Yukio Shirako

Evolutionary Relationships and Systematics of the Alphaviruses

ABSTRACT Partial E1 envelope glycoprotein gene sequences and complete structural polyprotein sequences were used to compare divergence and construct phylogenetic trees for the genus Alphavirus. Tree topologies indicated that the mosquito-borne alphaviruses could have arisen in either the Old or the New World, with at least two transoceanic introductions to account for their current distribution. The time frame for alphavirus diversification could not be estimated because maximum-likelihood analyses indicated that the nucleotide substitution rate varies considerably across sites within the genome. While most trees showed evolutionary relationships consistent with current antigenic complexes and species, several changes to the current classification are proposed. The recently identified fish alphaviruses salmon pancreas disease virus and sleeping disease virus appear to be variants or subtypes of a new alphavirus species. Southern elephant seal virus is also a new alphavirus distantly related to all of the others analyzed. Tonate virus and Venezuelan equine encephalitis virus strain 78V3531 also appear to be distinct alphavirus species based on genetic, antigenic, and ecological criteria. Trocara virus, isolated from mosquitoes in Brazil and Peru, also represents a new species and probably a new alphavirus complex.

Two Purified RNAs of Soil-borne Wheat Mosaic Virus are Needed for Infection

Summary RNAs of soil-borne wheat mosaic virus (SBWMV) from virions 281 nm, 138 nm and 92 nm long (designated here by relative lengths as 1.0L, 0.5L and 0.35L, respectively), were isolated and purified by three cycles of sucrose density gradient centrifugation. Infectivity assays with these RNAs proved the bipartite nature of SBWMV, the combination of 1.0L and either 0.5L or 0.35L RNAs being required for infection and for multiplication of progeny viruses. The 0.5L RNA underwent deletion mutation, producing smaller variants with various sizes, of which 0.4L and 0.35L RNAs were confirmed to be functional in combination with 1.0L RNA. The coat proteins of all isolates had mol. wt. of 19700. The mol. wt. of 1.0L, 0.5L, 0.4L and 0.35L RNAs, determined under denaturing conditions, were 2.28 × 106 (6500 bases), 1.23 × 106 (3500 bases), 0.97 × 106 (2800 bases) and 0.86 × 106 (2450 bases), respectively. A new virus group, furovirus (fungus-borne rod-shaped virus), is proposed for SBWMV.

Spontaneous Deletion Mutation of Soil-borne Wheat Mosaic Virus RNA II

Summary The wild-type (WT) isolate of soil-borne wheat mosaic virus has two species of rod-shaped virions 281 nm and 138 nm in length, which are designated as virions 1.0L and 0.5L, respectively. We reported previously that virions shorter than 0.5L arose in assay plants inoculated with separated and recombined 1.0L RNA and 0.5L RNA and suggested that these short virions were caused by deletion mutation of 0.5L RNA. We now report that these short virions arose after a period of several months in wheat plants that had been inoculated manually with unpurified WT isolate, and also when plants infected naturally in fields infested by the fungal vector, Polymyxa graminis, were grown at 17 °C. The sizes and relative proportions of virions shorter than 0.5L varied both from plant to plant and in the same plant sampled at different times. This indicates that the virions shorter than 0.5L arose by continued and spontaneous deletion mutation of 0.5L RNA.

Comparison of the in vitro Translation Products of Wild-type and a Deletion Mutant of Soil-borne Wheat Mosaic Virus

Summary A wild-type isolate (WT) of soil-borne wheat mosaic virus (SBWMV, isolate JT) (RNA I 6·9 kb and RNA II 3·6 kb) was successively transferred to wheat plants by mechanical inoculation and a deletion mutant (DM) (RNA I 6·9 kb and RNA II 2·1 kb) was produced. In wheat germ extracts, RNA I of both WT and DM directed synthesis of polypeptides having mol. wt. of 220000 (220K) and 150K, and RNA II of both WT and DM directed synthesis of 25K and 19K polypeptides. Incubation in wheat germ extracts of WT or DM virions purified with an alkaline buffer also gave 220K, 25K and 19K polypeptides as major products and a 150K polypeptide as a minor product. In rabbit reticulocyte lysates, RNA I of both WT and DM directed synthesis of only the 220K polypeptide, whereas WT RNA II produced 100K, 46K, 25K and 19K polypeptides and DM RNA II, 31K, 25K and 19K polypeptides. SBWMV DM antiserum precipitated polypeptides of 100K, 31K, 25K and 19K but not 220K and 46K. The 19K polypeptide was identified as the capsid protein from its electrophoretic mobility and its reaction with antiserum to virus particles. Thus, the differences between the in vitro translation products of WT and DM SBWMV were confined only to those coded by RNA II.

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.

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.

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.

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

ABSTRACT 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°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

ABSTRACT 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).

Reassortment between genetically distinct Japanese and US strains of Soil-borne wheat mosaic virus : RNA1 from a Japanese strain and RNA2 from a US strain make a pseudorecombinant virus

Summary Soil-borne wheat mosaic virus (SBWMV), the type species of the genus Furovirus, has a plus-sense bipartite RNA genome. Japanese and US strains of SBWMV are genetically distantly related, despite their biologically identical properties. Here we report formation of a pseudorecombinant virus consisting of RNA1 from a Japanese strain and RNA2 from a US strain, using infectious in vitro transcripts for both strains. Full-length infectious cDNA clones for a Japanese strain were previously constructed (Yamamiya and Shirako [38]). For RNA1 of a US strain, due to instability of full-length cDNA clones in Escherichia coli cells, it was necessary to prepare a full-length template DNA for in vitro transcription by combining overlapping 5′-terminal and 3′-terminal cDNAs individually cloned in two plasmids, whereas for RNA2 a full-length cDNA clone was the template. For infectivity assays, Chenopodium quinoa, a local lesion host, and wheat, a systemic host, were used. A mixture of Japanese RNA1 transcripts and US RNA2 transcripts caused formation of local lesions on C. quinoa leaves and systemic infection to wheat plants. The nucleotide sequence of the progeny viral RNA2 was identical to that of the US RNA2. The reciprocal combination was not infectious to either host. These results confirm that the Japanese and US SBWMV are genetically distantly related strains belonging to a single species.