al., 2013). In addition, HSP70 was found in ODV but not BV of Anticarsia gemmatalis NPV (AgMNPV) (Braconi et al., 2014). However, HSP70s were not reported to be associated with AcMNPV BV particles (Wang et al., 2010). Whereas BV can easily trap envelope associated host proteins when they acquire their envelope during budding from cell; it is possible that ODV could acquire HSP70s in their envelope if it is derived nuclear membrane components (Braunagel et al., 2009). It is unlikely that baculoviruses use different mechanisms for the assembly of viral particles in different insect cell lines. The insensitivity of BV production to the inhibitor of chaperoning activity of HSP/HSC70s (VER-155008) added at the mid-phase in viral replication cycle reported here suggests strongly that the virus maturation may proceed without assistance of cellular HSP/HSC70s. It is known that baculoviruses are capable of the nonspecific incorporation of alien proteins in polyhedra (Xiang et al., 2011) or in viral particles as was shown previously for
Please cite this article in press as: Lyupina, Y.V., et al., Egress of budded virions of Autographa californica nucleopolyhedrovirus does not require activity of Spodoptera frugiperda HSP/HSC70 chaperones. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.08.002
170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185
G Model VIRUS 96375 1–5 4
ARTICLE IN PRESS Y.V. Lyupina et al. / Virus Research xxx (2014) xxx–xxx
Fig. 3. Effect of VER-155008 on the budded virions (BV) production. Sf9 cells were infected with AcMNPV at a MOI of 10, and 100 M of VER-155008 (VER) was added to the media at 0 hpi, 6 hpi (A and B), 24 hpi (C and D), or 16 hpi (F). The BV production was determined at the indicated time points. The bars present the standard deviation for triplicate probes for each time point. Panels B and D present expression patterns of selected proteins for the samples analyzed in panels A and C, respectively. (E) The infected cells were harvested for microscopy at 48 hpi after incubation in the absence or in the presence of 100 M VER-155008 added at 24 hpi. The images were taken at a magnification of 40. Scale bars: 20 m.
Please cite this article in press as: Lyupina, Y.V., et al., Egress of budded virions of Autographa californica nucleopolyhedrovirus does not require activity of Spodoptera frugiperda HSP/HSC70 chaperones. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.08.002
G Model VIRUS 96375 1–5
ARTICLE IN PRESS Y.V. Lyupina et al. / Virus Research xxx (2014) xxx–xxx
205
bacterial chloramphenicol acetyltransferase (CAT) (Carbonell and Miller, 1987). This property is widely used for vaccine production (for review see Madhan et al., 2010; Mena and Kamen, 2011). Only one host protein, cyclophilin A, which catalyzes the isomerization of peptide bonds, has been predicted to play a specific role in baculovirus assembly so far (Hou et al., 2013). The ubiquitous HSPs are the most abundant proteins in eukaryotic cell comprising up to 10% of total protein. Non-specific trapping of nuclear HSP70s during the assembly of viral particles looks as a possible source for contamination with host HSPs. The egress of BV from infected cells may also proceed without participation of HSP/HSC70s. Microtubules may play major role in BV trafficking to the plasma membrane (Fang et al., 2009), and actin-based machinery may also be involved in this process (Ohkawa et al., 2010). The budding from plasma membrane is presumably mediated by the endosomal sorting complex required for transport (ESCRT) machinery (Li and Blissard, 2012). The results obtained in this report suggest an essential function of host HSP/HSC70 chaperones in supporting expression of viral products but a minor role in assembling and processing of budded virions and their trafficking outside the cell.
206
Acknowledgement
186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204
We thank George Rohrmann for critical reading of the 208 manuscript. This research was supported by grants from the 209 Q3 Russian Foundation for Basic Research to V.S.M. (12-04-00085) and 210 to S.N.B. (14-04-00792). 207
211
212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258
References Braconi, C.T., Ardisson-Araujo, D.M., Leme, A.F., Oliveira, J.V., Pauletti, B.A., GarciaMaruniak, A., Ribeiro, B.M., Maruniak, J.E., Zanotto, P.M., 2014. Proteomic analyses of baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus budded and occluded virus. J. Gen. Virol. 95, 980–989. Braunagel, S.C., Cox, V., Summers, M.D., 2009. Baculovirus data suggest a common but multifaceted pathway for sorting proteins to the inner nuclear membrane. J. Virol. 83, 1280–1288. Breitenbach, J.E., Popham, H.J., 2013. Baculovirus replication induces the expression of heat shock proteins in vivo and in vitro. Arch. Virol. 158, 1517–1522. Breitenbach, J.E., Shelby, K.S., Popham, H.J., 2011. Baculovirus induced transcripts in hemocytes from the larvae of Heliothis virescens. Viruses 3, 2047–2064. Carbonell, L.F., Miller, L.K., 1987. Baculovirus interaction with nontarget organisms: a virus-borne reporter gene is not expressed in two mammalian cell lines. Appl. Environ. Microbiol. 53, 1412–1417. Carinhas, N., Robitaille, A.M., Moes, S., Carrondo, M.J., Jenoe, P., Oliveira, R., Alves, P.M., 2011. Quantitative proteomics of Spodoptera frugiperda cells during growth and baculovirus infection. PLoS ONE 6, e26444. Chen, G.Y., Shiah, H.C., Su, H.J., Chen, C.Y., Chuang, Y.J., Lo, W.H., Huang, J.L., Chuang, C.K., Hwang, S.M., Hu, Y.C., 2009. Baculovirus transduction of mesenchymal stem cells triggers the toll-like receptor 3 pathway. J. Virol. 83, 10548–10556. Choi, J.Y., Roh, J.Y., Wang, Y., Zhen, Z., Tao, X.Y., Lee, J.H., Liu, Q., Kim, J.S., Shin, S.W., Je, Y.H., 2012. Analysis of genes expression of Spodoptera exigua larvae upon AcMNPV infection. PLOS ONE 7, e42462. Clem, R.J., 2007. Baculoviruses and apoptosis: a diversity of genes and responses. Curr. Drug Targets 8, 1069–1074. Clem, R.J., Fechheimer, M., Miller, L.K., 1991. Prevention of apoptosis by a baculovirus gene during infection of insect cells. Science 254, 1388–1390. Fang, M., Nie, Y., Theilmann, D.A., 2009. AcMNPV EXON0 (AC141) which is required for the efficient egress of budded virus nucleocapsids interacts with betatubulin. Virology 385, 496–504. Hou, D., Zhang, L., Deng, F., Fang, W., Wang, R., Liu, X., Guo, L., Rayner, S., Chen, X., Wang, H., Hu, Z., 2013. Comparative proteomics reveal fundamental structural and functional differences between the two progeny phenotypes of a baculovirus. J. Virol. 87, 829–839. Huang, N., Wu, W., Yang, K., Passarelli, A.L., Rohrmann, G.F., Clem, R.J., 2011. Baculovirus infection induces a DNA damage response that is required for efficient viral replication. J. Virol. 85, 12547–12556. Iwanaga, M., Shibano, Y., Ohsawa, T., Fujita, T., Katsuma, S., Kawasaki, H., 2014. Involvement of HSC70-4 and other inducible HSPs in Bombyx mori nucleopolyhedrovirus infection. Virus Res. 179, 113–118. Katsuma, S., Mita, K., Shimada, T., 2007. ERK- and JNK-dependent signaling pathways contribute to Bombyx mori nucleopolyhedrovirus infection. J. Virol. 81, 13700–13709. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227, 680–685. Langfield, K.K., Walker, H.J., Gregory, L.C., Federspiel, M.J., 2011. Manufacture of measles viruses. In: Merten, O.-W., Al-Rubeai, M. (Eds.), Viral
5
Vectors for Gene Therapy. Methods and Protocols. Humana Press, New York/Dordrecht/Heidelberg/London, pp. 345–366. Li, Z., Blissard, G.W., 2012. Cellular VPS4 is required for efficient entry and egress of budded virions of Autographa californica multiple nucleopolyhedrovirus. J. Virol. 86, 459–472. Liu, X., Chen, K., Cai, K., Yao, Q., 2008. Determination of protein composition and host-derived proteins of Bombyx mori nucleopolyhedrovirus by 2-dimensional electrophoresis and mass spectrometry. Intervirology 51, 369–376. Lyupina, Y.V., Abaturova, S.B., Erokhov, P.A., Orlova, O.V., Beljelarskaya, S.N., Mikhailov, V.S., 2013. Proteotoxic stress induced by Autographa californica nucleopolyhedrovirus infection of Spodoptera frugiperda Sf9 cells. Virology 436, 49–58. Lyupina, Y.V., Dmitrieva, S.B., Timokhova, A.V., Beljelarskaya, S.N., Zatsepina, O.G., Evgen’ev, M.B., Mikhailov, V.S., 2010. An important role of the heat shock response in infected cells for replication of baculoviruses. Virology 406, 336–341. Lyupina, Y.V., Zatsepina, O.G., Timokhova, A.V., Orlova, O.V., Kostyuchenko, M.V., Beljelarskaya, S.N., Evgen’ev, M.B., Mikhailov, V.S., 2011. New insights into the induction of the heat shock proteins in baculovirus infected insect cells. Virology 421, 34–41. Madhan, S., Prabakaran, M., Kwang, J., 2010. Baculovirus as vaccine vectors. Curr. Gene Ther. 10, 201–213. Massey, A.J., Williamson, D.S., Browne, H., Murray, J.B., Dokurno, P., Shaw, T., Macias, A.T., Daniels, Z., Geoffroy, S., Dopson, M., Lavan, P., Matassova, N., Francis, G.L., Graham, C.J., Parsons, R., Wang, Y., Padfield, A., Comer, M., Drysdale, M.J., Wood, M., 2010. A novel, small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells. Cancer Chemother. Pharmacol. 66, 535–545. Mena, J.A., Kamen, A.A., 2011. Insect cell technology is a versatile and robust vaccine manufacturing platform. Expert Rev. Vaccines 10, 1063–1081. Mitchell, J.K., Byers, N.M., Friesen, P.D., 2013. Baculovirus F-box protein LEF-7 modifies the host DNA damage response to enhance virus multiplication. J. Virol. 87, 12592–12599. Mitchell, J.K., Friesen, P.D., 2012. Baculoviruses modulate a proapoptotic DNA damage response to promote virus multiplication. J. Virol. 86, 13542–13553. Nguyen, Q., Nielsen, L.K., Reid, S., 2013. Genome scale transcriptomics of baculovirus–insect interactions. Viruses 5, 2721–2747. Nguyen, Q., Palfreyman, R.W., Chan, L.C., Reid, S., Nielsen, L.K., 2012. Transcriptome sequencing of and microarray development for a Helicoverpa zea cell line to investigate in vitro insect cell–baculovirus interactions. PLOS ONE 7, e36324. Nobiron, I., O’Reilly, D.R., Olszewski, J.A., 2003. Autographa californica nucleopolyhedrovirus infection of Spodoptera frugiperda cells: a global analysis of host gene regulation during infection, using a differential display approach. J. Gen. Virol. 84, 3029–3039. Ohkawa, T., Volkman, L.E., Welch, M.D., 2010. Actin-based motility drives baculovirus transit to the nucleus and cell surface. J. Cell Biol. 190, 187–195. Okano, K., Mikhailov, V.S., Maeda, S., 1999. Colocalization of baculovirus IE-1 and two DNA-binding proteins DBP and LEF-3, to viral replication factories. J. Virol. 73, 110–119. Popham, H.J., Grasela, J.J., Goodman, C.L., McIntosh, A.H., 2010. Baculovirus infection influences host protein expression in two established insect cell lines. J. Insect Physiol. 56, 1237–1245. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493–497. Rohrmann, G.F., 2013. Baculovirus Molecular Biology, third ed. http://www.ncbi.nlm.nih.gov/pubmed/24479205 Rosinski, M., Reid, S., Nielsen, L.K., 2002. Kinetics of baculovirus replication and release using real-time quantitative polymerase chain reaction. Biotechnol. Bioeng. 77, 476–480. Sagisaka, A., Fujita, K., Nakamura, Y., Ishibashi, J., Noda, H., Imanishi, S., Mita, K., Yamakawa, M., Tanaka, H., 2010. Genome-wide analysis of host gene expression in the silkworm cells infected with Bombyx mori nucleopolyhedrovirus. Virus Res. 147, 166–175. Salem, T.Z., Zhang, F., Xie, Y., Thiem, S.M., 2011. Comprehensive analysis of host gene expression in Autographa californica nucleopolyhedrovirus-infected Spodoptera frugiperda cells. Virology 412, 167–178. Schultz, K.L., Friesen, P.D., 2009. Baculovirus DNA replication-specific expression factors trigger apoptosis and shutoff of host protein synthesis during infection. J. Virol. 83, 11123–11132. Wang, R., Deng, F., Hou, D., Zhao, Y., Guo, L., Wang, H., Hu, Z., 2010. Proteomics of the Autographa californica nucleopolyhedrovirus budded virions. J. Virol. 84, 7233–7242. Wang, Y., Oberley, L.W., Murhammer, D.W., 2001. Evidence of oxidative stress following the viral infection of two lepidopteran insect cell lines. Free Radic. Biol. Med. 31, 1448–1455. Xiang, X.W., Yang, R., Chen, L., Hu, X.L., Yu, S.F., Wu, X.F., 2011. Co-occlusion of foreign protein into polyhedra with BmNPV polyhedrin. Bing Du Xue Bao 27, 366–371. Xiao, W., Yang, Y., Weng, Q., Lin, T., Yuan, M., Yang, K., Pang, Y., 2009. The role of the PI3K-Akt signal transduction pathway in Autographa californica multiple nucleopolyhedrovirus infection of Spodoptera frugiperda cells. Virology 391, 83–89. Xue, J., Qiao, N., Zhang, W., Cheng, R.L., Zhang, X.Q., Bao, Y.Y., Xu, Y.P., Gu, L.Z., Han, J.D., Zhang, C.X., 2012. Dynamic interactions between Bombyx mori nucleopolyhedrovirus and its host cells revealed by transcriptome analysis. J. Virol. 86, 7345–7359.
Please cite this article in press as: Lyupina, Y.V., et al., Egress of budded virions of Autographa californica nucleopolyhedrovirus does not require activity of Spodoptera frugiperda HSP/HSC70 chaperones. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.08.002
259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343