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PubMed1991

Cell-free expression of the coxsackievirus 3C protease using the translational initiation signal of an insect virus RNA and its characterization.

We have expressed the 3C protease of coxsackievirus B3 (CVB3) in a cell-free system. This expression system employs the translational initiation signal of an insect virus RNA, black beetle virus (BBV) RNA 1, to direct CVB3-specific protein synthesis. Using this expression system, we demonstrate that a biologically active 3C protease is synthesized which possesses both cis and trans processing capabilities. This in vitro-synthesized 3C protease is analogous to the native 3C, which was obtained from cytoplasmic extracts of CVB3-infected HeLa cells, in all biological parameters that were evaluated. In addition, antibody prepared against the 3C protease purified from extracts of CVB3-infected HeLa cells cross-reacts with the 3C protease produced in this cell-free system. Using the translational initiation signal from BBV RNA 1, we also have expressed the CVB3 capsid precursor and part of the P2 region in vitro, and have shown that the capsid precursor is cleaved between 1C (VP3) and 1D (VP1) by the proteolytic activity of in vitro-synthesized 3C in trans. Evidence also is presented to implicate the 2A protein of CVB3 as having proteolytic function.

3C Viral Proteases↗
PubMed2006

Genomic sequence of Spodoptera frugiperda Ascovirus 1a, an enveloped, double-stranded DNA insect virus that manipulates apoptosis for viral reproduction.

Ascoviruses (family Ascoviridae) are double-stranded DNA viruses with circular genomes that attack lepidopterans, where they produce large, enveloped virions, 150 by 400 nm, and cause a chronic, fatal disease with a cytopathology resembling that of apoptosis. After infection, host cell DNA is degraded, the nucleus fragments, and the cell then cleaves into large virion-containing vesicles. These vesicles and virions circulate in the hemolymph, where they are acquired by parasitic wasps during oviposition and subsequently transmitted to new hosts. To develop a better understanding of ascovirus biology, we sequenced the genome of the type species Spodoptera frugiperda ascovirus 1a (SfAV-1a). The genome consisted of 156,922 bp, with a G+C ratio of 49.2%, and contained 123 putative open reading frames coding for a variety of enzymes and virion structural proteins, of which tentative functions were assigned to 44. Among the most interesting enzymes, due to their potential role in apoptosis and viral vesicle formation, were a caspase, a cathepsin B, several kinases, E3 ubiquitin ligases, and especially several enzymes involved in lipid metabolism, including a fatty acid elongase, a sphingomyelinase, a phosphate acyltransferase, and a patatin-like phospholipase. Comparison of SfAV-1a proteins with those of other viruses showed that 10% were orthologs of Chilo iridescent virus proteins, the highest correspondence with any virus, providing further evidence that ascoviruses evolved from a lepidopteran iridovirus. The SfAV-1a genome sequence will facilitate the determination of how ascoviruses manipulate apoptosis to generate the novel virion-containing vesicles characteristic of these viruses and enable study of their origin and evolution.

Animals↗
PubMed1987

Microcarriers as a culturing system of insect cells and insect viruses.

There is an increasing interest in culturing of insect cells, which are host for arthropod-born (Arbo) viruses. The potential applications of Arbo viruses are in the following two main fields: 1. Medical applications (e.g. preparation of viral vaccines and viral antigens for diagnostic purposes). 2. As bioinsecticides in pest control in horticulture, agriculture and forestry. One of the potential cell substrates for these applications is an anchorage-dependent-mosquito cell line established from embryonic tissues of Aedes aegypti (AA). The following areas were investigated in the reported research with the AA cell line: The AA cells were successfully propagated in microcarrier (MC)-culturing-systems. Of the tested MC's the cellulose-based microgranular MC (DE-53 of Whatman, having an exchange capacity of 2 meq/g) was found to be the best MC. Cells grew in MCs-cells aggregates in submerged spinner culture. The AA-MC's culture was successfully scaled-up to 8 litre culture volume. "Trypsinization" of the AA cells from the MC surface is successfully done by RDB, a dispersion agent from a plant origin (produced and marketed at the author's Institute). Other known dispersion agents (trypsin, collagenase, pronase) failed to disperse the AA cells from the MC. A serum-free medium was developed for culturing the AA cells on the DE-53 MC's. Bovine serum albumin was the main serum substituant in the developed medium. Arboviruses, from the Toga group, were grown in the AA-MC culture. Sindbis virus (from alpha-group) and West Nile virus (from the Flavi group), chronically infected the AA cells, which continuously produce and liberate these two viruses.(ABSTRACT TRUNCATED AT 250 WORDS)

Aedes↗
PubMed1953

Insect viruses.

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Animals↗