Role of the zinc-finger and basic motifs of chrysanthemum virus B p12 protein in nucleic acid binding, protein localization and induction of a hypersensitive response upon expression from a viral vector.

The genomes of carlaviruses encode cysteine-rich proteins (CRPs) of unknown function. The 12 kDa CRP of chrysanthemum virus B (CVB), p12, has been shown previously to induce a hypersensitive response (HR) when expressed from potato virus X (PVX). This study demonstrated that a p12-induced HR was preceded by induction of a number of genes related to pathogenesis, stress and systemic acquired resistance. p12 localized predominantly to the nucleus. Interestingly, it was found that p12 bound both RNA and DNA in vitro, but notably exhibited a preference for DNA in the presence of Zn(2+) ions. Mutational analysis of the p12 conserved sequence motifs demonstrated that the basic motif is required for p12 translocation to the nucleus, thus representing part of the protein nuclear localization signal, whereas the predicted zinc finger motif is needed for both Zn(2+)-dependent DNA binding and eliciting an HR in PVX-infected leaves. Collectively, these results link, for the first time, nuclear localization of the protein encoded by a cytoplasmically replicating virus and its DNA-binding capacity with HR induction. Furthermore, these data suggest that p12 may mediate induction of the host genes by binding to the plant genomic DNA, and emphasize that CVB p12 is functionally distinct from other known nuclear-localized proteins encoded by the plant positive-stranded RNA viruses.

Expression, localization and effects on virulence of the cysteine-rich 8 kDa protein of Potato mop-top virus.

Potato mop-top virus (PMTV) RNA3 contains a triple gene block (TGB) encoding viral movement proteins and an open reading frame for a putative 8 kDa cysteine-rich protein (CRP). In this study, PMTV CRP was shown to be expressed in the course of virus infection, and a PMTV CRP-specific subgenomic RNA was mapped. CRP has previously been shown to be dispensable for infection of PMTV in Nicotiana benthamiana. In this study, PMTV CRP was found to increase the severity of disease symptoms when expressed from Potato virus X or Tobacco mosaic virus in N. benthamiana and Nicotiana tabacum, suggesting that the protein affects virulence of the virus or might suppress a host defence mechanism. However, PMTV CRP did not show RNA silencing suppression activity in three assays. Host responses to the PMTV CRP expression from different viral genomes ranged from an absence of response to extreme resistance at a single cell level and were dependent on the viral genome. These findings emphasized involvement of viral proteins and/or virus-induced cell components in the plant reaction to CRP. PMTV CRP was predicted to possess a transmembrane segment. CRP fused to the green fluorescent protein was associated with endoplasmic reticulum-derived membranes and induced dramatic rearrangements of the endoplasmic reticulum structure, which might account for protein functions as a virulence factor of the virus.

Sequence divergence of four soilborne sugarbeet-infecting viruses.

Soilborne viruses are among the most harmful pathogens of sugarbeet (Beta vulgaris L.ssp. vulgaris) but most of them lack information on genetic variability due to paucity of sequence data. Only one isolate of Beet soil borne virus (BSBV; genus Pomovirus), Beet virus Q (BVQ; genus Pomovirus) and Beet soil borne mosaic virus (BSBMV; genus Benyvirus) has been characterised for the coat protein (CP) gene. In this study, the CP gene sequences of three isolates each of BSBV and Beet necrotic yellow vein virus (BNYVV; genus Benyvirus) (France, Germany and USA), two isolates of BVQ (France and Germany), and one isolate of BSBMV (USA) were determined. Phylogenetic analyses including sequences from databanks indicated that the French BNYVV isolate of this study belongs to so-called P-type, the American isolate to A-type and the German isolate to B-type. The CP genes of the three BSBV isolates characterised in this study and the one available from databank were highly identical (98.4-99.0% at nucleotide level; one variable amino acid). The BSBMV isolate studied here differed from the previously characterised isolate for five nucleotides and four amino acids in the CP region. The two BVQ isolates characterised in this study contained three additional nucleotides resulting in an additional amino acid residue (arginine) at CP position 86, as compared to the only isolate available in databank.

Complete genome sequence and analyses of the subgenomic RNAs of sweet potato chlorotic stunt virus reveal several new features for the genus Crinivirus.

The complete nucleotide sequences of genomic RNA1 (9,407 nucleotides [nt]) and RNA2 (8,223 nt) of Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) were determined, revealing that SPCSV possesses the second largest identified positive-strand single-stranded RNA genome among plant viruses after Citrus tristeza virus. RNA1 contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyltransferase, helicase, and polymerase domains. RNA2 contains the Closteroviridae hallmark gene array represented by a heat shock protein homologue (Hsp70h), a protein of 50 to 60 kDa depending on the virus, the major coat protein, and a divergent copy of the coat protein. This grouping resembles the genome organization of Lettuce infectious yellows virus (LIYV), the only other crinivirus for which the whole genomic sequence is available. However, in striking contrast to LIYV, the two genomic RNAs of SPCSV contained nearly identical 208-nt-long 3' terminal sequences, and the ORF for a putative small hydrophobic protein present in LIYV RNA2 was found at a novel position in SPCSV RNA1. Furthermore, unlike any other plant or animal virus, SPCSV carried an ORF for a putative RNase III-like protein (ORF2 on RNA1). Several subgenomic RNAs (sgRNAs) were detected in SPCSV-infected plants, indicating that the sgRNAs formed from RNA1 accumulated earlier in infection than those of RNA2. The 5' ends of seven sgRNAs were cloned and sequenced by an approach that provided compelling evidence that the sgRNAs are capped in infected plants, a novel finding for members of the Closteroviridae.

Coat protein gene-mediated resistance to Potato virus A in transgenic plants is suppressed following infection with another potyvirus.

High levels of resistance to Potato virus A (PVA, genus Potyvirus), indicated by absence of detectable infection in inoculated leaves, were attained in Nicotiana benthamiana transformed with a construct expressing the PVA 5'-untranslated region fused with the coat protein (CP)-encoding sequence. Low steady-state levels of the transgene transcripts were detected. Resistance was PVA-specific and did not protect the plants against infection with Potato virus Y (PVY, genus Potyvirus). Consequently, the steady-state levels of the CP-transgene mRNA were greatly elevated in the plants infected with PVY, and plants became susceptible to infection with PVA. These data show that virus resistance obtained by expressing regions of a plant virus genome in transgenic plants may be suppressed following infection with another virus that evades the virus-specific resistance.

Potyviral helper-component proteinase expressed in transgenic plants enhances titers of Potato leaf roll virus but does not alleviate its phloem limitation.

Coinfection of Nicotiana benthamiana with Potato virus A (PVA, a potyvirus) and Potato leaf-roll virus (PLRV, a luteovirus) induces a synergistic interaction manifested by enhanced titers of PLRV. The helper component proteinase (HC-Pro) of potyviruses is involved in viral vascular movement and suppression of an antiviral defense mechanism in plants. Data of our study showed that accumulation of PLRV in transgenic N. benthamiana expressing the PVA HC-Pro was enhanced on average by 4.5-fold, as compared to a 6.0-fold enhancement in wild-type N. benthamiana plants doubly infected with PVA and PLRV. Enhancement of PLRV accumulation was directly proportional to the concentration of the HC-Pro in leaves. In the HC-Pro-transgenic plants and wild-type plants, PLRV was almost exclusively confined to the phloem, but the HC-Pro-transgenic plants had a fourfold greater number of PLRV-infected cells within the phloem tissues, as revealed by immunohistochemical staining. In the leaves doubly infected with PLRV and PVA, PLRV was found to exit the phloem in 25.0% of the veins, infecting all types of leaf cells, but, on average, PLRV accumulation was not enhanced more than by sixfold at the whole-leaf level. Therefore, potyviral/luteoviral synergism seems to be based on two mechanisms. One of them is mediated by the HC-Pro and increases luteovirus accumulation without allowing detectable egress from vascular tissue. The other mechanism probably depends on additional potyviral proteins and alleviates the normal phloem limitation of PLRV.

The readthrough region of Potato mop-top virus (PMTV) coat protein encoding RNA, the second largest RNA of PMTV genome, undergoes structural changes in naturally infected and experimentally inoculated plants.

Molecular data on Potato mop-top virus (PMTV), genus Pomovirus, is currently mostly based on analysis of two Scottish isolates, PMTV-S and PMTV-T. Here we report the complete sequence of "the coat protein (CP) encoding RNA" of an isolate of PMTV obtained from the field in Sweden. Our data show that this RNA (3134 nt) is the second largest of the three RNA species in the tripartite PMTV genome, and it should, therefore, be referred to as RNA 2. This nomenclature is consistent with other pomoviruses. The sequence of the readthrough domain (RT) of RNA 2 was determined also in two additional field isolates of PMTV from Finland and Denmark. All three isolates contained a novel, 109 nucleotides long sequence at the 3'-end of the RT, which has not been found in PMTV-S and PMTV-T. Hence, our data suggest that the RNA 2 sequences previously described for the isolates PMTV-T and PMTV-S may represent deletion derivatives. The C-proximal half of RT contained many amino acid (aa) differences among the isolates, in contrast to only few aa differences in the N-proximal part of RT. Deletion variants of RNA 2 were generated from the Nordic isolates in potato tubers infected in the field, and in the mechanically inoculated test plants. All deletions started within a short region (18 nt) and removed 558-940 nt from the 3'-end of RT region. This study for the first time describes the full-length sequence of the "CP-encoding RNA" (RNA2) of PMTV, and reveals considerable aa variability and occurrence of deletion variants of RT in the field isolates of PMTV.

Movement of hordeivirus hybrids with exchanges in the triple gene block.

The barley stripe mosaic virus (BSMV) triple gene block (TGB) coding for movement proteins (MPs) was replaced with the respective TGB genes from two other hordeiviruses, poa semilatent virus (PSLV) or lychnis ringspot virus (LRSV). The BSMV/LRSV recombinant did not exhibit infectivity on the plants tested, whereas the infection rate and host range of the BSMV/PSLV hybrid were similar to those of BSMV. In particular, the BSMV/PSLV hybrid infected Nicotiana benthamiana, a nonhost plant for PSLV, indicating a contribution of non-MP elements of BSMV genome to host specificity of virus transport. Assuming that the PSLV TGB was functional in the BSMV genome context, a further series of recombinants was constructed, in which smaller portions of the BSMV TGB were replaced by the corresponding PSLV sequences. Examination of the infectivity of the hybrid viruses suggested that the TGB-coded proteins could interact in a host-dependent manner to mediate cell-to-cell movement. Analysis of recombinants with hybrid sequences of the first gene in the TGB (beta b gene) indicated that (i) sequence-independent binding of beta b to viral RNAs could occur during formation of beta b-RNA complexes in vivo, and that (ii) the beta b MP is involved in virus long-distance movement, for which homologous N- and C-terminal beta b domains are required.

Genome sequences of poa semilatent and lychnis ringspot hordeiviruses.

The nucleotide sequences of the genomic RNAs of two hordeiviruses, poa semilatent virus (PSLV) and lychnis ringspot virus (LRSV), were determined. The genome organization of both viruses is similar to that of the type hordeivirus, barley stripe mosaic virus (BSMV). Comparisons of the amino acid sequences of the hordeivirus replicase components revealed a high level of sequence similarity to those of soil-borne wheat mosaic virus (SBWMV), peanut clump virus (PCV), and Indian peanut clump virus. The cysteine-rich gamma b proteins of hordeiviruses are similar to those of SBWMV, PCV, and tobraviruses. Analysis of cis-acting elements in the genomes of the three hordeiviruses revealed conservation of putative core elements of subgenomic RNA promoters. Complex patterns of conservation were also found in the 5'-untranslated regions of hordeivirus genomic RNAs alpha, beta, and gamma.

Host-controlled cell-to-cell movement of a hybrid barley stripe mosaic virus expressing a dianthovirus movement protein.

The triple gene block (TGB) of barley stripe mosaic virus (BSMV), coding for viral movement proteins (MPs), was replaced by the single MP gene of red clover necrotic mosaic virus (RCNMV). Accumulation of the hybrid virus in barley plants (the selective host for BSMV) was reduced compared to BSMV. The hybrid virus induced small necrotic local lesions on Chenopodium amaranticolor leaves and did not infect Nicotiana clevelandii (the selective host for RCNMV). The hybrid virus accumulated in the inoculated leaves of Nicotiana benthamiana, but not in the upper noninoculated leaves. Thus the RCNMV MP gene substituted for the BSMV TGB in cell-to-cell movement, but not in systemic spread. Hybrid virus movement was efficient only in N. benthamiana, the common host for BSMV and RCNMV. These data point to the involvement of host-specific factors in the function of virus-coded transport determinants.

Comparisons of the genomic cis-elements and coding regions in RNA beta components of the hordeiviruses barley stripe mosaic virus, lychnis ringspot virus, and poa semilatent virus.

Nucleotide sequences of the genomic RNA beta components of hordeiviruses poa semilatent virus (PSLV) and lychnis ringspot virus (LRSV) were determined. PSLV and LRSV closely resemble barley stripe mosaic virus (BSMV), type hordeivirus, in the gene arrangement of their RNAs beta, comprising 5'-proximal beta a (coat protein) gene and downstream triple gene block (TGB) coding for the beta b, beta c, and beta d putative transport proteins. The beta a, beta b, beta c, and beta d proteins of the three hordeiviruses showed significant sequence similarity, with the respective proteins of PSLV and BSMV being closer to each other than to their counterparts of LSRV. Comparisons of the TGB-encoded proteins of hordeiviruses, potexviruses, carlaviruses, and furoviruses indicate that the first and second TGB genes belong to the monophyletic groups, whereas the third gene may have multiple ancestry. LRSV, PSLV, and BSMV showed remarkable variation in the 3'-untranslated regions of their genomic RNAs. Among the three hordeiviruses, LRSV has the shortest 3'-noncoding region that lacks tentative pseudoknot-forming elements conserved upstream of the 3'-tRNA-like structure in the BSMV and PSLV genomes. On the other hand, LRSV RNA beta, like that of BSMV, contained the internal poly(A) sequence that is absent from PSLV RNA.

Movement of a barley stripe mosaic virus chimera with a tobacco mosaic virus movement protein.

The tobacco mosaic virus (TMV) 30K movement protein (MP) gene was inserted into a full-length cDNA clone of barley stripe mosaic virus (BSMV) RNA beta replacing the triple gene block (TGB). The resulting recombinant ND-MPT genome, consisting of infectious wt transcripts of BSMV RNAs alpha and gamma, together with the hybrid RNA beta transcript, was inoculated onto test plants to study the functional compatibility between the BSMV TGB-adapted genetic system and the tobamovirus transport gene. ND-MPT infected the inoculated leaves of Nicotiana benthamiana and Chenopodium amaranticolor, which are common hosts for the parental viruses; the size, growth rate, and morphology of local lesions on C. amaranticolor were influenced by the foreign MP gene. However, the hybrid virus failed to infect barley, N. tabacum (var. Samsun), and N. clevelandii, the selective hosts. Thus, the TMV MP was able to functionally substitute for the BSMV TGB-coded MPs, i.e., the 30K MP functioned independently of any other BSMV sequences. However, the TMV MP gene promoted the cell-to-cell movement in a host-dependent manner.

Genome characterization and taxonomy of Plantago asiatica mosaic potexvirus.

The complete nucleotide sequence of Plantago asiatica mosaic virus (P1AMV) genomic RNA has been determined. The 6128 nucleotide sequence contains five open reading frames (ORFs) coding for proteins of M(r) 156K (ORF1), 25K (ORF2), 12K (ORF3), 13K (ORF4) and 22K (ORF5). The sequences of these P1AMV proteins exhibit strong homology to the proteins of the other potexviruses. Phylogenetic trees based on the multiple sequence alignments of three conserved domains in ORF1 product and capsid protein reveal a close relationship of P1AMV to papaya mosaic virus and clover yellow mosaic virus. The P1AMV genomic RNA and a major subgenomic RNA (sgRNA) of 0.9 kb have been detected in infected leaves by Northern blot hybridization. The latter sgRNA is the messenger for virus capsid protein and its 5' terminus has been located 23 nucleotides upstream of the initiator codon of the coat protein gene. The P1AMV virion RNA and RNA transcript resembling the 0.9 kb sgRNA have been translated in vitro giving rise to a single major 170K product and a major 22K product, respectively.