Cloning and Expression of SpltMNPV Sl136 Gene and Functions of the Expressed Product.

By computer-assisted analysis, it was revealed that ORF136 gene product in SpltMNPV genome had the basic properties of membrane protein. A putative signal peptide was present at the N-terminal and a transmembrane region near the C-terminal of SL136 protein. In the N-terminal half region, there was a coiled-coil domain, which is a typical feature of a number of viral fusion proteins. After PCR amplification, a recombinant plasmid pBVSl136 and a recombinant AcMNPV containing Sl 136 were constructed, in order to express Sl136 gene in E.coli and insect Hi5 cells, respectively. The SDS-PAGE results showed that both expression levels were high. Cell membrane fusion was induced in the Sl-zsu-1 cells, which had been transfected with Sl136 gene alone, by lowering pH of the medium. These results suggested that SL136 protein may be an envelope fusion protein.

Cloning and Sequence Analysis of the Chitinase Gene of Spodoptera litura Nuclear Polyhedrosis Virus.

Using AcMNPV chiA-containing fragment as a probe, the chitinase gene of Spodoptera litura nuclear polyhedrosis virus (SpltNPV) was localized in two contiguous fragments, XbaI 5.1 kb and 2.1 kb, and the intact SpltNPV chiA gene was obtained by cloning and sequencing those two fragments. The complete open reading frame of the gene was 1 695 nucleotide long, encoding a putative protein of 564 amino acids with molecular weight of 62.9 kD. Sequence analysis further revealed that the 5' noncoding region had a baculovirus late promoter motif TAAG. A polyadenylation signal, AATAAA, was located dowmnstream of the translation stop codon. A putative signal peptide was present at the N-terminus of the protein. This gene shares 57% homology with AcMNPV chitinase gene.

Apoptosis of Spodoptera litura cells induced by AcMNPV ie-1 gene.

Apoptosis was induced in Spodoptera litura Sl-zsu-1 cells transfected with plasmid pAcie-1 DNA containing ie-1 gene of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Apoptotic bodies appeared 24 h post transfection and cell mortality peaked after 48 h. Electrophoresis of total DNA extract of Sl-zsu-1 cells showed a DNA ladder, indicating that transfection of ie-1 gene alone can trigger apoptosis directly or indirectly. It was shown by cytochalasin D (CD) and ammonium chloride inhibition assays that the entry of virions into cytoplasm was required for induction of apoptosis. Reverse transcriptase PCR (RT-PCR) was used to demonstrate that ie-1 gene had transcribed in Sl-zsu-1 cells immediately after infection. Inhibition of DNA replication could not block the cell death process, suggesting that this apoptosis was independent on viral late replication events. Apoptosis did not occur when Sl-zsu-1 cells were infected with an AcMNPV mutant tsB821 at its nonpermissive temperature. Therefore, we concluded that the ie-1 gene is at least one of the factors inducing apoptosis in Sl-zsu-1 cells infected with AcMNPV.

Entry of Bombyx mori cypovirus 1 into midgut cells in vivo.

In vivo entry of Bombyx mori cypovirus 1 into the silkworm midgut was studied by electron microscopy of ultrathin sections of midguts from silkworm larvae that had been administered virus-contaminated leaves. In 3 h, virions were observed outside and inside midgut cells, including columnar cells, goblet cells and muscle cells. Virions were seen adhering to the plasma membrane of microvilli, embedding in the membrane and settling themselves intact inside the microvilli of the columnar cells. These results suggested that intact virions entered columnar cells by means of direct penetration through the cell membrane. In 12, 24 and 48 h, virogenic stromata and progeny virions were observed in columnar cells, but not in other midgut cells.