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.

FIG. 1

Amino acid sequences of FBNYV Clink and its homologues in other nanoviruses. The GenBank accession numbers of the corresponding genomic DNAs are as follows: banana bunchy top virus (BBTV), L41518; FBNYV, AJ132187; milk vetch dwarf virus (MDV), AB000923; subterranean clover stunt virus (SCSV), U16732. Comparison was done using the PileUp program of the University of Wisconsin Genetics Computer Group. Conserved amino acids of the pRB-binding and F-box motifs are marked in black. Amino acids of FBNYV Clink altered by mutation are indicated by asterisks.

FIG. 2

In vitro interaction of Clink and its mutants with human pRB and MsSKP1 proteins. Western blots of protein complexes retained by glutathione-Sepharose beads incubated with different combinations of bacterial extracts expressing wild-type and mutant His₆-Clink and GST-pRB fusion proteins (A) or His₆-Clink and GST-MsSKP1 fusion proteins (B) are shown. The antibodies used are indicated at the bottom, and the respective proteins or protein combinations are indicated at the top. Lanes loaded with crude extracts from bacteria are labeled “extracts” or “ex.,” and those loaded with complexes retained on the beads are labeled “complexes” or “com.” The term “Clink^LxRxA” stands for Clink^{C112R, E114A}. Molecular masses of marker proteins (M) are indicated on the left in kilodaltons. The expected sizes of the fusion proteins are 44.3 kDa for GST-MsSKP1, 91 kDa for GST-pRB, and 21.1 kDa for His₆-Clink. Even though wild-type and two mutated Clink proteins have nearly identical molecular masses, faster migration of the Clink^LxRxA protein is observed and may be due to a higher isoelectric point resulting from the amino acid substitutions (6.28 for wild-type Clink and 6.58 for Clink^LxRxA).

FIG. 3

Interaction of Clink and its mutants with ZmRb1 and MsSKP1 proteins in yeast. (A) Growth of double transformants on selective medium lacking uracil. The respective combinations of plasmids encoding fusion proteins with the GAL4 DNA-binding domain (pGBT9 derivatives) and fusion proteins with the GAL4 activation domain (pAD and pGAD424 derivatives) are indicated. (B) β-Galactosidase (β-gal.) activity of yeast extracts containing the indicated plasmid combinations. Each value is the mean of at least three independent transformants.

FIG. 4

Replication enhancement of FBNYV rep DNAs by Clink. Southern hybridization assays of total DNA extracted from leaf discs of N. benthamiana after agroinoculation with pairwise combinations of agrobacteria carrying pBin19 with dimers of a respective rep component (indicated at the bottom [FBNYV DNA]), along with agrobacteria carrying the pBin19 empty vector (v) or wild-type or mutated Clink-encoding FBNYV DNA (indicated at the top) are shown. Leaf disc medium was supplemented (+) or not (−) with plant hormones (horm.). rep-specific probes used for hybridization are shown at the bottom. Replicative forms of viral DNA are marked by ccc (covalently closed circular DNA) and ss (ssDNA). “Input DNA” denotes hybridization with residual pBin19-rep plasmids from agrobacteria used as the inoculum. In each series of experiments, equal amounts of total DNA were loaded, as judged by ethidium bromide staining of the gels (data not shown). The intensities of all DNA bands were quantified upon analysis of the blots with a PhosphorImager (Molecular Dynamics), and stimulation of replication was normalized to the signal produced by the input DNA. (A) DNA was extracted at 3 days after agroinoculation. (B) Leaf discs were agroinoculated with rep2 component and wild-type or mutated Clink-encoding DNA (indicated at the top), and DNA was extracted 1, 2, 3, and 6 days after agroinoculation, as indicated at the bottom.