A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious as cloned DNA and purified virions

Here, we describe a tripartite circular single-stranded (ss) DNA mycovirus, named Fusarium graminearum gemytripvirus 1 (FgGMTV1). The genome of FgGMTV1 comprises three circular ssDNA segments (DNA-A, DNA-B, and DNA-C). Sequence alignments and phylogenetic analyses showed that FgGMTV1 is nested within the family Genomoviridae. We also constructed the first infectious DNA clones of a DNA mycovirus. Our results show that DNA-A and DNA-B are mutually interdependent for their replication and are associated with severely reduced colony growth and hypovirulence. DNA-C relies on DNA-A and DNA-B for replication and is necessary for the recovery of abnormal fungal phenotypes. DNA-C also enhances the accumulation of viral DNA in infected fungi and permits stable colonization and easy transmission via conidia. This is the first multipartite DNA virus isolated from a fungus. Our phylogenetic analyses also suggest that the multipartite genome of FgGMTV1 may have evolved from a monopartite genome of an ancient genomovirus.

Permeabilized mammalian cells as an experimental system for nuclear import of geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions

The plant-infecting geminiviruses deliver their genome and viral proteins into the host cell nucleus. Members of the family Geminiviridae possess either a bipartite genome composed of two ∼2.6 kb DNAs or a monopartite genome of ∼3.0 kb DNA. The bipartite genome of Bean dwarf mosaic virus (BDMV) encodes several karyophilic proteins, among them the capsid protein (CP) and BV1 (nuclear shuttle protein). A CP is also encoded by the monopartite genome of Tomato yellow leaf curl virus (TYLCV). Here, an in vitro assay system was used for direct demonstration of nuclear import of BDMV BV1 and TYLCV CP, as well as synthetic peptides containing their putative nuclear localization signals (NLSs). Full-length recombinant BDMV BV1 and TYLCV CP mediated import of conjugated fluorescently labelled BSA molecules into nuclei of permeabilized mammalian cells. Fluorescently labelled and biotinylated BSA conjugates bearing the synthetic peptides containing aa 3–20 of TYLCV CP (CP-NLS) or aa 84–106 of BDMV BV1 (BV1-NLS) were also imported into the nuclei of permeabilized cells. This import was blocked by the addition of unlabelled BSA–NLS peptide conjugates or excess unlabelled free NLS peptides. The CP- and BV1-NLS peptides also mediated nuclear import of fluorescently labelled BSA molecules into the nuclei of microinjected mesophyll cells of Nicotiana benthamiana leaves, demonstrating their biological function in intact plant tissue. BV1-NLS and CP-NLS were shown to mediate specific binding to importin α, both in vitro and in vivo. These results are consistent with a common nuclear-import pathway for CP and BV1, probably via importin α.