Tsuguo Matsumoto

SEQUENCES OF TEN CIRCULAR SSDNA COMPONENTS ASSOCIATED WITH THE MILK VETCH DWARF VIRUS GENOME

Milk vetch dwarf virus (MDV) is a member of the proposed genus Nanovirus, and its genome is composed of multiple, circular ssDNA components of about 1 kb. We have cloned and sequenced ten ssDNA components and designated them MDV-C1 to C10. Each DNA component contains one potential major open reading frame, and contains a putative stem-loop structure in the non-coding region. Notably, four components (C1, C2, C3 and C10) encode distinct replication-associated (Rep) proteins of 33 kDa, which show only limited (42-57%) amino acid identity. The six other components encode proteins with calculated molecular masses ranging from 12.7 to 19.7 kDa. Comparison of the sequences with those of other nanoviruses reveals that MDV is closely related to faba bean necrotic yellows virus (FBNYV) and subterranean clover stunt virus (SCSV). Six putative MDV genome products, including one Rep and five non-Rep proteins, show high (70.4-90.9%) amino acid identity to the corresponding six FBNYV proteins, whereas two other Rep proteins encoded by MDV-C2 and C3 are 82.3% and 73.0% identical to those encoded by SCSV-C2 and C6, respectively. These results indicate that MDV, FBNYV and SCSV have diverged from a common origin, which had multiple Rep components. In addition, the putative proteins encoded by MDV-C4 and its homologues contain a consensus retinoblastoma-binding motif, suggesting that they may be involved in controlling the host cell cycle.

Nucleotide sequences of genome segments 6 and 7 of Bombyx mori cypovirus 1, encoding the viral structural proteins V4 and V5, respectively.

Nucleotide sequence analyses of cDNAs derived from the double-stranded RNA genome segments 6 and 7 (S6 and S7) of Bombyx mori cypovirus 1 (BmCPV-1) have revealed that they consist of 1796 and 1501 nucleotides encoding putative proteins of 561 and 448 amino acids with molecular masses of 63604 and 49875 (p64 and p50), respectively. The amino acid sequence of p64, which has a high leucine residue content (10%), contains a leucine zipper motif. Antiserum raised against p64 specifically bound to a viral structural protein of ca. 68 kDa (V4), while antiserum against p50, which specifically bound to a protein of ca. 56 kDa in BmN4 cells infected with BmCPV-1, reacted with a cluster of four viral structural proteins ranging from ca. 34 to 40 kDa (V5). These observations indicate that p50 might be cleaved to V5 during the formation of virus particles.

The ecdysteroid UDP-glucosyltransferase gene of Autographa californica nucleopolyhedrovirus alters the moulting and metamorphosis of a non-target insect, the silkworm, Bombyx mori (Lepidoptera, Bombycidae)

The Autographa californica nucleopolyhedrovirus (AcMNPV) does not infect the silkworm and molecular studies on silkworm insusceptibility have not been performed. In cultured cells of the silkworm, the expression of viral genes has been reported. The expression of AcMNPV genes and their effect in vivo and in vitro was studied. In this study, the early gene, the ecdysteroid UDP-glucosyltransferase (egt) gene of AcMNPV, which inactivates the insect moulting hormone by sugar conjugation, was examined to determine whether it would alter the growth of the silkworm. Using wild-type (wt) AcMNPV, the egt gene deletion virus (vEGTDEL), and the virus carrying the egt promoter-lacZ cassette in vEGTDEL (vEGTZ), the egt promoter-driven expression in cultured cells and in nonproductive infection of the silkworm was characterized. Infection of cultured cells with vEGTZ at three different doses occurred in a single cell manner. When budded wt AcMNPV was injected into the fourth and fifth instar larvae, an increase in the amount of virus occurred and caused abnormal larval growth, which resulted in the prolongation or skipping of the larval instar, premature pupation, or death during the pupal stage. For infection of the fourth instar larvae, precocious metamorphosis was observed. When the same amount of vEGTDEL was injected, the alteration of growth did not occur. These results suggest that the egt gene was expressed in the primary infected cells of the silkworm, and that the EGT was secreted into the haemocoel, which significantly altered larval growth.

Production of an extracellular protease by Beauveria bassiana in the haemolymph of the silkworm, Bombyx mori

Protease production by Beauveria bassiana in the haemolymph of infected silkworms (Bombyx mori) were investigated by an enzyme‐linked immunosorbent assay. Results showed that B. bassiana produced a substantial amount of extracellular protease in the haemolymph of infected silkworms.

Electrophoretic karyotype of Metarhizium anisopliae

Abstract The chromosomal DNA molecules of Metarhizium anisopliae var. anisopliae have been separated into seven bands by pulsed-field gel electrophoresis. Using the Schizosaccharomyces pombe chromosomes as size standards, we estimate the size of the chromosomes to be between 1.6 and 7.4 megabase pairs (Mbp) and total genome sizes of the five isolates to be in the range 29.6 to 32.1 Mbp. Size differences between several chromosomes in the isolates were observed and electrophoretic karyotypes of the five isolates are readily distinguished from each other.

Isolation and characterization of a temperature-sensitive mutant of Bombyx mori nucleopolyhedrovirus for a putative RNA polymerase gene.

Temperature-sensitive (ts) mutants of Bombyx mori nucleopolyhedrovirus (BmNPV), which exhibited no polyhedra formation at the non-permissive temperature of 33 degrees C, were produced using the base analogue 5-bromodeoxyuridine. A unique ts mutant, designated ts-S1, was characterized in terms of gene mutation and virulence in cultured cells and silkworm larvae. Mutant-infected BmN4 cells at 33 degrees C showed normal viral DNA synthesis but defective budded virus production and polyhedrin synthesis, suggesting the absence of late and very late gene expression. Silkworm larvae were injected with ts-S1 and reared at 33.5 degrees C. At 7 days post-injection, none of the larvae exhibited nucleopolyhedrosis but some possessed viral DNA, detected by PCR using virus-specific primers. Continued rearing of the larvae at a permissive temperature of 25 degrees C showed that, while most developed into normal adults, some developed nucleopolyhedrosis, indicating that the former larvae had aborted the virus infection during the course of rearing at 33.5 degrees C. No viral DNA was detected in the adults. Marker rescue tests to identify the lesion involved in the ts phenotype of ts-S1, and nucleotide sequencing of the identified genome region, showed a single nucleotide mutation of a putative RNA polymerase gene, late expression factor-8 (lef-8). These results indicate that lef-8 is essential for BmNPV replication in vitro and in vivo.

Physical mapping and identification of interspersed homologous sequences in the Trichoplusia ni granulosis virus genome.

A restriction fragment library representing 89.3% of the genome of Trichoplusia ni granulosis virus (TnGV) was constructed. The library consisted of 13 of the 16 BamHI fragments, 18 of the 22 EcoRI fragments, and 6 of the 27 PstI fragments. By restriction endonuclease and Southern blot analysis of cloned or genomic viral DNA fragments, a complete physical map of TnGV was constructed for BamHI, EcoRI, PstI and XhoI. Three interspersed homologous regions (ihs1-ihs3) were identified from hybridization experiments and sequenced. Each TnGV ihs has an approximate size of 400 bp and shows homology to the other two. The orientation of ihs2 is inverted relative to ihs1 and ihs3. TnGV ihs regions do not have repetitive motifs or palindromic sequences, in contrast to homologous regions (hrs) of nuclear polyhedrosis viruses (NPVs). The genomic locations of TnGV ihs1-ihs3, represented in percentage map units, were very similar to those of ihs sequences previously reported in Bombyx mori NPV, suggesting that the ihs may be a novel type of cis-acting element common among baculoviruses. Additionally, an inverted repeat sequence, having overlapping, multiple inverted repeats of 400 bp, was identified to the left of ihs3 on the linearized genome map of TnGV.

Induction of a hemagglutinating activity in the hemolymph of the silkworm, Bombyx mori, infected with cytoplasmic polyhedrosis virus

Abstract The hemagglutinating activity increased significantly in the hemolymph of the fifth instar larvae of the silkworm, Bombyx mori, infected with cytoplasmic polyhedrosis virus (CPV). Analysis by immunoblotting clearly indicated the enhanced accumulation of a protein with hemagglutinating activity in the hemolymph of the silkworms infected with CPV, which is a lectin-like protein, possibly newly induced during infection with CPV. These results suggest the protein to be a candidate responsible in the silkworm for the immune defense against exogenous agents including CPV.

Electrophoretic karyotyping of the entomogenous fungus Paecilomyces fumosoroseus

Pulsed‐field gel electrophoresis was used to compare the electrophoretic karyotypes of isolates of Paecilomyces fumosoroseus. Electrophoretic karyotypes of P. fumosoroseus exhibit a high degree of similarity among the isolates. However, hybridization data indicated that similar sized chromosomes among the isolates did not always bear the same genetic information.

Isolation of cDNA clones coding for humoral lectin of silkworm, Bombyx mori, larvae.

In the hemolymph of the silkworm, Bombyx mori, lectin with hemagglutinating activity against sheep red blood cells increases at larval-larval ecdysis and at spinning stage (Suzuki and Natori, 1983) and is induced by infection with cytoplasmic polyhedrosis virus. A Bombyx lectin polypeptide with molecular weight approx 280K is responsible for hemagglutinating activity, since antiserum raised against this polypeptide inhibited hemagglutinating activity. The site of synthesis of Bombyx lectin was determined by primary tissue cultures of fat body and hemocytes. A hemagglutinating activity assay demonstrated that hemocyte is responsible for the release of hemagglutinin into the culture medium. Isolation of cDNA clones coding for Bombyx lectin was carried out on the cDNA library prepared in an expression vector lambda gt11 starting with poly(A)+ RNA from spinning larval hemocytes. As a result of immunoscreening, several positive clones were obtained, and the cDNA clones were characterized.