Role of the zinc-finger and basic motifs of chrysanthemum virus B p12 protein in nucleic acid binding, protein localization and induction of a hypersensitive response upon expression from a viral vector

Abstract

The genomes of carlaviruses encode cysteine-rich proteins (CRPs) of unknown function. The 12 kDa CRP of chrysanthemum virus B (CVB), p12, has been shown previously to induce a hypersensitive response (HR) when expressed from potato virus X (PVX). This study demonstrated that a p12-induced HR was preceded by induction of a number of genes related to pathogenesis, stress and systemic acquired resistance. p12 localized predominantly to the nucleus. Interestingly, it was found that p12 bound both RNA and DNA in vitro, but notably exhibited a preference for DNA in the presence of Zn 2+ ions. Mutational analysis of the p12 conserved sequence motifs demonstrated that the basic motif is required for p12 translocation to the nucleus, thus representing part of the protein nuclear localization signal, whereas the predicted zinc finger motif is needed for both Zn 2+ -dependent DNA binding and eliciting an HR in PVX-infected leaves. Collectively, these results link, for the first time, nuclear localization of the protein encoded by a cytoplasmically replicating virus and its DNA-binding capacity with HR induction. Furthermore, these data suggest that p12 may mediate induction of the host genes by binding to the plant genomic DNA, and emphasize that CVB p12 is functionally distinct from other known nuclear-localized proteins encoded by the plant positive-stranded RNA viruses.

An RNA Virus-Encoded Zinc-Finger Protein Acts as a Plant Transcription Factor and Induces a Regulator of Cell Size and Proliferation in Two Tobacco Species

“…Recently, we found that the p12 protein of chrysanthemum virus B (CVB) is implicated in host gene induction as a pathogenicity and virulence determinant (Lukhovitskaya et al, 2009). CVB belongs to the genus Carlavirus, which comprises 35 definitive species and 29 tentative members with conserved genome organization (Levay and Zavriev, 1991;Adams et al, 2004;Martelli et al, 2007).…”

“…Their positive-stranded genomic RNA encodes six genes, of which the gene for p12 occupies the extreme 39-terminal position (see Supplemental Figure 1A online; Adams et al, 2004;Martelli et al, 2007). CVB p12 and its homologs in other carlaviruses are Cys-rich proteins characterized by a central C-4 type zinc finger defined by its RCxRCxRxxPx 6-8 CDxxxC motif and an adjacent nuclear localization signal (NLS; see Supplemental Figure 1C online; Gramstat et al, 1990;Koonin et al, 1991;Lukhovitskaya et al, 2009; note that the carlavirus C-4 superfamily should not be confused with the C4 proteins of geminiviruses). The p12 protein binds both RNA and DNA but exhibits a preference for DNA in the presence of Zn 2+ and its pathogenic effects depend on translocation into the nucleus (Gramstat et al, 1990;Lukhovitskaya et al, 2009).…”

“…CVB p12 and its homologs in other carlaviruses are Cys-rich proteins characterized by a central C-4 type zinc finger defined by its RCxRCxRxxPx 6-8 CDxxxC motif and an adjacent nuclear localization signal (NLS; see Supplemental Figure 1C online; Gramstat et al, 1990;Koonin et al, 1991;Lukhovitskaya et al, 2009; note that the carlavirus C-4 superfamily should not be confused with the C4 proteins of geminiviruses). The p12 protein binds both RNA and DNA but exhibits a preference for DNA in the presence of Zn 2+ and its pathogenic effects depend on translocation into the nucleus (Gramstat et al, 1990;Lukhovitskaya et al, 2009). Since carlaviruses are positive-stranded RNA viruses and replicate in the cytoplasm, the role of the p12 protein nuclear localization and DNA binding in the virus-plant interactions has been enigmatic.…”

“…Since carlaviruses are positive-stranded RNA viruses and replicate in the cytoplasm, the role of the p12 protein nuclear localization and DNA binding in the virus-plant interactions has been enigmatic. We hypothesized that the p12 might act as a transcriptional activator localized to the plant nucleus (Lukhovitskaya et al, 2009).…”

Making sense of nuclear localization: A zinc-finger protein encoded by a cytoplasmically replicating plant RNA virus acts a transcription factor

“…1). 3,4 The ectopic expression of upp-L in the leaves results in cell proliferation and tissue growth phenotypically manifested as severe leaf malformation, the developmental alterations similar to the symptoms induced by CVB infection in the host species. The asymmetric changes in the expression pattern of key cell-cycle regulators (downregulation of cell-division inhibiting E2FC TF and upregulation of cell proliferation promoting E2FB) suggest that upp-L acts upstream of the core cell-cycle regulators conserved between animals and plants.…”