Journal of General Virology (1995), 76, 1025-1032. Printedin Great Britain
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Effect of methyltransferase inhibitors on the regulation of baculovirus protein synthesis Christian Bach, ~ A s t a Cramer 2 and Christoph S c h o l t i s s e k 1. 1Institutfiir Virologie, Justus-Liebig-Universitiit Giessen, Frankfurter Strasse 107, D-35392 Giessen and 2Institut fiir Virologie, Philipps-Universitiit, Robert Koch-Strasse 17, D-35037 Marburg, Germany
In the presence of the methyltransferase inhibitor 3deazaadenosine (3DA-Ado) the production of infectious Autographa californica nuclear polyhedrosis virus (AcMNPV) in tissue culture was only slightly affected, while the synthesis of very late proteins (polyhedrin and pl0) was abolished. The synthesis of the influenza virus proteins NS1 and HA, expressed under the polyhedrin promoter, was also abolished by 3DA-Ado. Further-
more, 3DA-Ado interfered with the shut-off of early and late AcMNPV proteins. Most of these results were also obtained with 5-azadeoxycytidine (5A-dCyt). In cells in which NS1 was produced abundantly, at least one specific AcMNPV protein was not synthesized. However, if the production of NS1 was inhibited by 3DAAdo, or if HA was synthesized instead, this AcMNPV protein showed up normally.
The genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) consists of a circular double-stranded DNA genome of 133 kbp which has the capacity to code for about 100 proteins. Its sequence has been published recently (Ayres et al., 1994). Protein synthesis of baculoviruses is highly regulated. One can roughly differentiate between early, late and very late viral proteins by using inhibitors for protein and DNA synthesis (Rice & Miller, 1986). The synthesis of early and late viral proteins is necessary for the production of virus particles that are able to infect cells, while the very late viral proteins are needed for the occlusion of virus particles into a protein capsule consisting mainly of polyhedrin. These polyhedral inclusion bodies protect the virus from an adverse environment and serve as vehicles for virus transmission between animals through the intestinal route (for reviews see Rohrmann, 1992; Kool & Vlak, 1993). Recently, we found that the methyltransferase inhibitor 3-deazaadenosine (3DA-Ado) specifically inhibited the replication of influenza viruses by retaining the mRNAs of only the late viral proteins, matrix protein M1, haemagglutinin (HA) and neuraminidase (NA), in the nuclei of the infected cells (Fischer et al., 1990; Vogel et al., 1994). This observation was of special interest, since influenza viruses are the only 'pure' RNA viruses with a clear nuclear phase, during which the synthesis of all eight viral mRNAs is started with the so-called capstealing process. The fully methylated cap of the cellular
premessenger RNA plus about 10 to 15 nucleotides downstream are cleaved off by the viral polymerase complex to function as a starter molecule (Krug, 1981). The viral nonstructural protein NS1 interfered in an artificial expression system with the nucleocytoplasmic transport of any poly(A)-containing mRNA (Fortes et al., 1994; Qiu & Krug, 1994). However, this observation did not explain the highly regulated synthesis of viral mRNAs and proteins. The protein kinase C inhibitor H7 [1-(5-isoquinolinylsulphonyl)-2-methylpiperazine] exhibited exactly the same spectrum of inhibition of late viral protein synthesis as 3DA-Ado did. There are indications that a viral protein may be involved in this kind of regulation (Vogel et al., 1994). To identify such a regulatory protein we wanted to singly or co-express individual influenza virus genes in insect cells using baculovirus vectors. As a first step in this direction we studied the effect of 3DA-Ado on baculovirus replication and on the expression of influenza virus genes under the control of the very late polyhedrin promoter. It was known that 3DA-Ado interfered with herpesvirus and vaccinia virus replication (Montgomery et al., 1982; Chiang, 1990); however, nothing was known about the mechanism of inhibition with respect to those two viruses. The effect of 3DA-Ado on baculovirus multiplication has not been studied yet. We did not necessarily expect that 3DA-Ado would interfere with baculovirus protein synthesis, since the DNA isolated from baculovirus particles does not contain measurable amounts of methyl groups on CG pairs, which normally regulate promoter activities on DNA (Eick et al., 1983; Tjia et al., 1979). However, during the course of our
* Author for correspondence. Fax +49 641 23960. 0001-2863 © 1995SGM
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Fig. 1. Autoradiograms of [35S]methionine-labelled proteins immunoprecipitated with polyclonal c¢-NS1. Sf9 cells were infected with Bac NS, and proteins were pulse-labelled at 48 h p.i. as described. (a) Cells were untreated ( - ) or treated ( + ) with 3DA-Ado. Left: - , untreated; + , 60 pg/ml 3DA-Ado were added to the medium of infected cells at the indicated times (0, 12, 24 and 36 h p.i.). Right: - , untreated; + , 3DA-Ado was added to the medium of infected cells 0 h p.i. at the indicated concentrations (20, 40, 60, 90 and 120 gg/ml). (b) Cells were untreated ( - ) or treated ( + ) with 5A-dCyt or H7. Left: - , untreated; + , 5A-dCyt was added to the medium of infected cells 0 h p.i. at the indicated concentrations (10, 20, 40 and 100 gM); Right: - , untreated; + , H7 was added to the medium of infected cells 0 h p.i. at 15 gg/ml. FPV: [35S]methionine-labelled viral proteins isolated from FPV (fowl plague virus)-infected chicken embryo fibroblasts (CEF) 5 h p.i. F P V - , infected cells were untreated; FPV + , infected cells were treated with 60 pg/ml 3DA-Ado (results in visible quantities o f NS2) (Fischer et al., 1990). FPV proteins (indicated on the right) served as molecular mass markers in all experiments. P, 85 kDa; NP, 56 kDa; M1, 28 kDa; NS1, 27 kDa; NS2, 14 kDa (not shown in all figures).
experiments we found that in the presence of 3DA-Ado the production of polyhedrin and p 10 protein (both very late proteins), and of influenza viral proteins under the control of the polyhedrin promoter was specifically inhibited, whereas the production of progeny virus was only slightly affected. Interestingly, 5-azadeoxycytidine (5A-dCyt) exhibited the same effect as 3DA-Ado. Therefore, methylation has to be considered as a regulatory element during baculovirus replication. 3DA-Ado is not significantly phosphorylated and is not incorporated into nucleic acids (Bader et al., 1978). It is converted to S-3DA-Ado-homocysteine (Backlund et al., 1986), which is the actual component interfering with influenza virus replication (Woyciniuk et al., 1995). 5A-dCyt is incorporated into DNA in place of dCyt and cannot be methylated. Furthermore, 5A-dCyt is a potent inhibitor of a specific methyltransferase (Creusot et aL, 1982). Other side-effects of these compounds are not known. Wild-type AcMNPV (originally obtained from Dr W. Doerfler, K61n, Germany) and recombinant baculoviruses were propagated in monolayer cultures of Spodoptera frugiperda (Sf) cells using TC 100 medium supplemented with 10% fetal calf serum (Gibco/Life
Technologies). Cells were infected at an m.o.i, of 10 p.f.u./cell and incubated at 27 °C for 3-5 days. Virus stocks were stored at 4 °C (Tjia et al., 1979). For the preparation of mRNA 3 × 107 Sf9 cells were infected at an m.o.i, of 10 p.f.u./cell with recombinant virus and left for the indicated times at 27 °C. 3-DA-Ado was used at a concentration of 60 gg/ml, except for the dose-response assay where 3DA-Ado was applied at concentrations of 20, 40, 60, 90 and 120 pg/ml. 5A-dCyt was used at a concentration of 40 pM or at 10, 20, 40 and 100 gM in the dose-response assay. The concentration of H7 was 15 pg/ml, a dose which inhibited influenza virus replication. The inhibitors were added to the medium either immediately [0 h p.i. (postinfection)] or at the indicated times after infection. To examine whether the effect of 3DA-Ado was reversible, the medium containing the inhibitor was replaced by fresh TC 100 lacking 3DA-Ado after washing the cells twice with PBS at room temperature. 3DA-Ado was obtained from Southern Research Institute, Birmingham, Ala., USA. 5A-dCyt and H7 were obtained from Sigma. Plasmid pBAC-NS was constructed by subcloning the NS-specific insert of the plasmid FPV4/NS into the
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Bac-NS FPV-
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Fig. 2. Northern blot analysis of mRNA isolated from Bac-NSinfected Sf9 cells 24 h and 48 h p.i. Cells were untreated ( - ) or treated ( + ) 0 h p.i. with 60 ~tg/ml 3DA-Ado. Northern blot analysis was directed against NS1 mRNA. Hybridization probes were [3~S]UTPlabelled. FPV: mRNA isolated from FPV-infected untreated CEF 5 h p.i. served as a control for hybridization quality and a length marker. Due to the noncoding region derived by vector construction of pBAC-NS the NS1 mRNA of recombinant Bac-NS virus migrated at a position corresponding to about 1000 additional nncleotides more slowly than 'native' NS 1 mRNA of FPV. The additional bands of the Bac-NS probes that migrated somewhat faster than the NS1 mRNA were not characterized but might be derived from NS1 mRNA by splicing. (NS2 mRNA, which is about 500 nt shorter than NS1 mRNA, is the splicing product of the NS1 precursor RNA and can be detected in FPV-infected CEF only in very small amounts.)
polylinker site of baculovector pVLI393, so that the flanking regions of the vector contained the DNA sequences which were necessary to obtain viable virus after recombination with BaculoGold DNA (Dianova). Furthermore, the region upstream of the polylinker site contained the wild-type AcMNPV sequence which was essential for high-level expression of genes that were under the control of the polyhedrin promoter (Luckow & Summers, 1988). FPV4/NS contained the 'full-length' cDNA of the NS gene (nt 13-890) of A/fowl plague/ Rostock/34 virus (FPV) in the Sinai restriction site of the p K S ( - ) Bluescript vector (Stratagene). Plasmid DNA, purified by standard procedures (Sambrook et al., 1989), was digested with B a m H I and E c o R I and was cloned into the pVL1393 polylinker site following
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standard protocols (Sambrook et al., 1989). The recombinant pBAC-NS plasmid DNA was transformed into competent XL1-Blue cells (Stratagene). The correct insertion was controlled by sequencing. A mixture of 2 lxg of the recombinant transfer vector pBAC-NS (containing the full-length cDNA of the influenza virus A/FPV/Rostock/34 NS gene) and 0-5 Ixg of BaculoGold baculovirus DNA was precipitated by adding Lipofectin (BRL/Life Technologies). Confluent monolayers of cells (2-8 x 106 cells per 50 mm dish) were transfected with the Lipofectin-DNA mixture and incubated at 27 °C for 6 days. The supernatant was collected and screened for recombinant viruses (which were identified by their polyhedrin-negative phenotype) and for the expression of the foreign gene. Purified recombinants were obtained after two cycles of plaque purification (Brown & Faulkner, 1977; Wood, 1977). Besides this Bac-NS recombinant virus a corresponding recombinant virus containing the influenza virus haemagglutinin gene of FPV (Bac-HA) (Kuroda et al., 1986) was used in a few experiments. Sf9 cells in 35 mm dishes (1-5 x 106 cells) were mockinfected or infected with recombinant or wild-type virus at an m.o.i, of 10 p.f.u./cell and incubated at 27 °C. At the indicated times p.i. cells were labelled with 50 p.Ci [3SS]methionine/culture for 3 h at 27°C. Labelled proteins in one aliquot of culture were analysed directly by PAGE as described by Bosch et al. (1979). Proteins in the other aliquot were immunoprecipitated prior to PAGE. These cells were lysed with RIPA buffer (Kistner et al., 1985) prior to immunoprecipitation with polyclonal NSl-specific antiserum (Young et al., 1983) (tiNS1; 1 ~tl/200 lal lysate), AcMNPV-specific antiserum (~-Ac; 2 ~tl/200 lal lysate) or polyhedrin-specific antiserum (~-PH; 2 ~tl/200 ~tl lysate) and Protein A Sepharose (2 mg/200 ~tl lysate) at 4 °C overnight. (The ctAc serum contains antibodies against structural proteins of AcMNPV.) Since we were interested mainly in protein patterns at different times after infection we did not try to assign various bands to specific viral or cellular proteins except for the influenza viral proteins and the polyhedrin protein of AcMNPV. For Northern blot analysis infected cells were washed twice with PBS at the indicated time, and total RNA was isolated by the method of Chomczynski & Sacchi (1987), followed by mRNA isolation using oligo(dT)-coated dynabeads (Dynal). The mRNAs were separated by agarose-formamide gel electrophoresis and blotted onto a nylon membrane (Hybond N, Amersham). For hybridization an NS1 mRNA specific [3~S]UTP-labelled probe was synthesized using the plasmid pAN3 (Fischer et al., 1990). The labelled probe was obtained by in vitro transcription of the linearized plasmid DNA using T7 polymerase (Stratagene). Northern blot hybridization
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was carried out as described by Wahl et al. (1979) as modified by Fischer et al. (1990). When Sf9 cells were infected with the recombinant Bac-NS virus the foreign protein was expressed abundantly (Figs 1 and 3). In contrast, when 3DA-Ado or 5AdCyt were added, immediately after infection, the production of NS1 was almost completely abolished (Fig. 1). However, we found that the later the inhibitors were added the weaker was the effect they had on NS 1 expression. That is, if added later than 12 h p.i. no significant decrease in the amount of protein was observed compared to the control experiment without drug. On the other hand, when 3DA-Ado was removed 12 or 24 h p.i. by washing the cells and replacing the medium with fresh medium lacking the inhibitor, the yields of NS1 were identical to those obtained from untreated cells (data not shown). With H7 we did not observe any effect (Fig. 1). We also studied NS1 m R N A synthesis by Northern blot analysis. As shown in Fig. 2 reasonable amounts of NSl-specific m R N A were detectable in Bac-NS-infected cells. However, in the presence of 3DA-Ado NS 1 m R N A synthesis was abolished. We conclude therefore that 3DA-Ado acted at the level of transcription. One possible explanation for the diminished protein synthesis in the presence of inhibitor was that the hostcell metabolism a n d / o r virus replication were disturbed in some way. To further investigate this question we had a closer look at cellular and viral growth under the influence of 3DA-Ado. Normally, insect cells double in about 24 h. When 3DA-Ado was added to freshly seeded cells the doubling time increased to 36 h. Thus, the inhibitor does not seem to interfere significantly with host-cell survival. When infected with wild-type AcMNPV, virus titres 24 h p.i. were similar with or without 3DA-Ado (6"8 × 10° vs 7"8 × 10° p.f.u./0"3 ml, respectively); 72 h p.i. the virus yield from drug-treated cells was still 14% of that (8.0 × 107 p.f.u./0-3 ml) from cells kept without inhibitor. After obtaining the above results it was interesting to see what effect 3DA-Ado had on the expression of polyhedrin. We infected cells with wild-type AcMNPV and added 3DA-Ado at 0, 12, 24 or 36 h p.i. Cells were
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examined optically for polyhedra 48 h after infection. In the control experiment without inhibitor every cell displayed large numbers of inclusion bodies. When added immediately or 12 h p.i. 3DA-Ado led to a dramatic decrease of polyhedra, although typical signs for virus infection could be observed. Later addition of 3DA-Ado resulted in numbers of polyhedra similar to those found in untreated cells (data not shown). This was a first hint that 3DA-Ado had the same effect on polyhedrin expression as seen for NS1. Studies at the protein level gave further evidence. Cells were infected with wild-type AcMNPV or Bac-NS in the presence or absence of 3DA-Ado, and proteins were labelled at different times p.i. to give a time course of protein expression during virus infection. Without inhibitor a distinct protein pattern emerged showing proteins which were expressed early in infection being replaced by other proteins as the infection proceeded. This process was delayed at an early stage if 3DA-Ado was present in the cells. Late proteins, especially polyhedrin and NSI were not synthesized (Fig. 3). 3DAAdo delayed the synthesis of early proteins and also interfered with their shut-off late in infection. This became especially clear when an antiserum was used to precipitate the baculovirus structural proteins (Fig. 4). The expression of pl0, which is another late baculovirus protein, showed the same susceptibility to 3DA-Ado. Because pl0 lacks methionine (Kuzio et al., 1984) this was seen only in Coomassie blue-stained gels. H7 had no effect on baculovirus protein synthesis (not shown). As shown in Fig. 3(a), besides the polyhedrin band one additional prominent protein band with a molecular mass of about 29 kDa appeared late in infection; this band was not found in Bac-NS-infected (Fig. 3 b) or in Bac-HA-infected Sf9 cells (data not shown). This extra protein could not be precipitated by a polyhedrin-specific antiserum (Fig. 3 c). If wild-type infected cells were pulselabelled for 1 h and chased for another 2 h the band was seen unchanged together with the polyhedrin. This indicated that the additional band consisted of an individual protein which was regulated in the same way as polyhedrin, and which was not expressed in Bac-NSor Bac-HA-infected Sf cells.
Fig. 3. Autoradiograms of total protein isolated from AcMNPV- or Bac NS-infected Sf9 cells harvested at different times after infection. Cells were untreated ( - ) or treated ( + ) with 3DA-Ado starting immediately after infection. Proteins were labelled with [35S]methionine at the indicated times and isolated as described. (a) Cells were infected with wild-type AcMNPV and left untreated ( - ) or treated ( + ) with 60 pg/ml 3DA-Ado. Polyhedrin (PH) is synthesized in untreated cells in large amounts from 24 h p.i. onward; in treated cells only low levels of polyhedrin are synthesized from 48 h p.i. onward. (b) Cells were infected with Bac-NS and left untreated ( - ) or treated ( + ) with 60 pg/ml 3DA-Ado. The foreign gene NS1 is expressed instead of polyhedrin and two other proteins. Comparable to the effect on polyhedrin synthesis in AcMNPV-infected cells (a), NS1 expression is strongly affected by 3DA-Ado. (c) Autoradiogram of [35S]methionine-labelled proteins isolated from AcMNPV-infected Sf9 cells. Cells were untreated and proteins were pulse-labelled 48 h p.i. and immunoprecipitated with a-PH as described. M, mock infected cells. FPV, see Fig. 1. In (a) and (b), at the left-hand side, the bands of the viral proteins mentioned in the text are indicated.
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Fig. 4. Autoradiograms of [35S]methionine-labelledproteins isolated from wild-type AcMNPV-infected Sf9 cells by immunoprecipitation with polyclonal ~-Ac. Cells were infected and left untreated ( - ) or treated (+) with 60~t/ml 3DA-Ado starting immediately after infection. Proteins werelabelledas describedat the indicatedtimes (12, 24, 36, 48 and 72 h p.i.). In untreated infectedcells synthesisof the viral proteins which migrate between NP and M1 (approx. 40 kDa), and between P and NP (approx. 70 kDa), and higher than P (approx. 88 kDa) increases until 24 h p.i. and decreases thereafter. In treated cells synthesis of these proteins begins somewhat later but is not switched off. Additionally,a significantprotein band (approx. 30 kDa) which migrates somewhat more slowly than M1 appears between 24 and 36 h p.i. in the absence but not in the presence of 3DA-Ado. There was another interesting observation concerning a protein which migrated in P A G E immediately above (34 kDa) the polyhedrin (32 kDa). In Bac-NS-infected cells (Fig. 3 b) this protein showed up only in the presence of 3DA-Ado, when the NS1 protein was not expressed. However, without 3DA-Ado, when the NS1 protein was abundantly expressed, this protein was not synthesized. In Bac-HA-infected cells this protein was produced in the absence of 3DA-Ado as well as in the presence of 3DA-Ado (data not shown) in the same way as could be seen in infected wild-type cells (Fig. 3 a). Most of the experiments as described above were also performed with 40 laM-5A-dCyt with exactly the same outcome (data not shown). Methylation of deoxycytidine of eukaryotic D N A has been implicated in promoter silencing, and the dis-
appearance of 5-methyldeoxycytidine from such sites by incubation with 5A-dCyt results in promoter reactivation (for review see Doerfler et al., 1983). Since baculovirus and insect (e.g. Drosophila) D N A does not contain measurable amounts of 5-methyldeoxycytidine (Doerfler et al., 1983), we did not expect that methyltransferase inhibitors like 5A-dCyt (Creusot et al., 1982) and 3DAAdo (Backlund et al., 1986) would interfere with very late baculovirus transcription. Therefore there might be another substrate for the enzyme involved in this kind of regulation. Influenza virus replication is also affected by 3DA-Ado by interfering with the switch from early to late viral protein synthesis (Fischer et al., 1990; Vogel et al., 1994). However, this can hardly be compared with its effect on regulation of baculovirus protein synthesis. Concerning influenza virus replication, H7 exhibits the same phenotype of inhibition as 3DA-Ado; however, H7 is without effect on baculovirus protein synthesis. In contrast, 5A-dCyt has no effect on influenza virus replication (unpublished results), while in the baculovirus system its effect resembles- as far as t e s t e d - t h a t of 3DA-Ado. 3DA-Ado affects only very late baculoviral transcription, and this only when added immediately after infection, but not when added later. Thus it seems not to interfere directly with very late transcription. On the other hand it prevents the shut-off of synthesis of at least several early a n d / o r late viral proteins (Fig. 4). It is interesting to note that this shut-off occurs at about the time when the (cellular) ~-amanitin-sensitive R N A polymerase is replaced by an a-amanitin-resistant enzyme (Fuchs et al., 1983). It might be speculated that for the shut-off of cellular and viral early and late protein synthesis and the activation of the polyhedrin promoter one of the cellular R N A polymerase components needs to be modified directly or indirectly by methylation, rendering it a-amanitin-resistant with a slightly different elution profile during DEAE-Sephadex chromatography (Fuchs et al., 1983). RNA-binding proteins, for example, are known to contain dimethylarginine (Wong et al., 1992). Alternatively C-methylation of the promoter of a late gene(s)- or even of cellular p r o m o t e r s - might be necessary for the switch-off, which might be the presupposition for very late transcription. Such low Cmethylation of D N A isolated from virus particles might have escaped detection (Tjia et al., 1979; Eick et al., 1983), and viral D N A isolated from infected cells has not been analysed. Knebel et al. (1985) have shown that a baculovirus promoter can be blocked by C-methylation. This at least means that C-methylated promoters are not recognized by the viral R N A polymerase. A-methylation does not play any role in baculovirus replication (Xia et al., 1993). The original aim of this study was to doubly express
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influenza viral genes in Sf9 cells in order to determine the effect of 3DA-Ado on the nucleocytoplasmic migration of the HA m R N A in the presence of NS1 (Vogel et al., 1994), which was shown by Qiu & Krug (1994) and Fortes et al. (1994) to interfere with mRNA transport in general. In doubly infected cells, to which 3DA-Ado was added 12 or 24 h p.i. in order to get substantial synthesis of NS 1 and HA, both mRNAs migrated normally to the cytoplasm as shown by in situ hybridization (preliminary observations). Therefore, it is interesting to note that specifically in the presence of NS1, of the many AcMNPV proteins expressed, one (visible in PAGE) specific protein (34 kDa, Fig. 3 b) is not synthesized. If the production of NS1 is inhibited, or if HA under the polyhedrin promoter is synthesized instead, this protein shows up normally. This might indicate that NS1 interferes in vivo only with the migration of a few specific mRNAs (or their translation), and not generally with all of them as claimed by Qiu & Krug (1994). The work was supported by the Sonderforschungsbereiche 272 and 286 of the Deutsche Forschungsgemeinschaft, the Bundesministerium ffir Forschung und Technologie O1KI9207, and the Fonds der Chemischen Industrie. This work is in partial fulfilment of the requirements for the degree Dr C. Bach, Fachbereich Chemie der Universit~it Giessen. The support of and discussion with Dr Dagmar Knebel-M6rsdorf of the Universit/it K61n, and with Dr Hans-Dieter Klenk of the Universitiit Marburg is highly appreciated. We are indebted to Dr Peter Palese, Mount Sinai School of Medicine, NY, USA, for providing us with the anti-NS1 antiserum; to Dr David Bishop, Institute of Virology and Environmental Microbiology, Oxford, UK, for anti-AcMNPV antiserum; to Hans Flipsen of the Agricultural University of Wageningen, Netherlands, for antipolyhedrin antiserum; and to Dr Stephan Ludwig, Institut fiir Virologie, Giessen, Germany, for the FPV4/NS plasmid.
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(Received 26 August 1994; Accepted 6 December 1994)