Comparison of the Nucleic Acid- and NTP-Binding Properties of the Movement Protein of Cucumber Mosaic Cucumovirus and Tobacco Mosaic Tobamovirus

Abstract

The cucumber mosaic virus (CMV) 3a movement protein (MP) was compared directly to the well-characterized tobacco mosaic virus (TMV) 30K MP by cloning the genes encoding these proteins into Escherichia coli, isolating the E. coli-expressed MPs, and characterizing them with regard to RNA- and NTP-binding activities. The two MPs were shown to bind single-stranded RNA and DNA cooperatively, but with no sequence specificity. However, discrete lengths of CMV RNA 3 could be protected against RNase digestion by the CMV 3a protein, indicating that the RNA was not uniformly covered by the MP after cooperative binding. The TMV 30K:RNA complex was more stable in NaCl than the CMV 3a:RNA complex; about 50% of the corresponding complexes were stable in 0.6 and 0.4 M NaCl, respectively. Both MPs could bind GTP strongly and UTP weakly, but not ATP or CTP. The CMV 3a protein expressed either in E. coli or in planta from RNA 3 of CMV was tagged at its C-terminus with six histidine residues, which facilitated its purification by affinity chromatography on a matrix containing Ni(2+)-nitrilotriacetate. The soluble, His-tagged 3a proteins, affinity-purified from E. coli and zucchini squash, both were able bind CMV RNA 3 in vitro.

Mapping of the RNA-binding domain of the cucumber mosaic virus movement protein.

“…The 30K protein of TMV comprises two domains : domain ' A ', which interacts with nucleic acids only in the context of the complete movement protein, and has a low surface probability, and the independent domain ' B ', which has a high surface probability (Citovsky et al, 1992). The CMV 3a protein also binds single-stranded (ss) nucleic acids in vitro (Li & Palukaitis, 1996). To define the domain of the 3a protein required to bind ssRNA, we employed deletion mutagenesis using mutated 3a proteins from inclusion bodies as well as soluble glutathione Stransferase (GST) fusion proteins.…”

Nucleic acid-binding properties and subcellular localization of the 3a protein of brome mosaic bromovirus.

“…4 B). Additionally, the RNA-binding activity of B3a was affected by heat treatment, suggesting that the activity is structure-dependent, similar to CMV (Li & Palukaitis, 1996).…”

“…The RNA-protein complexes formed by B3a were more sensitive to NaCl than are those formed by the TMV 30 kDa MP (Citovsky et al, 1990) and were almost as sensitive as those formed by the MPs of CaMV (Citovsky et al, 1991), alfalfa mosaic virus (AlMV) (Schoumacher et al, 1992) and cucumber mosaic virus (CMV) (Li & Palukaitis, 1996). B3a also bound ss DNA, ds RNA and ds DNA and, like AlMV MP (Schoumacher et al, 1992), showed higher affinity to ss than to ds nucleic acids.…”

The C-terminal 33 amino acids of the cucumber mosaic virus 3a protein affect virus movement, RNA binding and inhibition of infection and translation

“…The gels were dried and subjected to autoradiography. Treatment of MP-RNA complexes with RNase A (200 ng) and electrophoresis was done as described previously (Li & Palukaitis, 1996). The formation of complexes in different concentrations of NaCl and the detection of labelled RNA in complexes by filtration of the solutions and liquid scintillation counting were done as described previously (Li & Palukaitis, 1996).…”

“…Nevertheless, the 3a protein, expressed from RNA 3 of CMV, is designated the virus movement protein (MP), since this protein is involved in a number of functions associated with movement (Canto & Palukaitis, 1999a, b;Ding et al, 1995a;Gal-On et al, 1995;Kaplan et al, 1995;Li & Palukaitis, 1996;Li et al, 2001). The 3a MP binds ssRNA cooperatively (Li & Palukaitis, 1996), forming tightly packed nucleoprotein complexes (Nurkiyanova et al, 2001). This virus-encoded protein also interacts with plasmodesmata and facilitates the movement of viral RNA from one cell to another, as well as into and out of the vasculature (Blackman et al, 1998;Canto et al, 1997;Ding et al, 1995a).…”