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Infectious RNA Derived by Transcription from Cloned Cdna Copies of the Genomic RNA of an Insect Virus

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Infectious RNA Derived by Transcription from Cloned Cdna Copies of the Genomic RNA of an Insect Virus 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 ABSTRACT RNA transcripts of cloned cDNA of the MATERIALS AND METHODS genomic RNAs of BBV (black beetle virus) are infectious to We used BBV W17 (7), a viral strain that is highly cytolytic cultured cells of Drosophila melanogaster. Individual tran- to cultured cells of D. melanogaster. scripts had approximately 10% of the infectivity of the corre- Synthesis of Full-Length DNA Copies of BBV RNAs and sponding authentic virion RNA. Progeny virus resulting from Their Cloning into Transcription Vectors. RNAs were re- transcript infection was phenotypically indistinguishable from verse-transcribed into cDNA and converted to the double- the progenitor virus used to generate the original cDNA forms stranded form with reverse transcriptase (12). Specific as judged by sucrose density gradient sedimentation, specific oligonucleotides, used as primers, had extra nonviral bases at infectivity, plaque morphology, and serology. Although the their 5' ends, which resulted in the emergence of unique transcript RNAs used to produce this virus had 20 nonviral restriction sites at each end ofthe double-stranded DNA (Pst bases headed by a capping group at their 5' termini, these 20 I at the 5' end and Xba I at the 3' end). Full-length bases were absent in the progeny viral RNAs. The cDNA forms, double-stranded DNA copies ofBBV RNA1 and RNA2 were and therefore the resulting transcript RNAs, should be readily inserted into the Sma I site of the multicopy plasmid pUC13 modifiable by the techniques of recombinant DNA technology and were cloned in Escherichia coli JM 101. Complete BBV both for viral studies and for the insertion offoreign genes into DNA inserts were excised from pUC13 recombinant plas- the viral genome and thus into the host cytoplasm. mids and were inserted into transcription vectors pSP64 or pPM1 and cloned. The methods ofrecombinant DNA technology can be ofgreat In Vitro Transcription ofCloned BBV DNA. Selected pSP64 recombinant plasmids were cleaved with the restriction utility for studies of RNA viruses. Such methods are intrin- enzyme Xba I, and the resulting linear DNA templates (20 sically available for retroviruses, which have DNA as an ,ug/ml) were transcribed with SP6 RNA polymerase as intermediate in their synthesis. The methods have now described by Konarska et al. (13). Generally, 500 kLM become applicable to RNA phage QB (1) and to poliovirus (2), guanosine (5') triphospho(5')guanosine [G(5')ppp(5')G] or whose cDNAs have been shown to be infectious, and to the 7-methyguanosine derivative m7G(5')ppp(5')G was in- brome mosaic virus (3), for which infectious RNA has been cluded in the reaction mixture to provide capped transcripts. made by transcription from cloned viral cDNA. Infectious pPM1 recombinant plasmid DNA, linearized with Xba I, was cDNA and infectious transcript RNAs are also known for the transcribed with E. coli RNA polymerase as described by plant pathogens potato spindle tubor viriod (4) and hop stunt Ahlquist and Janda (12). viriod (5). To our knowledge there have not been published Clones were named according to the format pxBySPz or reports ofinfectious transcript RNAs for any insect or animal pxByPMz, where x = 1 or 2 indicates derivation from RNA1 virus. or RNA2, y is the isolate number of the pUC13 recombinant We show here that RNA transcripts derived from DNA plasmid containing the BBV insert, and z indicates the copies of the genomic RNAs of BBV are infectious to isolation number of either a pSP64 or a pPM1 recombinant cultured cells of Drosophila melanogaster; thus this virus, carrying the insert BBV DNA in the correct orientation. also, is modifiable by DNA methods. Infectivity Assays. Drosophila melanogaster cells (5 x 106) Black beetle virus (BBV) is an insect virus of the family in 80 Al ofPNKC buffer (35 mM Pipes/100 mM NaCl/10 mM Nodaviridae. Its genome consists of two single-stranded, KCl, 1 mM CaCl2/400 iLg of DEAE dextran per ml, pH 6.0), messenger-sense RNAs contained in a single virion (6, 7). were transfected (10) by addition of various amounts of Virion RNA1 (3106 bases) (8) codes for protein A (involved transcribed or virion RNAs in 20 ,ul ofPNKC buffer. After 10 in viral RNA synthesis) and protein B (function unknown), min at room temperature, cells (200 to 2 x 106) from each transfection mixture were added to untreated Drosophila whose cistron is silent. The protein B cistron is expressed by cells to give a total of 4 x 106 cells in 5 ml of NPKA buffer means ofa subgenomic messenger, RNA3 (389 bases) (9, 10), (25 mM Pipes, pH 6.75/100 mM NaCl/10 mM KCl/0.1% which is not encapsidated. Virion RNA2 (1399 bases) (11) bovine serum albumin). Cells were mixed and poured into encodes the virion coat protein precursor, a, which is 6-cm tissue culture dishes and, after 2-3 days at 26°C, proteolytically processed into the coat proteins /3 and y. Both infectious centers were counted (7). 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 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: BBV, black beetle virus; pfu, plaque-forming unit(s). 63 Downloaded by guest on September 29, 2021 64 Biochemistry: Dasmahapatra et al. Proc. Natl. Acad. Sci. USA 83 (1986) copies of BBV genomes. All such transcripts have the same Infectivity of Transcript RNA1 Plus Transcript RNA2. The four additional nonviral nucleotides at their 3' ends and the crucial test ofbiological activity is of course the infectivity of same 20 additional nonviral nucleotides at their 5' ends transcript RNA1 plus transcript RNA2 in the absence of any headed by a capping group, or in some experiments, by a authentic RNA. When cells were transfected with a mixture methylated capping group or a triphosphate. In several of transcripts that we had identified as infectious by the experiments we cloned full-length BBV insert DNAs in the procedures described above, plaque assays consistently transcription vector pPM1 and used E. coli RNA polymerase indicated infectivity under conditions that had been shown to to provide transcripts whose 5' termini are identical in be optimal for authentic RNAs. Infectivity required the sequence to authentic virion RNA. Table 1 shows the presence of both transcript RNA1 and transcript RNA2. We terminal structure of the various transcripts. were unable to detect infectivity with Xba I-linearized Infectivity of Transcript RNA1 Plus Authentic RNA2 and of p1B9SP DNA plus p2B1OSP DNA. The RNA input required Transcript RNA2 Plus Authentic RNA1. In order to initially for the maximal number of plaque-forming centers was identify infectious transcripts and to quantify their infectiv- always higher for transcript RNA than for virion RNA (Table ity, preparations of individual transcripts were combined 4). Fig. LA shows the results obtained when the input of with preparations of cognate RNAs derived from virions, and transcript RNA1 was varied and the input oftranscript RNA2 these mixtures were assayed for infectivity. To serve as a was held constant at 0.2 jig. Fig. 1B shows the results baseline for judging infectivity of the transcript RNAs, we obtained when the input of transcript RNA2 was varied and first determined the infectivity of combinations of authentic the input oftranscript RNA1 was held constant at 1.2 ,g (i.e., virion RNA1 and RNA2. Maximal infectivity was obtained approximately the optimal value found in Fig. LA). It is when 5 x 106 cells were transfected with a mixture of 0.5 ,ug evident from Fig. 1B that the highest infectivity is obtained at of virion RNA1 and 0.2 ,ug of virion RNA2. Under these approximately equal molarity oftranscript RNA1 and RNA2. conditions approximately 5% of the transfected cells formed At equimolarity, maximal infectivity is about 1% of that of plaques. authentic RNAs and is obtained at a transcript RNA con- Preparations of transcript RNA1 were assayed together centration approximately 3 times that required for optimal with virion RNA2 at concentrations optimal for virion RNAs. infectivity with authentic RNAs (Fig. 1C). Table 2 shows the infectivity of three transcripts from the The virus arising from the combination RNA1(plB9SP) same DNA [capped transcript p1B13SP, methylated capped and RNA2(p2BlOSP), designated BBV K1, has been studied transcript p1B13SP(M), and uncapped transcript p1B13SP- in detail. BBV K1 is phenotypically indistinguishable from (ppp)] and another transcript, p1B9SP, relative to virion BBV W17, the progenitor virus from which it was derived, as RNA1. The p1B13SP and p1B13SP(M) transcripts were each we describe below. approximately 10% as infectious as virion RNA1. The uncap- Plaque Analyses. Assay of W17 virions, W17 virion RNAs, ped transcript p1B13SP(ppp) had approximately 0.1% of the or W17 RNA1 plus any of our infectious transcript RNA2s infectivity ofvirion RNA1. Table 2 indicates infectivity at the resulted in mostly large (2-4 mm) clear plaques. Assay of level of 0.03% when no RNA1 was added. This infectivity is transcript RNA1 plus virion or transcript RNA2 resulted in due to the presence of a small admixture of virion RNA1 in small (0.5 mm) clear plaques.
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