Molecular characterization of three erbB transducing viruses generated during avian leukosis virus-induced erythroleukemia: extensive internal deletion near the kinase domain activates the fibrosarcoma- and hemangioma-inducing potentials of erbB.

Three new erbB transducing viruses generated during avian leukosis virus-induced erythroblastosis have been cloned and sequenced, and their transforming abilities have been analyzed. Provirus 9134 E1 expresses an amino-terminally truncated erbB product that is analogous to the proviral insertionally activated c-erbB gag-erbB fusion product. This virus efficiently induces erythroblastosis, but does not transform fibroblasts in vitro or induce sarcomas in vivo. In contrast, virus 9134 S3 expresses an erbB product identical to the erbB product of 9134 E1, with the exception of a large internal deletion located between the kinase domain and the putative autophosphorylation site, P1. Interestingly, this virus is no longer capable of inducing erythroblastosis, but can induce both fibrosarcomas and hemangiomas in vivo. Provirus 9134 F3 has sustained an approximately 23-amino-acid carboxy-terminal truncation and is capable of inducing both erythroblastosis and sarcomagenesis. This virus expresses an erbB product with the shortest carboxy-terminal truncation sufficient to reveal the sarcomagenic potential of this protein. The distinct transforming properties of these viruses indicate that different structural domains of the erbB product confer distinct disease specificities.     

Molecular cloning and characterization of the chicken DNA locus related to the oncogene erbB of avian erythroblastosis virus.

Chicken cell DNA contains sequences which are homologous to the avian erythroblastosis virus oncogene v-erb. These cellular sequences (c-erb) have been isolated from a library of chicken cell DNA fragments generated by partial digestion with AluI and HaeIII and shown to be shared by at least two loci in the chicken DNA. One of them, denoted c-erbB, contains approximately 1.8 kilobase pairs of chicken DNA homologous to the 3' part of the v-erb oncogene (v-erbB). Restriction mapping studies show that the c-erbB DNA sequences homologous to v-erbB are distributed among six EcoRI fragments located in a single genomic region. Heteroduplexes between v-erbB in viral RNA and cloned c-erbB DNA show that the chicken DNA sequences homologous to v-erbB are interrupted by 11 DNA sequences not present in the v-erb oncogene. We conclude from our data that the c-erbB locus might represent the cellular progenitor for the v-erbB domain of the v-erb oncogene.     

Mechanism of c-erbB transduction: newly released transducing viruses retain poly(A) tracts of erbB transcripts and encode C-terminally intact erbB proteins

We have previously shown that avian leukosis virus (ALV) induces erythroblastosis by insertional activation of the c-erbB gene. In 25% of the ALV-induced leukemic samples we have analyzed, acute retroviruses that have captured the activated erbB oncogene were released. The unusually high frequency at which erbB transduction occurs makes this an ideal system for studying the mechanism of oncogene transduction. In addition, these leukemic samples provide a rich source for the isolation of novel erbB-transducing viruses. We report here our characterization of several new erbB-transducing proviruses. The 5' recombination points of all these viruses mapped to the same intron in which proviral insertions cluster, supporting the hypothesis that transduction begins with proviral insertion near the oncogene. The 3' recombination points usually occurred within the 3' untranslated region downstream from the termination codon of the c-erbB gene. Three of the erbB-containing proviruses were molecularly cloned and analyzed in detail. Two of them were capable of releasing acute viruses, and interestingly, both retained poly(A) tracts of erbB messages in their genomes. A stretch of six adenosine residues in the ALV env gene appeared to mediate the 3' recombination events required for the generation of these viruses. These data provide further insight into the mechanism by which oncogenes are transduced into retroviral genomes.     

Ligand-independent dimerization of oncogenic v-erbB products involves covalent interactions.

Mutant v-erbB products of avian c-erbB1 have previously been used to correlate structural domains of the receptor encoded by this proto-oncogene with tissue-specific transformation potential. In these studies, deletion of the ligand-binding domain of the receptor has been shown to be required for transformation of erythroblasts, fibroblasts, and endothelial cells. It has, therefore, been postulated that deletion of this domain results in an allosteric change in the receptor analogous to the ligand-bound state of the epidermal growth factor receptor; i.e., it induces a receptor conformation that is constitutively active with respect to mitogenic signaling. While oncogenic v-erbB products have been shown to be expressed on the cell surface of both fibroblasts and erythroblasts, no comprehensive analysis of the oligomeric potential of these products has been conducted. Since the first event known to follow epidermal growth factor binding to its receptor is oligomerization, and receptor dimerization has been correlated with mitogenic signaling, we have carefully analyzed the ability of several v-erbB products to oligomerize in the three target cell types transformed by these oncogenes. In this report, we demonstrate the v-erbB products can efficiently homodimerize in all three target tissues, that this dimerization is ligand independent and occurs at the cell surface, and that there is no apparent correlation between v-erbB dimerization and transformation of avian fibroblasts. Furthermore, both oncogenic and nononcogenic v-erbB products can heterodimerize with the native c-erbB1 product in chicken embryo fibroblasts, suggesting that heterodimerization between v-erB and native c-erbB1 is not sufficient to result in c-erbB1-mediated sarcomagenesis.     

Chromosomal localisation of the human homologues to the oncogenes erbA and B.

Avian erythroblastosis virus (AEV) induces acute erythroleukemia and sarcomas in vivo and it transforms erythroblasts and fibroblasts in vitro. The virus has two host cell-derived genes, v-erbA and v-erbB. The latter encodes the oncogenic capacity of the virus, whereas v-erbA enhances the erythroblast transforming effects of v-erbB while being unable to induce neoplasms independently. Recently, human cellular homologues of these viral erb genes have been isolated. The chromosomal locations of two of these genes have been determined using EcoRI-digested DNA prepared from human-mouse somatic cell hybrids. The human c-erbA1 gene has been assigned to chromosome 17 and is located between 17p11 and 17q21. The human c-erbB sequence has been assigned to chromosome 7 and is located between 7pter and 7q22. Thus, in the human genome these genes are on two separate chromosomes. No evidence for the involvement of the human c-erb genes in neoplasia has been found.     

NS1 Protein of Influenza A Virus Down-Regulates Apoptosis

Wild-type (WT) influenza A/PR/8/34 virus and its variant lacking the NS1 gene (delNS1) have been compared for their ability to mediate apoptosis in cultured cells and chicken embryos. Cell morphology, fragmentation of chromatin DNA, and caspase-dependent cleavage of the viral NP protein have been used as markers for apoptosis. Another marker was caspase cleavage of the viral M2 protein, which was also found to occur in an apoptosis-specific manner. In interferon (IFN)-competent host systems, such as MDCK cells, chicken fibroblasts, and 7-day-old chicken embryos, delNS1 virus induced apoptosis more rapidly and more efficiently than WT virus. As a consequence, delNS1 virus was also more lethal for chicken embryos than WT virus. In IFN-deficient Vero cells, however, apoptosis was delayed and developed with similar intensity after infection with both viruses. Taken together, these data indicate that the IFN antagonistic NS1 protein of influenza A viruses has IFN-dependent antiapoptotic potential.     

Ultrastructural changes in the erythrocyte plasma membranes of chickens in the early stages of interaction with the influenza virus

Using freeze-fracture method it is shown that at the early stages of interaction with chicken erythrocytes the influenza virus induces generalized changes in the physical state of erythrocyte plasma membranes resulted from local disturbances of the cell surface in adsorption sites of virions. Ultrastructural changes seen in the membranes reach their maximum 5 minutes after infection which corresponds to the moment of maximum pinocytotic cell activity in relation to the viruses.     

A preliminary study on viral decontamination of chicken serum using gamma-irradiation

A preliminary experiment was carried out to determine whether a decontamination procedure using gamma irradiation, similar to that adopted in the European guideline for bovine serum contaminated by pestivirus, could be applied to chicken serum. Chicken sera spiked with known amounts of enveloped and non-enveloped chicken viruses were gamma irradiated. The remaining live viruses were then measured by titration and the virus reduction capacity of the irradiation process was established for both enveloped and non-enveloped virus models. In parallel with the irradiation procedure, a classical in vivo extraneous agent test was also evaluated in order to see if it has the capacity to detect low enough levels of live viruses to be used for testing irradiated serum. The results suggest that the principles of the bovine serum decontamination procedure may be applied to chicken serum. Further studies are required to determine if this process would provide an acceptable solution for the viral ‘decontamination’ of chicken serum.     

Chromatographic Analyses of Isoaccepting tRNAs from Avian Tumor Viruses

Low-molecular-weight RNA from transforming viruses (Rous sarcoma virus-Rous-associated virus 1, Schmidt-Ruppin strain of Rous sarcoma virus, and sarcoma-B(77)), from nontransforming viruses (Rous-associated virus 1 and sarcoma-NTB(77)), and from chicken liver, chicken embryo fibroblast, and Rous sarcoma virus-Rous-associated virus 1-transformed chicken embryo fibroblast was isolated and purified. To determine if there are modified, qualitatively or quantitatively different isoaccepting species of tRNA in these avian sarcoma viruses as compared with the cell of virus origin, chicken embryo fibroblast or normal chicken liver, methionyl-, arginyl-, and lysyl-tRNA (with high amino acid acceptance activity), and aspartyl- and glutamyl-tRNA from viral-trans-formed cells (with low viral amino acid acceptance activity) were co-chromatographed on reversed phase-5 chromatography columns, and elution profiles were compared. Although in each case the elution profile between a particular viral and host cell tRNA differed quantitatively, there was no qualitative difference in the profiles of corresponding tRNAs from either transforming or nontransforming viruses examined. Minor quantitative differences in the elution profiles might be a reflection of the metabolic state of the cells, since all evidence points to acceptor activity being of host rather than viral origin. Since, with the exception of selective packaging of methionyl-tRNA (IV) species by both transforming and nontransforming viruses, no selectivity was found for isoacceptor species of other tRNAs, it seems that such preferential packaging of methionyl-tRNA (IV) species has no bearing on the event of viral transformation.     

Modulation of cytokine networks by poxvirus: the myxoma virus model

Poxviruses are large eukaryotic DNA viruses that have evolved a variety of strategies to evade the host immune responses to virus infection. Myxoma virus is of particular interest because it is a rabbit-specific poxvirus that induces a defined systemic virus disease, myxomatosis, in the infected European rabbit. A variety of virus-encoded proteins that are important for subverting cellular immunity and inflammation have been identified, and many of these operate by modulating or subverting cellular cytokine networks. Studies on viral proteins which mediate these immune alterations reveal a multiplicity of strategies by which poxviruses evade clearance from immunocompetent hosts and also identify a new class of immunomodulatory proteins with which to investigate the effector functions of the immune system.     

Marek's disease virus: from miasma to model

Marek's disease virus (MDV) is an oncogenic herpesvirus that causes various clinical syndromes in its natural host, the chicken. MDV has long been of interest as a model organism, particularly with respect to the pathogenesis and immune control of virus-induced lymphoma in an easily accessible small-animal system. Recent advances in MDV genetics and the determination of the chicken genome sequence, aided by functional genomics, have begun to dramatically increase our understanding not only of lytic MDV replication, but also of the factors and mechanisms leading to latency and tumour formation. This new information is helping to elucidate cellular signalling pathways that have undergone convergent evolution and are perturbed by different viruses, and emphasizes the value of MDV as a comparative biomedical model. Furthermore, the door is now open for rational and efficient engineering of new vaccines against one of the most important and widespread infectious diseases in chickens.     

Rat C-Type Virus induced in Rat Sarcoma Cells by 5-Bromodeoxyuridine

HALOGENATED derivatives of uridine are highly effective inducers of latent C-type RNA viruses^1,2 and have been successfully used to induce viruses identical to, or similar to, the C-type RNA tumour viruses in mouse, rat and human cells^3–6. In previous experiments we used 5-bromodeoxyuridine (BrUdR) for induction of focus-forming virus in non-productive rat cells that have been transformed by mouse sarcoma virus². We describe here the induction of a C-type RNA virus in the cells of the rat tumour cell line XC, which contains the Rous sarcoma virus genome⁷. The induced virus possesses the group specific (gs) antigens of rat C-type viruses but not those of chicken C-type viruses.     

Determination of the baculovirus transducing titer in mammalian cells

Baculovirus has emerged as a promising vector for in vivo or ex vivo gene therapy. To date, the infectious titer and multiplicity of infection (MOI) based on the ability of baculovirus to infect insect cells are commonly adopted to indicate the virus dosage. However, the infectious titer and MOI do not reliably represent the baculovirus transducing ability, making the comparison of baculovirus-mediated gene transfer difficult. To determine the baculovirus transducing ability more rapidly and reliably, we developed a protocol to evaluate the transducing titers of baculovirus stocks. The virus was diluted twofold serially and used to transduce HeLa cells. The resultant transduction efficiencies were measured by flow cytometry for the calculation of transducing titers. Compared to the infectious titer, the determination of transducing titer is more reproducible as the standard deviations among measurements are smaller. Also, the transducing titers can be obtained in 24 h, which is significantly faster as opposed to 4-7 days to obtain the infectious titer. More importantly, we demonstrated that baculoviruses with higher transducing titers could transduce cells at higher efficiency and yield stronger and longer transgene expression, confirming that the transducing titer was representative of the baculovirus transducing ability. This finding is particularly significant for ex vivo gene delivery whereby unconcentrated viruses are used for transduction and long-term transgene expression is desired. In this regard, our titration protocol provides a simple, fast, and reliable measure to evaluate the quality of virus stocks during virus production and purification, and is helpful to predict the performance of vector supernatants and ensure reproducible gene delivery experiments.