A Viral Movement Protein as a Nuclear Shuttle (The Geminivirus BR1 Movement Protein Contains Domains Essential for Interaction with BL1 and Nuclear Localization)

Abstract

For the nuclear replicating bipartite geminiviruses such as squash leaf curl to systemically infect the host requires the active participation of two virus-encoded movement proteins, BR1 and BL1. These act in a cooperative manner to transport the viral singlestranded DNA genome from its site of replication in the nucleus to the cell periphery (A.A. Sanderfoot, S.C. Lazarowitz [1995] Plant Cell 7: 1185-1194). We have proposed that BR1 functions as a nuclear shuttle protein, transporting the viral single-stranded DNA to and from the nucleus as a complex that is recognized by BL1 for movement to adjacent cells. To further investigate this, we expressed BR1 mutants known to affect viral infectivity in Spodoptera frugiperda insect cells and Nicotiana tabacum L. cv Xanthi protoplasts and found these to be defective in either their nuclear targeting or their ability to be redirected to the cell periphery when co-expressed with BL1. Translational fusions to p-glucuronidase and alanine-scanning mutagenesis further demonstrated that the C-terminal 86 amino acids of BR1 contains a domain(s) essential for i t s interaction with B L I and identified two nuclear localization signals within the N-terminal 113 residues of BR1. These nuclear localization signals were precisely located within distinct 16-and 22-peptide segments of BR1. These studies support and extend our model for squash leaf curl virus movement, showing that BR1 has a domain structure, with an N-terminal region required for nuclear targeting and a C-terminal region required for i t s interaction with BL1. Department of Microbiology, University of lllinois 23Regulated trafficking across the nuclear membrane is an essential component of many signal transduction pathways in eukaryotic cells and a fundamental aspect of infection by animal and plant viruses that replicate in the nucleus. The transport of proteins across the nuclear membrane is an energy-requiring process mediated by proteins associated with nuclear pore complexes (Newmeyer and Forbes, 1988). Although nuclear pores have an effective size-exclusion limit of approximately 60 kD, proteins smaller than this appear not to diffuse efficiently across the nuclear membrane (reviewed by Silver, 1991). Karyophilic proteins contain NLSs, short basic regions of approximately 10 to 20 amino acids (reviewed

TheArabidopsisAux/IAA Protein Family Has Diversified in Degradation and Auxin Responsiveness

“…To facilitate the quantitative measurement of proteolytic rates, we fused the first 111 amino acids of IAA17 (Figure 1), which includes all residues N-terminal to conserved domain III, to nuclear-targeted LUC (LUC:NLS). Although amino acids shown to function as a bipartite nuclear localization signal (NLS) in Arabidopsis IAA3 and IAA9 and pea IAA4 are present in IAA17(1-111), an exogenous squash leaf curl virus NLS (Sanderfoot et al, 1996;Worley et al, 2000) was included to try to ensure the proper subcellular localization of the wild-type and mutant LUC fusion proteins. IAA17 (1-111):LUC:NLS showed a rapid rate of degradation under basal conditions of ;10 min ( Figure 2A) in a cycloheximidebased degradation assay (Worley et al, 2000).…”

The capsid protein of tomato yellow leaf curl virus binds cooperatively to single-stranded DNA.

“…Bipartite geminiviruses have been shown to encode a specific nonstructural protein, BR1, that shuttles the viral genome into and out of the nucleus (Noueiry et al, 1994 ;Sanderfoot & Lazarowitz, 1995 ;Sanderfoot et al, 1996). Since TYLCV, a monopartite geminivirus, lacks BR1, another viral protein must mediate nuclear transport of the TYLCV genome.…”

“…Unlike most other plant viruses, both mono-and bipartite geminiviruses need to be imported into the host-plant cell nucleus for their replication (Timmermans et al, 1994). Studies of the bipartite geminivirus squash leaf curl virus (SqLCV) indicated that a protein (BR1) encoded by the DNA B component binds the genomic viral ssDNA and shuttles it into and out of the host cell nucleus (Sanderfoot & Lazarowitz, 1995 ;Sanderfoot et al, 1996). Also, Noueiry et al (1994) have shown that the BR1 protein of another bipartite geminivirus, bean dwarf mosaic geminivirus (BDMV), is involved in the nuclear export of viral DNA.…”

The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights

“…Integration may induce small ssDNA; nuclear shuttle, cell-to-cell movement (Pascal et al, 1994;Sanderfoot and Lazarowitz, (SqLCV) 1995;Sanderfoot et al, 1996) Cucumber mosaic virus (CMV) 3a ssRNA, ssDNA; cell-to-cell movement (Ding et al, 1995;Li and Palukaitis, 1996 HC-Pro ssRNA (Maia and Bernardi, 1996) *Additional movement proteins with SSB activity are described in Mushegian and Koonin (1993), Carrington et al (1996), and Lazarowitz and Beachy (1999). deletions, less than 100 bp, of plant DNA at the insertion site. In addition, integration also generates`®ller' DNA, less than 50 bp homologous to nearby plant DNA, at the T-DNA-plant junction.…”