Walter Doerfler*. Institute of Genetics, University of ..... The medium used was TC-100 medium (Gardiner and Stockdale,. 1975) in the modification of W.H.R. ...
The EMBO Journal Vol.1 No. 12 pp. 1629- 1633, 1982
The translational map of the Autographa californica nuclear polyhedrosis virus (AcNPV) genome
Helmut Esche, Hermann Walter Doerfler*
Lubbert, Beate Siegmann, and
Institute of Genetics, University of Cologne, Cologne, FRG Communicated by W.Doerfler Received on I November 1982
We have mapped early and late viral gene products expressed in Autographa californica nuclear polyhedrosis virus (AcNPV)-infected Spodoptera frugiperda cells by cell-free translation of virus-specific RNA which was selected by hybridization to cloned restriction endonuclease fragments of AcNPV DNA. Proteins synthesized in vitro were labeled with [35S]methionine and analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography. At least four early AcNPV-specific polypeptides were found which mapped in two regions of the genome (9-25 and 43-59 map units). These early mRNAs are also synthesized at late times in the infection cycle. Cell-free translation of restriction fragmentselected late AcNPV-specific RNA (24 h post-infection) resulted in the identification and mapping of 24 viral proteins. Curiously, the region between 70 and 80 map units on the viral genome has been found silent with respect to mRNA which is translatable in a cell-free system. However, there may be RNA transcribed from this viral DNA segment. Key words: baculovirus/productive infection/Spodoptera frugiperda cells/early and late RNA/in vitro translation -
Introduction For the detailed study of the molecular biology of baculoviruses, Autographa californica nuclear polyhedrosis virus (AcNPV), a virus isolated from the alfalfa looper A. californica, has been used in several laboratories. This virus can serve as a model system for investigations on the molecular biology of insect cells in general and the viral DNA may be used to develop a cloning vector for this species. Moreover, baculoviruses have attracted interest because of their potential use for insect pest control. Baculoviruses contain a circular, double-stranded DNA molecule of mol. wt. 80 x 106-92 x 106 (Smith and Summers, 1978; Miller and Dawes, 1979; Tjia et al., 1979; Lubbert et al., 1981). Restriction maps of the viral genome have been determined by conventional methods (Smith and Summers, 1979; Vlak, 1980; Lubbert et al., 1981). In addition, various restriction fragments spanning the entire genome have been cloned in prokaryotic vectors (Lubbert et al., 1981; Cochran et al., 1982). By using pulse labeling experiments with [35S]methionine in AcNPV-infected cells, at least two classes of virus-induced proteins can be revealed, early polypeptides made prior to viral DNA synthesis and late polypeptides made after the onset of DNA replication (Carstens et al., 1979; Dobos and Cochran, 1980; Wood, 1980; Maruniak and Summers, 1981; Vlak et al., 1981). In vitro translation analyses of hybridselected mRNA obtained from AcNPV-infected cells have *To whom reprint requests should be sent. IRL Press Limited, Oxford, England. 0261-4189/82/0112-1629$2.00/0.
shown that at late times after infection essentially all of the cytoplasmic RNAs are viral specific (Vlak et al., 1981). RNA sequences complementary to the EcoRI-H restriction fragment (0-5.6 map units of the AcNPV genome) encode the polyhedrin polypeptide which constitutes the major protein component of the AcNPV polyhedra, the inclusion bodies found in the nuclei of infected cells. Here we describe experiments which map many of the early and late virus-coded proteins on the virion genome. In the approach chosen, the location of the AcNPV genes has been mapped by in vitro translation of preselected virus-specific RNAs prepared from AcNPV-infected Spodopterafrugiperda cells in messenger-dependent reticulocyte lysates. Cloned fragments of AcNPV DNA (Lubbert et al., 1981) were used to select specific viral mRNAs. The products of cell-free translation experiments using [35S]methionine were characterized by SDS-polyacrylamide gel electrophoresis followed by fluorography. In this way we have mapped four polypeptides expressed early and 24 polypeptides expressed late in infection. Peculiarly, RNA selected with fragments encompassing the region between 70 and 80 map units on the viral genome could not be translated into polypeptides. Other laboratories have also established translational maps of the AcNPV genome (Smith et al., 1982).
Results and Discussion We have employed in vitro translation of hybrid-selected mRNA from AcNPV-infected insect cells to analyze and to map early and late viral RNAs and proteins on the AcNPV genome. RNA was selected from cytoplasmic RNA preparations by hybridization to the cloned fragments of AcNPV DNA (Lubbert et al., 1981) immobilized on nitrocellulose filters. The mRNA eluted from specific hybrids was then translated in a messenger-dependent reticulocyte lysate in the presence of [35S]methionine, and the products of translation were identified by gel electrophoresis followed by fluorography. Adenovirus type 12 (Adl2) virion proteins were used as size markers. Translation in vitro of preselected early AcNPV RNA Early virus-specific RNA used to direct cell-free protein synthesis was prepared 6 h after the infection of S. frugiperda cells with AcNPV. The RNA was selected by hybridization to the total AcNPV genome, as well as to individual restriction endonuclease fragments of AcNPV DNA. The results of in vitro translation of preselected early AcNPV-specific RNA are shown in Figure 1. Viral RNA selected with total AcNPV DNA directs the synthesis of at least four polypeptides with mol. wts of 39, 38, 27, and 24 K (Figure 1, lane d). The mRNA coding for the 39-K polypeptide could be selected with fragments EcoRI-A (8.6- 19.6 map units; Figure 1, lane f) and EcoRI-I (19.6-24.5 map units; Figure 1, lane g), while the RNA for the 24-K polypeptide has sequences complementary only to the EcoRI-A fragment (Figure 1, lane f). The existence of other weak bands (Figure 1, lane g) could not be confirmed in other experiments. The 38-K polypeptide was encoded by sequences carried by fragments EcoRI-C 1629
H.Esche et al.
(42.5 -52.6 map units; Figure 1, lane i) and EcoRI-G (52.6-59.2 map units; Figure 1, lane j). RNA coding for the 27-K polypeptide was selected only with fragment EcoRI-G (Figure 1, lane j). In some experiments using RNA selected with the EcoRI-G fragment, we could detect, after longer exposures of the fluorograms, a minor polypeptide of 45 K in addition to the proteins described above (results not shown). The data indicate that the early viral mRNAs map within two regions, between -9-25 and 43-59 map units. All early mRNAs were present at higher levels also late (24 h) after infection. RNA isolated from mock-infected cells did not hybridize to AcNPV DNA (Figure 1, lane 1). Early viral polypeptides with similar mol. wts. to those expressed from the two early regions described above have been recently described by Smith et al. (1982). However, we have not been able to confirm these authors' observation that a polypeptide with a mol. wt. of 31 K is expressed from DNA sequences between -65 and 75 map units at early times after infection.
In a previously published study, we analyzed the synthesis of intracellular proteins early (3 h) after AcNPV infection of S. frugiperda cells and have found polypeptides with mol. wts. of 46, 30, and 29 K (Carstens et al., 1979). These mol. wts. agree with some but not all of the values described here. Translation in vitro of preselected late AcNPV RNA Late viral RNA used to program cell-free protein synthesis was isolated 24 h after infection and was selected by hybridization to individual EcoRI restriction endonuclease fragments of AcNPV DNA. The results of cell-free translation of late RNA selected with the different, cloned EcoRI fragments are shown in Figures 2 and 3. RNA selected by hybridization to the EcoRI-H fragment (O- 5.6 map units) directed the synthesis of a polypeptide with a mol. wt. of 33 K (Figure 2, lane e). Late viral RNAs selected by the EcoRI fragments, A, I, C, or G, which also encode sequences expressed early (6 h) after infection, directed the syntheses of
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Fig. 1. In vitro translation of AcNPV-specific RNA prepared from infected S. frugiperda cells by hybridization to the cloned EcoRI restriction endonuclease fragments of the AcNPV genome. Cells were infected with AcNPV (10 p.f.u./celL and RNA was prepared 6 h (early) after infection. Specific RNAs selected by hybridization to intact AcNPV DNA (d) or to the AcNPV DNA fragment EcoRI-P (e), EcoRI-A (f), EcoRI-I (g), EcoRl-K (h), EcoRI-C (i), EcoRI-G (j), or EcoRI-D (k). The [35S]methionine-labeled products of cell-free translation were analyzed by electrophoresis on 150/0 polyacrylamide gels which were dried and exposed for fluorography for 5 days. The results of cell-free translation experiments without added RNA (a), with unfractionated cytoplasmic RNA (b), or RNA from mock-infected cells selected with intact AcNPV-DNA (l) are also shown for comparison. Adl2 virion proteins used size markers (c). The results accumulated from numerous in vitro translation experiments are schematically summarized in the map on the bottom. were
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Translational map.of AcNPV genome
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Fig. 2. Proteins synthesized in vitro by the reticulocyte cell-free system programmed with AcNPV-specific RNA which was prepared from infected S. frugiperda cells and selected by hybridization to the cloned EcoRI restriction endonuclease fragments of AcNPV DNA. RNA prepared 24 h (late) after infection was used. mRNAs were selected by hybridization to intact AcNPV DNA (d) or to the AcNPV fragments EcoRI-H (e), EcoRI-P (f), EcoRI-A (g), EcoRI-I (h), EcoRl-K (i), EcoRI- (j) or EcoRI-F (1). The [3S]methionine-labeled products of cell-free translation were analyzed by electrophoresis on 150%o polyacrylamide gels followed by fluorography (3 days). Results of in vitro translation experiments without added RNA (a, l) or with unselected cytoplasmic RNA (b) are also shown for comparison. Adl2 virion proteins were used as size markers (c). All results of in vitro translation experiments shown in the fluorograms of Figures 2 and 3 are schematically summarized in part A of the bottom map. Part B shows the results of Smith et al. (1982). 1631
H.Esche et at.
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Fig. 3. In vitro translation of AcNPV-specific RNA which was prepared from S. frugiperda cells 24 h Oate) after infection with AcNPV. mRNA was selected by hybridization to intact AcNPV DNA (b) or to the AcNPV fragments EcoRI-C (d), EcoRl-G (e), EcoRl-D (f), EcoRI-R (g), EcoRl-L (h), EcoRI-E (i), EcoRI-N (j), EcoRI-Q (k) or EcoRI-B (). The [35S]methionine-labeled products of cell-free translation of the selected RNAs were analyzed by electrophoresis on 15% polyacrylamide gels which were dried and exposed for fluorography (3 days). The results of cell-free translation experiments without added RNA (a, m) are also shown for comparison. Adl2 virion proteins were used as size markers (c). The results of the in vitro translation experiments shown in the fluorograms are schematically summarized in the map (A) at the bottom of Figure 2.
additional polypeptides with mol. wts. of 37 K (EcoRI-A, Figure 2, lane g), 37 K, 22 K, 21 K (EcoRI-I, Figure 2, lane h), 35 K, and 21 K (EcoRI-C, Figure 3, lane d). All the results accumulated from numerous in vitro translation experiments using hybrid-selected AcNPV RNA have been schematically summarized for late viral gene products in Figure 2 (part A of bottom map). The circular AcNPV genome has been linearized in this scheme such that the EcoRI-H fragment encoding the polyhedrin polypeptide is located at the left terminus (Lubbert et al., 1981; Vlak and Smith, 1982). The late proteins map in regions distributed over nearly the entire viral genome. In part B of the map in Figure 2, we have included the results of Smith et al. (1982) on the mapping of late AcNPV-specific polypeptides for direct
comparison.
RNAs coding for some of the polypeptides (52 K, 39 K, 38 K, 25 K, 19 K) were selected on two adjacently located EcoRI fragments, e.g., for the 52-K and 25-K proteins on EcoRI fragments Q and B, for the 39-K protein on fragments A and I. This finding suggests that the DNA sequences coding for each of these polypeptides is located on adjacent EcoRI fragments. RNAs transcribed from AcNPV sequences located on fragment EcoRI-D (59.6-68.2 map units) codes for at least four polypeptides (50 K, 49 K, 34 K, 21 K; Figure 3, lane f), those transcribed from EcoRI-B sequences (89.7-100 map units) encode five proteins (52 K, 40 K, 36 K, 25 K, 16 K; Figure 3, lane 1). It is not known to what extent each of the polypeptides encoded by the EcoRI fragments D and B may be derived from spliced or unspliced mRNA. However, the observation that the RNAs coding for the 52-K and 25-K polypeptides can be selected with the adjacent DNA fragments EcoRI-Q (Figure 3, lane k) and EcoRlB (Figure 3, lane 1) could be explained, e.g., by splicing of 1632
RNA derived from this region, by overlapping coding regions, or by transcription from opposite strands. Fragments EcoRI-L (69.7-72.8 map units) and EcoRI-E (72.8- 79.8 map units) could not be used successfully in selecting translatable viral mRNA (Figure 3, lanes h, i). However, blotting of RNA from AcNPV-infected cells and hybridization to the nick-translated 32P-labeled EcoRI-E fragment of AcNPV DNA has shown two RNA species transcribed from this region (H.Lubbert, unpublished data). One possible explanation for this apparent discrepancy might be that proteins encoded by this region lack L-methionine altogether or are not efficiently labeled by this amino acid. The finding that a 21-K polypeptide can be selected by the EcoRI-F fragment and not by intact AcNPV DNA (Figure 3) can be explained by the different temperatures used in the hybridization reactions. Materials and methods Cells and virus S. frugiperda cells are an established cell line from ovarian tissue of the fall armyworm (Vaughn et al., 1977). These cells (a gift from K.Harrap, Oxford University) were grown in monolayers as described previously (Tjia et al., 1979) or in suspension cultures at 28°C in conventional, water-jacketed spinner vessels. The medium used was TC-100 medium (Gardiner and Stockdale, 1975) in the modification of W.H.R. Langridge (personal communication) supplemented with 10% fetal calf serum. Monolayers or suspension cultures of S. frugiperda cells were inoculated with extracellular AcNPV at multiplicities of 10 p.f.u /cell as described previously (Tjia et al., 1979; Carstens et al., 1979). Usually, infectious non-occluded virus was prepared 28 h after infection from the medium of infected cell cultures. The virus was titered by plaque assay (Wood, 1977) as described elsewhere (Tjia et al., 1979). Viral DNA and cloned viral DNA fragments Extracellular AcNP virions were purified and viral DNA was extracted as previously described (Tjia et al., 1979). The restriction map of the DNA from a particular isolate (E) (Tjia et al., 1979; Lubbert et al., 1981) differed slightly
Translational map of AcNPV genome from that of virus preparations used in other laboratories (Vlak and Smith, 1982). Recently, the isolation and characterization of molecular clones of most of the EcoRI fragments of AcNPV DNA has been described (Lubbert et al., 1981). Plasmid pBR322, XgtWES )XB and X Charon 4A DNAs have been used as cloning vectors. Cloned DNA fragments were propagated and purified by conventional methods. Inoculation of S. frugiperda cells with AcNPV and isolation of cytoplasmic RNA S. frugiperda cells were inoculated with non-occluded AcNPV at a multiplicity of 10 p.f.u./cell. At 6 h (early RNA) or 24 h (late RNA) after inoculation, cytoplasmic RNA was isolated as outlined previously (Esche et al., 1979). Briefly, cells were lysed in 10 mM Tris-HCl, pH 7.0, 100 mM NaCl, 2 mM MgCl2, 0.750o Nonidet P-40 for 10 min on ice. The nuclei were removed by centrifugation, and the cytoplasmic extract was made 0.5%!7o in SDS and 5 mM in EDTA. The mixture was extracted three times with phenol-chloroform-isoamylalcohol (25:24:1), previously saturated with 10 mM Tris-hydrochloride, pH 6.5. The RNA was precipitated from the aqueous phase twice with 3 volumes of ethanol at -20°C, resuspended in water, and stored at - 800C. Selection of specific populations of viral RNA by hybridization to cloned fragments of AcNPV DNA This technique has been described in detail for adenovirus-specific RNA and cloned adenoviral DNA fragments (Ricciardi et al., 1979; Esche and Siegmann, 1982). In brief, RNA complementary to a certain region of the AcNPV genome was selected by hybridizing - 3 mg of cytoplasmic RNA to -40 Ag of each of the cloned fragments of AcNPV DNA immobilized as plasmid DNA on nitrocellulose filters. The hybridization reaction was carried out in 50% formamide, 0.01 M PIPES (1,4-piperazinediethane sulfonic acid), pH 6.8, 0.4 M NaCl for 6 h, usually at 43 °C. Since the G-C contents varied in different fragments of AcNPV DNA, the optimal temperature for hybridization had to be determined empirically for most of the cloned fragments. Temperatures ranging from 400C to 46°C were used. Upon hybridization, filters were washed extensively with 1 x SSC (0.15 M NaCl, 0.015 M Na citrate), 0.1 % SDS, 0.002 M EDTA and preheated to 50°C. AcNPV-specific RNA was eluted by heating the filters in H20 to 100°C for 1 min. Subsequently, the RNA was precipitated with 2.5 volumes of ethanol at - 20°C. In vitro translation of hybrid-selected AcNPV RNA Selected AcNPV RNA was translated in a micrococcal nuclease-treated reticulocyte lysate (Pelham and Jackson, 1976). Polypeptides synthesized in the presence of [35S]methionine (Amersham) were analyzed on 10% or 15% (w/v) polyacrylamide gels (Laemmli, 1970) and treated for fluorography (Bonner and Laskey, 1974). Adenovirion proteins were used as size markers (Esche et al., 1979; Esche and Siegmann, 1982).
Wood,H.A. (1977) J. Invertebr. Pathol., 29, 304-307. Wood,H.A. (1980) Virology, 102, 21-27.
Acknowledgements We are indebted to Gerti Meyer zu Altenschildesche for media production. This research was supported by a grant (PTB 8334) from the Federal Ministry of Research and Technology, Bonn, FRG.
References Bonner,W.M., and Laskey,R.A. (1974) Eur. J. Biochem., 46, 83-88. Carstens,E.B., Tjia,S.T., and Doerfler,W. (1979) Virology, 99, 386-398. Cochran,M.A., Carstens,E.B., Eaton,B.T., and Faulkner,P. (1982) J. Virol., 41, 940-946. Dobos,P., and Cochran,M.A. (1980) Virology, 103, 446-464. Esche,H., and Siegmann,B. (1982) J. Gen. Virol., 60, 99-113. Esche,H., Schilling,R., and Doerfler,W. (1979) J. Virol., 30, 21-31. Gardiner,G.R., and Stockdale,H. (1975) J. Invertebr. Pathol., 25, 363-370. Laemmli,U.K. (1970) Nature, 227, 680-685. Lubbert,H., Kruczek,I., Tjia,S., and Doerfler,W. (1981) Gene, 16, 343-345. Maruniak,J.E., and Summers,M.D. (1981) Virology, 109, 25-34. Miller,L.K., and Dawes,K.P. (1979) J. Virol., 29, 1044-1055. Pelham,H.R.B., and Jackson,R.J. (1976) Eur. J. Biochem., 67, 247-256. Ricciardi,R., Miller,J.S., and Roberts,R.E. (1979) Proc. NatI. Acad. Sci. USA, 76, 4927-4931. Smith,G.E., and Summers,M.D. (1978) Virology, 89, 517-527. Smith,G.E., and Summers,M.D. (1979) J. Virol., 30, 828-838. Smith,G.E., Vlak,J.M., and Summers,M.D. (1982) J. ViroL, 44, 199-208. Tjia,S.T., Carstens,E.B., and Doerfler,W. (1979) Virology, 99, 399-409. Vaughn,J.L., Goodwin,R.H., Thomkins,G.J., and McCawley,P. (1977) In Vitro, 13, 213-217. Vlak,J.M. (1980) J. Invertebr. Pathol., 36, 409-414. Vlak,J.M., and Smith,G.E. (1982) J. Virol., 41, 1118-1121. Vlak,J.M., Smith,G.E., and Summers,M.D. (1981) J. Virol., 40, 762-771.
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