Processing and subcellular localization of the leader papain-like proteinase of Beet yellows closterovirus

Roman A. Zinovkin1, Tatyana N. Erokhina2, Dietrich E. Lesemann3, Wilhelm Jelkmann4 and Alexey A. Agranovsky1

1 Department of Virology and Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia
2 Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, 117871 Moscow, Russia
3 Department of Plant Virology, Microbiology and Biosafety, BBA, Messeweg 11-12, D-38104 Braunschweig, Germany
4 Institute for Plant Protection in Fruit Crops, BBA, Schwabenheimer Str. 101, D-69221 Dossenheim, Germany

Correspondence
Alexey Agranovsky
aaa{at}genebee.msu.su


   ABSTRACT
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
ORF 1a of Beet yellows closterovirus (BYV) encodes the domains of the papain-like proteinase (PCP), methyltransferase (MT) and RNA helicase. BYV cDNA inserts encoding the PCP–MT region were cloned in pGEX vectors next to the glutathione S-transferase gene (GST). In a ‘double tag’ construct, the GST–PCP–MT cDNA was flanked by the 3'-terminal six histidine triplets. Following expression in E. coli, the fusion proteins were specifically self-cleaved into the GST–PCP and MT fragments. MT-His6 was purified on Ni–NTA agarose and its N-terminal sequence determined by Edman degradation as GVEEEA, thus providing direct evidence for the Gly588/Gly589 bond cleavage. The GST–PCP fragment purified on glutathione S–agarose was used as an immunogen to produce anti-PCP monoclonal antibodies (mAbs). On Western blots of proteins from virus-infected Tetragonia expansa, the mAbs recognized the 66 kDa protein. Immunogold labelling of BYV-infected tissue clearly indicated association of the PCP with the BYV-induced membranous vesicle aggregates, structures related to closterovirus replication.


   MAIN TEXT
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Some positive-strand RNA viruses belonging to distantly related genera encode the domain(s) of the papain-like cysteine proteinase (PCP) responsible for the release of mature non-structural proteins (Gorbalenya et al., 1991). PCP domains show only marginal sequence conservation and may occupy different positions in the polyproteins of viruses, even those that are closely related (Koonin & Dolja, 1993). Some PCPs perform a single C-terminal cleavage in cis (so-called leader proteinases, L-PCPs), whereas others retain proteolytic activity after their release from a precursor and are able to cleave the virus and cell proteins in trans (reviewed in Dougherty & Semler, 1993). Mature proteins with PCP domains may have additional non-proteolytic activities: alphavirus nsp2 is an RNA helicase (Strauss & Strauss, 1994); the leader PCPs of coronaviruses and arteriviruses are implicated in subgenomic RNA synthesis (Herold et al., 1999; Tijms et al., 2001); and the potyvirus helper component proteinase (HC-Pro) is involved in virus transmission by insects, long-distance transport in plants, RNA amplification and suppression of RNA silencing (Maia et al., 1996; Carrington et al., 2001).

Closteroviridae, a family of elongated insect-transmissible viruses of plants, belongs to the Sindbis-like superfamily characterized by the conserved array of methyltransferase (MT), helicase (HEL) and RNA polymerase (POL) domains of RNA replicase (Goldbach et al., 1991; Rozanov et al., 1992; Koonin & Dolja, 1993). Closterovirus ORF 1a encodes one or two L-PCPs followed by MT and HEL; ORF 1b encodes POL (reviewed in Agranovsky, 1996). In the genome of Beet yellows closterovirus (BYV), ORF 1a encodes a 295 kDa polyprotein. Using monoclonal antibodies (mAbs) to MT and HEL, the methyltransferase-like and helicase-like proteins have been identified in vivo as 63 and 100 kDa products, respectively (Erokhina et al., 2000). In situ, these proteins are co-localized at the membranes of the BYV-induced vesicle aggregates (Erokhina et al., 2000, 2001), the complex ultrastructures consisting of vesicle clusters (each comprising several 100 nm vesicles surrounded by a common membrane) with the cytoplasm strands between them (Esau & Hoefert, 1971; Lesemann, 1988, 1991; D.-E. Lesemann, unpublished observations).

The BYV L-PCP was first identified by its limited sequence similarity with potyvirus HC-Pro. In line with the cleavage site prediction, a point substitution Gly588/Asp inhibited cleavage in a cell-free translation system, thus providing experimental support, albeit circumstantial, to the identification of the scissile bond (Agranovsky et al., 1994). Although BYV L-PCP and potyvirus HC-Pro show no similarity apart from the ~140-residue PCP domains, both are multifunctional proteins with common activities. Like HC-Pro, BYV L-PCP influences virus RNA amplification and cell-to-cell movement (Peremyslov et al., 1998; Peng & Dolja, 2000; Peng et al., 2003).

In this study, we wanted to gain more insight into the processing and subcellular localization of BYV L-PCP. Bacterial expression vectors were constructed, which allowed us to express L-PCP fusion proteins, monitor their self-cleavage and purify the N- and C-terminal cleavage products for microsequencing and for mAb production.

For L-PCP cloning, the insert from the BYV cDNA clone 154 (Agranovsky et al., 1994) was excised with EcoRI/Eco52I and ligated between the same sites of pGEX-4T3 (Amersham Pharmacia Biotech). The EcoRI–Eco72I fragment of the BYV 1518 clone was inserted between the EcoRI and SmaI sites of pGEX-4T1. The resulting clones, pGEX-1518 and pGEX-154, carried the glutathione S-transferase (GST) gene fused in frame with portions of the BYV ORF1a encoding, respectively, aa 259–720 and aa 302–683 (BYV 1a numbering; Fig. 1). To produce pGEX-1518{Delta}mt (encoding a C-terminally truncated protein, aa 259–600), pGEX-1518 was digested with BspTI and Eco52I, treated with Klenow fragment and religated. Plasmid pGEX-1518Cys509 was constructed by replacing an Eco47III–Eco52I fragment in pGEX-1518 with the same fragment from pB515C509 (containing the point substitution Cys509/Thr) (Agranovsky et al., 1994). To obtain a BYV cDNA flanked by the 5'-terminal GST gene and the 3'-terminal six histidine triplets (pGEX-1518-His6), the XhoI–HindIII fragment from the pQE-p65-C6H vector (Agranovsky et al., 1997) was inserted between the SalI and NotI sites of pGEX-1518. The plasmids were used for transformation of the E. coli strain BL-21. The following conditions were found to be optimal for bacterial expression of BYV L-PCP fusion proteins: E. coli cultures were allowed to grow to OD600=0·6 at 33 °C, followed by induction with 0·2 mM IPTG and further growth for 4 h at 26 °C.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1. Schematic representation of the BYV ORF 1a (numbers on the upper scale, triplets from the 5' end) and the expressed cDNA inserts in the pGEX clones. The glutathione S-transferase (GST) gene and the BYV-specific inserts are shown as grey and white areas within each box, respectively, with the calculated molecular masses of the encoded polypeptides indicated. Insert pGEX-1518Cys508 contained a point mutation of the catalytic cysteine (black diamond). Insert pGEX-1518-His6 encoded an extra tag consisting of the 15 kDa portion of the BYV p65 protein histidinylated at the C terminus (p65C-His6; cross-hatched area). The conserved domains are the papain-like proteinase (PCP), methyltransferase (MT) and RNA helicase (HEL). The PCP cleavage site is denoted by a broken line. Drawn approximately to scale.

 
The IPTG-induced cells harbouring pGEX-154 accumulated proteins migrating as 55 and 70 kDa entities, which were absent from the non-induced control (Fig. 2A, lanes 1 and 2). Likewise, the 62 and 76 kDa proteins accumulated in the induced pGEX-1518-containing cells (Fig. 2A, lane 3). The single 62 or 76 kDa products were detected in the cells harbouring pGEX-1518{Delta}mt (cDNA with a deletion of the MT sequence) or pGEX-1518Cys509 (cDNA with a mutation of catalytic Cys509 abolishing the PCP activity in vitro; Agranovsky et al., 1994) (Fig. 2A, lanes 4 and 5). The sizes of the 55 and 62 kDa proteins were consistent with self-processing of the BYV PCP in bacteria transformed by the respective pGEX-154 or pGEX-1518 vectors, whereas the 70 and 76 kDa proteins apparently represented uncleaved products. The respective C-terminal 10 and 15 kDa cleavage products could not be confidently identified, presumably because of their instability and/or masking by the abundant low molecular mass E. coli proteins (Fig. 2A).



View larger version (41K):
[in this window]
[in a new window]
 
Fig. 2. (A, B) 15 % SDS-PAGE of BYV PCP fusion ptoteins expressed in E. coli. (A) Lanes 1 and 2, total protein from the pGEX-154-transformed non-induced and IPTG-induced cells, respectively. Lanes 3–5, total protein from the induced pGEX-1518 cells (lane 3), pGEX-1518{Delta}mt cells (lane 4) and pGEX-1518C509 cells (lane 5). Lanes 6 and 7, recombinant fusion proteins purified by glutathione S–agarose chromatography from the cells harbouring pGEX-1518 and pGEX-1518{Delta}mt, respectively. Lane 8, molecular mass standards (Bio-Rad, Low Range: 97·4, 66·2, 45·0, 31·0, 21·5 and 14 kDa). (B) Lanes 1 and 2, total protein from non-induced and IPTG-induced cells harbouring the pGEX-1518-His6 vector; arrowheads indicate the virus-specific protein bands of 90, 62 and 28 kDa. Lane 3, recombinant proteins purified by Ni-NTA chromatography. The N-terminal sequence of the 28 kDa protein was determined by Edman degradation. Lane 4, molecular mass standards. (C) Western blot analysis of total protein extracted from healthy and BYV-infected T. expansa (lanes H and I, respectively). The blot was developed with the anti-PCP mAb 3C1 as primary antibody. Positions of the protein markers (kDa) are indicated.

 
Following affinity purification on glutathione S–agarose (Smith & Johnson, 1988), a single 62 kDa protein band was detected in the bound-and-eluted fractions from both the pGEX-1518- and pGEX-1518{Delta}mt-transformed cell extracts (Fig. 2A, lanes 6 and 7). The absence of the uncleaved 76 kDa protein in the bound fraction of the GST–1518 proteins (Fig. 2A, lane 6) may be explained by continued self-processing of the protein following chromatography in native conditions.

We wanted to identify the BYV L-PCP cleavage site directly by microsequencing the N terminus of an MT-containing cleavage product. The pGEX-1518-His6 vector was constructed encoding a fusion of GST, PCP–MT and the C-terminal portion of the BYV p65 protein with the His6 tag (Fig. 1). E. coli BL-21 cells containing pGEX-1518-His6 accumulated proteins of 90, 62 and 28 kDa, whose apparent molecular masses were consistent with those calculated for the respective uncleaved fusion, the N-terminal GST–PCP fragment and the C-terminal MT–p65 fragment (Fig. 1; Fig. 2B, lane 2). In line with this, the protein fraction purified on Ni–NTA agarose in denaturing conditions (Agranovsky et al., 1997), contained only the C-terminal 28 kDa fragment and the 90 kDa uncleaved protein (Fig. 2B, lane 3). Following transfer to membrane, the 28 kDa protein band was excised and subjected to automated Edman degradation. The N-terminal sequence of the protein fragment was determined as Gly-Val-Asp-Asp-Asp-Ala, thus confirming the BYV PCP cleavage of the Gly588/Gly589 bond in the 1a polyprotein.

Immunization of mice with GST–1518{Delta}mt, the fusion protein purified from the pGEX-1518{Delta}mt-transformed E. coli (Fig. 2A), and screening of hybridomas by indirect ELISA resulted in five clones (4A1, 4A2, 2B3, 1C3 and 3C1) reacting positively with the recombinant immunogen but not with GST. All five mAbs recognized the GST–1518{Delta}mt protein on Western blots of total protein from the IPTG-induced cells (data not shown). On Western blot analysis of total phenol-extracted protein from infected Tetragonia expansa plants, mAbs 4A1, 4A2, 2B3 and 3C1 recognized the major 66 kDa protein (Fig. 2C), whose apparent size agrees with that of BYV L-PCP released in vitro (Agranovsky et al., 1994) and with the established cleavage site (Fig. 2B). This result was not unexpected, yet was important, as the possibility of additional cleavages within the leader protein had not been excluded – especially in view of the fact that the 1a polyprotein processing in vivo appears to be more sophisticated than it seemed (Erokhina et al., 2000).

In immunogold labelling, all five mAbs reacted with the BYV-induced vesicle aggregates in the infected T. expansa cell sections (Fig. 3A and Table 1). Most of the gold label was observed on the membranes and cytoplasm strands separating the vesicle clusters (Fig. 3B). The labelling was statistically significant for the specimens embedded in Lowicryl as well as in Epon (Table 1). Specific labelling of similar intensity has been recorded for anti-MT and anti-HEL mAbs (Erokhina et al., 2001). mAbs 4A1 and 4A2 showed an elevated reaction with the nuclei and chloroplasts; however, this was probably non-specific, as a comparable (or higher) labelling was recorded on the healthy tissue sections (Table 1). No mAb reaction with the cell walls and plasmodesmata was observed (data not shown). No labelling of infected cell ultrastructures was seen in the controls with heterologous mAbs (Table 1) or the gold-conjugated secondary antibody alone (not shown).



View larger version (126K):
[in this window]
[in a new window]
 
Fig. 3. Immunogold labelling and electron microscopy of the BYV-induced vesicle aggregates in the virus-infected Tetragonia expansa tissue cuts embedded in Lowicryl. Labelling was carried out with the PCP panel mAb 1C3 as primary antibody. (A) Overview showing a specifically labelled vesicle aggregate (VA) in contrast to unlabelled virus particle aggregates (VP), chloroplasts (C), mitochondrion (M) and cell wall (W). Bar, 500 nm. (B) Enlarged part of a vesicle aggregate showing specific labelling on the dark membrane/cytoplasm strands (arrows), which surround the unlabelled, low-contrast centres of the vesicle clusters (VC). Bar, 500 nm.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Statistics for immunogold labelling of various ultrastructures in healthy and BYV-infected T. expansa cells probed with mAbs to the PCP domain of BYV or with heterologous mAbs

(a) Tissue embedded in Epon

 

View this table:
[in this window]
[in a new window]
 
(b) Tissue embedded in Lowicryl

 
Taken together, the data of this work clearly show that BYV L-PCP, when expressed in a foreign (prokaryotic) cell context, mediates its specific self-release from a polyprotein. Expression in E. coli and affinity purification of the C-terminal cleavage products suitable for microsequencing have previously been reported for human coronavirus 3C-like proteinase (Ziebuhr & Siddell, 1999) and birnavirus VP4 proteinase (Lejal et al., 2000). We constructed L-PCP fusion proteins with affinity tags at one or both flanks, which allowed purification of the N- and C-terminal cleavage products for antibody production and microsequencing. The pGEX/BL-21 system also obviated the problems encountered in expression of ‘difficult’ proteins, such as BYV L-PCP (Erokhina et al., 2000).

Our electron microscopic analysis of the BYV-infected tissues subjected to immunogold labelling with anti-PCP mAbs indicated association of the leader protein with the closterovirus-induced membranous vesicle aggregates. Replication of positive-strand RNA viruses of animals and plants is connected with vesicles or spherules derived from various membranous organelles of the cell (reviewed in Buck, 1996). In Semliki Forest virus and probably in other Sindbis-like superfamily viruses, the MTR IV motif in the MT domain is responsible for anchoring the replicase complex to membranes (Ahola et al., 1999). On infection of Brome mosaic bromovirus, the MT–HEL-containing 1a protein induces budding of the vesicles from endoplasmic reticulum membranes, thus creating secluded replication sites (Schwartz et al., 2002). The BYV methyltransferase-like and helicase-like proteins also reside on the membranes of multivesicular aggregates, thus indicating that these ultrastructures are replication compartments (Erokhina et al., 2001). Co-localization of L-PCP with closterovirus replication-associated proteins agrees with its involvement in RNA accumulation (Peremyslov et al., 1998; Peng & Dolja, 2000). However, the possibility that the BYV leader protein is also involved in fleeting interactions with other cell compartments and/or virus products to perform activities such as virus long-distance transport (Peng et al., 2003) cannot be excluded.


   ACKNOWLEDGEMENTS
 
We are grateful to Christina Maass for excellent technical assistance and to Alexander Galkin for critical reading of the manuscript. This study has been supported by grants 00-04-48252 and 01-04-49189 from the Russian Foundation for Basic Research. The work of R. A. Zinovkin was supported by the Young Scientist Fellowship YSF 2002-0061 from INTAS.


   REFERENCES
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Agranovsky, A. A. (1996). The principles of molecular organization, expression and evolution of closteroviruses: over the barriers. Adv Virus Res 47, 119–158.[Medline]

Agranovsky, A. A., Koonin, E. V., Boyko, V. P., Maiss, E., Froetschl, R., Lunina, N. A. & Atabekov, J. G. (1994). Beet yellows closterovirus: complete genome structure and identification of a leader papain-like thiol protease. Virology 198, 311–324.[CrossRef][Medline]

Agranovsky, A. A., Folimonova, S. Y., Folimonov, A. S., Denisenko, O. N. & Zinovkin, R. A. (1997). The beet yellows closterovirus p65 homologue of HSP70 chaperones has ATPase activity associated with its conserved N-terminal domain but does not interact with unfolded protein chains. J Gen Virol 78, 535–542.[Abstract]

Ahola, T., Lampio, A., Auvinen, P. & Kääriäinen, L. (1999). Semliki Forest virus mRNA capping enzyme requires association with anionic membrane phospholipids for activity. EMBO J 18, 3164–3172.[Abstract/Free Full Text]

Buck, K. (1996). Comparison of the replication of positive-stranded RNA viruses of plants and animals. Adv Virus Res 47, 159–251.[Medline]

Carrington, J. C., Kasschau, K. D. & Johansen, L. K. (2001). Activation and suppression of RNA silencing by plant viruses. Virology 281, 1–5.[CrossRef][Medline]

Dougherty, W. G. & Semler, B. L. (1993). Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes. Microbiol Rev 57, 781–822.[Medline]

Erokhina, T. N., Zinovkin, R. A., Vitushkina, M. V., Jelkmann, W. & Agranovsky, A. A. (2000). Detection of beet yellows closterovirus methyltransferase-like and helicase-like proteins in vivo using monoclonal antibodies. J Gen Virol 81, 597–603.[Abstract/Free Full Text]

Erokhina, T. N., Vitushkina, M. V., Zinovkin, R. A., Lesemann, D. E., Jelkmann, W., Koonin, E. V. & Agranovsky, A. A. (2001). Ultrastructural localization and epitope mapping of beet yellows closterovirus methyltransferase-like and helicase-like proteins. J Gen Virol 82, 1983–1994.[Abstract/Free Full Text]

Esau, K. & Hoefert, L. L. (1971). Cytology of beet yellows virus infection in Tetragonia. I. Parenchyma cells in infected leaf. Protoplasma 72, 255–273.

Goldbach, R., Le Gall, O. & Wellink, J. (1991). Alpha-like viruses in plants. Semin Virol 2, 19–25.

Gorbalenya, A. E., Koonin, E. V. & Lai, M. M. (1991). Putative papain-related thiol proteases of positive-strand RNA viruses. Identification of rubi- and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha- and coronaviruses. FEBS Lett 288, 201–205.[CrossRef][Medline]

Herold, J., Siddell, S. G. & Gorbalenya, A. E. (1999). A human RNA viral cysteine proteinase that depends upon a unique Zn2+-binding finger connecting the two domains of a papain-like fold. J Biol Chem 274, 14918–14925.[Abstract/Free Full Text]

Koonin, E. V. & Dolja, V. V. (1993). Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Crit Rev Biochem Mol Biol 28, 375–430.[Abstract]

Lejal, N., Da Costa, B., Huet, J.-C. & Delmas, B. (2000). Role of Ser-652 and Lys-692 in the protease activity of infectious bursal disease virus VP4 and identification of its substrate cleavage sites. J Gen Virol 81, 983–992.[Abstract/Free Full Text]

Lesemann, D.-E. (1988). Cytopathology. In The Plant Viruses, vol. 4, pp. 179–235. Edited by R. G. Milne. New York: Plenum.

Lesemann, D.-E. (1991). Specific cytological alterations in virus-infected plant cells. In Electron Microscopy of Plant Pathogens, pp. 147–159. Edited by K. Mendgen & D.-E. Lesemann. Berlin/Heidelberg/New York: Springer.

Maia, I. G., Haenni, A.-L. & Bernardi, F. (1996). Potyviral HC-Pro: a multifunctional protein. J Gen Virol 77, 1335–1341.[Medline]

Peng, C. W. & Dolja, V. V. (2000). Leader proteinase of the beet yellows closterovirus: mutation analysis of the function in genome amplification. J Virol 74, 9766–9770.[Abstract/Free Full Text]

Peng, C. W., Napuli, A. J. & Dolja, V. V. (2003). Leader proteinase of beet yellows virus functions in long-distance transport. J Virol 77, 2843–2849.[Abstract/Free Full Text]

Peremyslov, V. V., Hagiwara, Y. & Dolja, V. V. (1998). Genes required for replication of the 15·5-kilobase RNA genome of a plant closterovirus. J Virol 72, 5870–5876.[Abstract/Free Full Text]

Rozanov, M. N., Koonin, E. V. & Gorbalenya, A. E. (1992). Conservation of the putative methyltransferase domain: a hallmark of the ‘Sindbis-like’ supergroup of positive-strand RNA viruses. J Gen Virol 73, 2129–2134.[Abstract]

Schwartz, M., Chen, J., Janda, M., Sullivan, M., den Boon, J. & Ahlquist, P. (2002). A positive-strand RNA virus replication complex parallels form and function of retrovirus capsids. Mol Cell 9, 505–514.[Medline]

Smith, D. B. & Johnson, K. S. (1988). Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione-S-transferase. Gene 67, 31–40.[CrossRef][Medline]

Strauss, J. H. & Strauss, E. G. (1994). The alphaviruses: gene expression, replication and evolution. Microbiol Rev 58, 491–562.[Medline]

Tijms, M. A., van Dinten, L. C., Gorbalenya, A. E. & Snijder, E. J. (2001). A zinc finger-containing papain-like protease couples subgenomic mRNA synthesis to genome translation in a positive-stranded RNA virus. Proc Natl Acad Sci U S A 98, 1889–1894.[Abstract/Free Full Text]

Ziebuhr, J. & Siddell, S. G. (1999). Processing of the human coronavirus 229E replicase polyproteins by the virus-encoded 3C-like proteinase: identification of proteolytic products and cleavage sites common to pp1a and pp1ab. J Virol 73, 177–185.[Abstract/Free Full Text]

Received 7 February 2003; accepted 5 May 2003.