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.

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 common strategy of DNA viruses is the creation of an environment favorable for efficient replication of their genome by subverting the cell cycle control of the host and forcing cells into DNA synthesis or S phase. In mammalian cells, a key cell cycle regulator is the retinoblastoma tumor suppressor protein pRB, which represses onset and progression into S phase by interacting with a wide range of cell cycle-related proteins. Among those are transcription factors of the E2F family that form complexes with hypophosphorylated pRB. During the G 1 /S transition, pRB is progressively phosphorylated by the action of cyclin-dependent kinases, and as a result, E2F is released from the complex and becomes available to activate the expression of S-phase-specific genes (14, 30).Oncoproteins of certain mammalian DNA tumor viruses, such as E1A of adenovirus type 6, E7 of human papillomavirus type 16, or the large T antigen protein of simian virus 40, stimulate the entry of cells into S phase by interaction with pRB through a short protein sequence comprising essentially the sequence motif LxCxE (10,45). This interaction abrogates the pRB-mediated block of cell cycle progression and may contribute to tumor formation (24). In addition, these or other viral proteins are involved in the neutralization of additional cell cycle regulators, in particular of the growth suppressor p53 (24,39). The interaction between the papillomavirus E7 and E6 proteins with pRB and p53, respectively, mediates the degradation of the latter by the ubiquitin-proteasome pathway (8,25,44). Hence, in addition to the interaction with growth suppressors, papillomaviruses make use of the protein degradation machinery to target these proteins to the 26S proteasome.Ubiquitination of proteins destined for degradation by the 26S proteasome is mediated by the action of the enzymatic complexes E1, E2, and E3 (21). E1 and E2 activate ubiquitin and catalyze the polyubiquitination of the substrate, which is thus marked for degradation by the 26S proteasome. A diverse class of complexes, the E3 ubiquitin-ligases, contains the elements of specificity for the substrates to be ubiquitinated. The

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.

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.

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.

Murine monoclonal antibodies (MAbs) were produced for the detection of faba bean necrotic yellows virus (FBNYV), an isometric ssDNA virus belonging to a new, yet unnamed genus of plant viruses. A total of 19 FBNYV-specific MAbs were obtained from three fusion experiments and characterised by determining their immunoglobulin types and titres as well as their corresponding epitopes. At least six distinct epitopes were revealed on FBNYV particles of different virus isolates. Only two MAbs reacted with SDS-dissociated FBNYV virions in triple antibody sandwich (TAS)-ELISA and with viral capsid protein in Western blots. Almost all MAbs were more sensitive in detecting FBNYV in viruliferous aphids by TAS-ELISA than polyclonal anti-FBNYV IgG by double antibody sandwich ELISA and permitted virus detection in individual aphids even following short acquisition access feeding periods.Coat protein variation among FBNYV isolates and serological relatedness to taxonomically similar viruses was studied by determining the cross reactivity of these MAbs with several field isolates of FBNYV as well as with milk vetch dwarf (MDV), banana bunchy top (BBTV), and subterranean clover stunt (SCSV) viruses. Whereas none of the MAbs reacted with BBTV, only one reacted with SCSV, indicating that FBNYV and SCSV share a common epitope. By contrast, 16 of the 19 MAbs reacted with MDV, suggesting that FBNYV and MDV are serologically closely related and strains of the same virus. When all 19 MAbs produced were tested against a total of 107 samples of FBNYV collected during virus surveys in Egypt, Ethiopia, Jordan, Morocco and Syria, five MAbs showed differential reactions. While the majority of the samples reacted with all 19 MAbs, about 20% of the 107 FBNYV samples did not react with one and/or other of these live MAbs, permitting the differentiation of seven serotypes of FBNYV and suggesting a considerable coat protein variation in FBNYV isolates from the countries surveyed. The MDV isolate from Japan and five FBNYV samples from Ethiopia appeared to be the least closely related to typical FBNYV isolates by not reacting with three and four, respectively, of the five differentiating Mabs.

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.

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 fiverep 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.