Pseudotypes of vesicular stomatitis virus determined by exogenous and endogenous avian RNA tumor viruses

Vesicular stomatitis virus (VSV) forms pseudotypes with envelope components of avian RNA tumor viruses. The VSV pseudotypes possess the specific host range, interference, and antigenic properties of the tumor virus. Growth of VSV in cells expressing chick cell-associated helper factor coded by an endogenous viral genome yields pseudotypes with the envelope specificity of the helper factor.     

High specific infectivity avian RNA tumor viruses

Conditions for obtaining avian RNA tumor viruses of high specific infectivity were investigated. The following results were obtained:

1. (1) Pr-C-RSV showed highest specific infectivity when harvested from virus-producing chick embryo fibroblasts at 20-min intervals. Such virus was 5 times more infectious than virus harvested at 30-sec intervals, and 10 times more infectious than virus harvested at 24-hr intervals. Other cloned nondefective RNA tumor viruses showed similar patterns.

2. (2) Prompt chilling of virus collected at frequent intervals was essential because otherwise it was heat-inactivated rapidly. Virus harvested at 24-hr intervals was heat-inactivated much more slowly. However, this difference was not due to differences in the virus particles, but to differences in media that had been in contact with cells for short or long periods of time, respectively.

     

Electrophoretic analysis of the RNA of avian tumor viruses

The heat-dissociated RNA of avian leukosis and sarcoma viruses of several subgroups was analyzed by polyacrylamide gel electrophoresis. Heterogeneous patterns obtained in early experiments appeared to be due primarily to RNA degradation during steps of RNA characterization by sucrose gradient centrifugation rather than procedures of virus purification, virus incubation at 37°, or RNA extraction. All the viruses studied contained a major RNA species with an estimated size between 2.5 and 2.8 × 10⁶ daltons and at least one minor RNA species of slightly smaller size. This suggests that if the genome of avian tumor viruses is indeed segmented, the subunits are of a similar size. The heat-dissociated RNA of RAV-2, RAV-60, RAV-50, and Schmidt-Ruppin RSV (subgroup A) was generally less complex than that of Schmidt-Ruppin RSV (subgroup D). No significant difference was found in the RNA of RAV-2 grown in chicken cells that were positive or negative for the expression of latent viral genes (chf). However, in the absence of other known leukosis viruses, the pattern of heat-dissociated RNA of Bryan RSV grown in cells expressing chf functions was consistently broader than that of Bryan RSV grown in cells not expressing chf functions.     

Genomes of endogenous and exogenous avian retroviruses

The endogenous viruses of chickens are closely related to the exogenous avian leukosis viruses (ALV) yet as a group differ from these viruses in their host range, growth rate, and oncogenicity. The present study was undertaken to determine the patterns of relationship among the genomes of endogenous and exogenous ALVs. Complete or partial T1 oligonucleotide maps were prepared from the genomes of endogenous viruses that reside at eight distinct loci in chickens. Selected endogenous viruses and recombinants of endogenous or endogenous and exogenous viruses were characterized for host range and growth rate. From these data we could infer the following: (1) Endogenous viruses form a distinct lineage of ALVs with the most distinctive differences occurring in the portion of env that encodes host range and the U3 portion of the long terminal repeat; (2) The U3 sequences of endogenous ALVs determine the low growth rates of these viruses; and (3) Endogenous ALVs have distinctive oligonucleotide markers that allow them to be subclassified into distinct lineages. Our results suggest that endogenous viruses are derived from one another and not from exogenous field strains of ALV. This phenomenon may be related to the unique env encoded host range of endogenous ALVs, their unique U3 encoded growth rates, or perhaps their unique access, as residents of germ line DNA, to germ line cells.     

A study of plaque formation with avian RNA tumor viruses.

Avian leukosis viruses of subgroups B, D, F, and one strain of reticuloendotheliosis virus (REV) are able to make plaques in several lines of chick embryo fibroblasts. Moreover, plaque formation with some strains of subgroup A is obtained only in chicken fibroblasts of $\text{C}\text{C}$ phenotype. One exception is RAV-3 which produces plaque in line 6 ($\text{C}\text{E}$) only. Several important steps for the reproducibility of the assay are described. Of all the vital stains tested, only neutral red incorporated into the agar overlay is capable of inducing the formation of plaques. Several lysosome stabilizers and labilizers are examined and their role in the mechanism of plaque formation is discussed.     

Susceptibility and resistance of chicken macrophages to avian RNA tumor viruses.

A set of trans dominant mutations in the cI repressor gene of λ bacteriophage was mapped with the aid of a set of deletions. The clustering of all the mutants in a restricted region of the repressor gene suggests that these mutants may alter a site of the repressor responsible for the recognition of the operator.     

Subgroup-specific antigenic determinants of avian RNA tumor virls structural proteins: analysis of virus recombinants.

Two nonglycosylated structural proteins of avian RNA tumor viruses, p15 and p19, were examined for the presence of subgroup-specific antigenic determinants by using competition radioimmunoassays. A comparison of viruses from subgroups A, B, and C revealed that subgroup B and C virus proteins were equally efficient in inhibiting the homologous radioimmunoassays which used antiserum against B77 and iodinated p15 and p19 from B77. On the other hand, subgroup A virus proteins were less efficient than subgroup C viruses in causing inhibition in these assays. That these differences in inhibition were due to true immunological differences was confirmed by using heterologous competition radioimmunoassays. Since it was possible to distinguish the p19 or p15 of subgroup A viruses from the corresponding proteins of either subgroup B or subgroup C virus, recombinant viruses from crosses between leukosis viruses of subgroup A and sarcoma viruses of subgroups B or C were examined. The recombinant viruses have the envelope glycoprotein gene (env) of the subgroup A virus and the sarcoma gene (src) of the subgroup B or C virus. The results show that 14 clones out of 17 examined had p19 from the sarcoma virus. RNA fingerprinting has shown that the src gene is located close to the 3′ end of the genome and is closely followed towards the 5′ end by the env gene. The observed linkage between src and p19 can be explained by postulating that the gene for p19 is located close to the 5′ end of the genome and that recombination takes place between circular forms of the virus genome.     

Genetic diversity among avian influenza viruses

The RNAs of a series of avian influenza viruses of the subtype Hav7Neg2 were examined to determine if their antigenic similarity reflected an overall conservation of their RNA sequences. Genetic analysis by gel electrophoresis showed a marked variability in all of the RNA segments of the isolates. Analysis by competitive hybridization indicated that with some genome segments this variability represented major genetic differences. These differences were present even among concurrent isolates from one geographical area. In contrast, similar analysis of human H3N2 influenza virus isolates showed that viruses isolated 9 years apart were much more similar than the cocirculating avian viruses. The genetic diversity in avian influenza viruses may result from the cocirculation of many different influenza A viruses in ducks and their ability to recombine in nature.     

RNA tumor viruses of pheasants: Characterization of avian leukosis subgroups F and G

Endogenous leukosis-like viruses of ring-necked pheasants (Phasianus colchicus) and golden pheasants (Chrysolophus pictus) have been isolated and characterized. The majority of the normal pheasant embryo cultures contain helper activity for the defective Bryan high titer strain of Rous sarcoma virus. The Rous sarcoma pseudotypes produced with endogenous helper activity from ring-necked pheasants belong to subgroup F. The pseudotypes from golden pheasant cells constitute subgroup G. Subgroup F and G pseudotypes can infect all known genetic types of chicken fibroblasts as well as pheasant and Japanese quail cells, but do not plate on goose cells. Duck cells are resistant to subgroup G but not to F.The subgroup F and G helper viruses isolated from Rous sarcoma viral pseudotypes show interference with their homologous subgroup. RAV-61, a standard of subgroup F, interferes with pseudotypes produced with endogenous helper activity from ring-necked pheasant cells but not with subgroup G pseudotypes.Subgroups F and G do not cross-react with subgroup A to E in neutralization tests. Some normal ring-necked pheasant sera have anti-F activity.Subgroup F and probably also G leukosis-like viruses can undergo genetic recombination with nondefective avian sarcoma viruses.     

Identification of a tRNA nucleotidyltransferase and its substrates in virions of avian RNA tumor viruses

Purified preparations of avian RNA tumor viruses contain a nucleotidyltransferase which catalyzes the addition of adenosine monophosphate and cytosine monophosphate at the 3′-end of certain transfer RNAs, probably in the sequence pCpCpAoh. The reaction can be carried out with physically intact virions treated with a low concentration of nonionic detergent. Under these circumstances, a number of the distinct tRNAs associated with virions of RSV, but not those associated with the viral genome, serve as substrate for the enzyme. Disruption of the virions with higher concentrations of nonionic detergent permits the transferase to utilize exogenously added tRNA as substrate.     

Genetic susceptibility of chicken times quail hybrid embryos to avian RNA tumour viruses.

An attempt was made to hybridize the chicken (Gallus domesticus) male with Japanese quail (Coturnix coturnix japonica) female in order to study the genetic susceptibility of hybrid embryos to avian RNA tumour viruses of subgroups, A, B, D and E. In the hybrids the results supported the prevailing concept that susceptibility is dominant over resistance regardless of the dominant trait contributed by either parent. It was also observed that the Ie gene of the chicken was unable to suppress the 'quail-coded' susceptibility to subgroup E virus in the hybrid system, suggesting the lack of penetrance of the Ie gene. Despite the fact that some hybrids were resistant to viruses of subgroups B and D, they were susceptible to subgroup E virus, which was not expected on the basis of the concept that subgroup B-resistant cells cannot be E-susceptible. Also, the hybrids were susceptible to E virus regardless of gs antigen expression and presence of the Ie gene in the genome. This indicates that our earlier suggestion that the Ie gene is another expression of the gs antigen-determining gene is inconsistent.