The ORF0 product of Potato leafroll virus is indispensable for virus accumulation

Ewa Sadowy1,2, Anna Maasen1, Marek Juszczuk1, Chantal David2, Wlodzimierz Zagórski-Ostoja1, Bruno Gronenborn2 and M. Danuta Hulanicka1

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. Chelmska 30/34, 00-725 Warszawa, Poland. Fax +48 22 841 29 49. e-mail sadowy{at}ibbrain.ibb.waw.pl


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Using a cDNA expression cassette in combination with agroinoculation of potato leaf discs we have investigated the role the protein encoded by ORF0 of Potato leafroll virus (PLRV) and have shown its importance for virus accumulation. Two mutations introduced into ORF0 by site-directed mutagenesis prevented expression of the corresponding protein and completely abolished virus accumulation in plant cells. They did not, however, affect translation of ORF1 and ORF2. We therefore conclude that ORF0 of PLRV produces a protein essential for virus accumulation, a hitherto undescribed finding.


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Potato leafroll virus (PLRV) is the type member of the genus Polerovirus and belongs to the family Luteoviridae, which also contains the genera Luteovirus and Enamovirus (Pringle, 1998 ). PLRV has a monopartite, non-polyadenylated RNA genome of ~6 kb with a small protein, VPg, linked to its 5' end (Mayo et al., 1982 ). PLRV RNA encodes six main open reading frames (ORF; Fig. 1A) that are expressed by a variety of mechanisms (reviewed in Miller et al., 1997 ). The three 5'-proximal ORFs are translated directly from the genomic RNA and include ORF1, encoding the 70 kDa viral proteinase (Hulanicka et al., 1999 ) and ORF2, which is translated via a ribosomal frameshift within ORF1 to yield the 118 kDa viral replicase. Three other ORFs are expressed via a subgenomic RNA synthesized in infected cells and include ORF3, encoding the 23 kDa viral capsid protein, ORF4, encoding a 17 kDa putative movement protein and ORF5, expressed by translational readthrough as a fusion protein with the capsid protein. It encodes a 56 kDa protein that presumably is involved in aphid transmission of the virus.



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Fig. 1. (A) Schematic structure of the PLRV genome. ORFs are represented by boxes. The catalytic centre of the putative viral proteinase is marked by an asterisk; the VPg coding sequence is represented by a black box in ORF1. The VPg at the 5' end of the genomic RNA is represented by a closed circle; the GDD motif of the putative replicase is indicated by an open square, oblique and horizontal arrows symbolize ribosomal frameshift and readthrough sites, respectively. (B) Schematic representation of the pBOK plasmid. Dashed rectangles marked 35S-p and ter denote the 35S promoter and terminator sequences, respectively; arrowheads marked LB and RB denote the T-DNA left and right border, respectively. Relevant restriction endonuclease sites are symbolized as follows: A, ApaI; K, KpnI; T, Tth111I; thick black line symbolizes viral cDNA.

 
The function of the putative 28 kDa translation product of ORF0 in the PLRV life-cycle is unknown. Apart from its counterparts in other poleroviruses (reviewed in Mayo & Ziegler-Graff, 1996 ), it shares no homology with known proteins. An analogous protein, P1, is also encoded by the genome of sobemoviruses, enamoviruses and barnaviruses (reviewed in Tamm & Truve, 2000a ). Transgenic plants expressing PLRV ORF0 show symptoms similar to those observed in plants infected with the virus and the phenotype of such transgenic plants appears to correlate with the amount of mRNA for ORF0 (van der Wilk et al., 1997 ). Although a translation product of ORF0 was undetectable in transgenic plants, other constructs producing untranslatable ORF0 mRNA resulted in symptomless transgenic plants. Inactivation of ORF0 in an infectious cDNA clone of Beet western yellows virus (BWYV) resulted in a reduction of virus replication and a delay in onset of symptoms upon plant infection (Reutenauer et al., 1993 ). Rice yellow mottle virus (RYMV) deficient in P1 was unable to spread in host plants although some level of replication was maintained (Bonneau et al., 1998 ). This observation, together with the fact that P1 of Cocksfoot mottle virus is an RNA-binding protein, led to the suggestion that P1 of sobemoviruses might be a movement protein (Tamm & Truve, 2000a ).

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 208–210) 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 1–369 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 ORF1–ORF2 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 ORF1–ORF2 frameshift.



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Fig. 2. Proteins synthesized by ORF0 mutants in an in vitro translation system. Lane 1, translation of TMV RNA; lanes 2, 3 and 4, translation of pJFT, pQ47stop and pJF transcripts, respectively; lane 5, translation of PLRV virion RNA; lane 6, translation without exogenous RNA. The calculated sizes of the proteins are indicated on the right.

 
The biological importance of ORF0 expression was assessed by agroinoculation with plasmids pBOK, pBOK-T and pBQ47stop. Agroinoculation of potato leaf discs followed by RNA and protein isolation and their analysis by Northern hybridization and immunoblotting, respectively, were done as described (Sadowy et al., 2001 ). Both pBOK-T and pBQ47stop failed to produce any genomic or subgenomic viral RNA (Fig. 3A, lanes 4 and 5, respectively), whereas in RNA preparations from leaf discs agroinoculated with pBOK (wild-type control) the genomic and subgenomic RNAs were readily detected (Fig. 3A, lane 3). Consistent with the results of the Northern analyses, viral capsid protein was absent from protein preparations obtained from leaf discs agroinoculated with pBOK-T or pBQ47stop (Fig. 3B, lanes 4 and 5, respectively) whereas it was readily detected in extracts prepared from discs agroinoculated with pBOK (Fig. 3B, lane 3).



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Fig. 3. RNA and proteins synthesized by ORF0 mutants in leaf discs. (A) Northern analysis of RNA isolated from leaf discs agroinoculated with pBOK, pBOK-T and pB-Q47stop. Lane 1, RNA isolated from Physalis floridana infected with PLRV; lane 2, RNA isolated from mock-inoculated leaf discs; lanes 3, 4 and 5, RNA isolated from leaf discs agroinoculated with pBOK, pBOK-T and pB-Q47stop, respectively. Methylene blue staining of 28S rRNA as a control of equal loading of RNA (10 µg per lane except lane 1) as shown below. (B) Western analysis of capsid protein in total proteins isolated from leaf discs agroinoculated with pBOK, pBOK-T and pB-Q47stop. Lane 1, proteins isolated from mock-inoculated leaf discs; lane 2, virions of PLRV; lanes 3, 4 and 5, proteins isolated from leaf discs agroinoculated with pBOK-T, pB-Q47stop and pBOK, respectively.

 
To exclude the possibility that the mutations that abolish expression of a functional ORF0 product also influence expression of the overlapping ORF1 and the ORF1–ORF2 frameshift protein, we verified their expression by in vitro translation. The results of leaf disc agroinoculations with pBOK-T and pBQ47stop demonstrate that a functional ORF0 product is required for PLRV accumulation. This finding raises questions about the role of an ORF0 protein in this process. Considering its location in the viral genome, ORF0 may be expressed early during infection and it seems plausible that its product may play a direct role in PLRV replication. Analyses of the amino acid sequence of the ORF0 translation product revealed the presence of a potential transmembrane domain and potential phosphorylation sites (Mayo et al., 1989 ). The relatively high content of hydrophobic amino acids in the ORF0 protein suggests that it may serve as a membrane anchor for the replication complex, as was shown for the 6 kDa protein of Tobacco etch virus (Schaad et al., 1997 ) and the 3A protein of Poliovirus (Datta & Dasgupta, 1994 ; Towner et al., 1996 ). The ORF0 protein might also act as a cofactor of the PLRV proteinase or replicase. Protein cofactors are required for activity of numerous virus-encoded proteinases (de Groot et al., 1990 ; Arias et al., 1993 ; Amberg et al., 1994 ; Molla et al., 1994 ; Webster et al., 1994 ; Wassenaar et al., 1997 ) and stimulate replicase activity (Lama et al., 1994 ; Richards & Ehrenfeld, 1998 ).

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.


   Acknowledgments
 
We thank Anne-Lise Haenni for critical reading of manuscript. This work was supported by grant 6P04B01615 from the State Committee for Scientific Research (KBN). E.S. was a recipient of a UNESCO short-term fellowship in Biotechnology (SC/BSC/LS/97/BAC).


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Received 17 November 2000; accepted 14 February 2001.