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Published on: March 24, 2017
Detection of beet yellows closterovirus methyltransferase-like and helicase-like proteins in vivo using monoclonal
T N Erokhina1, R A Zinovkin, M V Vitushkina
1Department of Virology, Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia.
Abstract:
In the positive-stranded RNA genome of beet yellows closterovirus (BYV), the 5'-terminal ORF 1a encodes a 295 kDa polyprotein with the domains of papain-like cysteine proteinase, methyltransferase (MT) and helicase (HEL), whereas ORF 1b encodes an RNA-dependent RNA polymerase. Eleven and five hybridoma cell lines secreting monoclonal antibodies (MAbs) were derived from mice injected with the bacterially expressed fragments of the BYV 1a product encompassing the MT and HEL domains, respectively. On immunoblots of protein from BYV-infected Tetragonia expansa plants, four MAbs against the MT recognized a approximately 63 kDa protein, and two MAbs against the HEL recognized a approximately 100 kDa protein. Both the methyltransferase-like protein and the helicase-like protein were found mainly in the fractions of large organelles (P1) and membranes (P30) of the infected plants. These data clearly indicate that (i) the BYV methyltransferase-like and helicase-like proteins, like other related viral enzymes, are associated with membrane compartments in cells, and (ii) the 1a protein, apart from the cleavage by the leader papain-like proteinase that is expected to produce the 66 kDa and 229 kDa fragments, undergoes additional processing by a virus-encoded or cellular proteinase.
Insights
Beet yellows virus (BYV) methyltransferase and helicase proteins are associated with cellular membranes. These proteins, encoded by the BYV 1a gene, undergo further processing beyond initial cleavage.
Area of Science:
- Plant virology
- Molecular biology
- Proteomics
Background:
- The beet yellows closterovirus (BYV) genome is positive-stranded RNA.
- ORF 1a encodes a large polyprotein containing methyltransferase (MT) and helicase (HEL) domains.
- ORF 1b encodes the RNA-dependent RNA polymerase.
Purpose of the Study:
- To characterize the BYV 1a protein domains, specifically the methyltransferase and helicase.
- To investigate the cellular localization of these BYV proteins.
- To understand the processing of the BYV 1a polyprotein.
Main Methods:
- Generation of hybridoma cell lines producing monoclonal antibodies (MAbs) against BYV 1a fragments (MT and HEL domains).
- Immunoblot analysis of proteins from BYV-infected plants using generated MAbs.
- Subcellular fractionation to isolate large organelles (P1) and membrane fractions (P30).
Main Results:
- MAbs recognized specific protein sizes: ~63 kDa for MT-specific MAbs and ~100 kDa for HEL-specific MAbs.
- Both methyltransferase-like and helicase-like proteins were primarily found in large organelle and membrane fractions.
- Data indicate association of these viral proteins with cellular membrane compartments.
Conclusions:
- The BYV methyltransferase-like and helicase-like proteins localize to membrane compartments within infected cells.
- The BYV 1a polyprotein undergoes additional proteolytic processing, in addition to cleavage by its leader proteinase.
