Cowpea Mosaic Virus RNA Replication in Crude Membrane Fractions from Infected Cowpea and Chenopodium amaranticolor

Eggen, Rik I.L.; Kaan, Anita; Goldbach, Rob; Kammen, A. van

doi:10.1099/0022-1317-69-11-2711

Cited by 25 publications

(16 citation statements)

References 31 publications

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“…protoplasts were harvested to determine expression of sequences under control of the CaMV 35S promoter. Transfected protoplasts were stained by the indirect fluorescent antibody technique (Hibi et al, 1975;Maule et al, 1980), using one of the following antisera: anti-24K (Wellink et al, 1987), anti-VPg (Eggen et al, 1988), anti-32K (Franssen et al, 1984b), anti-ll0K (van Bokhoven et al, 1992 or anti-poliovirus 3D (kindly provided by Dr O. C. Richards, University of Utah Medical Center). Upon treatment with goat antirabbit antibody conjugated to fluorescein isothiocyanate (FITC; Nordic) the protoplasts were examined by fluorescence microscopy.…”

Section: Methodsmentioning

confidence: 99%

Protoplasts transiently expressing the 200K coding sequence of cowpea mosaic virus B-RNA support replication of M-RNA

Bokhoven

Verver

Wellink

et al. 1993

Journal of General Virology

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In order to identify the viral polymerase involved in cowpea mosaic virus (CPMV) RNA replication the 87K, ll0K and 170K proteins as well as the complete 200K polyprotein of CPMV B-RNA have been produced in cowpea protoplasts, using expression vectors based on the 35S promoter of cauliflower mosaic virus. CPMVspecific proteins were obtained that were indistinguishable from proteins found in CPMV-infected protoplasts. Proteolytic processing of precursor proteins synthesized from the expression vectors proved that the 24K protease contained within these proteins is active.Moreover, it was established that protoplasts transfected with the expression vector containing the entire 200K coding sequence, but not those transfected with vectors containing the 170K, ll0K or 87K coding sequences, were able to support replication of co-inoculated M-RNA. Despite the ability to support replication of M-RNA for protoplasts transiently expressing the 200K coding region, CPMV-specific RNA polymerase activity dependent on exogenous added template RNA could not be detected in extracts of these protoplasts in assays using poly(A)' oligo(U) or other template/primer combinations. In contrast, extracts of protoplasts in which poliovirus polymerase was produced exhibited RNA polymerase activity in such assays. These results indicate that the CPMV polymerase, unlike the poliovirus polymerase, is not able to use oligo(U) as a primer or cannot function on exogenous template and primer RNA.

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Section: Methodsmentioning

confidence: 99%

Protoplasts transiently expressing the 200K coding sequence of cowpea mosaic virus B-RNA support replication of M-RNA

Bokhoven

Verver

Wellink

et al. 1993

Journal of General Virology

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“…A strong hybridization was detected with minus-strand synthetic RNA2, indicating that the ToRSV plus strand was synthesized from the endogenous templates. In contrast, the labeled RdRp products hybridized very weakly to ToRSV plus-strand RNA2, suggesting that the ToRSV minus strand was synthesized at very low levels (if at all (17), and a potyvirus (Plum pox virus) (33). Analysis of the RdRp products of other plant picornavirus-like viruses has revealed that plus-strand RNA is synthesized predominantly; i.e., the minus strand is synthesized at very low levels or not at all (17,31,33,47).…”

mentioning

confidence: 99%

Tomato Ringspot Virus Proteins Containing the Nucleoside Triphosphate Binding Domain Are Transmembrane Proteins That Associate with the Endoplasmic Reticulum and Cofractionate with Replication Complexes

Han

Sanfaçon²

2003

J Virol

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Replication of all known positive-strand RNA viruses occurs in replication complexes associated with intracellular membranes. The putative nucleoside triphosphate binding (NTB) protein of Tomato ringspot virus (ToRSV) contains a stretch of hydrophobic residues at its C terminus, suggesting that it may act as a membrane anchor for the replication complex. Anti-NTB antibodies detected two predominant proteins in membrane-enriched fractions (the 66-kDa NTB and 69-kDa NTB-VPg proteins) along with other, larger proteins. The proteins containing the NTB domain cofractionated with markers of the endoplasmic reticulum (ER) and with ToRSV-specific RNA-dependent RNA polymerase activity in sucrose gradients. ToRSV infection induced severe changes in the morphology of the ER in plants expressing an ER-targeted green fluorescent protein (ER-GFP), and proteins containing the NTB domain colocalized with ER-GFP in indirect immunofluorescence assays. The proteins containing the NTB domain have properties of integral membrane proteins. Proteinase K protection assays using purified membranes from infected plants revealed that although the central portion of the NTB domain is exposed to the cytoplasmic face of the membranes, an 8-kDa fragment, recognized by anti-VPg antibodies, is protected by the membranes. This fragment probably consists of the 3-kDa VPg and the 5-kDa stretch of hydrophobic residues at the C terminus of the NTB protein, suggesting a luminal location for the VPg in at least a portion of the molecules. These results provide evidence that proteins containing the NTB domain are transmembrane proteins associated with ER-derived membranes and support the hypothesis that one or several of the proteins containing the NTB domain anchor the replication complex to the ER.Replication of the genomes of all characterized positivestrand RNA viruses occurs in large complexes which are associated with intracellular membranes (5). Many positive-strand RNA viruses induce extensive proliferation and modification of intracellular membranes in their hosts, which often results in the accumulation of numerous membranous vesicles. Doublestranded RNA (dsRNA) replication intermediates and viral replication factors are associated with the membranous vesicles, which are thought to be the site of the replication (6,7,18,25,48,49,52). The requirement for intact membranes for successful virus replication has also been demonstrated using cell-free replication systems (2, 35, 59). Different viruses associate with and modify different types of intracellular membranes (5). Although the importance of the association of viral replication factors with intracellular membranes is well documented, the mechanisms by which replication complexes are fixed on specific membrane sites remain poorly understood.Picornaviruses and several plant viruses with similar genomic organization have been shown to replicate in association with membranes derived from the endoplasmic reticulum (ER) (6,42,47,51). One or several viral proteins are thought to act as membrane an...

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“…As described in Methods, these fractions were obtained according to the protocol of Eggen et al (1988), which allows the separation of a SF and of a CMF. These fractions were prepared either from inoculated leaves or from the systemically infected apical leaves of the plants.…”

Section: Detection and Subcellular Localization Of Viral Protein In Imentioning

confidence: 99%

“…Two fractions enriched in different subcellular compartments were obtained essentially as described by Eggen et al (1988) from inoculated or from apical, systemically infected C. quinoa or C. murale leaves. Fresh tissue (2 g) was ground with a Dounce homogenizer in 3 ml of buffer (50 mM-Tris-acetate pH 7-4, 10 mMpotassium acetate, 1 mM-EDTA, 5 mM-DTT and 0.5 mM-PMSF) and the homogenate was centrifuged for 15 min at 1000 g. The supernatant was adjusted to 20~ glycerol and centrifuged at 30000 g for 30 min at 4 °C.…”

Section: Cloning Of Gcmv Cdnas In the Expression Vectormentioning

confidence: 99%

Immunodetection of the proteins encoded by grapevine chrome mosaic nepovirus RNA2

Hibrand

Gall

Candresse

et al. 1992

Journal of General Virology

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Fragments of the putative non-structural proteins (44K and 46K) encoded by RNA2 of grapevine chrome mosaic nepovirus (GCMV) were expressed as fusion proteins in Escherichia coli and used to raise specific antisera. All three proteins encoded by GCMV RNA2 (viral coat protein, and the 44K and 46K proteins) were detected by immunoblotting in subcellular fractions prepared from the leaves of infected Chenopodium quinoa plants, confirming a previously proposed model of the GCMV RNA2-encoded polyprotein. In addition to the 44K protein, one of the antisera detected a 90K protein presumably representing a precursor of the 44K and 46K proteins. Whereas the 44K and coat proteins could be detected in both soluble and membrane fractions, the 46K protein was found to be specific to the membrane fraction. Analysis of the kinetics of accumulation of the proteins showed that the 44K and 46K proteins were very transient whereas the coat protein was more stable and could be detected up to 21 days after inoculation. These results provide the first direct in vivo data supporting the maturation map of the GCMV RNA2 polyprotein deduced from in vitro experiments.

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Cowpea Mosaic Virus RNA Replication in Crude Membrane Fractions from Infected Cowpea and Chenopodium amaranticolor

Cited by 25 publications

References 31 publications

Protoplasts transiently expressing the 200K coding sequence of cowpea mosaic virus B-RNA support replication of M-RNA

Protoplasts transiently expressing the 200K coding sequence of cowpea mosaic virus B-RNA support replication of M-RNA

Tomato Ringspot Virus Proteins Containing the Nucleoside Triphosphate Binding Domain Are Transmembrane Proteins That Associate with the Endoplasmic Reticulum and Cofractionate with Replication Complexes

Immunodetection of the proteins encoded by grapevine chrome mosaic nepovirus RNA2

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