Department of Biological Sciences, SAF Building, South Kensington Campus, Imperial College, London SW7 2AZ, UK
Correspondence
Julie Olszewski
j.olszewski{at}imperial.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: INRA, Unité de Virologie et Immunologie moléculaires, 78350 Jouy-en-Josas, France.
Present address: Syngenta, Jealotts Hill International Research Centre, Bracknell, Berkshire RG42 6EY, UK.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
AcMNPV carries about 150 genes (Ayres et al., 1994), which encode proteins required for processes such as viral RNA transcription and DNA replication. For instance, AcMNPV encodes its own DNA polymerase and RNA polymerase activities (Tomalski et al., 1988
; Guarino et al., 1998
). Despite such a large array of viral genes, AcMNPV is dependent upon host-encoded protein activities for successful infection. For example, AcMNPV genes expressed at early times of infection (i.e. prior to viral DNA replication) are transcribed by the host RNA polymerase II proteins. Expression of late and very late baculoviral genes entails a shift from host to viral RNA polymerase activity, the mechanism of which is not well understood.
In addition to stealing host proteins for completion of its life-cycle, baculoviruses have also been shown to physically reorganize host cells and to reprogramme host gene expression. For example, following entry into Trichlopusia ni TN-368 cells, AcMNPV induces the formation of actin cables as a result of the activity of a viral protein known as actin rearrangement-inducing factor (Arif-1) (Roncarati & Knebel-Morsdorf, 1997). These actin cables are believed to assist viral nucleocapsid transport to the nucleus where the baculovirus replicates (Lanier & Volkman, 1998
). Cytoskeletal alteration occurs again toward the end of the infection when cathepsin like protease involved in actin degradation, encoded by a a viral gene, causes breakdown of the host cell cytoskeleton, facilitating virus spread (Lanier et al., 1996
).
It was shown that AcMNPV infection of Sf9 cells also results in an arrest of the host cell-cycle at the G2/M transition phase. One hypothesis is that the virus intentionally halts its host cell at a time when nuclear membrane permeability is optimal to allow virus import into the nucleus (Braunagel et al., 1998; Ikeda & Kobayashi, 1999
). Additionally, to prevent infected cells from undergoing programmed cell death (apoptosis), which leads to reduced levels of virus replication, baculoviruses encode anti-apoptotic proteins such as p35 or inhibitor of apoptosis proteins (IAPs) (Clem & Miller, 1993
; Birnbaum et al., 1994
) to prevent premature cell death.
Baculovirus infection alters both host protein and host mRNA levels. AcMNPV infection causes a global shutoff of host protein synthesis in Sf cells beginning around 18 h, as demonstrated by protein metabolic labelling experiments at various times during infection (Carstens et al., 1979). Re-directing the translation machinery from host protein synthesis to viral protein synthesis is thought to allow for better virus production levels.
Ooi & Miller (1988) demonstrated that there is also a shutoff of several host mRNAs following AcMNPV infection of Sf21 cells. They showed by Northern blot analysis that mRNA levels of actin, histone and heat shock protein 70 were substantially reduced from 12 to 18 h following infection with AcMNPV. More recently, expression levels of Sf21 translation initiation factors eIF4E (Van Oers et al., 2001
) and eIF5A (Van Oers et al., 1999
) were also shown to be down-regulated at the mRNA level following infection with AcMNPV, despite the reliance of the virus on the host translation machinery to produce its viral proteins. This decrease in mRNA levels was found to begin between 12 and 24 h after infection. The mechanism for this down-regulation of host mRNA levels mediated by baculovirus infection has not been elucidated and could be due to scenarios such as a degradation of host mRNA late in infection, inactivation of the host RNA polymerase or specific inhibition of host mRNA splicing. Other large DNA viruses like herpes simplex virus (HSV) also reduce levels of functional host mRNAs upon infection. A virion host shutoff (Vhs) protein was identified in HSV (Fenwick & McMenamin, 1984
; Scheck & Bachenheimer, 1985
; Strom & Frenkel, 1987
; Oroskar & Read, 1989
). This protein accelerates the degradation of host mRNA, while another HSV protein inhibits pre-mRNA splicing (Hardy & Sandri-Goldin, 1994
). These two activities redirect protein synthesis towards viral proteins.
Despite the worldwide use of AcMNPV-based expression systems and associated research to understand and manipulate the virus replication cycle, there are no published studies which examine at the RNA level the host genome-wide response to AcMNPV infection. We adopted a global approach to look at differential expression of Sf9 genes following AcMNPV infection by using differential display technology. Our aim was to confirm the general shutoff of host Sf9 mRNAs following AcMNPV infection, and to screen for any host genes which were not subjected to this shutoff. Additionally, we were interested in determining whether any host mRNAs were actually up-regulated at early times of infection, before the more general shutoff began. By doing so, we wanted to determine the extent to which Sf9 cells need to be reprogrammed for virus replication, and to identify Sf9 genes which are crucial for successful baculovirus infection. We describe here the results of this two-part experiment examining differential expression of Sf9 genes both early and late after AcMNPV infection.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Infection time-course.
Sf9 cells were plated in six-well plates (106 cells per dish) and left to attach for 30 min. They were incubated with AcMNPV at a m.o.i. of 10 or medium only for uninfected controls (0·7 ml per dish). Supernatants were replaced by fresh medium after 1 h incubation and from then cells were left to incubate at 27 °C for 6, 12, 18, 24 and 36 h for the first time-course, and 3, 6, 9, 12 and 24 h for the second one.
RNA extraction.
Cells were harvested in RNA stat 60 (Ams Biotechnology) (1 ml per 35 mm dish) according to the manufacturer's instructions and stored at -80 °C after homogenization. Total RNA extraction was performed with a 0·2 vol. of chloroform. RNA was precipitated from the aqueous phase with a 0·5 vol. of isopropanol, washed with 75 % ethanol, dried and resuspended in 20 µl DEPC-treated water. Quantification was done both on a 1 % agarose gel and with a spectrophotometer (two readings at both A260 and A280). Polyadenylated mRNA was extracted from total RNA with an Oligotex mRNA mini kit (Qiagen).
Differential display.
The DisplayPROFILE kit (Display Systems Biotech) was used to identify differentially expressed RNA transcripts. Reverse transcription was carried out according to the manufacturer's instructions. Briefly, first strand cDNA synthesis was performed with displayTHERMO-RT (Display Systems) using an oligo(dT) TnV primer (where V=A, C or G). For the first time-course, total RNA was used (1 µg per time-point). For the second time-course, mRNA (300 ng) was extracted from mock-infected cells and cells at 3 and 6 h post-infection (p.i.) only. After second strand cDNA synthesis, cDNA was phenol/chloroform extracted, ethanol precipitated and digested with TaqI, a four-base cutter restriction endonuclease that leaves 5'-overhangs. The so-called standard and EP adaptors were ligated for 3 h at 37 °C to prevent religation of TaqI-digested cDNA fragments. The 5' overhang of the EP adaptor has a dideoxynucleotide to prevent DNA synthesis and therefore amplification of fragments with EP adaptors at both ends. To perform the PCR, primers complementary to the adaptor sequences were used. The O-extension 5' primer annealed to the EP adaptor, while a set of 64 3' displayPROBES annealed to the standard adaptor. These displayPROBES included a 3 nucleotide sequence (NNN, where N=A, T, G or C) to be complementary to the cDNA and different for each primer, allowing amplification of all cDNA variants. The O-extension primer was end-labelled with [-33P]dATP (ICN) for 30 min at 37 °C and further used in a touchdown PCR according to the manufacturer's instructions, using MasterTaq (Eppendorf) as the DNA polymerase. PCR products were separated on an 8 % urea/polyacrylamide gel (40x35 cm) with 0·6x Tris/borate/EDTA electrophoresis buffer. The gel was run for 150 min at 60 W, transferred to Whatman paper, dried and exposed for 48 h on film (Biomax MR, Kodak). To recover differentially expressed cDNA bands, the film was carefully realigned to the gel and marked fragments were extracted with a blade and dissolved in 50 µl TE buffer at 95 °C for 15 min. This template (1 µl) was used for reamplification with the specific displayPROBE. A primer similar to the O-extension primer lacking the extension protection group was synthesized (5'-ACTGGTCTCGTAGACTGCGT-3') to perform the PCRs and to further sequence the fragments. PCR products were purified prior to direct sequencing using a Qiaquick gel extraction kit (Qiagen). Sequencing was done on an ABI 3700 sequencer, using the Big Dye Terminator Cycle sequencing kit (ABI Prism, Applied Biosystems). When direct PCR sequencing was not possible, the cDNA was cloned into the pGEM-T Easy plasmid vector (Promega) and several of the resulting cloned inserts were then sequenced. The sequences were compared to those in the GenBank databases using the Basic Local Alignment Search Tool (BLAST; Altschul et al., 1990
). For RpL19 sequence analysis, the low complexity default filter was removed.
Northern blot analysis.
Total RNA (10 µg per lane) was separated on a 1 % agarose/formaldehyde gel and blotted onto nylon membranes (Hybond-N+, Amersham Pharmacia Biotech) after a 3 h capillary transfer, using standard protocols (Sambrook et al., 1989). The RNA was cross-linked at 1200 W (254 nm for 30 s) and membranes were incubated for 2 h at 42 °C in prehybridization buffer (40 % formamide, 10x SSPE, 5x Denhardt's solution, 0·1 % SDS and 100 µg herring sperm DNA ml-1). Purified PCR products (25 ng), amplified with the restriction fragment differential display kit (RFDD), were used as DNA probes. They were labelled with 50 µCi [
-32P]dCTP nucleotides (3000 Ci mmol-1, ICN) using the High Prime DNA labelling kit (Roche). Probes were added to the prehybridization buffer after removal of unincorporated nucleotides (ProbeQuant G-50, Amersham Pharmacia Biotech) and hybridization was carried out for 20 h at 42 °C. After washing at 65 °C (in succession with 2x, 1x and 0·1x SSC and 0·1 % SDS), membranes were exposed wet (to allow further stripping) on BAS films overnight. Radioactivity was visualized on a BAS 1500 phosphorimager (Fujifilm) and on Kodak films (Biomax MR).
Southern blot.
To discriminate up-regulated cellular transcripts from viral transcripts, isolated and amplified RFDD fragments were electrophoresed through 1·5 % agarose gels, transferred to membranes and hybridized with random primer-labelled AcMNPV viral DNA (High Prime kit) as previously described (Sambrook et al., 1989). Fragments that were negative for this assay were further analysed in a semi-quantitative RT-PCR.
Semi-quantitative RT-PCR.
Sf9 RFDD transcripts were analysed in a duplex semi-quantitative RT-PCR (Quantum RNA 18S kit, Ambion) according to the manufacturer's instructions. Briefly, total RNA (2 µg) was extracted at different times p.i. (0, 3, 6, 9 and 12 h), reverse transcribed and used in duplex PCR reactions (see primer design in Table 1). PCR products were quantified by ethidium bromide fluorescence using the Kodak 1D 3.5.2 program. Universal 18S rRNA primers were used to amplify ribosomal cDNA and serve as an internal control in a duplex PCR reaction. To modulate the amplification efficiency of the 18S template relative to the gene-specific template, Quantum RNA (Ambion) provides 18S competimers primers which are modified at their 3' ends to block extension by DNA polymerase. To allow similar amplification levels for the 18S target and the gene of interest, appropriate ratios of 18S primers and 18S competimers had to be determined. They were 4 : 6 for actin and elongation factor 1-
, 3 : 7 for Hsc70, 1 : 9 for Rho GTPase and 2 : 8 for the remaining transcripts. To determine the linear range of PCR reactions for each gene, preliminary amplifications were performed for 16, 18, 20, 22, 24, 26, 28 and 30 cycles. The cycle number in the middle of the linear range was picked for the final optimized semi-quantitative PCRs in Figs 3 and 4
; this was 20 for actin, elongation factor 1-
and ADP/ATP translocase; 21 for Hsc70; 22 for poly(A) binding protein; 26 for Rho GTPase and 23 for the remaining PCR reactions.
|
|
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the first RFDD time-course experiment we performed, total RNA was extracted from mock-infected cells, and cells at 6, 12, 18, 24 and 36 h p.i. The resulting RFDD fragments were screened in the following way: fragments (visualized as bands) which were present in the mock-infected cells were examined for their continued presence, as well as their relative abundance at each time-point, throughout the time-course of infection by AcMNPV. With a conservative average of approximately 50 bands which could be resolved per lane (i.e. per primer pair) for each of the 64 primer sets tested, more than 3000 cDNA fragments were examined (data not shown). We observed the shutoff of a majority of mRNA transcripts between 12 and 18 h p.i. This was in contrast to some RFDD products, which were not present in the mock-infected lanes but were first detected between 6 and 18 h p.i., and accumulated to high levels. We hypothesized that the former category of mRNAs would correspond to host Sf9 sequences, while the latter corresponded to the expression of baculoviral genes (Fig. 1A).
|
Despite the relatively small number of Spodoptera frugiperda genomic sequences available in the database, several hits' with significant homologies to S. frugiperda sequences were recovered, as well as homologies to other invertebrate or vertebrate sequences, from the down-regulated transcripts (Table 2; see RpS2 to Twinstar). A subset of fragments identified from the RFDD screen as potential viral sequences was also amplified and sequenced. Each fragment, as expected, corresponded to AcMNPV sequences such as p6.9, chitinase, Lef-5 and egt (data not shown). Since we were interested in studying the expression levels of host and not viral sequences, other fragments fitting this profile were not explored further in this experiment.
|
|
Restriction fragment differential display: 2nd time-course
We were also interested in determining whether any host transcripts were actually up-regulated at early times of infection, before the general shutoff of host RNA synthesis. Therefore, we performed a second RFDD experiment. In this second time-course, total RNA was extracted from Sf9 cells early after infection (3 and 6 h p.i.) or from mock-infected cells, and mRNA was isolated from these total RNA samples. There was a good correlation between the quantity of total RNA (mostly ribosomal RNA) amongst samples with the amount of mRNA isolated early after viral infection (3, 6 p.i.), indicating that overall levels of messenger RNA were constant at this stage (Fig. 1C). The use of mRNA for this RFDD experiment increased the number of amplified products for each primer pair from
50 per lane for total RNA to
120 per lane for mRNA, on average (Fig. 1B
). In this second RFDD experiment, we looked for up-regulation events between mock-infected cells and those at 3 or 6 h p.i. We did this by identifying fragments which were present in the mock-infected samples and apparently up-regulated during infection. We also looked for fragments which were present only at 3 or 6 h p.i., but were not seen in the mock lane. The latter situation would correspond either to expression of viral genes or to the up-regulation of host mRNAs which are not produced, or at very low abundance, in uninfected cells. An estimated 7600 cDNA fragments were examined in this screen.
Eighty-five potentially up-regulated RFDD fragments were isolated and re-amplified. To differentiate between up-regulated host transcripts and AcMNPV transcripts, the PCR products were subjected to agarose gel electrophoresis and Southern blot analysis, using random-primer labelled probes from the entire AcMNPV genome. Out of these 85 up-regulated transcripts, 75 were of viral origin (data not shown). The remaining 10 cellular RFDD fragments were cloned and then sequenced (Table 2; see Actin to 14-3-3). They were then subjected to verification by Northern blot analysis or semi-quantitative RT-PCR (sQRT-PCR) or both, using total RNAs collected from independent time-course infections of Sf9 cells with AcMNPV (Figs 3 and 4
). Nine of the ten cellular mRNAs did not show reproducible or significantly up-regulated levels during time-course infections, using these methods (Fig. 3
). The general decline of mRNA levels observed for other host transcripts was also confirmed for all of them by 24 h p.i. Consistent although moderate up-regulation of a cellular sequence at early times of infection was only confirmed for one RFDD fragment that from a heat shock protein 70 cognate (Hsc70) sequence (Fig. 4
). Both sQRT-PCR (Fig. 4A
) and Northern blot analysis (Fig. 4C
) showed an increase in the abundance of the transcript at 3 or 6 h p.i. not observed in mock infected cells (Fig. 4B
). However, this Hsc70 mRNA was eventually down-regulated between 12 and 24 h p.i. Only one heat shock family protein sequence had been published to date for Spodoptera frugiperda Hsp83 (Landais et al., 2001
). Therefore, we amplified and sequenced the full coding sequence of Sf9 Hsc70 using 5'/3' RACE (Table 2
). The resulting 2723 bp gene sequence had a best hit with a Bombyx mori Hsc70 sequence in GenBank. Their amino acid sequences were 94 % identical.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Actin, the typical eukaryotic mRNA used for controlling recovery/loading of cellular RNA, could not be used as control in this experiment as its level is significantly decreased as a result of baculovirus infection, as was confirmed by RFDD and sQRT-PCR in this work. This had been reported previously (Ooi & Miller, 1988; Wei & Volkman, 1992
). AcMNPV has an important interaction with the cell cytoskeleton. As early as 30 min after cell entry, the virus induces the formation of filamentous F-actin cables (Charlton & Volkman, 1993
) which is hypothesized to facilitate nucleocapsid transport into the nucleus (Lanier & Volkman, 1998
). Actin skeleton reorganization is also required for endocytosis of another family of large DNA viruses of animals, the adenoviruses. This reorganization is mediated by Rho GTPases (Li et al., 1998
; Krijnse Locker et al., 2000
). It was therefore of interest to look at the levels of a rho gene transcript throughout baculoviral infection, especially as the RhoA signal transduction pathway was shown to regulate the replication capabilities of viruses such as HIV-1 (Wang et al., 2000
) and respiratory syncytial virus (Gower et al., 2001
). The small GTPase rho1 that we amplified, however, showed the same regulation pattern as that of other host transcripts analysed. The up-regulation observed in RFDD analysis was not confirmed by sQRT-PCR. A profilin host transcript, an actin regulatory gene that stimulates actin filament assembly, was found to be up-regulated in the RFDD (data not shown). However, no further analysis was performed as the RFDD sequence was too short (80 bp) for verification by our other methods.
Similarly, up-regulation of the cellular 14-3-3 transcript observed in the RFDD analysis was not confirmed by sQRT-PCR. This gene would be an interesting candidate to explore at the protein level, as it is involved in eukaryotic cellular signalling events which control the cell cycle and apoptosis (reviewed in Yaffe, 2002), probably via phospho-dependent proteinprotein interactions. Given that the 14-3-3 proteins were found to associate with other DNA viruses proteins like polyomavirus (Pallas et al., 1994
) and parvovirus (Brockhaus et al., 1996
), and that baculoviruses are known to modulate their host's cell cycle as well in order to avoid cellular apoptosis, this protein is likely to play some role in the infectious process.
A discrepancy between apparently up-regulated RFDD fragments and the sQRT-PCR results was encountered frequently in this study (9 of 10 transcripts). This is mostly attributed to simultaneous extraction of several closely migrating PCR products from the RFDD polyacrylamide gel. Cloning of the extracted product and analysis of several clones allowed identification of a recurring insert for further analysis in most cases. However, it may also be possible that the use of radioactive labelling in the differential display technology allowed identification of slight up-regulation events overlooked in the sQRT-PCR where ethidium bromide staining, a less sensitive method, was used. If this is the case, up-regulation patterns observed for other genes like Hsp60, integrin and cadherin in the RFDD (but not investigated further), could be of some significance, but would probably require Northern blot analysis, using longer probe sequences for each cDNA. The biological significance to baculovirus infection of any small increase in the amount of a cellular mRNA would have to be proven by functional studies.
For more abundant transcripts, like ribosomal protein genes, false positives caused by mixed populations of PCR products was not an issue. Ribosomal proteins are typically produced at a rate that maintains a constant ratio to rRNA under physiological conditions in an uninfected cell. In AcMNPV-infected Sf9 cells, several ribosomal protein mRNAs decreased dramatically at 24 h p.i. (Fig. 1), showing a dissociation in the regulation pattern of rRNA and ribosomal protein mRNAs. The mRNA levels of other proteins involved in cellular translation, such as poly(A) binding protein (PABP), elongation factor 1-
and glutaminyl-tRNA synthetase, were assessed and found to decrease after 12 h p.i.
The interaction of host chaperone proteins at different stages of virus life-cycles is the subject of numerous studies (reviewed by Sullivan & Pipas, 2001). The heat shock protein cognate 70, a constitutively expressed member of the Hsp70 family, is the chaperone that is most commonly involved with virus interactions. As well as protein folding and unfolding, it plays a role in protein translocation (Zimmermann, 1998
) in association with several co-factors such as Hsp40 (Hohfeld, 1998
). Although it has not been found in association with baculovirus infection to date, it was shown to be involved in replication and assembly of other DNA viruses. Following SV40 or adenovirus type 5 infection, Hsc70 was found to be the main virus-induced member of the Hsp70 family (Sainis et al., 1994
). The same authors established that the chaperone protein translocated to the nucleus late in infection only in permissive cell lines. In addition, Hsc70 co-immunoprecipitated with the viral capsid protein VP1, suggesting a role in virus packaging (Sainis et al., 2000
). Polyomavirus capsid protein was also found to be associated with Hsc70 during virus infection (Cripe et al., 1995
). The authors suggested a role in preventing premature assembly of capsids in the cytosol or in nuclear transport. Hsc70 was shown to also be involved in adenovirus DNA nuclear import by interacting either with nuclear localization sequences of viral proteins or in the disassembly of the nucleocapsid prior to nuclear import (Saphire et al., 2000
). Hsc70 was found to interact with the L protein of hepatitis B virus, playing a crucial role in virus morphogenesis by regulating L protein translocation (Prange et al., 1999
). When interacting with RNA viruses, other functions were found for Hsc70. It was involved in syncytium formation by human T-cell lymphotropic virus type I (Fang et al., 1999
; Sagara et al., 1998
, 2001
). It was also shown to be part of a complex receptor for rotavirus entry into the cell (Guerrero et al., 2002
). In the light of these studies, the early up-regulation of Hsc70 following baculovirus infection demonstrated in this paper suggests that the chaperone could play an important role in the baculovirus life-cycle. It would be an interesting protein to target for functional studies during baculovirus infection.
From this study, up-regulation of host mRNA transcript levels does not appear to play a pivotal role in reprogramming the host Sf9 cell upon baculovirus infection. Two possibilites, not mutually exclusive, remain: either the virus is capable of manipulating the host cellular environment via the viral proteins that it encodes without much alteration of host gene expression, or host gene expression is regulated to facilitate infection, but primarily at the protein level. Altering the phosphorylation state of host proteins, a common mechanism for protein regulation in the cell, could provide such a mechanism. AcMNPV does encode two viral protein kinases and a tyrosine phosphatase, which could participate in such activities (reviewed in Miller et al., 1997). In support of the hypothesis that differential regulation of host genes can occur at the protein level is a recent report (Quadt et al., 2002
) that Spodoptera frugiperda TATA-binding protein levels rise throughout an AcMNPV time-course infection to 72 h p.i., but mRNA levels for the protein begin to fall between 16 and 24 h p.i. Finally, although we have shown the absence of marked up-regulation events at the RNA level following viral infection, this has to be analysed within the limits of an in vitro study. We are now interested in studying the effects of baculovirus infection upon host gene expression of caterpillars in vivo.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ayres, M. D., Howard, S. C., Kuzio, J., Lopez-Ferber, M. & Possee, R. D. (1994). The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology 202, 586605.[CrossRef][Medline]
Birnbaum, M. J., Clem, R. J. & Miller, L. K. (1994). An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs. J Virol 68, 25212528.[Abstract]
Braunagel, S. C., Parr, R., Belyavskyi, M. & Summers, M. D. (1998). Autographa californica nucleopolyhedrovirus infection results in Sf9 cell cycle arrest at G2/M phase. Virology 244, 195211.[CrossRef][Medline]
Brockhaus, K., Plaza, S., Pintel, D. J., Rommelaere, J. & Salome, N. (1996). Nonstructural proteins NS2 of minute virus of mice associate in vivo with 14-3-3 protein family members. J Virol 70, 75277534.[Abstract]
Carstens, E. B., Tija, S. T. & Doerfler, W. (1979). Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus. I. Synthesis of intracellular proteins after virus infection. Virology 99, 386396.
Charlton, C. A. & Volkman, L. E. (1993). Penetration of Autographa californica nuclear polyhedrosis virus nucleocapsids into IPLB Sf 21 cells induces actin cable formation. Virology 197, 245254.[CrossRef][Medline]
Clem, R. J. & Miller, L. K. (1993). Apoptosis reduces both the in vitro replication and the in vivo infectivity of a baculovirus. J Virol 67, 37303738.[Abstract]
Cripe, T. P., Delos, S. E., Estes, P. A. & Garcea, R. L. (1995). In vivo and in vitro association of hsc70 with polyomavirus capsid proteins. J Virol 69, 78077813.[Abstract]
Fang, D., Haraguchi, Y., Jinno, A., Soda, Y., Shimizu, N. & Hoshino, H. (1999). Heat shock cognate protein 70 is a cell fusion-enhancing factor but not an entry factor for human T-cell lymphotropic virus type I. Biochem Biophys Res Commun 261, 357363.[CrossRef][Medline]
Fenwick, M. L. & McMenamin, M. M. (1984). Early virion-associated suppression of cellular protein synthesis by herpes simplex virus is accompanied by inactivation of mRNA. J Gen Virol 65, 12251228.[Abstract]
Gower, T. L., Peeples, M. E., Collins, P. L. & Graham, B. S. (2001). RhoA is activated during respiratory syncytial virus infection. Virology 283, 188196.[CrossRef][Medline]
Guarino, L. A., Xu, B., Jin, J. & Dong, W. (1998). A virus-encoded RNA polymerase purified from baculovirus-infected cells. J Virol 72, 79857991.
Guerrero, C. A., Bouyssounade, D., Zarate, S., Isa, P., Lopez, T., Espinosa, R., Romero, P., Mendez, E., Lopez, S. & Arias, C. F. (2002). Heat shock cognate protein 70 is involved in rotavirus cell entry. J Virol 76, 40964102.
Hardy, W. R. & Sandri-Goldin, R. M. (1994). Herpes simplex virus inhibits host cell splicing, and regulatory protein ICP27 is required for this effect. J Virol 68, 77907799.[Abstract]
Hohfeld, J. (1998). Regulation of the heat shock conjugate Hsc70 in the mammalian cell: the characterization of the anti-apoptotic protein BAG-1 provides novel insights. Biol Chem 379, 269274.[Medline]
Ikeda, M. & Kobayashi, M. (1999). Cell-cycle perturbation in Sf9 cells infected with Autographa californica nucleopolyhedrovirus. Virology 258, 176188.[CrossRef][Medline]
Krijnse Locker, J., Kuehn, A., Schleich, S., Rutter, G., Hohenberg, H., Wepf, R. & Griffiths, G. (2000). Entry of the two infectious forms of vaccinia virus at the plasma membane is signaling-dependent for the IMV but not the EEV. Mol Biol Cell 11, 24972511.
Landais, I., Pommet, M. J., Mita, K., Nohata, J., Gimenez, S., Fournier, P., Devauchelle, G., Duonor-Cerutti, M. & Ogliastro, M. (2001). Characterization of the cDNA encoding the 90 kDa heat-shock protein in the lepidoptera Bombyx mori and Spodoptera frugiperda. Gene 271, 223231.[CrossRef][Medline]
Lanier, L. M. & Volkman, L. E. (1998). Actin binding and nucleation by Autographa california M nucleopolyhedrovirus. Virology 243, 167177.[CrossRef][Medline]
Lanier, L. M., Slack, J. M. & Volkman, L. E. (1996). Actin binding and proteolysis by the baculovirus AcMNPV: the role of virion-associated V-CATH. Virology 216, 380388.[CrossRef][Medline]
Lee, H. H. & Miller, L. K. (1978). Isolation of genotypic variants of Autographa californica nuclear polyhedrosis virus. J Virol 27, 754767.[Medline]
Liang, P. & Pardee, A. B. (1993). Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967970.
Li, E., Stupack, D., Bokoch, G. M. & Nemerow, G. R. (1998). Adenovirus endocytosis requires actin cytoskeleton reorganization mediated by Rho family GTPases. J Virol 72, 88068812.
Miller, L. K. (1997). The Baculoviruses. New York: Plenum Press.
Okano, K., Shimada, T., Mita, K. & Maeda, S. (2001). Comparative expressed-sequence-tag analysis of differential gene expression profiles in BmNPV-infected BmN cells. Virology 282, 348356.[CrossRef][Medline]
Ooi, B. G. & Miller, L. K. (1988). Regulation of host RNA levels during baculovirus infection. Virology 166, 515523.[Medline]
O'Reilly, D. R., Miller, L. K. & Luckow, V. A. (1992). Baculovirus Expression Vectors: a Laboratory Manual. New York: W. H. Freeman.
Oroskar, A. A. & Read, G. S. (1989). Control of mRNA stability by the virion host shutoff function of herpes simplex virus. J Virol 63, 18971906.[Medline]
Pallas, D. C., Fu, H., Haehnel, L. C., Weller, W., Collier, R. J. & Roberts, T. M. (1994). Association of polyomavirus middle tumor antigen with 14-3-3 proteins. Science 265, 535537.[Medline]
Prange, R., Werr, M. & Loffler-Mary, H. (1999). Chaperones involved in hepatitis B virus morphogenesis. Biol Chem 380, 305314.[Medline]
Quadt, I., Mainz, D., Mans, R., Kremer, A. & Knebel-Morsdorf, D. (2002). Baculovirus infection raises the level of TATA-binding protein that colocalizes with viral DNA replication sites. J Virol 76, 1112311127.
Roncarati, R. & Knebel-Morsdorf, D. (1997). Identification of the early actin-rearrangement-inducing factor gene, arif-1, from Autographa californica multicapsid nuclear polyhedrosis virus. J Virol 71, 79337941.[Abstract]
Sagara, Y., Ishida, C., Inoue, Y., Shiraki, H. & Maeda, Y. (1998). 71-kilodalton heat shock cognate protein acts as a cellular receptor for syncytium formation induced by human T-cell lymphotropic virus type 1. J Virol 72, 535541.
Sagara, Y., Inoue, Y., Kojima, E., Ishida, C., Shiraki, H. & Maeda, Y. (2001). Phosphatidylglycerol participates in syncytium formation induced by HTLV type 1-bearing cells. AIDS Res Hum Retroviruses 17, 125135.[CrossRef][Medline]
Sainis, I., Angelidis, C., Pagoulatos, G. & Lazaridis, I. (1994). The hsc70 gene which is slightly induced by heat is the main virus inducible member of the hsp70 gene family. FEBS Lett 355, 282286.[CrossRef][Medline]
Sainis, L., Angelidis, C., Pagoulatos, G. N. & Lazaridis, L. (2000). HSC70 interactions with SV40 viral proteins differ between permissive and nonpermissive mammalian cells. Cell Stress Chaperones 5, 132138.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Saphire, A. C., Guan, T., Schirmer, E. C., Nemerow, G. R. & Gerace, L. (2000). Nuclear import of adenovirus DNA in vitro involves the nuclear protein import pathway and hsc70. J Biol Chem 275, 42984304.
Schek, N. & Bachenheimer, S. L. (1985). Degradation of cellular mRNAs induced by a virion-associated factor during herpes simplex virus infection of Vero cells. J Virol 55, 601610.[Medline]
Strom, T. & Frenkel, N. (1987). Effects of herpes simplex virus on mRNA stability. J Virol 61, 21982207.[Medline]
Sullivan, C. S. & Pipas, J. M. (2001). The viruschaperone connection. Virology 287, 18.[CrossRef][Medline]
Tomalski, M. D., Wu, J. G. & Miller, L. K. (1988). The location, sequence, transcription, and regulation of a baculovirus DNA polymerase gene. Virology 167, 591600.[Medline]
Van Oers, M. M., Van Marwijk, M., Kwa, M. S., Vlak, J. M. & Thomas, A. A. (1999). Cloning and analysis of cDNAs encoding the hypusine-containing protein eIF5A of two lepidopteran insect species. Insect Mol Biol 8, 531538.[CrossRef][Medline]
Van Oers, M. M., Van der Veken, L. T., Vlak, J. M. & Thomas, A. A. (2001). Effect of baculovirus infection on the mRNA and protein levels of the Spodoptera frugiperda eukaryotic initiation factor 4E. Insect Mol Biol 10, 255264.[CrossRef][Medline]
Vaughn, J. L., Goodwin, R. H., Tompkins, G. J. & McCawley, P. (1977). The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro 13, 1317.
Wang, L., Zhang, H., Solski, P. A., Hart, M. J., Der, C. J. & Su, L. (2000). Modulation of HIV-1 replication by a novel RhoA effector activity. J Immunol 164, 53695374.
Wei, N. & Volkman, L. E. (1992). Hyperexpression of baculovirus polyhedrin and p10 is inversely correlated with actin synthesis. Virology 191, 4248.[Medline]
Yaffe, M. B. (2002). How do 14-3-3 proteins work? Gatekeeper phosphorylation and the molecular anvil hypothesis. FEBS Lett 513, 5357.[CrossRef][Medline]
Zimmermann, R. (1998). The role of molecular chaperones in protein transport into the mammalian endoplasmic reticulum. Biol Chem 379, 275282.[Medline]
Received 3 April 2003;
accepted 1 July 2003.