Expression and characterisation of the ryegrass mottle virus non-structural proteins
The Ryegrass mottle virus (RGMoV) single-stranded RNA genome is organised into four open reading frames (ORF) which encode several proteins: ORF1 encodes protein P1, ORF2a contains the membrane-associated 3C-like serine protease, genome-linked protein VPg and a P16 protein gene. ORF2b encodes replicase RdRP and the only structural protein, coat protein, is synthesised from ORF3. To obtain the non-structural proteins in preparative quantities and to characterise them, the corresponding RGMoV gene cDNAs were cloned in pET- and pColdI-derived expression vectors and overexpressed in several E. coli host cells. For protease and RdRP, the best expression system containing pColdI vector and E. coli WK6 strain was determined. VPg and P16 proteins were obtained from the pET- or pACYC- vectors and E. coli BL21 (DE3) host cells and purified using Ni-Sepharose affinity chromatography. Attempts to crystallize VPg and P16 were unsuccessful, possibly due to non-structured amino acid sequences in both protein structures. Methods based on bioinformatic analysis indicated that the entire VPg domain and the C-terminal part of the P16 contain unstructured amino acid stretches, which possibly prevented the formation of crystals.
If the inline PDF is not rendering correctly, you can download the PDF file here.
Balke I. Resevica G. Zeltins A. (2007). The ryegrass mottle virus genome codes for a sobemovirus 3C-like serine protease and RNA-dependent RNA polymerase translated via -1 ribosomal frameshifting. Virus Genes35(2) 395-398.
Bates H.J. Farjah M. Osman T.A.M. Buck K.W. (1995). Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus. J. Gen. Virol.76 1483-1491.
Callaway A.S. George C.G. Lommel S.A. (2004). A Sobemovirus coat protein gene complements long-distance movement of a coat protein-null Dianthovirus. Virology330 186-195.
Dunker A.K. Brown C.J. Lawson J.D. Iakoucheva L.M. Obradovic Z. (2002). Intrinsic disorder and protein function. Biochemistry41(21) 6573-6582.
Gayathri P. Satheshkumar P.S. Prasad K. Nair S. Savithri H.S. Murthy M.R. (2006). Crystal structure of the serine protease domain of Sesbania mosaic virus polyprotein and mutational analysis of residues forming the S1-binding pocket. Virology346(2) 440-451.
Govind K. Savithri H.S. (2010). Primer-independent initiation of RNA synthesis by SeMV recombinant RNA-dependent RNA polymerase. Virology401(2) 280-292.
Hebrard E. Bessin Y. Michon T. Longhi S. Uversky V.N. Delalande F. Van Dorsselaer A. Romero P. Walter J. Declerck N. Fargette D. (2009). Intrinsic disorder in Viral Proteins Genome-Linked: Experimental and predictive analyses. Virol. J.6 23.
Kalinina N.O. Rakitina D.V. Solovyev A.G. Schiemann J. Morozov S.Y. (2002). RNA helicase activity of the plant virus movement proteins encoded by the first gene of the triple gene block. Virology296(2) 321-329.
Luft J.R. Collins R.J. Fehrman N.A. Lauricella A.M. Veatch C.K. DeTitta G.T. (2003). A deliberate approach to screening for initial crystallization conditions of biological macromolecules. J. Struct. Biol.142 170-179.
Makinen K. Makelainen K. Arshava N. Tamm T. Merits A. Truve E. Zavriev S. Saarma M. (2000). Characterization of VPg and the polyprotein processing of cocksfoot mottle virus (genus Sobemovirus). J. Gen. Virol.81(11) 2783-2789.
Mandahar C. (2006). Positive-sense viral RNA. In: Multiplication of RNA Plant Viruses. Mandahar C. (ed.). Springer Verl Dordrecht pp. 29-70.
Meier M. Paves H. Olspert A. Tamm T. Truve E. (2006). P1 protein of Cocksfoot mottle virus is indispensable for the systemic spread of the virus. Virus Genes32(3) 321-326.
Meier M. Truve E. (2007). Sobemoviruses possess a common CfMV-like genomic organization. Arch. Virol.152(3) 635-640.
Nair S. Savithri H.S. (2010a). Natively unfolded nucleic acid binding P8 domain of SeMV polyprotein 2a affects the novel ATPase activity of the preceding P10 domain. FEBS Lett.584(3) 571-576.
Nair S. Savithri H.S. (2010b). Processing of SeMV polyproteins revisited. Virology396(1) 106-117.
Pantazatos D. Kim J.S. Klock H.E. Stevens R.C. Wilson I.A. Lesley S.A. Woods V. L. Jr. (2004). Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS. Proc. Natl. Acad. Sci. USA101(3) 751-756.
Plevka P. Tars K. Zeltins A. Balke I. Truve E. Liljas L. (2007). The three-dimensional structure of ryegrass mottle virus at 2.9 A resolution. Virology369(2) 364-374.
Prilusky J. Felder C.E. Zeev-Ben-Mordehai T. Rydberg E.H. Man O. Beckmann J.S. Silman I. Sussman J.L. (2005). FoldIndex: A simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics21(16) 3435-3438.
Romero P. Obradovic Z. Kissinger C.R. Villafranca J.E. Dunker A.K. (1997). Identifying Disordered Regions in Proteins from Amino Acid Sequences. Proc. I.E.E.E. International Conference on Neural Networks pp. 90-95.
Satheshkumar P.S. Gayathri P. Prasad K. Savithri H.S. (2005). "Natively unfolded" VPg is essential for Sesbania mosaic virus serine protease activity. J. Biol. Chem.280(34) 30291-30300.
Satheshkumar P.S. Lokesh G.L. Savithri H.S. (2004). Polyprotein processing: Cis and trans proteolytic activities of Sesbania mosaic virus serine protease. Virology318(1) 429-438.
Sereme D. Lacombe S. Konate M. Pinel-Galzi A. Traore V.S. Hebrard E. Traore O. Brugidou C. Fargette D. Konate G. (2008). Biological and molecular characterization of a putative new sobemovirus infecting Imperata cylindrica and maize in Africa. Arch. Virol.153(10) 1813-1820.
Sire C. Bangratz-Reyser M. Fargette D. Brugidou C. (2008). Genetic diversity and silencing suppression effects of Rice yellow mottle virus and the P1 protein. Virol. J.5 55.
Tamm T. Truve E. (2000). Sobemoviruses. J. Virol.74(14) 6231-6241.
Vacic V. Oldfield C.J. Mohan A. Radivojac P. Cortese M.S. Uversky V.N. Dunker A. K. (2007). Characterization of molecular recognition features MoRFs and their binding partners. J. Proteome Res.6(6) 2351-2366.
Weinheimer I. Boonrod K. Moser M. Zwiebel M. Fullgrabe M. Krczal G. Wassenegger M. (2010). Analysis of an autoproteolytic activity of rice yellow mottle virus silencing suppressor P1. Biol. Chem.391(2-3) 271-281.
Zell R. Fritz H.J. (1987). DNA mismatch-repair in Escherichia coli counteracting the hydrolytic deamination of 5-methyl-cytosine residues. EMBO J.6 1809-1815.
Zhang F.Y. Toriyama S. Takahashi M. (2001). Complete nucleotide sequence of ryegrass mottle virus: A new species of the genus sobemovirus. J. Gen. Plant Pathol.67 63-68.