David C. Kelly

The detection of intracellular retrovirus-like entities in Drosophila melanogaster cell cultures.

A Drosophila melanogaster cell line has been examined for the presence of retrovirus particles. When these cells were disrupted and analysed on sucrose density gradients a subcellular fraction with a density of 1.22 g/ml was found to possess endogenous DNA polymerase activity and could catalyse polymerization of deoxynucleotide triphosphates in response to added template primers. The latter activity had the cation and template primer responses expected for reverse transcriptase. A high mol. wt. polyadenylic acid-containing RNA was also purified from this fraction and could be dissociated by heat treatment into 30 to 35S and smaller species. Electron microscopy revealed the presence of torroidal forms reminiscent of intracytoplasmic A-type retrovirus particles within the Drosophila cells. Similar forms were found associated with the subcellular fraction of 1.22g/ml. We concluded that our D. melanogaster cell line contains retroviruses similar, but not identical, to the A-type particles previously described in mammalian and avian cells.

A Small Iridescent Virus (Type 29) Isolated from Tenebrio molitor: a Comparison of its Proteins and Antigens with Six Other Iridescent Viruses

Summary A small iridescent virus (type 29) has been isolated from the meal worm Tenebrio molitor. The virus is distinct from a number of previous isolates of small iridescent viruses (types 2, 6, 21, 22, 23 and 28) judged by polyacrylamide gel electrophoresis of its structural polypeptides. Immunodiffusion and immunoprecipitation tests showed that iridescent virus type 29 is related to types 22 and 23 but not types 2, 6, 21 and 28. The relationships between iridescent virus types 22, 23 and 29 were further studied by complement fixation and kinetic neutralization. Complement fixation tests confirmed that these viruses were related and showed that type 29 is distantly related to types 22 and 23 (which were more closely related to each other) though type 23 shares a closer relationship with type 29 than type 22. Kinetic neutralization experiments suggested a close relationship between types 29 and 23, and that both these viruses were remotely related to type 22. Low neutralization rate constants were obtained with these sera.

Three African isolates of small iridescent viruses: Type 21 from Heliothis armigera (Lepidoptera: Noctuidae), type 23 from Heteronychus arator (Coleoptera: Scarabaeidae), and type 28 from Lethocerus columbiae (Hemiptera Heteroptera: Belostomatidae)

Abstract Three small (130-nm) iridescent viruses have been found in invertebrates indigenous to Africa: Heliothis armigera, Heteronychus arator , and Lethocerus columbiae . Assessed by polyacrylamide-gel electrophoresis of the structural polypeptides and by serological tests (immunoneutralisation, immunoprecipitation, and complement fixation), the viruses represent related but not identical isolates.

Molecular cloning and physical mapping of the genome of simian herpes B virus and comparison of genome organization with that of herpes simplex virus type 1

The molecular structure of the genome of simian herpes B virus (SHBV) was determined by restriction endonuclease mapping studies. Genomic DNA was cleaved with restriction endonucleases BamHI and SalI into 41 and 58 fragments, respectively. Most of these fragments were cloned into the plasmid vector pACYC184; uncloned fragments were identified following isolation from agarose gels. Terminal fragments were identified by exonuclease digestion and radioactive end-labelling, and linkage of fragments was deduced by a combination of single and double digest experiments and cross-blot hybridizations. The genome is larger than that of herpes simplex virus type 1 (HSV-1), being approximately 165 kilobase pairs. Like that of HSV-1, the SHBV genome is composed of a long and a short unique region each flanked by inverted repeat sequences, which allow the unique regions to invert relative to one another, resulting in four possible isomeric arrangements of the molecule. Genome locations of several SHBV genes were compared with their HSV-1 homologues.

Baculovirus replication: phosphorylation of polypeptides synthesized in Trichoplusia ni nuclear polyhedrosis virus-infected cells

A number of polypeptides synthesized specifically in Trichoplusia ni multiple nucleocapsid nuclear polyhedrosis virus (T. ni MNPV)-infected Spodoptera frugiperda cells are phosphorylated both early and late in infection. Certain non-structural proteins and the major basic internal protein are the main phosphoproteins detected in infected cells. The polyhedron protein was not phosphorylated. Many cell proteins continue to be phosphorylated throughout infection. Pulse-chase experiments have shown that some polypeptides are stably phosphorylated whereas other polypeptides (including the major basic protein) have phosphates which cycle on and off. One polypeptide was substantially labelled only after a chase with unlabelled orthophosphate. Fractionation of cells into nucleus and cytoplasm showed that polypeptides located in both the cytoplasm and nucleus were phosphorylated.

Nucleotide sequence analysis of a homologue of herpes simplex virus type 1 gene US9 found in the genome of simian herpes B virus

The 10K gene of simian herpes B virus (SHBV) has been located and the nucleotide sequence determined. Its relationship to homologous genes in other herpes-viruses has been examined. The SHBV 10K gene exhibits a closer relationship to its homologue in HSV-1 than to those in the other viruses studied. Nucleotide sequence identity of 61% was found between the HSV-1 and SHBV 10K genes, and 57% identity was found between the corresponding predicted protein sequences. A comparison of the amino acid sequences of the herpesvirus 10K proteins has revealed a number of conserved features. These are examined in relation to possible functions of the 10K proteins. Implications for evolutionary relationships between SHBV and other herpesviruses are discussed.

Baculovirus Replication: Glycosylation of Polypeptides Synthesized in Trichoplusia ni Nuclear Polyhedrosis Virus-infected Cells and the Effect of Tunicamycin

Summary Infected cell-specific polypeptides (ICSP) in Trichoplusia ni nuclear polyhedrosis virus (MNPV)-infected cells could be radiolabelled with various sugar precursors including mannose, N-acetyl glucosamine, N-acetyl mannosamine and glucose. Glycosylation occurred mainly late in infection. Eleven polypeptides were glycosylated including the major envelope protein, the polyhedron protein (very late in infection) and a major low molecular weight non-structural polypeptide (also very late in infection). Tunicamycin inhibited T. niMNPV replication and prevented the addition of N-acetyl glucosamine and, to a lesser extent, mannose to the appropriate proteins. This had the effect of enhancing the migration in SDS-polyacrylamide gels of some glycopolypeptides but not that of the polyhedron protein. Tunicamycin prevented the envelopment of nucleocapsids both within the nucleus and at the plasma membrane. Polyhedron formation did, however, occur in the presence of tunicamycin.

A comparative study of the polypeptides of three iridescent viruses by N-terminal analysis, amino acid analysis, and surface labeling

Abstract Polyacrylamide gel analysis of the structural proteins of three types of iridescent viruses (2, 6, and 9) demonstrated that the purified virions had one major and more than 20 minor polypeptides. Surface labeling procedures performed on pure intact virions, using 125 I in the presence of lactoperoxidase and chloramine T (at low iodine concentrations), demonstrated that the major and two or three minor polypeptides were located on the outside. The major structural polypeptide was isolated from each virus type by preparative polyacrylamide gel electrophoresis. Amino acid analysis indicated that this protein was very similar in the three iridescent viruses. The three polypeptides had an identical N terminal (proline). While the major polypeptide of each virus has a slightly different molecular weight as determined by polyacrylamide gel electrophoresis, the similarities in iodine labeling, N terminals, and amino acids suggests a common function for this protein.

The Replication and Titration of Iridescent Virus Type 22 in Spodoptera frugiperda Cells

A plaque assay for iridescent virus type 22 (from Simulium sp.) using Spodoptera frugiperda cells has been devised, and the kinetics of growth of the virus in this cell line have been determined. The virus particle/p.f.u. ratio was 75 +/- 8, and the p.f.u./TCID50 ratio was 0.56 +/- 0.11.

Isolation of a Bisegmented Double-stranded RNA Virus from Thirlmere Reservoir

A novel bisegmented double-stranded RNA virus has been isolated from water processed from Thirlmere reservoir. The virus is icosahedral, 58 nm in diam., has a buoyant density of 1.32 g/ml in CsCl, has an S value of 400 and a RNA/protein ratio of 0.087. The two linear segments of RNA have approx. mol. wt. of 2.26 X 10(6) and 2.09 X 10(6). The virus contains six polypeptides. The virus was isolated in Drosophila melanogaster cells and fails to replicate in other insect, amphibian, avian, piscine, mammalian and plant cells tested. The virus is biochemically different from infectious pancreatic necrosis virus (IPNV) and Drosophila X virus (DXV). The virus is also serologically unrelated to IPNV (strain Sp) and another invertebrate pathogenic virus, Tellina virus 1. The virus shares common antigens with DXV but is not completely identical.