Role of the Sonchus Yellow Net Virus N Protein in Formation of Nuclear Viroplasms

Abstract

Plant rhabdoviruses are classified into the genera Cytorhabdovirus and Nucleorhabdovirus on the basis of their sites of replication, morphogenesis, and maturation (for a review, see reference 17). Sonchus yellow net nucleorhabdovirus (SYNV) replicates in the nucleus and is the most extensively characterized among the plant rhabdoviruses. SYNV encodes six genes in a negative-sense orientation; these six genes encode a nucleocapsid protein (N), a phosphoprotein (P), a putative movement protein (sc4), a matrix protein (M), a glycoprotein (G), and a large polymerase protein (L). The N, P, and L proteins are components of an infectious nucleocapsid core (15) with RNA-dependent RNA polymerase activity that can be purified from the nuclei of virus-infected cells (34,35). These core components form viroplasm-like structures within the nucleus that are thought to be the sites of viral replication (22). The N protein contains a carboxy (C)-terminal bipartite nuclear localization signal (NLS) that is required for nuclear import (7). The P protein when expressed alone localizes both inside and outside of the nucleus, but coexpression of the N and P proteins in plant and yeast (Saccharomyces cerevisiae) cells results in formation of compact subnuclear foci that are reminiscent of viroplasms (7). Sedimentation and immunological analyses have shown that in vivo associations of the N, P, and L proteins are required for RNA-dependent RNA polymerase activity (35). Yeast two-hybrid analyses and affinity chromatography experiments have also verified homologous (N-N) and heterologous (N-P) binding that is mediated by a region near the amino (N) terminus of the N protein (7). These results indicate that complex interactions of the N, P, and L proteins are required for subnuclear viroplasm formation and that the nucleocapsid cores within the viroplasms function in replication of genomic and antigenomic RNAs and in mRNA transcription (17).In the current study, we have conducted experiments to define the contributions of the N protein to the formation of viroplasms. These experiments include refined mapping to identify amino acids in an N-terminal helix-loop-helix motif of the N protein that result in subnuclear localization and N-N and N-P protein binding. We have also shown that mutations within the helix-loop-helix motif that disrupt N-N and N-P interactions interfere with the formation of subnuclear foci. The C-terminal NLS that is required for nuclear import of the N protein (7) mediates binding to importin ␣ homologues from yeast (ySrp1) and plant Arabidopsis thaliana (AtSrp1), but the P protein fails to bind to the importin ␣ homologues. These

Construction of a Sonchus Yellow Net Virus Minireplicon: a Step toward Reverse Genetic Analysis of Plant Negative-Strand RNA Viruses

“…Plasmids harboring the SYNV N, P, and L genes used in these experiments were inserted into pGD vectors as described previously (29). Mutant N proteins with various nuclear localization and interaction phenotypes were described by Deng et al (30). During the course of evaluating the P protein in pGD (29), we discovered a single nucleotide error in the initial sequence (39).…”

“…The N protein mutant derivatives (F42A, Y51A, and YIY) that affect N-N and N-P heterologous interactions and subnuclear localization (30), as well as the nuclear localization signal (NLS) mutant (KKRR) constructed by Goodin et al (40), were also analyzed in planta to assess their ability to function in MR amplification and reporter gene expression.…”

“…Proteins were extracted from agroinfiltrated N. benthamiana leaves harvested at 5 to 10 days postinfiltration (dpi) and evaluated by Western blotting (30). Proteins separated by SDS-PAGE were either stained with Coomassie blue or transferred to nitrocellulose membranes and detected with polyclonal antiserum specific to the SYNV N, P, or L protein (47) or monoclonal antibodies elicited against GFP (Clontech Laboratories, Mountain View, CA) and red fluorescent protein (RFP) (Huada, Beijing, China).…”

“…The requirement of the N, P, and L genes for MR replication should permit analyses of biological activity of core protein mutants that we have been previously able to characterize only by their biochemical properties and cytological effects. Among these, we have shown that mutations within the N protein are important for N and P protein interactions and for nuclear localization (30,40). To determine the transcriptional activities of these mutants, bacteria containing individual N protein mutant plasmids were mixed with bacteria harboring plasmids for expression of the P and L proteins, the pSYNV-MR eGFP-DsRed derivative, and the p19 and ␥b proteins.…”

“…Our approach relies on our previous studies with Agrobacterium tumefaciens-mediated expression of Sonchus yellow net nucleorhabdovirus (SYNV) core N, P, and L proteins in Nicotiana benthamiana (29,30). We have applied this methodology for delivery of the viral core protein genes into agroinfiltrated leaf cells in conjunction with plasmids containing a Cauliflower mosaic virus (CaMV) double 35S (35S 2 ) promoter for transcription of SYNV agRNA derivatives.…”