Institute of Biochemistry and Biophysics PAS, Ul. Pawiskiego 5A, 02-106 Warsaw, Poland1
Institut des Sciences Végétales CNRS, Av. de la Terrasse, 91 198 Gif-sur-Yvette, France2
Author for correspondence: Ewa Sadowy. Present address: Sera & Vaccine Central Research Laboratory, Ul. Chemska 30/34, 00-725 Warszawa, Poland. Fax +48 22 841 29 49. e-mail sadowy{at}ibbrain.ibb.waw.pl
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Abstract |
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Infectious cDNA clones and their modification by reverse genetics represent important tools to study RNA viruses (reviewed in Boyer & Haenni, 1994 ). We used an infectious cDNA clone of the Polish isolate of PLRV (Sadowy et al., 2000
) to study the role of ORF0 in the virus life-cycle. For this purpose, two mutations that abolished ORF0 expression were constructed and their consequences were followed in vitro and in vivo.
Two plasmids, pJF (Sadowy et al., 1998 ) and pBOK [Fig. 1B
; Sadowy et al., 2001
(accompanying paper)] were used to construct mutants of ORF0. pJF was mutagenized by Tth111I-cleavage at nt position 81, end-filling and re-ligation. The resulting plasmid (pJF-T) contains an additional C residue at nt 81 which causes a frameshift and, as a consequence, terminates translation of ORF0 at position 106. In addition, a mutation that changes Gln-47 of the putative ORF0 product (nt 208210) into a stop codon was introduced by site-directed mutagenesis (QuikChange, Stratagene) using primers 208-1 and 208-2 (5' GTTATAATCATGAATAGATTTACCGCATATGC and 5'ATATGCGGTAAATCTATTCATGATTATAACCG; altered nucleotides in bold) to yield plasmid pQ47stop. The sequence of viral cDNA in the clone was verified.
Plasmid pBOK contains a full-length cDNA of PLRV flanked by the 35S promoter and terminator sequences of Cauliflower mosaic virus (CaMV) in pBin19 (Bevan, 1988). It drives efficient virus replication upon agroinoculation of potato leaf discs (Sadowy et al., 2001 ). The P0 mutations described above were introduced into pBOK using a smaller derivative thereof, pOK, as an intermediate. pOK is a derivative of pBluescript KS(+) (Stratagene) that contains the 35S promoter and the cDNA corresponding to nt 1369 of PLRV. Plasmids carrying the P0 mutations were digested with KpnI (cuts upstream of the 35S promoter) and ApaI (cuts at nt 369 of the PLRV genome), and the mutant fragments were exchanged for the corresponding wild-type DNA of pBOK, yielding pBOK-T and pBQ47stop, respectively.
The impact of these ORF0 mutations on the synthesis of ORF1 and ORF2 proteins was studied by in vitro translation of transcripts derived from plasmids pJF, pJF-T and pQ47stop. Plasmid DNAs linearized with ScaI (nt position 5882) were used for in vitro transcription (Kujawa et al., 1993 ); 200 ng of the uncapped transcripts were then translated in a rabbit reticulocyte lysate (Boehringer Mannheim) supplemented with L-[35S]methionine. Radioactively labelled products were separated on discontinuous denaturing 12·5% polyacrylamide gels (Laemmli, 1970
) and visualized by autoradiography (Fig. 2
). Translation of the wild-type transcript (lane 4) and PLRV gRNA (lane 5) resulted in the same pattern of three major proteins (28, 70 and 118 kDa), corresponding to the translation products of ORF0, ORF1 and the natural ORF1ORF2 frameshift. Two proteins of 70 and 118 kDa were detected as translation products of pJF-T and pQ47stop (lanes 2 and 3). Therefore, the introduced mutations prevent ORF0 synthesis without affecting translation of overlapping ORF1 or influencing efficiency of the ORF1ORF2 frameshift.
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In addition, the ORF0 protein may be involved in an interaction with a host specificity factor. Considering the relatively low sequence conservation of ORF0 among poleroviruses (Mayo & Ziegler-Graff, 1996 ) and the infection-like phenotype of ORF0-transgenic plants, such a role for ORF0 protein appears possible. Such a function has indeed been demonstrated for P1 of RYMV (Voinnet et al., 1999
). Additional experiments to identify viral or cellular targets of the ORF0 protein will be required to further clarify its role in the PLRV life-cycle.
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References |
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Received 17 November 2000;
accepted 14 February 2001.