ICTV Virus Taxonomy Profile: Nanoviridae.

Nanoviridae is a family of plant viruses (nanovirids) whose members have small isometric virions and multipartite, circular, single-stranded (css) DNA genomes. Each of the six (genus Babuvirus) or eight (genus Nanovirus) genomic DNAs is 0.9-1.1 kb and is separately encapsidated. Many isolates are associated with satellite-like cssDNAs (alphasatellites) of 1.0-1.1 kb. Hosts are eudicots, predominantly legumes (genus Nanovirus), and monocotyledons, predominantly in the order Zingiberales (genus Babuvirus). Nanovirids require a virus-encoded helper factor for transmission by aphids in a circulative, non-propagative manner. This is a summary of the ICTV Report on the family Nanoviridae, which is available at ictv.global/report/nanoviridae.

Taxonomy update for the family Alphasatellitidae: new subfamily, genera, and species.

Alphasatellites (family Alphasatellitidae) are circular, single-stranded DNA molecules (~1-1.4 kb) that encode a replication-associated protein and have commonly been associated with some members of the families Geminiviridae, Nanoviridae, and Metaxyviridae (recently established). Here, we provide a taxonomy update for the family Alphasatellitidae following the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021. The taxonomic update includes the establishment of the new subfamily Petromoalphasatellitinae. This new subfamily includes three new genera as well as the genus Babusatellite, which previously belonged to the subfamily Nanoalphasatellitinae. Additionally, three new genera and 14 new species have been established in the subfamily Geminialphasatellitinae, as well as five new species in the subfamily Nanoalphasatellitinae.

Identification of a novel nanovirus in parsley.

Using next-generation sequencing to characterize agents associated with a severe stunting disease of parsley from Germany, we identified a hitherto undescribed virus. We sequenced total RNA and rolling-circle-amplified DNA from diseased plants. The genome sequence of the virus shows that it is a member of the genus Nanovirus, but it lacks DNA-U4. In addition to the seven genomic DNAs of the virus, we identified a second DNA-R and seven distinct alphasatellites associated with the disease. We propose the name "parsley severe stunt associated virus" (PSSaV) for this novel nanovirus.

Genome diversity and evidence of recombination and reassortment in nanoviruses from Europe.

The recent identification of a new nanovirus, pea necrotic yellow dwarf virus, from pea in Germany prompted us to survey wild and cultivated legumes for nanovirus infections in several European countries. This led to the identification of two new nanoviruses: black medic leaf roll virus (BMLRV) and pea yellow stunt virus (PYSV), each considered a putative new species. The complete genomes of a PYSV isolate from Austria and three BMLRV isolates from Austria, Azerbaijan and Sweden were sequenced. In addition, the genomes of five isolates of faba bean necrotic yellows virus (FBNYV) from Azerbaijan and Spain and those of four faba bean necrotic stunt virus (FBNSV) isolates from Azerbaijan were completely sequenced, leading to the first identification of FBNSV occurring in Europe. Sequence analyses uncovered evolutionary relationships, extensive reassortment and potential remnants of mixed nanovirus infections, as well as intra- and intercomponent recombination events within the nanovirus genomes. In some virus isolates, diverse types of the same genome component (paralogues) were observed, a type of genome complexity not described previously for any member of the family Nanoviridae. Moreover, infectious and aphid-transmissible nanoviruses from cloned genomic DNAs of FBNYV and BMLRV were reconstituted that, for the first time, allow experimental reassortments for studying the genome functions and evolution of these nanoviruses.

High variability and rapid evolution of a nanovirus.

Nanoviruses are multipartite single-stranded DNA (ssDNA) plant viruses that cause important diseases of leguminous crops and banana. Little has been known about the variability and molecular evolution of these viruses. Here we report on the variability of faba bean necrotic stunt virus (FBNSV), a nanovirus from Ethiopia. We found mutation frequencies of 7.52 x 10(-4) substitutions per nucleotide in a field population of the virus and 5.07 x 10(-4) substitutions per nucleotide in a laboratory-maintained population derived thereof. Based on virus propagation for a period of more than 2 years, we determined a nucleotide substitution rate of 1.78 x 10(-3) substitutions per nucleotide per year. This high molecular evolution rate places FBNSV, as a representative of the family Nanoviridae, among the fastest-evolving ssDNA viruses infecting plants or vertebrates.

Twenty years of evolution and diversification of digitaria streak virus in Digitaria setigera.

Within the family Geminiviridae, the emergence of new species results from their high mutation and recombination rates. In this study, we report the variability and evolution of digitaria streak virus (DSV), a mastrevirus isolated in 1986 from the grass Digitaria setigera in an island of the Vanuatu archipelago. Viral DNA of DSV samples was amplified from D. setigera specimens, derived from the naturally infected original plant, which were propagated in different laboratories in France and Italy for more than 20 years. From the consensus sequences, the nucleotide substitution rate was estimated for the period between a sample and the original sequence published in 1987, as well as for the period between samples. In addition, the intra-host genetic complexity and diversity of 8 DSV populations with a total of 165 sequenced haplotypes was characterized. The evolutionary rate of DSV was estimated to be between 1.13 × 10-4 and 9.87 × 10-4 substitutions/site/year, within the ranges observed in other single-stranded DNA viruses and RNA viruses. Bioinformatic analyses revealed high variability and heterogeneity in DSV populations, which confirmed that mutant spectra are continuously generated and are organized as quasispecies. The analysis of polymorphisms revealed nucleotide substitution biases in viral genomes towards deamination and oxidation of single-stranded DNA. The differences in variability in each of the genomic regions reflected a dynamic and modular evolution in the mutant spectra that was not reflected in the consensus sequences. Strikingly, the most variable region of the DSV genome, encoding the movement protein, showed rapid fixation of the mutations in the consensus sequence and a concomitant dN/dS ratio of 6.130, which suggests strong positive selection in this region. Phylogenetic analyses revealed a possible divergence in three genetic lineages from the original Vanuatu DSV isolate.

Plant geminivirus rep protein induces rereplication in fission yeast.

The replication-associated protein (Rep) of geminiviruses, single-stranded DNA viruses of higher plants, is essential for virus replication. Since these viruses do not encode their own polymerases, Rep induces differentiated plant cells to reenter the cell cycle by interacting with the plant homologues of retinoblastoma proteins in order to activate the host DNA synthesis machinery. We have used fission yeast (Schizosaccharomyces pombe) as a model organism to analyze the impact of ectopically expressed African cassava mosaic virus Rep protein on the cell division cycle in closer detail. Upon expression, Rep showed its characteristic DNA cleavage activity, and about 10% of the cells exhibited morphological changes. They were elongated threefold, on average, and possessed a single but enlarged and less compact nucleus in comparison to noninduced or vector-only control cells. Flow cytometry of Rep-expressing cultures revealed a distinct subpopulation of Rep protein-containing cells with aberrant morphology. The other 90% of the cells were indistinguishable from control cells, and no Rep was detectable. Rep-expressing cells exhibited DNA contents beyond 2C, indicating ongoing replication without intervening mitosis. Because a second open reading frame (ORF), AC4, is present within the Rep gene, the role of AC4 was examined by destroying its start codon within the AC1 ORF. The results confirmed that Rep is necessary and sufficient to induce rereplication in fission yeast. The unique potential of this well-investigated model for dissecting the cell cycle control by geminiviral proteins is discussed.

Reconstitution of authentic nanovirus from multiple cloned DNAs.

We describe a new plant single-stranded DNA (ssDNA) virus, a nanovirus isolate originating from the faba bean in Ethiopia. We applied rolling circle amplification (RCA) to extensively copy the individual circular DNAs of the nanovirus genome. By sequence analyses of more than 208 individually cloned genome components, we obtained a representative sample of eight polymorphic swarms of circular DNAs, each about 1 kb in size. From these heterogeneous DNA populations after RCA, we inferred consensus sequences of the eight DNA components of the virus genome. Based on the distinctive molecular and biological properties of the virus, we propose to consider it a new species of the genus Nanovirus and to name it faba bean necrotic stunt virus (FBNSV). Selecting a representative clone of each of the eight DNAs for transfer by T-DNA plasmids of Agrobacterium tumefaciens into Vicia faba plants, we elicited the development of the typical FBNSV disease symptoms. Moreover, we showed that the virus thus produced was readily transmitted by two different aphid vector species, Aphis craccivora and Acyrthosiphon pisum. This represents the first reconstitution of a fully infectious and sustainably insect-transmissible nanovirus from its cloned DNAs and provides compelling evidence that the genome of a legume-infecting nanovirus is typically comprised of eight distinct DNA components.

Subterranean Clover Stunt Virus Revisited: Detection of Two Missing Genome Components.

Subterranean clover stunt virus (SCSV) is a type species of the genus Nanovirus in the family Nanoviridae. It was the first single-stranded DNA plant virus with a multipartite genome, of which genomic DNA sequences had been determined. All nanoviruses have eight genome components except SCSV, for which homologs of two genome components present in all other nanovirus genomes, DNA-U2 and DNA-U4, were lacking. We analysed archived and more recent samples from SCSV-infected legume plants to verify its genome composition and found the missing genome components. These results indicated that SCSV also has eight genome components and is a typical member of the genus Nanovirus.

Solution structure, divalent metal and DNA binding of the endonuclease domain from the replication initiation protein from porcine circovirus 2.

Circoviruses are the smallest circular single-stranded DNA viruses able to replicate in mammalian cells. Essential to their replication is the replication initiator, or Rep protein that initiates the rolling circle replication (RCR) of the viral genome. Here we report the NMR solution three-dimensional structure of the endonuclease domain from the Rep protein of porcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome in swine. The domain comprises residues 12-112 of the full-length protein and exhibits the fold described previously for the Rep protein of the representative geminivirus tomato yellow leaf curl Sardinia virus. The structure, however, differs significantly in some secondary structure elements that decorate the central five-stranded beta-sheet, including the replacement of a beta-hairpin by an alpha-helix in PCV2 Rep. The identification of the divalent metal binding site was accomplished by following the paramagnetic broadening of NMR amide signals upon Mn(2+) titration. The site comprises three conserved acidic residues on the exposed face of the central beta-sheet. For the 1:1 complex of the PCV2 Rep nuclease domain with a 22mer double-stranded DNA oligonucleotide chemical shift mapping allowed the identification of the DNA binding site on the protein and aided in constructing a model of the protein/DNA complex.

Nanovirus DNA-N encodes a protein mandatory for aphid transmission.

Nanoviruses possess a multipartite single-stranded DNA genome and are naturally transmitted to plants by various aphid species in a circulative non-propagative manner. Using the cloned genomic DNAs of faba bean necrotic stunt virus (FBNSV) for reconstituting nanovirus infections we analyzed the necessity of different virus components for infection and transmission by aphids. We found that in the absence of DNA-U1 and DNA-U2 symptom severity decreased, and in the absence of DNA-U1 the transmission efficiency decreased. Most significantly, we demonstrated that the protein encoded by DNA-N (NSP) is mandatory for aphid transmission. Moreover, we showed that the NSP of FBNSV could substitute for that of a distantly related nanovirus, pea necrotic yellow dwarf virus. Altering the FBNSV NSP by adding 13 amino acids to its carboxy-terminus resulted in an infectious but non-transmissible virus. We demonstrate that the NSP acts as a nanovirus transmission factor, the existence of which had been hypothesized earlier.

Analysis of DNAs associated with coconut foliar decay disease implicates a unique single-stranded DNA virus representing a new taxon.

The unique ecology, pathology and undefined taxonomy of coconut foliar decay virus (CFDV), found associated with coconut foliar decay disease (CFD) in 1986, prompted analyses of old virus samples by modern methods. Rolling circle amplification and deep sequencing applied to nucleic acid extracts from virion preparations and CFD-affected palms identified twelve distinct circular DNAs, eleven of which had a size of about 1.3 kb and one of 641 nt. Mass spectrometry-based protein identification proved that a 24 kDa protein encoded by two 1.3 kb DNAs is the virus capsid protein with highest sequence similarity to that of grabloviruses (family Geminiviridae), even though CFDV particles are not geminate. The nine other 1.3 kb DNAs represent alphasatellites coding for replication initiator proteins that differ clearly from those encoded by nanovirid DNA-R. The 641 nt DNA-gamma is unique and may encode a movement protein. Three DNAs, alphasatellite CFDAR, capsid protein encoding CFDV DNA-S.1 and DNA-gamma share sequence motifs near their replication origins and were consistently present in all samples analysed. These DNAs appear to be integral components of a possibly tripartite CFDV genome, different from those of any Geminiviridae or Nanoviridae family member, implicating CFDV as representative of a new genus and family.

Arabidopsis thaliana is a host of the legume nanovirus Faba bean necrotic yellows virus.

We report infection of Arabidopsis thaliana with the legume nanovirus Faba bean necrotic yellows virus (FBNYV) by its insect vector Aphis craccivora. Symptoms of FBNYV infection on A. thaliana include stunting and reduced apical dominance, and are rather mild, compared to the severe necrosis and early plant death induced by the virus in the natural host Vicia faba. An inoculation access period of 6h is sufficient to transmit FBNYV to A. thaliana. FBNYV is readily transmitted back from A. thaliana to V. faba, where it induces the characteristic severe disease symptoms. Hence, passage through A. thaliana does not affect FBNYV pathogenicity. FBNYV accumulates to the highest levels in roots and stems, compared to cauline and rosette leaves. In cauline leaves, the kinetics of virus accumulation correlates with the amount of master Rep protein accumulation.

Molecular diversity of Chickpea chlorotic dwarf virus in Sudan: high rates of intra-species recombination - a driving force in the emergence of new strains.

In Sudan Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus, family Geminiviridae) is an important pathogen of pulses that are grown both for local consumption, and for export. Although a few studies have characterised CpCDV genomes from countries in the Middle East, Africa and the Indian subcontinent, little is known about CpCDV diversity in any of the major chickpea production areas in these regions. Here we analyse the diversity of 146 CpCDV isolates characterised from pulses collected across the chickpea growing regions of Sudan. Although we find that seven of the twelve known CpCDV strains are present within the country, strain CpCDV-H alone accounted for ∼73% of the infections analysed. Additionally we identified four new strains (CpCDV-M, -N, -O and -P) and show that recombination has played a significant role in the diversification of CpCDV, at least in this region. Accounting for observed recombination events, we use the large amounts of data generated here to compare patterns of natural selection within protein coding regions of CpCDV and other dicot-infecting mastrevirus species.

Nanoviruses: genome organisation and protein function.

Nanoviruses, single-stranded DNA (ssDNA) plant viruses with a multipartite genome, share similarities with members of the Circoviridae family that infect mammals or birds as well as with the Geminiviridae, the only other plant virus family with circular ssDNA genomes. Although the virions of the latter are unique and different from that of the circoviruses, the mode of replication of viruses with monopartite or multipartite circular ssDNA genomes is strikingly similar. They multiply by rolling circle replication using virus-encoded multifunctional replication initiator proteins (Rep proteins) that catalyse initiation of ssDNA replication and resolution of replicative ssDNA into circular single-stranded virion DNA. All these ssDNA viruses exploit host polymerases for DNA synthesis and code for proteins that modulate the host's cell cycle favourably for virus multiplication. Recent three-dimensional structure analyses of a geminivirus and a parvovirus Rep protein have revealed an intriguing similarity between the catalytic domains of their respective Rep proteins. Furthermore, these structural data revealed that ssDNA virus replication initiator proteins might represent evolutionary intermediates between certain RNA-binding proteins and some multifunctional origin-binding proteins of papovaviruses.

The RXL motif of the African cassava mosaic virus Rep protein is necessary for rereplication of yeast DNA and viral infection in plants.

Geminiviruses, single-stranded DNA plant viruses, encode a replication-initiator protein (Rep) that is indispensable for virus replication. A potential cyclin interaction motif (RXL) in the sequence of African cassava mosaic virus Rep may be an alternative link to cell cycle controls to the known interaction with plant homologs of retinoblastoma protein (pRBR). Mutation of this motif abrogated rereplication in fission yeast induced by expression of wildtype Rep suggesting that Rep interacts via its RXL motif with one or several yeast proteins. The RXL motif is essential for viral infection of Nicotiana benthamiana plants, since mutation of this motif in infectious clones prevented any symptomatic infection. The cell-cycle link (Clink) protein of a nanovirus (faba bean necrotic yellows virus) was investigated that activates the cell cycle by binding via its LXCXE motif to pRBR. Expression of wildtype Clink and a Clink mutant deficient in pRBR-binding did not trigger rereplication in fission yeast.

Gene copy number is differentially regulated in a multipartite virus.

Multipartite viruses have a genome divided into several nucleic acid segments, each encapsidated separately. An evident cost for these viral systems, particularly if some segments are rare, is the difficulty of gathering one copy of each segment to ensure infection. Here, we investigate the segment frequency-related cost by monitoring the copy number of the eight single-gene segments composing the genome of a plant nanovirus. We show that some viral genes accumulate at low frequency, whereas others dominate. We further show that the relative frequency of viral genes impacts both viral accumulation and symptom expression, and changes specifically in different hosts. Earlier proposed benefits of viral genome segmentation do not depend on the segments' frequency and cannot explain our observations. We propose that the differential control of gene/segment copy number may represent an unforeseen benefit for multipartite viruses, which may compensate for the extra costs induced by the low-frequency segments.

Transcripts encoding the nanovirus master replication initiator proteins are terminally redundant.

The multicomponent single-stranded DNA plant nanoviruses encode unique master replication initiator (Rep) proteins. We have mapped the 5' and 3' termini of the corresponding polyadenylated mRNAs from faba bean necrotic yellows virus (FBNYV) and subterranean clover stunt virus and found that these are terminally redundant by up to about 160 nt. Moreover, the origin of viral DNA replication is transcribed into RNA that is capable of folding into extended secondary structures. Other nanovirus genome components, such as the FBNYV DNA encoding the protein Clink or an FBNYV DNA encoding a non-essential para-Rep protein, are not transcribed in such a unique fashion. Thus, terminally redundant mRNAs and the resulting transcription of the replication origin appear to be restricted to nanovirus master Rep DNAs. We speculate that this may be a way to regulate the expression of the essential master Rep protein.

The nanovirus-encoded Clink protein affects plant cell cycle regulation through interaction with the retinoblastoma-related protein.

Nanoviruses, multicomponent single-stranded DNA plant viruses, encode a unique cell cycle link protein, Clink, that interacts with retinoblastoma-related proteins (RBR). We have established transgenic Arabidopsis thaliana lines that conditionally express Clink or a Clink variant deficient in RBR binding. By controlled induction of Clink expression, we demonstrated the capacity of the Clink protein to alter RBR function in vivo. We showed that transcription of both S-phase-specific and G2/M-phase-specific genes was up-regulated depending on the RBR-binding proficiency of Clink. Concomitantly, ploidy levels increased in a substantial fraction of leaf cell nuclei. Also, leaf epidermis cells of transgenic plants producing Clink were smaller and more numerous, indicating additional cell divisions in this tissue. Furthermore, cytogenetic analyses following induction of Clink expression in mature leaves revealed the presence of metaphasic and anaphasic nuclei, clear evidence that Clink-mediated RBR inactivation is sufficient to induce quiescent cells to reenter cell cycle progression and, for at least a fraction of them, to pass through mitosis. Expression of Clink had no effect on genes transcribed by RNA polymerases I and III, suggesting that, in contrast to its mammalian homologue, A. thaliana RBR is not involved in the repression of polymerase I and polymerase III transcription. The results of these in vivo analyses firmly establish Clink as a member of the diverse class of multifunctional cell cycle modulator proteins encoded by small DNA viruses.

Solution structure of the endonuclease domain from the master replication initiator protein of the nanovirus faba bean necrotic yellows virus and comparison with the corresponding geminivirus and circovirus structures.

Nanoviruses are a family of plant viruses that possess a genome of multiple circular single-stranded DNA (ssDNA) components and are strikingly similar in their replication mode to the plant geminiviruses and to the circoviruses that infect birds or mammals. These viruses multiply by rolling circle replication using virus-encoded multifunctional replication initiator proteins (Rep proteins) that catalyze the initiation of replication on a double-stranded DNA (dsDNA) intermediate and the resolution of the ssDNA into circles. Here we report the solution NMR three-dimensional structure of the endonuclease domain from the master Rep (M-Rep) protein of faba bean necrotic yellows virus (FBNYV), a representative of the nanoviruses. The domain comprises amino acids 2-95 (M-Rep2-95), and its global fold is similar to those previously described for the gemini- and circovirus Rep endonuclease domains, consisting of a central 5-stranded antiparallel beta-sheet covered on one side by an alpha-helix and irregular loops and on the other, more open side of the domain, by an alpha-helix containing the catalytic tyrosine residue (the catalytic helix). Longer domain constructs extending to amino acids 117 and 124 were also characterized. They contain an additional alpha-helix, are monomeric, and exhibit catalytic activity indistinguishable from that of M-Rep2-95. The binding site for the catalytic metal was identified by paramagnetic broadening and maps to residues on the exposed face of the central beta-sheet. A comparison with the previously determined Rep endonuclease domain structures of tomato yellow leaf curl Sardinia virus (TYLCSV), a geminivirus, and that of porcine circovirus type 2 (PCV2) Rep allows the identification of a positively charged surface that is most likely involved in dsDNA binding, and reveals common features shared by all endonuclease domains of nanovirus, geminivirus, and circovirus Rep proteins.