Interaction between RNA-Dependent RNA Polymerase of Alfalfa Mosaic Virus and Its Template: Oxidation of Vicinal Hydroxyl Groups Blocksin VitroRNA Synthesis

Graaff, M. de; Thorburn, Christine; Jaspars, E.M.J.

doi:10.1006/viro.1995.0036

Cited by 7 publications

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“…Although the AMV coat protein co-purifies in polymerase preparations (Quadt et al, 1991), and coat protein has been described as an integral component of the polymerase protein (Degraaff, Thorburn, and Jaspars, 1995), the coat protein-polymerase interaction was not detected in yeast two-hybrid experiments (Van Der Heijden et al, 2001). Technical issues with the yeast two hybrid assay might explain the results, because it was also found that yeast growth was not observed when full-length P1 and P2 proteins were used in the binding and activation domain constructs (Van Der Heijden et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

Alfalfa mosaic virus coat protein bridges RNA and RNA-dependent RNA polymerase in vitro

et al. 2007

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Alfalfa mosaic virus (AMV) RNA replication requires the viral coat protein (CP). AMV CP is an integral component of the viral replicase; moreover, it binds to the viral RNA 3'-termini and induces the formation of multiple new base pairs that organize the RNA conformation. The results described here suggest that AMV coat protein binding defines template selection by organizing the 3'-terminal RNA conformation and by positioning the RNA-dependent RNA polymerase (RdRp) at the initiation site for minus strand synthesis. RNA-protein interactions were analyzed by using a modified Northwestern blotting protocol that included both viral coat protein and labeled RNA in the probe solution ("far-Northwestern blotting"). We observed that labeled RNA alone bound the replicase proteins poorly; however, complex formation was enhanced significantly in the presence of AMV CP. The RNA-replicase bridging function of the AMV CP may represent a mechanism for accurate de novo initiation in the absence of canonical 3' transfer RNA signals.

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Section: Discussionmentioning

confidence: 99%

Alfalfa mosaic virus coat protein bridges RNA and RNA-dependent RNA polymerase in vitro

et al. 2007

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The 3′ End of Hepatitis E Virus (HEV) Genome Binds Specifically to the Viral RNA-Dependent RNA Polymerase (RdRp)

2001

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Hepatitis E virus (HEV) is the major cause of acute epidemic and sporadic hepatitis in the developing world. It is a positive-strand RNA virus with a genome length of about 7.2 kb. The replication mechanism of this virus is virtually unexplored. Identification of the regulatory elements involved in initiation of replication may help in designing specific inhibitors for therapy. In the positive-stranded RNA viruses the initiation of replication requires interaction of the 3' end of genome with its RNA-dependent RNA polymerase (RdRp) and possibly host-derived cofactors for synthesis of the minus-strand replicative intermediate. Secondary structure prediction of the conserved 3' end of the infectious HEV genome was carried out to identify possible stem-loop structures necessary for RNA-protein interaction and the model was confirmed by structure probing experiments. Electrophoretic mobility-shift assays showed specific binding of purified and refolded recombinant HEV RdRp protein to the 3' end of its RNA genome containing the poly(A) stretch. Mutations at the 3' end, in which the stem-loop structures were partially or completely destroyed or recreated revealed that the two stem-loop structures SL1 and SL2 at the 3' end and the poly(A) stretch are necessary for this binding. The interacting nucleotides in such an interaction were further identified by generating footprints of the complex by Pb(II)-induced hydrolysis. This specific binding of viral RdRp to the 3' end of HEV RNA directs the synthesis of complementary-strand RNA and thus such a binding domain might assume the role of a possible cis-acting element as a potential site for the initiation of replication.

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Activation of the alfalfa mosaic virus genome by viral coat protein in non-transgenic plants and protoplasts. The protection model biochemically tested

Houwing

Jaspars

2000

Archives of Virology

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In non-transgenic host plants and protoplasts alfalfa mosaic virus displays a strong need for coat protein when starting an infection cycle. The "protection model" states that the three viral RNAs must have a few coat protein subunits at their 3' termini in order to protect them in the host cell against degradation by 3'- to- 5' exoribonucleases [Neeleman L, Van der Vossen EAG, Bol JF (1993) Virology 196: 883-887]. We demonstrated that the naked genome RNAs are slightly infectious, if the inoculation is done at very high concentrations, or if it is preceded by an additional inoculation with the RNAs 1 and 2 (encoding subunits for the viral RNA polymerase). This could mean that the necessity for protection by coat protein is lost if the RNAs in large quantities can overcome the activity of the degrading enzymes, or are protected by association with the RNA polymerase, respectively. However, after having tested in protoplasts the survival of separately preinoculated naked RNA 1 during several hours before RNA 2 was inoculated, on the one hand, or of simultaneously inoculated RNAs 1 and 2, with cycloheximide in the medium during the first hours after inoculation, on the other hand, we had to conclude that the viral genome RNAs are quite stable in the cell in the absence of coat protein or RNA polymerase, respectively. This invalidates the protection model. Accommodation of the above findings by our published "messenger release model" for genome activation [Houwing CJ, Jaspars EMJ (1993) Biochimie 75: 617-621] is discussed.

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Interaction between RNA-Dependent RNA Polymerase of Alfalfa Mosaic Virus and Its Template: Oxidation of Vicinal Hydroxyl Groups Blocksin VitroRNA Synthesis

Cited by 7 publications

References 0 publications

Alfalfa mosaic virus coat protein bridges RNA and RNA-dependent RNA polymerase in vitro

Alfalfa mosaic virus coat protein bridges RNA and RNA-dependent RNA polymerase in vitro

The 3′ End of Hepatitis E Virus (HEV) Genome Binds Specifically to the Viral RNA-Dependent RNA Polymerase (RdRp)

Activation of the alfalfa mosaic virus genome by viral coat protein in non-transgenic plants and protoplasts. The protection model biochemically tested

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