Roger J. Avery

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.

The RNAs of defective-interfering influenza virus.

Abstract Defective interfering progeny virus was produced by one or two high multiplicity passages of an avian influenza (fowl plaque) virus in chick embryo fibroblast cells, and the degree of defectiveness increased in proportion to the m.o.i. Analysis of RNA from virus preparations of increasing defectiveness showed a progressive decrease in the amount of virion RNA segments 1, 2, and 3. Their absence was corroborated by the inability of defective virus to direct the synthesis of Pl, P2, and P3 proteins in an in vitro-coupled transcription-translation system. The loss of RNA segments 1–3 was associated with an increase of six RNA segments smaller than virion RNA segment 8. The significance of the changes in virus particle RNA content are discussed in relation to the loss of infectivity.

The DNA content of four small iridescent viruses: genome size, redundancy, and homology determined by renaturation kinetics.

Abstract Fragmented DNA preparations from iridescent virus types 2, 6, 9, and 18 have been characterised for GC content and fragment size as a prelude to hybridisation studies. The genome size of the DNAs have been found to be 147 × 106, 152 × 106, 114 × 106, and 114 × 106 daltons for iridescent virus types 2, 6, 9, and 18, respectively, by studies of the kinetics of renaturation. Iridescent virus types 2 and 6 were found to contain a small proportion of DNA reannealing more rapidly than the bulk of the DNA, indicative of the presence of repeated sequences. In addition, the nucleotide sequence homology among the viruses has been investigated and found to range from 0 to 100%.

Expression and transmission of a rodent retrovirus-like (VL30) gene family☆

A transcriptionally active sub-set of the dispersed mouse VL30 family of proviral genetic elements was shown to be highly transmissible as a murine leukaemia virus pseudotype. Newly acquired VL30 proviruses (present at 1 to 2 copies per cell) were shown to be transcriptionally active. These data substantiate the hypothesis that this process of duplicative transposition may have played a major role in the evolution of the gene family and also demonstrate that VL30 elements would be capable of mediating oncogene activation by a promoter-insertion-type mechanism during leukaemia virus-induced tumourgenesis.

The Isolation and Characterization of a Clonally Related Series of Murine Retrovirus-infected Mouse Cells

Starting with cloned NIH 3T3 mouse cells we have isolated a series of related lines infected with the Kirsten murine sarcoma/leukaemia (MSV/MLV) virus complex. These lines exhibit all three possible infected cell phenotopes: (i) transformed MSV/MLV producers; (ii) non-transformed MLV producers; (iii) transformed non-producers. We have also selected non-transformed revertants from one of the non-producer clones. This series of lines allows the study of the expression of the virus genome against a constant background of cellular gene expression. We have further characterized the lines with regard to anchorage dependence of growth, tumorigenicity and the presence of a rescuable sarcoma genome. The non-producer clones are uniform in their transformed properties. The revertants contain rescuable sarcoma virus, biologically indistinguishable from the original transforming virus, implying that the reversion is due to a change in cellular rather than viurs genetic information.

Oligonucleotide Fingerprinting of the RNA Species Obtained from Six Drosophila C Virus Isolates

Summary The virion RNAs of six independent isolates of Drosophila C virus (DCV) have been characterized by ribonuclease T1 oligonucleotide fingerprinting. All six isolates share common large oligonucleotides. Two of the isolates, from Vigier and Charolles, are closely related while a third French isolate from Gif is more distantly related. The other three isolates, two from Morocco (Taroudant and Ouarzazate) and one from the French Antilles were mixtures of more than one variant of DCV, but were clearly related to the three French isolates.

Camptothecin: an Inhibitor of Influenza Virus Replication

Summary Camptothecin, an inhibitor of cellular nucleic acid synthesis, prevents fowl plague virus replication in BHK 21/13 cells. In the presence of camptothecin the synthesis of haemagglutinin and neuraminidase antigens does not occur; the ribonucleoprotein antigen is synthesized and accumulates only in the cell nucleus; and the synthesis of virus-specific RNA (of which complementary RNA predominates) is reduced considerably.

Influence of the host cell on the genomic and subgenomic RNA content of defective-interfering influenza virus.

Clonally derived stocks of defective-interfering (DI) influenza virus prepared by chick embryo fibroblast (CEF) cells were all deficient in genomic RNAs 1, 2 and 3 but had different patterns of subgenomic RNAs. A single passage at high multiplicity in L cells altered the pattern of genomic RNAs independently of the subgenomic species, while in BHK cells the opposite situation prevailed. Therefore, there is no simple relationship in DI influenza virus between the loss of genomic RNA segments and the presence of subgenomic RNAs.

The subcellular localization of virus-specific RNA in influenza virus-infected cells.

Summary The synthesis of complementary RNA (cRNA) precedes that of virus particle RNA (vRNA) in fowl plague virus-infected cells. Early cRNA synthesis occurs in the cytoplasm, while the later vRNA synthesis is a nuclear event. Influenza RNA synthesized in infected cells does not contain poly A sequences, as judged by binding to nitrocellulose filters under controlled conditions.

Genetic organization and cloning of kirsten murine sarcoma virus DNA

Summary The Kirsten murine sarcoma virus genome is a recombinant molecule derived from the Kirsten murine leukemia virus genome and sequences transduced from a normal rat cell. We show here that leukemia virus derived sequences comprise the 5′ terminal 0.5 kilobasepairs (kbp) and the 3′ terminal 1.2 kbp of the linear 6.5 kbp sarcoma virus DNA. The parental leukemia virus thus contributes the long terminal repeat sequences (0.5 kbp) and some additional 3′ sequences to the sarcoma virus genome. Using DNA synthesised by detergent-disrupted virions a 4.6 kbp SmaI fragment containing the rat-specific sequences of the sarcoma virus was cloned into the plasmid pAT153. This clone was then used as a sarcoma virus specific probe to clone full sized circular viral DNA from infected NIH 3T3 cells.