Biological and genetic diversity of Wheat yellow mosaic virus (Genus Bymovirus).

The biological and genetic diversity of Wheat yellow mosaic virus (WYMV) isolates in Japan was characterized. On the basis of wheat cultivar reactions, 14 WYMV isolates from various places were classified into pathotypes I, II, or III. These were distributed in central, northern, and southern areas of Japan, respectively. WYMV isolates comprised three genotypes (A, A' and B) based on amino acid differences in RNA1 and two genotypes (a and b) based on amino acid differences in RNA2. A correlation was found between the WYMV RNA1-based genotype and pathotype, suggesting that factors associated with pathogenicity map to RNA1. Genotype Aa and A'a were distributed mainly in the central to southern areas of Japan, and genotype Bb was found in northern areas of Japan, as shown by reverse-transcription polymerase chain reaction restriction fragment length polymorphism analysis. Chinese isolates YA and YZ were closely related to genotypes Bb and Aa, respectively. Wheat was introduced from China to Japan in the 4th and 5th centuries, and the two genotypes of WYMV might also have been introduced with the crop from China and later adapted to local wheat cultivars in Japan.

Chromosomal integration of a binding domain:bait gene into yeast enhances detection in the two-hybrid system.

We have developed a modified yeast two-hybrid system using the GAL4 transcription activator by integrating a BD:bait gene (GAL4 binding domain:bait gene) into the host chromosome. Locus-specific integration by homologous recombination and use of a strong transcription promoter enabled uniform expression of the integrated BD:bait gene in all host cells. Moreover, after introduction of an AD:prey (GAL4 activation domain:prey) plasmid, false positives were nearly undetectable and false negatives were decreased significantly. Using this improved method a cDNA library could be efficiently screened using roughly 1/10 of the materials required for conventional sequential two-hybrid screen.

Barley Yellow Mosaic Virus VPg Is the Determinant Protein for Breaking eIF4E-Mediated Recessive Resistance in Barley Plants.

In this study, we investigated the barley yellow mosaic virus (BaYMV, genus Bymovirus) factor(s) responsible for breaking eIF4E-mediated recessive resistance genes (rym4/5/6) in barley. Genome mapping analysis using chimeric infectious cDNA clones between rym5-breaking (JT10) and rym5-non-breaking (JK05) isolates indicated that genome-linked viral protein (VPg) is the determinant protein for breaking the rym5 resistance. Likewise, VPg is also responsible for overcoming the resistances of rym4 and rym6 alleles. Mutational analysis identified that amino acids Ser-118, Thr-120, and His-142 in JT10 VPg are the most critical residues for overcoming rym5 resistance in protoplasts. Moreover, the rym5-non-breaking JK05 could accumulate in the rym5 protoplasts when eIF4E derived from a susceptible barley cultivar was expressed from the viral genome. Thus, the compatibility between VPg and host eIF4E determines the ability of BaYMV to infect barley plants.

Association of VPg and eIF4E in the host tropism at the cellular level of Barley yellow mosaic virus and Wheat yellow mosaic virus in the genus Bymovirus.

Barley yellow mosaic virus (BaYMV) and Wheat yellow mosaic virus (WYMV) are separate species in the genus Bymovirus with bipartite plus-sense RNA genomes. In fields, BaYMV infects only barley and WYMV infects only wheat. Here, we studied the replicative capability of the two viruses in barley and wheat mesophyll protoplasts. BaYMV replicated in both barley and wheat protoplasts, but WYMV replicated only in wheat protoplasts. The expression of wheat translation initiation factor 4E (eIF4E), a common host factor for potyviruses, from the WYMV genome enabled WYMV replication in barley protoplasts. Replacing the BaYMV VPg gene with that of WYMV abolished BaYMV replication in barley protoplasts, whereas the additional expression of wheat eIF4E from BaYMV genome restored the replication of the BaYMV mutant in barley protoplasts. These results indicate that both VPg and the host eIF4E are involved in the host tropism of BaYMV and WYMV at the replication level.

Differential detection of Wheat yellow mosaic virus, Japanese soil-borne wheat mosaic virus and Chinese wheat mosaic virus by reverse transcription loop-mediated isothermal amplification reaction.

A differential detection method for three wheat viruses: Wheat yellow mosaic virus (WYMV), Japanese soil-borne mosaic virus (JSBWMV) and Chinese wheat mosaic virus (CWMV) using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction was developed. All three primer sets, which were designed from the genome sequences of WYMV, JSBWMV and CWMV respectively, worked most efficiently at 65 °C and could detect each virus RNA within 10 min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. Furthermore, these primer sets showed unique annealing curves. The peak denaturing temperatures of WYMV, JSBWMV and CWMV primer sets were 87.6 °C, 84.8 °C and 86.4 °C, respectively and were clearly distinguished by the isothermal DNA amplification and fluorescence detection device. The RT-LAMP assay including all three primer sets was found to be 100 times more sensitive than RT-PCR for WYMV and JSBWMV and as sensitive as RT-PCR for CWMV. The RT-LAMP method was validated for the simultaneous detection of these viruses in wheat and barley leaves.

Influence of amino acid at position 132 in VPg on replication and systemic infection of Barley yellow mosaic virus.

A substitution of Lys with Asn or His at the amino-acid position 132 in VPg (VPg132) correlates with overcoming rym4-gene resistance by European strain 2 of Barley yellow mosaic virus (BaYMV-2). From the full-length cDNA clones for a Japanese BaYMV isolate JK05 (BaYMV-JK05) we generated virus mutants with Tyr, Lys, Asn, and Ala substituted for wild-type His at the VPg132. Only Tyr and Asn mutants replicated efficiently in protoplasts from barley varieties that are susceptible to wild-type virus. The Tyr mutant also infected susceptible barley plants systemically with the emergence of virus populations with partial or complete reversion to His, whereas the Asn mutant did not cause systemic infection. Thus, the VPg132 amino acid is essential for both efficient replication and systemic infection. Neither wild-type virus nor any of the mutants replicated in protoplasts from a rym4 barley genotype. Therefore, substitution of the VPg132 amino acid alone cannot enable breaking rym4-mediated resistance in the BaYMV-JK05 background.

Bymovirus reverse genetics: requirements for RNA2-encoded proteins in systemic infection.

Barley yellow mosaic virus (BaYMV), the type species of the genus Bymovirus in the family Potyviridae in the picornavirus-like superfamily, causes a yellow mosaic disease of winter barley with significant yield losses in Europe and East Asia. Until now, infectious in vitro transcripts for the bipartite plus-sense RNA genome of any bymovirus species have not been available, rendering molecular analyses of bymovirus pathogenicity and the host resistance mechanisms difficult. In this study, we constructed the first cDNA clones of BaYMV RNA1 and RNA2, from which infectious RNA can be transcribed in vitro. Using in vitro transcripts, we showed that RNA1, which encodes eight proteins, including a viral proteinase NIa-Pro, the RNA-dependent RNA polymerase NIb, genome-linked viral protein VPg and the capsid protein CP, replicated autonomously in barley mesophyll protoplasts in the absence of RNA2 optimally at 15 degrees C, a temperature similar to the optimum for causing disease in barley fields. For systemic infection of barley plants, RNA1 alone was not sufficient and RNA2 was also required. Of the two proteins encoded on RNA2 (P1 with cysteine proteinase activity and P2 with unknown functions), P1 was essential and P2 was dispensable for systemic infectivity. The expression of both P1 and P2, but not the precursor polyprotein, together with RNA1 increased systemic infection and caused mosaic leaf symptoms. The infectious cDNA clones of BaYMV will be vital for future studies of bymovirus-host-vector interactions at the molecular level.

Orchid fleck virus is a rhabdovirus with an unusual bipartite genome.

Orchid fleck virus (OFV) has an unusual bipartite negative-sense RNA genome with clear sequence similarities to those of nucleorhabdoviruses. The OFV genome consists of two single-stranded RNA molecules, RNA1 and RNA2 that are 6413 and 6001 nt long, respectively, with open reading frame (ORF) information in the complementary sense. RNA1 encodes 49 (ORF1), 26 (ORF2), 38 (ORF3), 20 (ORF4) and 61 kDa (ORF5) proteins, and RNA2 encodes a single protein of 212 kDa (ORF6). ORF1, ORF5 and ORF6 proteins had significant similarities (21-38 % identity) to the nucleocapsid protein (N), glycoprotein (G) and polymerase (L) gene products, respectively, of other rhabdoviruses, especially nucleorhabdoviruses, whereas ORF2, ORF3 and ORF4 proteins had no significant similarities to other proteins in the international databases. Similarities between OFV and rhabdoviruses were also found in the sequence complementarity at both termini of each RNA segment (the common terminal sequences are 3'-UGUGUC---GACACA-5'), the conserved intergenic sequences and in being negative sense. It was proposed that a new genus Dichorhabdovirus in the family Rhabdoviridae of the order Mononegavirales should be established with OFV as its prototype member and type species.

RNase E maintenance of proper FtsZ/FtsA ratio required for nonfilamentous growth of Escherichia coli cells but not for colony-forming ability.

Inactivation or deletion of the RNase E-encoding rne gene of Escherichia coli results in the growth of bacterial cells as filamentous chains in liquid culture (K. Goldblum and D. Apirion, J. Bacteriol. 146:128-132, 1981) and the loss of colony-forming ability (CFA) on solid media. RNase E dysfunction is also associated with abnormal processing of ftsQAZ transcripts (K. Cam, G. Rome, H. M. Krisch, and J.-P. Bouché, Nucleic Acids Res. 24:3065-3070, 1996), which encode proteins having a central role in septum formation during cell division. We show here that RNase E regulates the relative abundances of FtsZ and FtsA proteins and that RNase E depletion results in decreased FtsZ, increased FtsA, and consequently an altered FtsZ/FtsA ratio. However, while restoration of the level of FtsZ to normal in rne null mutant bacteria reverses the filamentation phenotype, it does not restore CFA. Conversely, overexpression of a related RNase, RNase G, in rne-deleted bacteria restores CFA, as previously reported, without affecting FtsZ abundance. Our results demonstrate that RNase E activity is required to maintain a proper cellular ratio of the FtsZ and FtsA proteins in E. coli but that FtsZ deficiency does not account for the nonviability of cells lacking RNase E.

Engineering of a Sagiyama alphavirus RNA-based transient expression vector.

Sagiyama virus (SAGV), a strain of Getah virus in the genus Alphavirus in the family Togaviridae, has a broad host range in vertebrates and invertebrates but is not pathogenic for humans. We engineered the SAGV genome as an efficient transient expression vector using the full-length infectious cDNA clone pSAG2 as the background. A green fluorescent protein (GFP) gene was used as a reporter gene and expressed from a subgenomic mRNA. When the GFP gene was placed downstream of the intact capsid protein gene or an internally deleted capsid protein gene encoding the N-terminal 9 amino acids and C-terminal 149 amino acids, autoproteolysis occurred efficiently at the boundary site to release GFP from the N-terminally-fused capsid-protease domain. GFP was also expressed efficiently without the 5'-terminal region of the capsid protein gene, suggesting that SAGV capsid protein gene does not have a translation enhancer sequence. To provide structural proteins for pseudovirion formation, a nonviable mutant construct, pSAG2.3L, which contains a Gly-to-Leu substitution at the - 2 position of the nsP3/4 cleavage site, was used as a helper. GFP was expressed up to 50 pg from 1 X 10(6) BHK21 cells after inoculation of pseudovirions. The C6/36 mosquito cell was also a suitable host for a large scale expression of GFP using pseudovirions. In addition to high-level transient expression, safeness of SAGV should give an advantage over other alphavirus expression vectors.

The optimal temperature for RNA replication in cells infected by Soil-borne wheat mosaic virus is 17 degrees C.

Systemic infection of wheat plants with Soil-borne wheat mosaic virus (SBWMV) requires temperatures below 20 degrees C. Here we examine the cause of the temperature sensitivity by inoculating infectious in vitro transcripts of SBWMV RNA1 and RNA2 to barley mesophyll protoplasts. After RNA inoculation, protoplasts were incubated at temperatures between 15 and 25 degrees C for up to 48 h. Western blot analysis showed that the capsid protein accumulated most abundantly at 17 degrees C but was not detectable at 25 degrees C. Northern blot analysis showed that the wild-type RNA1 and RNA2 and their subgenomic RNAs accumulated most abundantly at 17 degrees C but were barely detectable at 25 degrees C. An RNA1 mutant in which the p152 and p211 replicase genes were placed between the 5'- and 3'-untranslated regions also replicated most efficiently at 17 degrees C but not at 25 degrees C. Thus, the requirement for temperatures lower than 20 degrees C for SBWMV infection is primarily determined by replication of RNA1, which encodes the viral RNA replicase.

Modification of the 5' terminus of Sindbis virus genomic RNA allows nsP4 RNA polymerases with nonaromatic amino acids at the N terminus to function in RNA replication.

We have previously shown that Sindbis virus RNA polymerase requires an N-terminal aromatic amino acid or histidine for wild-type or pseudo-wild-type function; mutant viruses with a nonaromatic amino acid at the N terminus of the polymerase, but which are otherwise wild type, are unable to produce progeny viruses and will not form a plaque at any temperature tested. We now show that such mutant polymerases can function to produce progeny virus sufficient to form plaques at both 30 and 40 degrees C upon addition of AU, AUA, or AUU to the 5' terminus of the genomic RNA or upon substitution of A for U as the third nucleotide of the genome. These results are consistent with the hypothesis that (i) 3'-UA-5' is required at the 3' terminus of the minus-strand RNA for initiation of plus-strand genomic RNA synthesis; (ii) in the wild-type virus this sequence is present in a secondary structure that can be opened by the wild-type polymerase but not by the mutant polymerase; (iii) the addition of AU, AUA, or AUU to the 5' end of the genomic RNA provides unpaired 3'-UA-5' at the 3' end of the minus strand that can be utilized by the mutant polymerase, and similarly, the effect of the U3A mutation is to destabilize the secondary structure, freeing 3'-terminal UA; and (iv) the N terminus of nsP4 may directly interact with the 3' terminus of the minus-strand RNA for the initiation of the plus-strand genomic RNA synthesis. This hypothesis is discussed in light of our present results as well as of previous studies of alphavirus RNAs, including defective interfering RNAs.

Rice grassy stunt tenuivirus nonstructural protein p5 interacts with itself to form oligomeric complexes in vitro and in vivo.

We investigated the interaction of Rice grassy stunt tenuivirus (RGSV) nonstructural protein p5, a protein of 22 kDa encoded on vRNA 5, with all 12 RGSV proteins by using a GAL4 transcription activator-based yeast two-hybrid system. The p5 protein interacted only with itself and not with any other viral protein; the interacting domains were localized within the N-terminal 96 amino acids of p5. The p5-p5 interaction was reproduced in an Sos recruitment-mediated yeast two-hybrid system as well in by far-Western blots. Native p5 protein extracted from RGSV-infected rice tissue was detected in a large complex with a molecular mass of approximately 260 kDa composed of 12 molecules of p5 or a p5 oligomer with an unidentified host factor(s).