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 |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
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; D.-E. Lesemann, unpublished observations).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 EcoRIEco72I 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 259720 and aa 302683 (BYV 1a numbering; Fig. 1
). To produce pGEX-1518
mt (encoding a C-terminally truncated protein, aa 259600), pGEX-1518 was digested with BspTI and Eco52I, treated with Klenow fragment and religated. Plasmid pGEX-1518Cys509 was constructed by replacing an Eco47IIIEco52I 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 XhoIHindIII 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.
|
|
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, PCPMT 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 GSTPCP fragment and the C-terminal MTp65 fragment (Fig. 1
; Fig. 2B
, lane 2). In line with this, the protein fraction purified on NiNTA 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 GST1518mt, the fusion protein purified from the pGEX-1518
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 GST1518
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).
|
|
|
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 MTHEL-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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
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, 311324.[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, 535542.[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, 31643172.
Buck, K. (1996). Comparison of the replication of positive-stranded RNA viruses of plants and animals. Adv Virus Res 47, 159251.[Medline]
Carrington, J. C., Kasschau, K. D. & Johansen, L. K. (2001). Activation and suppression of RNA silencing by plant viruses. Virology 281, 15.[CrossRef][Medline]
Dougherty, W. G. & Semler, B. L. (1993). Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes. Microbiol Rev 57, 781822.[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, 597603.
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, 19831994.
Esau, K. & Hoefert, L. L. (1971). Cytology of beet yellows virus infection in Tetragonia. I. Parenchyma cells in infected leaf. Protoplasma 72, 255273.
Goldbach, R., Le Gall, O. & Wellink, J. (1991). Alpha-like viruses in plants. Semin Virol 2, 1925.
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, 201205.[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, 1491814925.
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, 375430.[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, 983992.
Lesemann, D.-E. (1988). Cytopathology. In The Plant Viruses, vol. 4, pp. 179235. 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. 147159. 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, 13351341.[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, 97669770.
Peng, C. W., Napuli, A. J. & Dolja, V. V. (2003). Leader proteinase of beet yellows virus functions in long-distance transport. J Virol 77, 28432849.
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, 58705876.
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, 21292134.[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, 505514.[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, 3140.[CrossRef][Medline]
Strauss, J. H. & Strauss, E. G. (1994). The alphaviruses: gene expression, replication and evolution. Microbiol Rev 58, 491562.[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, 18891894.
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, 177185.
Received 7 February 2003;
accepted 5 May 2003.