Comparison of in vivo and in vitro translation of Cowpea Mosaic Virus RNAs

Goldbach, Rob; Schilthuis, J.G.; Rezelman, G.

doi:10.1016/0006-291x(81)91716-2

Cited by 39 publications

(26 citation statements)

References 18 publications

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Section: Discussionmentioning

confidence: 99%

“…Each RNA has a small protein (VPg) covalently attached to its 5" terminus (Stanley et al, 1978: Daubert et al, 1978 and is polyadenylated at the 3' end (El Manna & Bruening, 1973). Translation in vitro and protoplast studies have shown that each RNA is initially translated into a large polypeptide which is subsequently cleaved by virusspecific proteases to functional virus proteins (Davies et al, 1977;Pelham, 1979;: Rottier et al, 1980Goldbach et al, 1981).Replication of CPMV RNA in infected plants takes place in a membrane-associated replication complex containing a 110K polypeptide encoded by the B RNA and two hostspecified polypeptides (Dorssers et al, 1984: Franssen et al, 1984. As intermediates in the replication process, partially double-stranded RNA molecules, termed replicative intermediates (RI), and completely double-stranded RNA molecules, termed replicative forms (RF), are produced (van Griensven & van Kammen, 1969: van Griensven et al, 1973.…”

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Double-stranded, Replicative Form RNA Molecules of Cowpea Mosaic Virus Are Not Infectious

Shanks

Lomonossoff

Evans

1985

Journal of General Virology

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SUMMARYVirus-specific double-stranded replicative form (RF) RNA was isolated from cowpeas infected with cowpea mosaic virus. The RF was assayed for infectivity in a local lesion host and a systemic host. In neither host was undenatured RF infectious, although infectivity was restored upon denaturation.Cowpea mosaic virus (CPMV) is a virus of legumes whose genome consists of two separately encapsidated, positive-sense RNA molecules, both of which are required for infection (van Kammen, 1967;de Jager, 1976). Both RNA molecules have recently been sequenced, the larger, B RNA having 5889 nucleotides, the smaller, M RNA having 3481 nucleotides (Lomonossoff & Shanks, 1983 ;van Wezenbeek et al., 1983). Each RNA has a small protein (VPg) covalently attached to its 5" terminus (Stanley et al., 1978: Daubert et al., 1978 and is polyadenylated at the 3' end (El Manna & Bruening, 1973). Translation in vitro and protoplast studies have shown that each RNA is initially translated into a large polypeptide which is subsequently cleaved by virusspecific proteases to functional virus proteins (Davies et al., 1977;Pelham, 1979;: Rottier et al., 1980Goldbach et al., 1981).Replication of CPMV RNA in infected plants takes place in a membrane-associated replication complex containing a 110K polypeptide encoded by the B RNA and two hostspecified polypeptides (Dorssers et al., 1984: Franssen et al., 1984. As intermediates in the replication process, partially double-stranded RNA molecules, termed replicative intermediates (RI), and completely double-stranded RNA molecules, termed replicative forms (RF), are produced (van Griensven & van Kammen, 1969: van Griensven et al., 1973. In this short communication we describe the isolation of RF molecules from CPMV-infected cowpeas and assays of their infectivity.The method of purification of the two RF forms of CPMV, RF-M and RF-B, involving DNase I treatment and CF-11 and Sepharose column chromatography, is essentially a standard method for preparing double-stranded RNA and has been widely used in the preparation of plant virus RFs (Zelcer et al., 1981: Jackson et al., 1971 including those from CPMV (Dorssers et al., 1984). Primary leaves of Vigna unguiculata var. 'Blackeye Early Ramshorn' were harvested 5 or 6 days after inoculation with CPMV strain SB. The mid-ribs were excised and the leaf tissue was frozen in liquid nitrogen and the RNA extracted and treated with DNase I as described by Zelcer et al. (1981). After the elimination of the DNase I by phenol extraction, the RNA was precipitated with ethanol and redissolved in water. An equal volume of 4 M-LiC1 was added and the solution was kept at -20 °C overnight. After centrifugation, the supernatant containing double-stranded RNA and tRNA was precipitated with ethanol. Partially double-stranded RNA (RI) could be recovered from the LiCl-insoluble precipitate.The LiCl-soluble RNA was dissolved in 100 mM-NaCI, 50 mM-Tris-HC1 pH 7.4, 1 mM-EDTA (STE) and ethanol was added to a final concentration of 17.5 % (v/v). The solution was loaded on to a...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Double-stranded, Replicative Form RNA Molecules of Cowpea Mosaic Virus Are Not Infectious

Shanks

Lomonossoff

Evans

1985

Journal of General Virology

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“…This cleavage gives rise to several polypeptides. One of them, a 60-kDa protein, is a precursor of both capsid proteins VP23 and VP37 [16].…”

Section: Discussionmentioning

confidence: 99%

Translational Stability of Plant Viral RNAs Microinjected into Living Cells

Huez

Cleuter

Bruck

et al. 1983

European Journal of Biochemistry

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Three different alternative structural features have been shown to be present at the 3′ terminus of plant viral RNAs: (a) a poly(A) track, (b) a tRNA‐like structure, (c) no special structural or sequence characteristic. We have compared the translational stability after injection into frog oocytes of a representative of each type: (a) the small genomic RNA (M‐RNA) of cowpea mosaic virus (CPMV), (b) the subgenomic mRNA for coat protein (RNA 4) of brome mosaic virus (BMV), (c) the subgenomic mRNA for coat protein (RNA 4) of alfalfa mosaic virus (AIMV). It has been shown that CPMV M‐RNA exhibits the highest translational stability. However, the stability of AIMV RNA 4 is remarkably high and moreover significantly higher than that of BMV RNA 4. We demonstrate that, for all three viral RNA species considered, the presence of a poly(A) segment at the 3′ end of the molecules improves the translational stability. From a comparative investigation in which AIMV RNA 4 was also injected into HeLa cells, it is concluded that the stability of a given non‐adenylylated mRNA depends on the nature of the cytoplastic environment.

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“…Both genomic RNAs are separately encapsidated into icosahedral particles, built up by two coat proteins (Fribourg et al, 1977). The comovirus genome is translated into a large polyprotein from which the mature proteins are released upon cleavage by a virusencoded protease (Franssen et al, 1984;Goldbach et al, 1981). In cowpea mosaic virus (CPMV), type member of the comovirus group, the RNA polymerase is located at the C terminus of the polyprotein encoded by the B RNA and is released upon cleavage at a Q/G site (Franssen et al, 1984).…”

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confidence: 99%