Clink, a Nanovirus-Encoded Protein, Binds both pRB and SKP1

Abstract

Clink, a 20-kDa protein of faba bean necrotic yellows virus, a single-stranded DNA plant virus, interacts with pRB family members and a SKP1 homologue from Medicago sativa. An LxCxE motif and an F-box of Clink mediate the interactions with the respective proteins. The capacity of Clink to bind pRB correlates with its ability to stimulate viral replication. Interaction of a single protein with the cell cycle regulator pRB and SKP1, a constituent of the ubiquitin-protein turnover pathway, appears to be a novel feature. Hence, Clink may represent a new class of viral cell cycle modulators.A common strategy of DNA viruses is the creation of an environment favorable for efficient replication of their genome by subverting the cell cycle control of the host and forcing cells into DNA synthesis or S phase. In mammalian cells, a key cell cycle regulator is the retinoblastoma tumor suppressor protein pRB, which represses onset and progression into S phase by interacting with a wide range of cell cycle-related proteins. Among those are transcription factors of the E2F family that form complexes with hypophosphorylated pRB. During the G 1 /S transition, pRB is progressively phosphorylated by the action of cyclin-dependent kinases, and as a result, E2F is released from the complex and becomes available to activate the expression of S-phase-specific genes (14, 30).Oncoproteins of certain mammalian DNA tumor viruses, such as E1A of adenovirus type 6, E7 of human papillomavirus type 16, or the large T antigen protein of simian virus 40, stimulate the entry of cells into S phase by interaction with pRB through a short protein sequence comprising essentially the sequence motif LxCxE (10,45). This interaction abrogates the pRB-mediated block of cell cycle progression and may contribute to tumor formation (24). In addition, these or other viral proteins are involved in the neutralization of additional cell cycle regulators, in particular of the growth suppressor p53 (24,39). The interaction between the papillomavirus E7 and E6 proteins with pRB and p53, respectively, mediates the degradation of the latter by the ubiquitin-proteasome pathway (8,25,44). Hence, in addition to the interaction with growth suppressors, papillomaviruses make use of the protein degradation machinery to target these proteins to the 26S proteasome.Ubiquitination of proteins destined for degradation by the 26S proteasome is mediated by the action of the enzymatic complexes E1, E2, and E3 (21). E1 and E2 activate ubiquitin and catalyze the polyubiquitination of the substrate, which is thus marked for degradation by the 26S proteasome. A diverse class of complexes, the E3 ubiquitin-ligases, contains the elements of specificity for the substrates to be ubiquitinated. The

Geminiviruses Subvert Ubiquitination by Altering CSN-Mediated Derubylation of SCF E3 Ligase Complexes and Inhibit Jasmonate Signaling inArabidopsis thaliana

“…In plants, the Polerovirus P0 protein carries an F-box domain that allows its incorporation into an SCF complex to mediate degradation of AGO1, modulating gene silencing (Baumberger et al, 2007;Bortolamiol et al, 2007). The nanovirus Clink protein is also an F-box protein and can bind to both SKP1 and the cell cycle protein pRBR, affecting cell cycle regulation (Aronson et al, 2000).…”

ArabidopsisRETINOBLASTOMA-RELATED Is Required for Stem Cell Maintenance, Cell Differentiation, and Lateral Organ Production

“…In addition, viral RBR binding proteins, such as geminivirus AL or AC, are multifunctional , and the role of the RepA/RBR complex in the regulation of transcription of E2F site-containing promoters is not well understood (Munoz-Martin et al, 2003). Likewise, the nanovirus Clink protein binds RBR but also SKP1, which functions in the proteolytic degradation pathway (Aoyama and Chua, 1997;Aronson et al, 2000).…”

“…In addition, viral RBR binding proteins, such as geminivirus AL or AC, are multifunctional , and the role of the RepA/RBR complex in the regulation of transcription of E2F site-containing promoters is not well understood (Munoz-Martin et al, 2003). Likewise, the nanovirus Clink protein binds RBR but also SKP1, which functions in the proteolytic degradation pathway (Aoyama and Chua, 1997;Aronson et al, 2000).To establish conditional mutants that only affect RBR function, we used a chemically inducible RBRi approach to rapidly downregulate RBR expression. Our results show that conditional downregulation and loss of RBR function uncouples division and differentiation of meristemoid cells in leaves and disrupts the appropriate division and maintenance of meristem stem cells.…”

Advances in Small Isometric Multicomponent ssDNA Viruses Infecting Plants

“…Clink significantly increases replication of FBNYV DNA. The interaction of Clink with pRB and SKP1 has been demonstrated in yeast two-hybrid system and in vitro by pull-down assays with the proteins expressed in E. coli [4]. Similar interaction occurring in plant was demonstrated by FBNYV-based expression vector system [5].…”

“…In many of the Nanovirus or Babuvirus, several ssDNA components have been shown to encode Rep related proteins, of which only one Rep component that controls replication of other ssDNA components is known as master Rep (M-Rep) [94]. Replication of the DNAs is facilitated and enhanced by the action of 19 kDa Clink, a cell cycle linked protein [4]. Up to 12 distinct DNA components have been isolated from plants infected by different nano-or babuviruses.…”