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In eukaryotes, the major nuclear export pathway for mature mRNAs uses the dimeric receptor TAP/p15, which is recruited to mRNAs via the multisubunit TREX complex, comprising the THO core and different export adaptors. Viruses that replicate in the nucleus adopt different strategies to hijack cellular export factors and achieve cytoplasmic translation of their mRNAs. No export receptors are known in plants, but Arabidopsis TREX resembles the mammalian complex, with a conserved hexameric THO core associated with ALY and UIEF proteins, as well as UAP56 and MOS11. The latter protein is an orthologue of mammalian CIP29. The nuclear export mechanism for viral mRNAs has not been described in plants. To understand this process, we investigated the export of mRNAs of the pararetrovirus CaMV in Arabidopsis and demonstrated that it is inhibited in plants deficient in ALY, MOS11 and/or TEX1. Deficiency for these factors renders plants partially resistant to CaMV infection. Two CaMV proteins, the coat protein P4 and reverse transcriptase P5, are important for nuclear export. P4 and P5 interact and co-localise in the nucleus with the cellular export factor MOS11. The highly structured 5′ leader region of 35S RNAs was identified as an export enhancing element that interacts with ALY1, ALY3 and MOS11 in vitro., Graphical Abstract Graphical AbstractThe nuclear export of CaMV 35S RNA in Arabidopsis.

Datamining and metagenomic analyses of 277 open reading frame sequences of bipartite RNA viruses and variants in the genus Nepovirus documented how delicate it can be to unequivocally identify species, in particular subgroup A and C species, based on some of the currently adopted taxonomic demarcation criteria. It suggests a possible need for their amendment to accommodate pangenome information. In addition, we revealed a host-dependent structure of arabis mosaic virus (ArMV) populations at a cladistic level and confirmed a phylogeographic structure of grapevine fanleaf virus (GFLV) populations. We also identified new putative recombinant events for species of subgroups A, B and C. The evolutionary specificity of some capsid regions of ArMV and GFLV that were previously described and biologically validated as vector determinant was circumscribed in silico. Furthermore, a C-terminal segment of the RNA-dependent RNA polymerase of subgroup A species was predicted as a putative host range determinant based on statistically supported higher π values for GFLV and ArMV isolates infecting Vitis spp. compared to non-Vitis infecting ArMV isolates. This study illustrated how sequence information obtained via high throughput sequencing can increase our understanding of mechanisms that modulate virus diversity and evolution and create new opportunities for advancing studies on the biology of economically important plant viruses.

The nuclear export of cellular mRNAs is a complex process that requires the orchestrated participation of many proteins that are recruited during the early steps of mRNA synthesis and processing. This strategy allows the cell to guarantee the conformity of the messengers accessing the cytoplasm and the translation machinery. Most transcripts are exported by the exportin dimer Nuclear RNA export factor 1 (NXF1)–NTF2-related export protein 1 (NXT1) and the transcription–export complex 1 (TREX1). Some mRNAs that do not possess all the common messenger characteristics use either variants of the NXF1–NXT1 pathway or CRM1, a different exportin. Viruses whose mRNAs are synthesized in the nucleus (retroviruses, the vast majority of DNA viruses, and influenza viruses) exploit both these cellular export pathways. Viral mRNAs hijack the cellular export machinery via complex secondary structures recognized by cellular export factors and/or viral adapter proteins. This way, the viral transcripts succeed in escaping the host surveillance system and are efficiently exported for translation, allowing the infectious cycle to proceed. This review gives an overview of the cellular mRNA nuclear export mechanisms and presents detailed insights into the most important strategies that viruses use to export the different forms of their RNAs from the nucleus to the cytoplasm.

The nuclear export of mRNAs is a complex process, involving the participaton of numerous proteins, the recruitement of which starts during the early steps of mRNAs biosynthesis and maturation. This strategy allows the cell to export only mature and non-defective transcripts to the cytoplasm where they are directed to the translational machinery. The vast majority of mRNAs is exported by the dimeric transport receptor TAP-p15, which is mainly recruited by the large multiprotein complex TREX-1. Other mRNAs that do not display all typical features of a mature transcript use variants of the TAP-p15 export pathway or recruit the alternative export receptor CRM1. Most DNA viruses, retroviruses, and influenza viruses, the mRNAs of which are synthesized in the nucleus, also use TAP-p15 and/or CRM1 to export their mRNAs. The highjacking of the cellular export machinery by viral mRNAs usually involves the presence of constitutive structural elements that directly load cellular export factors and/or viral adaptor proteins. Associated with the host export machinery, viral mRNAs escape host surveillance, are efficiently exported in the cytoplasm in order to be translated, and thus make possible the progress toward the later events of the virus life cycles.

Polyethylene glycol transfection of plant protoplasts represents an efficient method to incorporate foreign DNA and study transient gene expression. Here, we describe an optimized protocol to deliver small noncoding RNAs into Arabidopsis thaliana protoplasts. An example of application is provided by demonstrating the incorporation of a 20 nt long small noncoding RNA deriving from the 5' extremity of an A. thaliana cytosolic alanine tRNA into freshly isolated protoplasts.

Polyethylene glycol transfection of plant protoplasts represents an efficient method to incorporate foreign DNA and study transient gene expression. Here, we describe an optimized protocol to deliver small noncoding RNAs into Arabidopsis thaliana protoplasts. An example of application is provided by demonstrating the incorporation of a 20 nt long small noncoding RNA deriving from the 5' extremity of an A. thaliana cytosolic alanine tRNA into freshly isolated protoplasts.