Shigemi Kawase

TERMINAL STRUCTURE OF A DENSOVIRUS IMPLIES A HAIRPIN TRANSFER REPLICATION WHICH IS SIMILAR TO THE MODEL FOR AAV

We cloned the complete sequence of Bombyx DNV (Ina isolate; Bm DNV-1) genome in a bacterial plasmid pUC 119 and determined the nucleotide sequences of both termini, resulting in elucidation of the nucleotide sequence of the complete genomic DNA of DNV. The complete sequence of the DNV DNA (5048 nucleotides) has inverted repeats of 225 nucleotides and the terminal 153 nucleotides are palindromic. The palindromes can fold back on themselves to form a hairpin structure but, unlike AAV, the small internal palindrome which forms a T-shaped conformation was not observed. End-label analysis demonstrated that the palindromic sequences at both termini can exist in either of two orientations (flip or flop) in virion DNA with different frequencies. These data suggest that the hairpin transfer model for AAV replication must be modified to explain the DNV replication. Additionally, a comparison study on the terminal structures of insect, human, and rodent parvoviruses allowed a prediction on the ancestral terminal structure of parvovirus genome.

DNA of a new parvo-like virus isolated from the silkworm, Bombyx mori

Abstract Parvo-like virus, which was designated as “Ina-flacherie virus (Ina-FV),” was isolated from the silkworm, Bombyx mori, and the properties of its DNA were characterized. Purified Ina-FV had a diameter of 22 ± 0.5 nm and a sedimentation coefficient of 102 S. On density gradient separation in CsCl, particles were found at densities of 1.40 and 1.45 g/ml. The DNA content of Ina-FV was 28 ± 2%. The DNA in low-salt buffer possessed properties typical of a single-stranded (ss) molecule. Double-stranded (ds) DNA was extracted under conditions of appropriate high salt and elevated temperature. Electron microscopical examination revealed that the ds DNA was composed of linear molecules with an average length of 1.7 μm and other less well-defined structures. The linear ds molecule had a molecular weight of about 3.4 × 106 determined by electron microscopy (EM) and agarose gel electrophoresis. When the ds DNA was alkali-denatured and examined in an EM, linear ss molecules with approximate length of 1.7 μm were observed, indicating that the linear ds molecule was formed from the annealing of the linear ss molecules of unit length. These data suggest that Ina-FV is closely related to members of the densovirus subgroup.

Structural analysis on the single-stranded genomic DNAs of the virus newly isolated from silkworm: the DNA molecules share a common terminal sequence

SummaryRecently, a parvo-like virus was newly isolated from silkworm larvae and the two viral DNAs (VD1 and VD2) with different electro-mobilities were identified. We cloned the viral DNAs in a plasmid pUC119 and demonstrated that these two DNAs were not a bimorphic molecules though they shared a common terminal sequence of 53 nucleotides. In addition, the sequence at the 5′ terminus of each strand of the viral DNA was located in inverted form at its 3′ terminus. On the other hand, the nucleotide sequences of VD1 and VD2 were different from that of the Bombyx densovirus (Ina isolate) DNA.

Effect of high temperature on the development of nuclear polyhedrosis virus in the silkworm, Bombyx mori

Abstract Effect of a high temperature on the development of nuclear polyhedrosis and nuclear polyhedrosis virus (NPV) was studied employing pupae and isolated pupal abdomens of the silkworm, Bombyx mori. It was shown that pupae inoculated with an NPV and incubated at 35°C survived longer than those incubated at 25°C. At lower dosages of virus, pupae at 35°C escaped death from NPV. When inoculated pupae were incubated at 35°C for varying periods and then transferred to 25°C, the longer the pupae had been kept at 35°C the longer they survived. In contrast, when inoculated pupae were transferred from 25° to 35°C, the longer the pupae had been kept at 25°C the sooner after inoculation they died. Essentially the same results were obtained in isolated abdomens which were in an arrested state of development, excluding the possibility that observed thermal inhibition of viral diseases is dependent upon the altered developmental processes at high temperatures. Virus titration experiments showed that, under experimental conditions utilized, no detectable accumulation of infectious NPV was present in abdomens inoculated with an NPV and incubated at 35°C. When inoculated abdomens were shifted up from 25° to 35°C at 3 days postinoculation, NPV accumulation was inhibited almost immediately, and when inoculated abdomens were shifted down from 35° to 25°C, infectious NPV started to accumulate as early as 1 day after the shift. It was also shown that the pattern of infectious NPV accumulation and that of nucleic acid increase in infected abdomens gave a rough correlation. These results indicate that the thermal inhibition of viral diseases is attributed, at least in part, to the restricted accumulation of infectious progeny and suggest that the virus replication mechanism itself is more sensitive to high temperatures than that related to other events necessary for viral replication to be initiated.

Characteristics of structural proteins of infectious flacherie virus from the silkworm, Bombyx mori.

Abstract Structural proteins and the characteristics of infectious flacherie virus (IFV) purified from the silkworm, Bombyx mori, are described. The purified IFV had four major structural proteins, which were detected only in high concentration gels of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a few minor ones. Molecular weights of the major proteins were 35,200 (VP 1), 33,000 (VP 2), 31,200 (VP 3), and 11,600 (VP 4), and numbers per virion were 62, 57, 54, and 31, respectively. Amino acid compositions of VP 1, VP 2, and VP 3 were similar to each other but that of VP 4 was somewhat different. By isoelectric focusing and two-dimensional electrophoresis, high resolution of the structural proteins was obtained with silver staining. The isoelectric points of the four major proteins were determined as 7.7(VP 1), 6.7(VP 2), 4.8(VP 3), and 5.5(VP 4). This work is the first report on insect picornaviruses that presents some discriminative properties of each viral protein that was compared to those of mammalian picornaviruses.

Structural proteins of densonucleosis virus isolated from the silkworm, Bombyx mori, infected with the flacherie virus

Abstract Four structural proteins were found in highly purified Bombyx densonucleosis virus particles which were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight was estimated from the relative mobility and the retardation coefficient. The major viral protein (VP1), accounting for 65% of the total virion protein, had a molecular weight of about 50,000, and the other three minor proteins (VP2, VP3, VP4) had molecular weights of about 57,000, 70,000, and 77,000, respectively. The Bombyx densonucleosis virus particle contains about 60 molecules of VP1, and VP1 is believed to be capsid protein.

Tyrosinase in the silkworm during the pupation period

Abstract The change in tyrosinase activity in silkworm blood from the full-grown larval stage to pupation follows a V-shaped curve. In the integument the curve is almost reversed, and the activity is lowest just after pupation. Tyrosinase activity in the blood plasma gives an almost similar curve to that of whole blood, whereas in the haemocytes the activity is rather constant and the ratio of the activity in blood plasma to whole blood was very low at pupation compared with any other time. Tyrosinase could be observed only in oenocytoids (and/or phagocytes of the pupal type); the other types of haemocytes scarcely stained with Nadi reagent or catechol solution. During pupation the darkly stained haemocytes amount to several per cent of all haemocytes. The change of tyrosinase activity in acetone powder of the blood is fairly different from that of intact blood, and the amount of copper found in blood plasma is higher than in the whole blood during pupation.

Nucleotide sequence of the polyhedrin gene of Bombyx mori cytoplasmic polyhedrosis virus A strain with nuclear localization of polyhedra

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) strain H produces many hexahedral polyhedra (inclusion bodies) in the cytoplasm of insect midgut epithelial cells. The mutant A strain, however, produces polyhedra in the nucleus. We determined cDNA sequences of the polyhedrin genes, the smallest of the 10 genome segments, of these two strains. The polyhedrin genes of the H and A strains were 944 bp long, and encoded polypeptides of 248 amino acids (Mr 28,500) and 252 amino acids (Mr 29,000), respectively. The extra four amino acid residues at the carboxy terminus of the strain A polyhedrin (Arg-Leu-Leu-Val) were the result of a single nucleotide substitution at an opal stop codon (TGA----CGA). A further amino acid substitution of the histidine residue at position 101 (His----Tyr) was seen. The carboxy-terminal extension revealed a considerable similarity to the consensus amino acid sequence of the DNA-binding domain of many DNA-binding proteins. We discuss the relationship between the intracellular localization of polyhedrins and mutations in their amino acid sequences.

Molecular cloning and characterization of Bombyx densovirus genomic DNA

SummaryA map of theBombyx densovirus (DNV) genomic DNA has been constructed by cleaving it with several restriction endonucleases. This map shows that this virus and the mammalian parvoviruses are distinct. A restriction fragment containing more than 85 per cent of the complete viral genome has been cloned in a bacterial plasmid.

In vitro translation of infectious flacherie virus RNA in a wheat germ and a rabbit reticulocyte system

Infectious flacherie virus is an insect picornavirus isolated from the silkworm, Bombyx mori. Its RNA was found to act as an efficient mRNA in a wheat germ extract and a rabbit reticulocyte lysate translation system. In either system the sum of molecular weights of translation products far exceeded the coding capacity of the virus genome, which suggests the occurrence of proteolytic cleavage of large primary products to smaller polypeptides as reported for other picornaviruses and/or premature termination of translation. The highest molecular weight product of 200 000 (polyprotein-like product) could be translated in both systems. One of the antigenic products common to both systems had a molecular weight of 130 000, which corresponds to the sum of molecular weights of the four major viral proteins. Another product, which comigrated with viral protein 0, the largest viral structural protein in SDS-polyacrylamide gel electrophoresis, also showed antigenicity. Peptide mapping of these polypeptides showed that the two in vitro systems translated the same cistron in the viral RNA and that the smaller polypeptide was a part of the 130 000 Da product.