Orchid Fleck Virus Structural Proteins N and P Form Intranuclear Viroplasm-Like Structures in the Absence of Viral Infection

Abstract

bOrchid fleck virus (OFV) has a unique two-segmented negative-sense RNA genome that resembles that of plant nucleorhabdoviruses. In infected plant cells, OFV and nucleorhabdoviruses induce an intranuclear electron-lucent viroplasm that is believed to be the site for virus replication. In this study, we investigated the molecular mechanism by which OFV viroplasms are produced in vivo. Among OFV-encoded proteins, the nucleocapsid protein (N) and the putative phosphoprotein (P) were present in nuclear fractions of OFV-infected Nicotiana benthamiana plants. Transient coexpression of N and P, in the absence of virus infection, was shown to be sufficient for formation of an intranuclear viroplasm-like structure in plant cells. When expressed independently as a fluorescent protein fusion product in uninfected plant cells, N protein accumulated throughout the cell, while P protein accumulated in the nucleus. However, the N protein, when coexpressed with P, was recruited to a subnuclear region to induce a large viroplasm-like focus. Deletion and substitution mutagenesis demonstrated that the P protein contains a nuclear localization signal (NLS). Artificial nuclear targeting of the N-protein mutant was insufficient for formation of viroplasm-like structures in the absence of P. A bimolecular fluorescence complementation assay confirmed interactions between the N and P proteins within subnuclear viroplasm-like foci and interactions of two of the N. benthamiana importin-␣ homologues with the P protein but not with the N protein. Taken together, our results suggest that viroplasm formation by OFV requires nuclear accumulation of both the N and P proteins, which is mediated by P-NLS, unlike nucleorhabdovirus viroplasm utilizing the NLS on protein N. R habdoviruses (family Rhabdoviridae, order Mononegavirales) have a broad range of hosts, including humans, livestock, fish, plants, and insects (1). They have bacilliform or bulletshaped virions containing a nonsegmented negative-sense RNA [(Ϫ)RNA] as the genome (2). All of the well-characterized animal rhabdoviruses replicate in the cytoplasm (1). However, the plant rhabdoviruses are divided into two groups, depending on their sites of replication and morphogenesis. Members of the genus Cytorhabdovirus replicate and undergo morphogenesis in the cytoplasm, whereas members of the genus Nucleorhabdovirus multiply in plant cell nuclei and their virions bud through the inner nuclear envelopes (3, 4).Orchid fleck virus (OFV), a Brevipalpus (Acari: Tenuipalpidae) mite-transmitted virus, has a unique two-segmented (Ϫ)RNA genome that resembles that of plant rhabdoviruses (5, 6). OFV is distributed worldwide and infects many kinds of orchids (5,7,8). OFV RNA1 (GenBank accession number AB244417; 6,413 nucleotides [nt]) contains five genes in the order 3=-N-P-3-M-G-5=, in which N encodes the nucleocapsid protein (open reading frame 1 [ORF1]), P encodes the phosphoprotein (ORF2), gene 3 encodes the candidate movement protein (ORF3), M encodes the matrix protein (ORF4), and G encodes the can...

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“…7b in Martin et al, 2012). In this study, interaction between ADV N and P was also demonstrated by BiFC, where the location of the N:P complex was subnuclear, similar to that reported for SYNV, PYDV, CoRSV and OFV (Bandyopadhyay et al, 2010;Min et al, 2010;Kondo et al, 2013;Ramalho et al, 2014), but different to that observed for LNYV, which was in the cytoplasm . The BiFC results for ADV N and P proteins confirmed the earlier observed co-localization results for these proteins, and showed that the interaction was similar to that reported for these two proteins from nucleorhabdoviruses rather than cytorhabdoviruses.…”

“…The interaction of N and P proteins is a common feature among not only the already characterized LNYV , PYDV (Bandyopadhyay et al, 2010) and SYNV (Min et al, 2010) but also the recently characterized dichorhaviruses coffee ringspot virus (CoRSV) (Ramalho et al, 2014) and orchid fleck virus (OFV) (Kondo et al, 2013), which have bipartite, negative-strand RNA genomes with clear sequence similarities to those of nucleorhabdoviruses, and the vertebrate rhabdovirus, vesicular stomatitis virus (VSV) (Moerdyk-Schauwecker et al, 2009; Fig. 7b in Martin et al, 2012).…”

Complete genome sequence and intracellular protein localization of Datura yellow vein nucleorhabdovirus

“…In contrast, MFSV N and P proteins co-localize in the nucleolus (Tsai et al, 2005) and PYDV N and P form large sub-nuclear aggregates (Bandyopadhyay et al, 2010;Martin et al, 2012). OFV and CoRSV N and P proteins interact and form large sub-nuclear foci with CoRSV N and P also co-localised throughout the cell (Kondo et al, 2013;Ramalho et al, 2014). The nuclear viroplasm-focussed N and P protein interaction and co-localization seen for dichorha-and nucleorhabdoviruses is in stark contrast to the co-localization and interaction of cytorhabdovirus N and P proteins in the cytoplasm outside the nucleus (Martin et al, 2012).…”

“…P proteins of OFV and CoRSV are also exclusively nuclear (Kondo et al, 2013;Ramalho et al, 2014). For all nucleorhabdoviruses where viral protein localization and interactions have been studied, N and P proteins have been shown to interact and co-localize in the nucleus (Goodin et al, 2001(Goodin et al, , 2002Tsai et al, 2005;Bandyopadhyay et al, 2010;Martin et al, 2012).…”

“…The N proteins of the nucleorhabdoviruses PYDV and maize fine streak virus (MFSV) also have an exclusive nuclear localization (Bandyopadhyay et al, 2010;Tsai et al, 2005), unlike N protein of the cytorhabdovirus lettuce necrotic yellows virus (LNYV), which is localized exclusively in the cell periphery as component of the cytoplasmic viroplasm (Martin et al, 2012). On the other hand, N proteins of the dichorhaviruses orchid fleck virus (OFV) and coffee ringspot virus (CoRSV) which are related to nucleorhabdoviruses, but have a bipartite genome, are distributed throughout the plant cell and imported into the nucleus through interactions with the exclusively nuclear P proteins (Kondo et al, 2013;Ramalho et al, 2014).…”