THOMAS GALLAGHER, AND PAUL KAESBERG. Biophysics Laboratory and Biochemistry ... Ahlquist and Janda (12). Clones were named according to the ...
Proc. Nati. Acad. Sci. USA Vol. 83, pp. 63-66, January 1986
Biochemistry
Infectious RNA derived by transcription from cloned cDNA copies of the genomic RNA of an insect virus (black beetle virus/Nodaviridae/viral gene expression/in vitro transcription/Drosophila melanogaster)
BIMALENDU DASMAHAPATRA, RANJIT DASGUPTA, KEITH SAUNDERS, BERNARD SELLING, THOMAS GALLAGHER, AND PAUL KAESBERG Biophysics Laboratory and Biochemistry Department, University of Wisconsin, Madison, WI 53706
Communicated by Robert H. Burris, August 30, 1985
RNA transcripts of cloned cDNA of the ABSTRACT genomic RNAs of BBV (black beetle virus) are infectious to cultured cells of Drosophila melanogaster. Individual transcripts had approximately 10% of the infectivity of the corresponding authentic virion RNA. Progeny virus resulting from transcript infection was phenotypically indistinguishable from the progenitor virus used to generate the original cDNA forms as judged by sucrose density gradient sedimentation, specific infectivity, plaque morphology, and serology. Although the transcript RNAs used to produce this virus had 20 nonviral bases headed by a capping group at their 5' termini, these 20 bases were absent in the progeny viral RNAs. The cDNA forms, and therefore the resulting transcript RNAs, should be readily modifiable by the techniques of recombinant DNA technology both for viral studies and for the insertion of foreign genes into the viral genome and thus into the host cytoplasm.
MATERIALS AND METHODS We used BBV W17 (7), a viral strain that is highly cytolytic to cultured cells of D. melanogaster. Synthesis of Full-Length DNA Copies of BBV RNAs and Their Cloning into Transcription Vectors. RNAs were reverse-transcribed into cDNA and converted to the doublestranded form with reverse transcriptase (12). Specific oligonucleotides, used as primers, had extra nonviral bases at their 5' ends, which resulted in the emergence of unique restriction sites at each end of the double-stranded DNA (Pst I at the 5' end and Xba I at the 3' end). Full-length double-stranded DNA copies of BBV RNA1 and RNA2 were inserted into the Sma I site of the multicopy plasmid pUC13 and were cloned in Escherichia coli JM 101. Complete BBV DNA inserts were excised from pUC13 recombinant plasmids and were inserted into transcription vectors pSP64 or pPM1 and cloned. In Vitro Transcription of Cloned BBV DNA. Selected pSP64 recombinant plasmids were cleaved with the restriction enzyme Xba I, and the resulting linear DNA templates (20 ,ug/ml) were transcribed with SP6 RNA polymerase as described by Konarska et al. (13). Generally, 500 kLM guanosine (5') triphospho(5')guanosine [G(5')ppp(5')G] or the 7-methyguanosine derivative m7G(5')ppp(5')G was included in the reaction mixture to provide capped transcripts. pPM1 recombinant plasmid DNA, linearized with Xba I, was transcribed with E. coli RNA polymerase as described by Ahlquist and Janda (12). Clones were named according to the format px By SPz or pxByPMz, where x = 1 or 2 indicates derivation from RNA1 or RNA2, y is the isolate number of the pUC13 recombinant plasmid containing the BBV insert, and z indicates the isolation number of either a pSP64 or a pPM1 recombinant carrying the insert BBV DNA in the correct orientation. Infectivity Assays. Drosophila melanogaster cells (5 x 106) in 80 Al of PNKC buffer (35 mM Pipes/100 mM NaCl/10 mM KCl, 1 mM CaCl2/400 iLg of DEAE dextran per ml, pH 6.0), were transfected (10) by addition of various amounts of transcribed or virion RNAs in 20 ,ul of PNKC buffer. After 10 min at room temperature, cells (200 to 2 x 106) from each transfection mixture were added to untreated Drosophila cells to give a total of 4 x 106 cells in 5 ml of NPKA buffer (25 mM Pipes, pH 6.75/100 mM NaCl/10 mM KCl/0.1% bovine serum albumin). Cells were mixed and poured into 6-cm tissue culture dishes and, after 2-3 days at 26°C, infectious centers were counted (7).
The methods of recombinant DNA technology can be of great utility for studies of RNA viruses. Such methods are intrinsically available for retroviruses, which have DNA as an intermediate in their synthesis. The methods have now become applicable to RNA phage QB (1) and to poliovirus (2), whose cDNAs have been shown to be infectious, and to brome mosaic virus (3), for which infectious RNA has been made by transcription from cloned viral cDNA. Infectious cDNA and infectious transcript RNAs are also known for the plant pathogens potato spindle tubor viriod (4) and hop stunt viriod (5). To our knowledge there have not been published reports ofinfectious transcript RNAs for any insect or animal virus. We show here that RNA transcripts derived from DNA copies of the genomic RNAs of BBV are infectious to cultured cells of Drosophila melanogaster; thus this virus, also, is modifiable by DNA methods. Black beetle virus (BBV) is an insect virus of the family Nodaviridae. Its genome consists of two single-stranded, messenger-sense RNAs contained in a single virion (6, 7). Virion RNA1 (3106 bases) (8) codes for protein A (involved in viral RNA synthesis) and protein B (function unknown), whose cistron is silent. The protein B cistron is expressed by means of a subgenomic messenger, RNA3 (389 bases) (9, 10), which is not encapsidated. Virion RNA2 (1399 bases) (11) encodes the virion coat protein precursor, a, which is proteolytically processed into the coat proteins /3 and y. Both RNA1 and RNA2 have a 5'-terminal capping group and a blocking moiety, presumably a protein (8, 11), at their 3' terminus.
RESULTS DNA from pSP64 clones was cleaved with Xba I and transcribed with SP6 RNA polymerase to produce complete
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Abbreviations: BBV, black beetle virus; pfu, plaque-forming unit(s).
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Proc. Natl. Acad. Sci. USA 83
Infectivity of Transcript RNA1 Plus Transcript RNA2. The crucial test of biological activity is of course the infectivity of transcript RNA1 plus transcript RNA2 in the absence of any authentic RNA. When cells were transfected with a mixture of transcripts that we had identified as infectious by the procedures described above, plaque assays consistently indicated infectivity under conditions that had been shown to be optimal for authentic RNAs. Infectivity required the presence of both transcript RNA1 and transcript RNA2. We were unable to detect infectivity with Xba I-linearized p1B9SP DNA plus p2B1OSP DNA. The RNA input required for the maximal number of plaque-forming centers was always higher for transcript RNA than for virion RNA (Table 4). Fig. LA shows the results obtained when the input of transcript RNA1 was varied and the input of transcript RNA2 was held constant at 0.2 jig. Fig. 1B shows the results obtained when the input of transcript RNA2 was varied and the input of transcript RNA1 was held constant at 1.2 ,g (i.e., approximately the optimal value found in Fig. LA). It is evident from Fig. 1B that the highest infectivity is obtained at approximately equal molarity of transcript RNA1 and RNA2. At equimolarity, maximal infectivity is about 1% of that of authentic RNAs and is obtained at a transcript RNA concentration approximately 3 times that required for optimal infectivity with authentic RNAs (Fig. 1C). The virus arising from the combination RNA1(plB9SP) and RNA2(p2BlOSP), designated BBV K1, has been studied in detail. BBV K1 is phenotypically indistinguishable from BBV W17, the progenitor virus from which it was derived, as we describe below. Plaque Analyses. Assay of W17 virions, W17 virion RNAs, or W17 RNA1 plus any of our infectious transcript RNA2s resulted in mostly large (2-4 mm) clear plaques. Assay of transcript RNA1 plus virion or transcript RNA2 resulted in small (0.5 mm) clear plaques. Nevertheless, assay of K1 virions or K1 virion RNAs resulted in large plaques indistinguishable in morphology from those of W17 virions. We judge that the plaque morphology engendered by RNA1 transcripts is a transient consequence of their terminal structure. However, we do not rule out the possibility that viral sequence changes were introduced in the course of construction of transcript RNA1 and that these changes were reversed by mutation during the proliferation of K1. Virion and Virion RNA Comparisons. The virions of K1 and its progenitor virus, W17 sedimented identically in sucrose gradients. Their specific infectivities were similar [220 and 290 particles required per plaque-forming unit (pfu) for W17 and K1, respectively]. Antiserum raised against wild-type BBV neutralized both viruses. Their virion RNA1 and RNA2 migrated identically upon agarose gel electrophoresis. Their
copies of BBV genomes. All such transcripts have the same four additional nonviral nucleotides at their 3' ends and the same 20 additional nonviral nucleotides at their 5' ends headed by a capping group, or in some experiments, by a methylated capping group or a triphosphate. In several experiments we cloned full-length BBV insert DNAs in the transcription vector pPM1 and used E. coli RNA polymerase to provide transcripts whose 5' termini are identical in sequence to authentic virion RNA. Table 1 shows the terminal structure of the various transcripts. Infectivity of Transcript RNA1 Plus Authentic RNA2 and of Transcript RNA2 Plus Authentic RNA1. In order to initially identify infectious transcripts and to quantify their infectivity, preparations of individual transcripts were combined with preparations of cognate RNAs derived from virions, and these mixtures were assayed for infectivity. To serve as a baseline for judging infectivity of the transcript RNAs, we first determined the infectivity of combinations of authentic virion RNA1 and RNA2. Maximal infectivity was obtained when 5 x 106 cells were transfected with a mixture of 0.5 ,ug of virion RNA1 and 0.2 ,ug of virion RNA2. Under these conditions approximately 5% of the transfected cells formed plaques. Preparations of transcript RNA1 were assayed together with virion RNA2 at concentrations optimal for virion RNAs. Table 2 shows the infectivity of three transcripts from the same DNA [capped transcript p1B13SP, methylated capped transcript p1B13SP(M), and uncapped transcript p1B13SP(ppp)] and another transcript, p1B9SP, relative to virion RNA1. The p1B13SP and p1B13SP(M) transcripts were each approximately 10% as infectious as virion RNA1. The uncapped transcript p1B13SP(ppp) had approximately 0.1% of the infectivity of virion RNA1. Table 2 indicates infectivity at the level of 0.03% when no RNA1 was added. This infectivity is due to the presence of a small admixture of virion RNA1 in our preparations of virion RNA2 (10). Similarly, several preparations of transcript RNA2 were assayed together with virion RNA1 at concentrations optimal for virion RNAs. Table 3 shows that two RNA2 transcripts independently derived from the pSP64 vector and one derived from the pPM1 vector had equal infectivity, about 8% of that obtained with virion RNA2. Their similar infectivity suggests that under these conditions the additional 20 nonviral bases on the 5' ends of the transcripts derived from pSP64 did not have a strong negative influence on infectivity. We have not yet checked the validity of this conclusion for pPM1-based RNA1 transcripts. Again it may be noted that the virion RNA1 preparation is slightly infectious in the absence of added RNA2 because of the presence of a small admixture of virion RNA2. Table 1. Terminal sequences of RNA1 and RNA2
RNA1 W17 vRNA1
p1B13SP p1B13SP(M) p1B13SP(ppp) K1 vRNA1
7mGpppGUUUU .......... AGGU (protein) GpppGaauacaagcuugggcugcaGUUUU ........... AGGUcuagOH M7GpppGaauacaagcuugggcugcaGUUUU .......... AGGUcuagOH pppGaauacaagcuugggcugcaGUUUU .......... AGGUcuagOH M7GpppGUUUU .............. (protein) RNA 2
m7GpppGUAAA .......... AGGU (protein) GpppGaauacaagcuugggcugcaGUAAA .......... AGGUcuagOH m7GpppGUAAA .......... AGGUcuagOH m7GpppGUAAA ............... (protein) Terminal sequences of the RNAs we have used are shown, with capital letters signifying viral nucleotides and lower-case letters signifying nonviral nucleotides. The existence of a protein at the 3' termini of the virion RNAs is an inference from the fact that the terminus is blocked and that, in wild-type BBV RNA, the block is partially removable by protease. p1B13SP(M) refers to a transcript having a methylated cap; plB13SP-(ppp) refers to a transcript having a noncapped 5'-terminal triphosphate. vRNA1 and vRNA2, virion RNA1 and RNA2.
W17 vRNA2
p2B1OSP p2B1OPM8 K1 vRNA2
(1986)
Biochemistry: Dasmahapatra et al.
Proc. Natl. Acad. Sci. USA 83 (1986)
Table 2. Infectivity of transcript RNA1 in the presence of virion RNA2 pfu x 10-3 per RNA1 5 x 106 cells Relative infectivity None 0.063 0.03 p1B13SP 18.8 10.0 p1B13SP(M) 18.3 10.0 p1B13SP(ppp) 0.200 0.1 p1B9SP 18.0 10.0 vRNA1 188 100 Drosophila cells (5 x 106) in 100 1.l of buffer were transfected with 0.5 1Lg of the RNA1 preparations in conjunction with 0.2 ,ug of virion RNA2. After transfection, productively infected cells were scored by plaque assay. RNA infectivities are normalized to the virion RNA1 (vRNA1) plus RNA2 combination. p1B13SP(M) refers to a transcript having a methylated cap. p1B13SP(ppp) refers to a transcript having a noncapped 5'-terminal triphosphate.
virion proteins a and 8 migrated identically upon NaDodSO4/polyacrylamide gel electrophoresis. W17 and K1 virion RNA1 and RNA2 each have a 5'terminal cap and a 3'-terminal blocking group. Sequence analysis (Fig. 2) demonstrates that the additional 20 nonviral bases present at the 5' ends of the transcript RNAs are not reproduced in the K1 virion RNAs. Except for base 15 in K1 RNA1, both RNA1 and RNA2 of the K1 virion RNAs have 5'-terminal sequences identical to their W17 counterparts. Position 15 in K1 RNA1 and the corresponding position in transcript RNA1 (position 35) is a uridine residue while W17 RNA1 has a cytidine residue, indicating a transitional mutation presumably introduced during cloning. Thus, 5' nonviral sequences, existent in transcripts, are not reproduced in progeny viral RNA, but internal viral sequence changes introduced into transcripts are reproduced in the progeny virus they generate. We have not yet sequenced the 3' proximal regions of these RNAs, but we have determined that the 3' termini are resistant to addition of pCp with T4 RNA ligase, suggesting that the termini are blocked as are the W17 RNA 3' termini. Thus by our present criteria, virus K1 and its progenitor virus W17 are phenotypically indistinguishable but have at least one genotypic difference.
Table 4. Infectivity of transcript RNA1 (plB9SP) plus transcript RNA2 (p2B1OSP) pfu per 5 x Relative 106 cells RNA1, ,ug RNA2, Ag infectivity 0.5 None 0 0 None 0.2 0 0 0.5 0.2 115 0.07 0.75 0.2 160 0.09 1.0 0.2 283 0.16 0.75 0.3 210 0.12 1.0 0.5 450 0.25 0.5 (vRNA1) 0.2 (vRNA2) 175 x 103 100 Drosophila cells were transfected with transcript RNA1 plus transcript RNA2 at the indicated concentrations. Productively infected cells were scored as described in Table 2. In later experiments, higher infectivity was obtained (see Fig. 1). vRNA1 and vRNA2, virion RNA1 and RNA2. consequence of structural differences at their 5' and 3' termini. Nevertheless, they should provide an effective vehicle for introduction of defined sequence changes into the viral genome by the methods of recombinant DNA technology. Moreover, the fact that the RNA1 transcripts are functional allows study of their replication in the complete absence of RNA2. Previously, studies of transfection with RNA1 alone were limited by the difficulty of completely eliminating RNA2, since even a minute admixture of virion RNA2 led to completion of the infectious process (10). The RNA1 terminal structures may be involved in the viral infectious cycle at both the level of translation and of nucleic acid replication. RNA1 codes for protein A, which is essenA
400
200 2
4
B
0
C.) 200)o
DISCUSSION We have shown that DNA forms of the genomic RNAs of BBV W17 can be transcribed into RNA that is infectious to cultured cells of D. melanogaster, yielding virus indistinguishable from W17. The transcript RNAs are less infectious than their W17 virion RNA counterparts, most likely as a
65
x
0
lOC)o
tn 0.
0
0.5
1.0
1.5
2.0
Table 3. Infectivity of transcript RNA2 in the presence of virion RNA1 pfu x 10-3 per 5 x 106 cells RNA2 Relative infectivity None 0.15 0.09
p2B1OSP p2B48SP
p2B1OPM8
14 13 15 180
8.3 7.4 8.6 100
vRNA2 Drosophila cells were transfected with the 0.2Ig of the RNA2 preparations in conjunction with 0.5 /ig of virion RNA1. Productively infected cells were scored as described in Table 2. In later experiments, higher infectivity was obtained (see Fig. 1). vRNA2, virion RNA2.
2 RNA,
ug
FIG. 1. Effect of varying the input of transcript RNA1 (plB9SP) and transcript RNA2 (p2BlOSP). (A) Input of transcript RNA1 was varied, whereas input of transcript RNA2 was 0.2 ,&g. (B) Input of transcript RNA2 was varied, whereas input of transcript RNA1 was 1.2 jig. (C) Input of transcript RNA1 and transcript RNA2 was
equimolar.
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Biochemistry: Dasmahapatra et al. C -I I I I ACGlT ACGlT
FIG. 2. Sequence analysis of the 5' end of BBV RNA1 transcript p1B9SP (lanes Tr) and RNA2 isolated from virus K1 (lanes K1) and its progenitor, W17 (lanes W17). A 32P-5'-end-labeled single-stranded DNA primer, complementary to bases 70-123 of BBV RNA1, was used to prime cDNA synthesis from the RNAs in the presence of dideoxynucleotides A, C, G, or T (14, 15). The arrow points to position 15, the site of the base that is different in the RNA1 of K1 and W17.
tial for viral RNA replication; thus, the RNA1 that initiates the infectious process must be translated effectively in order to provide a functional protein A. Therefore, RNA1 transcripts p1B13SP, p1B13SP(M), and to a small extent p1B13SP(ppp) must have this capability, and, in particular, they are not fatally flawed because of the existence of 20 extra 5' nonviral bases. These extra bases separate the 5' ends from the initiation codon by 58 nucleotides. Transcripts p1B13SP and p1B13SP(M) are equally infectious, but transcript p1B13SP(ppp) has comparatively low infectivity. Evidently, the existence of cap is important, but its methylation state is not. Possibly, the principal need for the capping group in these assays is to provide a barrier against nucleolytic degradation.
The first functional opportunity for the 3' termini of the incoming RNAs probably exists when an RNA-replicating complex has been established and synthesis of negativestrand RNA is initiated. All of our transcripts carry four additional nonviral bases, the terminus of which has a free hydroxyl group. We conclude that a blocked 3' terminus is not essential for initiation of infectivity and that the additional bases do no substantial harm. We are unable to state unequivocally that the four bases are not copied. However, the fact that the eventual virion RNAs have the precisely correct 5'-terminal structure suggests that there is no copying of the 20 nonviral bases putatively existing at the 3' termini of the RNA negative strands. Since virion protein synthesis is a late event in virus multiplication (16), input RNA2 need not be translated directly. However, newly synthesized RNA2 must serve as a template for synthesis of functional messenger. The 5' terminus of RNA2 transcript p2B1OPM8 is the same as that of W17 virion RNA2. Thus, its lower infectivity than virion RNA2 (Table 3) results from its imperfect 3' terminus (or possibly from adventitious sequence changes introduced in its construction). The similarity in infectivity between p2B1OPM8 and p2B1OSP suggests that the 20 nonviral bases present at the 5' terminus of the latter do not confer an additional loss in infectivity.
Proc. Natl. Acad. Sci. USA 83
(1986)
It is clear that it is possible to produce BBV by presenting susceptible Drosophila cells with RNA having appropriate viral sequences flanked with nonviral structures existent only for ease in construction. Evidently, BBV and its host have a mechanism for identifying the initiation sites for minus- and plus-strand synthesis, even when these sites are not terminal. Thus, for viruses such as BBV, genome modification by means of a DNA intermediate can involve transcription systems that yield RNA transcripts lacking the precise terminal structures that exist on authentic viral RNA. With the availability of a readily modifiable genome, BBV and its host Drosophila comprise a system of enormous potential for elucidating the molecular biology of RNA viruses and for introduction of new proteins into a eukaryotic host. BBV grows to exceptionally high titer in cultured cells of D. melanogaster (17), an organism whose genetic characteristics are well understood. A reliable plaque assay is available (7). Crystallography of BBV is well underway (18). Since RNA1 can replicate independently of RNA2 (10), replicative functions can be studied in the absence of RNA2 or with extensively modified RNA2. Similarly, modification of RNA2 can lead to an understanding of the functions of coat protein and the process of assembly. Furthermore, it should be possible to replicate suitably engineered foreign messenger RNAs in the presence of RNA1. Note Added in Proof. Mizutani and Colonno (19) have very recently
reported synthesis of infectious RNA of human rhinovirus type 14. We thank Prof. Roland R. Rueckert for discussions throughout the course of this work. We thank Prof. Paul Ahlquist for discussions and for providing his vector, pPM1. We also wish to acknowledge the conscientious technical assistance of Chris Nelson. This research was supported by National Institutes of Health Grants AI1466, A115342, and CA08662 and National Institutes of Health Career Award A121942. 1. Taniguchi, T., Palmieri, M. & Weissmann, C. (1978) Nature (London) 274, 223-228. 2. Racaniello, V. & Baltimore, D. (1981) Science 214, 916-919. 3. Ahlquist, P., French, R., Janda, M. & Loesch-Fries, S. (1984) Proc. Natl. Acad. Sci. USA 81, 7066-7070. 4. Cress, D., Kiefer, M. & Owens, R. (1983) Nucleic Acids Res. 11, 6821-6835. 5. Ohno, T., Ishikawa, M., Takamatsu, N., Meshi, T., Okada, Y., Sano, T. & Shikata, E. (1983) Proc. Jpn. Acad. Ser. B 59, 251-254. 6. Longworth, J. & Carey, G. (1976) J. Gen. Virol. 33, 31-40. 7. Selling, B. & Rueckert, R. (1984) J. Virol. 51, 251-253. 8. Dasmahapatra, B., Dasgupta, R., Ghosh, A. & Kaesberg, P.
(1985) J. Mol. Biol. 182, 183-190.
9. Guarino, L., Ghosh, A., Dasmahapatra, B., Dasgupta, R. & Kaesberg, P. (1984) Virology 139, 199-203. 10. Gallagher, T. M., Friesen, P. D. & Rueckert, R. (1983) J. Virol. 46, 481-489. 11. Dasgupta, R., Ghosh, A., Dasmahapatra, B., Guarino, L. & Kaesberg, P. (1984) Nucleic Acids Res. 12, 7215-7223. 12. Ahlquist, P. & Janda, M. (1984) Mol. Cell. Biol. 4, 2876-2882. 13. Konarska, M., Padgett, R. & Sharp, P. (1984) Cell 38, 731-736. 14. Zimmern, D. & Kaesberg, P. (1978) Proc. Natl. Acad. Sci. USA 75, 4257-4261. 15. Ahlquist, P., Dasgupta, R. & Kaesberg, P. (1981) Cell 23, 183-189. 16. Friesen, P. D. & Rueckert, R. (1984) J. Virol. 49, 116-124. 17. Friesen, P., Scotti, P., Longworth, J. & Rueckert, R. (1980) J. Virol. 35, 741-747. 18. Hosur, M. V., Schmidt, T., Tucker, C., Johnson, J. E., Selling, B. & Rueckert, R. R. (1984) Virology 133, 119-127. 19. Mizutani, S. & Colonno, R. (1985) J. Virol. 56, 628-632.
