The molecular characterization of the squash leaf curl virus BR1 movement protein: Defining BR1's role in systemic movement and host range determination

Bipartite geminiviruses such as squash leaf curl virus (SqLCV) encode two movement proteins (MPs) essential for systemic infection of host plants. The BR1 MP is the focus of the work presented here. My work implicates BR1 in determining the host range of the virus through the characterization of a naturally occurring mutant B component (B$\sb{\rm R}\sp{tdl}$) which has lost the ability to infect Nicotiana benthamiana, but remains fully infectious for pumpkin and squash. The mutation responsible for the host range alteration is a single missense mutation within the BR1 coding sequence. To more precisely examine the role of BR1 in host range and viral movement, I have introduced a series of missense and deletion mutations throughout the coding sequence and examined the effects of these mutations on viral infectivity in pumpkin, squash, and N. benthamiana. For BR1, a range of mutant phenotypes from partially to fully defective was observed that affected the overall level and rate of infectivity. In contrast to mutations in the BL1 MP, however, mutations in BR1 never affected the appearance of disease symptoms. Several mutations produced host specific phenotypes, generally retaining high levels of infectivity for pumpkin and squash while losing infectivity for N. benthamiana. Interestingly, functional SqLCV coat protein (CP) was found to specifically mask defective phenotypes of certain BR1 mutations. This result suggests a possible redundancy of function between the CP and BR1, and places the CP within the SqLCV movement pathway. Previous studies have shown BR1 to be a nuclear localized single-stranded (ss)DNA binding protein. To characterize the mode of action of BR1 in these processes further, I investigated the effects of mutations in BR1 on its nuclear localizing and ssDNA binding activities. In a collaborative effort with Anton Sanderfoot, I was able to identify two nuclear localization signals in the N-terminus of BR1, an SV40 and a bipartite type signal. Also, utilizing a variety of experimental approaches, I localized a ssDNA binding region of ${\sim}$70 amino acids to the N-terminus. This region shares homology to binding motifs identified for other ssDNA binding proteins from animal and bacterial cells and viruses.U of I OnlyETDs are only available to UIUC Users without author permissio