Summary
Complementary DNA of the middle-component RNA of the melon strain of squash mosaic comovirus (SqMV) was cloned. Clones containing the coat protein genes were identified by hybridization with a degenerate oligonucleotide synthesized according to the amino acid sequence of a purified peptide fragment of the SqMV large coat protein. A clone containing of 2.5 kbp cDNA insert of SqMV M-RNA was sequenced. The total insert sequence of 2510 bp included a 2373 bp open reading frame (ORF) (encoding 791 amino acids), a 123 bp 3′-untranslated region, and a poly(A) region. This ORF is capable of encoding both the 42 and 22 k SqMV coat proteins. Direct N-terminal sequence analysis of the 22 k coat protein revealed its presence at the 3′ end of this ORF and the position of the proteolytic cleavage site (Q/S) used to separate the large and small coat proteins from each other. A putative location of the N-terminal proteolytic cleavage site of the 42 k coat protein (Q/N) was predicted by comparisons with the corresponding coat proteins of cowpea mosaic virus, red clover mottle virus, and bean-pod mottle virus. Although the available nucleotide sequences of these viruses revealed little similarity, their encoded coat proteins shared about 47% identity. The identity of the encoded 42 k and 22 k peptides was confirmed by engineering the respective gene regions for expression followed by transfer into tobacco protoplasts using the polyethylene glycol method. Both SqMV coat proteins were expressed in vivo as determined by their reactivity to SqMV coat protein specific antibodies.
Similar content being viewed by others
References
Bachmair A, Finley D, Varshavsky A (1986) In vitro half-life of a protein in function of its amino-terminal residue. Science 234: 179–286
Beachy RN, Loesch-Fries S, Tumer NE (1990) Coat protein-mediated resistance against virus infection. Annu Rev Phytopathol 28: 451–474
Bergman T, Jornvall H (1987) Electroblotting of individual polypeptides from SDS/polyacrylamide gels for direct sequence analysis. Eur J Biochem 169: 9–12
Bruening G (1978) Comovirus group. CMI/AAB Descriptions of Plant Viruses no 199
Campbell RN (1971) Squash mosaic virus. CMI/AAB Descriptions of Plant Viruses no 43
Chee PP (1991) Somatic embryogenesis and plant regeneration of squashCucurbito pepo L. cv. YC 60. Plant Cell Rep 9: 620–622
Chee PP (1992) Initiation and maturation of somatic embryos of squash (Cucurbito pepo). HortScience 27: 59–60
Chen Z, Stauffacher C, Li Y, Schmidt T, Wu B, Kamer G, Shanks M, Lomonossoff G, Johnson JE (1989) Protein-RNA interactions in an icosahedral virus at 3.0 Å resolution. Science 245: 154–159
Cleveland DW, Fischer SG, Krischner MW, Laemmli UK (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem 252: 1102–1106
Dayhof MO, Schwarz RM, Orcutt BC (1979) A model of evolutionary change in proteins. In: Dayhoff MO (ed) Atlas of protein sequence and structure, vol 5, suppl 3. National Biomedical Research Foundation, Washington, DC, pp 345–352
Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12: 387–395
Fang G, Grumet R (1990)Agrobacterium tumefaciens mediated transformation and regeneration of muskmelon plants. Plant Cell Rep 9: 160–164
Garcia JA, Schrijvers L, Tan A, Vos P, Wellink J, Goldbach R (1987) Proteolytic activity of cowpea mosaic virus encoded 24 K protein synthesized inEscherichia coli. Virology 159: 67–75
Goldbach R, Krijt J (1982) Cowpea mosaic virus-encoded protease does not recognise primary translation products of M RNAs of other comoviruses. J Virol 43: 1151–1154
Goldbach R, van Kammen A (1985) Structure, replication and expression of the bipartite genome of cowpea mosaic virus. In: Davies JW (ed) Molecular plant virology, vol 2. CRC Press, Boca Raton, pp 83–120
Gubler U, Hoffman BJ (1983) A simple and very efficient method for getting cDNA libraries. Gene 25: 263–269
Hanahan D (1986) Studies of transformation ofE. coli with plasmids. J Mol Biol 166: 557–562
Hiebert E, Purcifull DE (1981) Mapping of the two coat protein genes on the middle RNA component of squash mosaic virus (comovirus group). Virology 113: 630–636
Maniatis T, Fritsch E, Sambrook J (1982) Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor
Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinyllidene difluoride membranes. J Biol Chem 262: 10035–10038
Moghal SM, Francki RIB (1976) Towards a system for the identification and classification of potyviruses I. Serology and amino acid composition of six distinct viruses. Virology 73: 350–362
Moore DD (1987) Construction of recombinant DNA libraries. In: Ausubel FM et al (ed) Current protocols in molecular biology. Wiley, New York, pp 501–563
Negrutiu I, Shillito R, Potrykus I, Biasini G, Sala F (1987) Hybrid genes in the analysis of transformation conditions. Plant Mol Biol 8: 363–373
Negy JI, Maliga P (1976) Callus induction and plant regeneration from mesophyll protoplasts ofNicotiana sylvestris. Z Pflanzenphysiol 78: 453–455
Nelson MR, Kunhtsen HK (1973) Squash mosaic virus variability: review and serological comparisons of six biotypes. Phytopathology 63: 920–926
Nida DL, Anjos JR, Lomonossoff GP, Ghabrial SA (1992) Expression of cowpea mosaic virus coat protein precursor in transgenic tobacco plants. J Gen Virol 73: 157–163
Sanger F (1981) Determination of nucleotide sequences in DNA. Science 214: 1205–1210
Shanks S, Stanley J, Lomonossoff GP (1987) The primary structure of red clover mottle virus middle component RNA. Virology 155: 697–706
Slightom JL, Theisen TW, Koop BF, Goodman M (1987) Orangutan fetal globin genes: nucleotide sequences reveal multiple gene conversions during hominid phylogeny. J Biol Chem 262: 7472–7483
Slightom JL (1991) Custom polymerase-chain-reaction engineering of a plant expression vector. Gene 100: 251–255
Slightom JL, Drong RF, Sieu LC, Chee PP (1991) Custom polymerase chain reaction engineering plant expression vectors and genes for plant expression. Plant Mol Biol Manual B16: 1–55
van Wezenbeek P, Verver J, Harmsen J, Vos P, van Kammen A (1983) Primary structure and gene organization of the middle-component RNA of cowpea mosaic virus. EMBO J 2: 941–946
Vos P, Verver J, Jaegle M, Wellink J, van Kammen A, Goldbach R (1988) Two viral proteins involved in the proteolytic processing of cowpea mosaic virus polyproteins. Nucleic Acids Res 16: 1967–1985
Wang M, Gonsalves D (1990) ELISA detection of various tomato spotted wilt virus isolates using specific antisera to structural proteins of the virus. Plant Dis 74: 154–158
Weickert MJ, Chambliss GH (1989) Acid-phenol minipreps make excellent sequencing templates. USB Editorial Comments 16(2): 5–6
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M 13 mp 18 and pUC 19 vectors. Gene 33: 103–119
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Hu, J.S., Pang, S.Z., Nagpala, P.G. et al. The coat protein genes of squash mosaic virus: cloning, sequence analysis, and expression in tobacco protoplasts. Archives of Virology 130, 17–31 (1993). https://doi.org/10.1007/BF01318993
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01318993