Infectivity of nanovirus DNAs: induction of disease by cloned genome components of Faba bean necrotic yellows virus.

Circumstantial evidence suggests that the genome of Faba bean necrotic yellows virus (FBNYV), a nanovirus, consists of eight distinct, circular, single-stranded DNAs, each of about 1 kb and encoding only one protein. Here, the use of cloned full-length FBNYV DNAs for reproducing FBNYV-like symptoms in Vicia faba, the principal natural host of FBNYV, is reported. Characteristic symptoms of FBNYV infection were obtained in faba bean plants following biolistic DNA delivery or agroinoculation with all eight FBNYV DNAs. Although the eight different DNAs have been invariably detected in field samples infected with the various geographical FBNYV isolates, experimental infection with different combinations of fewer than eight DNAs also led to typical FBNYV symptoms. Even only five genome components, DNA-R, DNA-S, DNA-M, DNA-U1 and DNA-U2, were sufficient for inducing disease symptoms in V. faba upon agroinoculation. Symptomatic plants agroinoculated or bombarded with eight DNAs contained typical FBNYV virions; however, the virus was not transmitted by Aphis craccivora or Acyrthosiphon pisum, two efficient aphid vectors of FBNYV.

Dual-colour imaging of membrane protein targeting directed by poa semilatent virus movement protein TGBp3 in plant and mammalian cells.

The movement function of poa semilatent hordeivirus (PSLV) is mediated by the triple gene block (TGB) proteins, of which two, TGBp2 and TGBp3, are membrane proteins. TGBp3 is localized to peripheral bodies in the vicinity of the plasma membrane and is able to re-direct TGBp2 from the endoplasmic reticulum (ER) to the peripheral bodies. For imaging of TGBp3-mediated protein targeting, PSLV TGBp3 tagged with a red fluorescent protein (DsRed) was used. Coexpression of DsRed-TGBp3 with GFP targeted to the ER lumen (ER-GFP) demonstrated that ER-GFP was contained in typical ER structures and peripheral bodies formed by TGBp3 protein, suggesting an ER origin for these bodies. In transient coexpression with viral membrane proteins tagged with GFP, DsRed-TGBp3 directed to the peripheral bodies the homologous TGBp2 protein and two unrelated membrane proteins, the 6 kDa movement protein of beet yellows closterovirus and the putative movement protein encoded by the genome component 4 of faba bean necrotic yellows nanovirus. However, coexpression of TGBp3 with GFP derivatives targeted to the ER membranes by artificial hydrophobic tail sequences suggested that targeting to the ER membranes per se was not sufficient for TGBp3-directed protein trafficking to peripheral bodies. TGBp3-induced targeting of TGBp2 also occurred in mammalian cells, indicating the universal nature of the protein trafficking signals and the cotargeting mechanism.

The master rep concept in nanovirus replication: identification of missing genome components and potential for natural genetic reassortment.

Faba bean necrotic yellows virus (FBYNV), Milk vetch dwarf virus (MDV), and Subterranean clover stunt virus (SCSV) are nanoviruses that infect leguminous plants. From MDV- and SCSV-infected tissue we identified viral DNAs that encode a replication initiator protein (Rep), essential for replication of the multiple circular single-stranded DNAs of these viruses. These previously undescribed Rep proteins of MDV and SCSV are strikingly similar in sequence and functionally equivalent to the master Rep protein of FBYNV. Moreover, we demonstrated that the master Rep proteins of the three viruses are able to trigger replication of heterologous nanovirus DNAs. Such cross-species replication may reflect a considerable potential for genetic reassortment among nanoviruses in nature and be of significance for their evolution.

Clink, a nanovirus-encoded protein, binds both pRB and SKP1.

Clink, a 20-kDa protein of faba bean necrotic yellows virus, a single-stranded DNA plant virus, interacts with pRB family members and a SKP1 homologue from Medicago sativa. An LxCxE motif and an F-box of Clink mediate the interactions with the respective proteins. The capacity of Clink to bind pRB correlates with its ability to stimulate viral replication. Interaction of a single protein with the cell cycle regulator pRB and SKP1, a constituent of the ubiquitin-protein turnover pathway, appears to be a novel feature. Hence, Clink may represent a new class of viral cell cycle modulators.

A single rep protein initiates replication of multiple genome components of faba bean necrotic yellows virus, a single-stranded DNA virus of plants.

Faba bean necrotic yellows virus (FBNYV) belongs to the nanoviruses, plant viruses whose genome consists of multiple circular single-stranded DNA components. Eleven distinct DNAs, 5 of which encode different replication initiator (Rep) proteins, have been identified in two FBNYV isolates. Origin-specific DNA cleavage and nucleotidyl transfer activities were shown for Rep1 and Rep2 proteins in vitro, and their essential tyrosine residues that catalyze these reactions were identified by site-directed mutagenesis. In addition, we showed that Rep1 and Rep2 proteins hydrolyze ATP, and by changing the key lysine residue in the proteins' nucleoside triphosphate binding sites, demonstrated that this ATPase activity is essential for multiplication of virus DNA in vivo. Each of the five FBNYV Rep proteins initiated replication of the DNA molecule by which it was encoded, but only Rep2 was able to initiate replication of all the six other genome components. Furthermore, of the five rep components, only the Rep2-encoding DNA was always detected in 55 FBNYV samples from eight countries. These data provide experimental evidence for a master replication protein encoded by a multicomponent single-stranded DNA virus.

Ten distinct circular ssDNA components, four of which encode putative replication-associated proteins, are associated with the faba bean necrotic yellows virus genome.

Four further circular ssDNA components (C7-C10), about 1 kb in size and structurally similar to the previously described components (C1-C6) found associated with a Syrian (Sy) isolate of faba bean necrotic yellows virus (FBNYV), have been identified. Similar to C1 and C2, two of the new components (C7 and C9) encode putative replication-associated (Rep) proteins of 33.2 and 32.7 kDa, respectively, the former of which is 90% identical to the C10 Rep protein of milk vetch dwarf virus (MDV). C8 encodes a putative protein (17.4 kDa) whose function is unknown, but which is highly conserved between FBNYV and the other nanoviruses MDV, subterranean clover stunt virus and banana bunchy top virus. The putative protein (19.7 kDa) encoded by C10 contains an LXCXE motif, which is also present in the homologues of its relatives, suggesting that they may all interact with plant retinoblastoma-like proteins. Sequence information for seven components of an Egyptian (Eg) FBNYV isolate indicated that six of them share > 96% identity with FBNYV-Sy. However, the C1 Rep protein of FBNYV-Eg was only 63.5% identical to that of FBNYV-Sy, but was 88.3 % identical to the MDV-C2 Rep protein. We conclude that the FBNYV genome consists of seven to ten components, six of which encode non-Rep proteins. All ten components of the FBNYV genome, except the Rep components C2 and C9, had closely related counterparts in the MDV genome. In spite of this similarity, FBNYV and MDV appear to be distinct virus species.

Analysis of six DNA components of the faba bean necrotic yellows virus genome and their structural affinity to related plant virus genomes.

Faba bean necrotic yellows virus (FBNYV) has a multicomponent circular ssDNA genome. In addition to a previously described genome component (C1) coding for a replicase-associated protein (Rep), five further components (C2 to C6) have now been identified. Each of the six components is about 1 kb in size, contains one major open reading frame (ORF) in the virion sense with a TATA box and polyadenylation signal, and has a noncoding region containing a highly conserved sequence possibly forming a stem-loop structure. Similar to C1, C2 encodes another putative Rep of 33.1 kDa, which is closely related to the Rep of banana bunchy top virus (BBTV). Based on bacterial expression and immunoblot analysis, the ORF of C5 encodes the capsid protein (CP) with a deduced molecular mass of 19 kDa. The FBNYV CP shares the highest amino acid (aa) identity (56.2%) with that of subterranean clover stunt virus (SCSV). The ORF of C4 potentially codes for a hydrophobic protein which appears to be structurally and functionally similar to the BBTV-C4 and SCSV-C1 proteins. No protein sequence similarities were found in databases for the C3 and C6 ORFs of FBNYV. FBNYV is clearly distinct from any known virus but is taxonomically related to BBTV and SCSV.

Sequence analysis of a faba bean necrotic yellows virus DNA component containing a putative replicase gene.

Faba bean necrotic yellows virus (FBNYV) has a circular ssDNA genome possibly consisting of several components of about 1 kb each. The complete nucleotide sequence of one component of FBNYV (FBNYV DNA 1) containing a putative replicase gene is presented. This component consists of 1002 nucleotides and, in the virion orientation, contains one large open reading frame (ORF1) potentially encoding a 32.3 kDa replicase with the NTP-binding motif GGEGKS. No obvious functions could be assigned to two smaller ORFs (7.4 and 9.3 kDa) occurring in the complementary orientation. Amino acid sequence comparisons of the putative replicase of FBNYV with that of other similar ssDNA viruses yielded higher homologies to subterranean clover stunt virus than to banana bunchy top and coconut foliar decay viruses. A potential stem-loop structure and a TATA box were identified within the noncoding region. Two oligonucleotides derived from FBNYV DNA 1 were used for direct sequencing of the virion ssDNA to determine its virion polarity and for amplifying part of this component by immunocapture PCR in extracts from from FBNYV-infected plants.