Analysis of Putative Interactions Between Potyviral Replication Proteins and Plant Retinoblastoma Proteins

Sequence comparisons suggest that the RNA-dependent RNA polymerase (NIb) of potyviruses and bymoviruses, as well as the viral polymerase of potexviruses may contain a putative retinoblastoma protein (pRb) binding motif. The possibility that the potyviral NIb may function in the nucleus through interactions with plant pRb-related (RBR) proteins, and the modifications of the cell cycle was investigated by a combination of mutagenesis of the NIb and yeast two-hybrid system (YTHS). Mutation of a highly conserved glutamic acid residue in the putative pRb-binding motif of the NIb had no detectable phenotypic effect on replication of Potato virus A (PVA). Furthermore, the NIb proteins from Potato virus V and PVA failed to interact with maize or tobacco RBR proteins in yeast. Although the conservation of the motif for pRb interaction in plant RNA viruses is intriguing, these proteins from plant RNA viruses appear not to interact with plant RBR proteins.     

Regulation of MSV and WDV virion-sense promoters by WDV nonstructural proteins: a role for their retinoblastoma protein-binding motifs

In this work we demonstrate that wheat dwarf virus (WDV) RepA can activate WDV and maize streak virus (MSV) virion (V)-sense expression in plant tissues. Rep alone does not have any effect on the silent WDV promoter and it represses the basal MSV promoter activity. MSV promoter activation by RepA depends on an intact RepA retinoblastoma protein (RB)-binding domain. Promoter repression by Rep also depends on this domain to some extent. Mutation of the RepA RB-binding domain has no effect on WDV promoter activation. The WDV promoter contains two sites that fit the consensus E2F-binding site. One, WDV1, binds human E2F-1 in one-hybrid assays in yeast. It also binds specifically to maize and wheat proteins in vitro and, when fused to a minimal 35S promoter, it confers responsiveness to RepA only when the RepA RB-binding domain and the WDV1 site are intact. In the whole WDV V-sense promoter context, mutations of this sequence have no effect, suggesting that additional sequences are important for RepA-mediated promoter activation.     

Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures

RepA, the replication initiator protein of Pseudomonas pPS10 plasmid, is made of two winged-helix (WH) domains. RepA dimers undergo a structural transformation upon binding to origin DNA sequences (iterons), resulting in monomerization and alpha-helix into beta-strand conversion. This affects the N-terminal domain (WH1) and generates a metastable intermediate. Here it is shown that the interaction of short dsDNA oligonucleotides, including iteron or operator RepA targets, with the isolated WH1 domain promotes the assembly of different nanostructures. These range from irregular aggregates to amyloid spheroids and fibers. Their intrinsic order inversely correlates with the extent of the transformation induced by each DNA sequence on RepA. However, DNA is not a constituent of the assembled fibers, in agreement with the protein-only principle for amyloid structure. Thus, the RepA-WH1 domain on DNA binding mimics the behavior of the mammalian prion protein. The stretch of amino acids responsible for WH1 aggregation has been identified, leading to the design of mutants with enhanced or reduced amyloidogenicity and the synthesis of a peptide that assembles into a cross-beta structure. RepA amyloid assemblies could have a role in the negative regulation of plasmid replication. This article underlines the potential of specific nucleic acid sequences in promoting protein amyloidogenesis at nearly physiological conditions.