Generation of high-titer RCAS virus from DF1 chicken fibroblasts.

RCAS viruses are replication-competent in avian cells, but are replication-deficient in mammalian cells. Therefore, high-titer RCAS virus stocks can be generated only in avian cells. The chicken fibroblast cell line DF1 is well suited for this purpose. Successful infection of target mammalian cells, particularly in vivo, is dependent on the production of high titer viruses by DF1 cells. Moreover, consistency in viral titer helps to ensure uniformity in results produced following the use of independent lots of virus producer cells. Therefore, it is critical to determine the viral titer before initiating these experiments. Because several factors, including insert size and the effect of the inserted gene product on the viability of DF1 cells, influence viral titer, the production of high virus titers cannot be assumed. For RCASBP-A-based viruses, a titer of >1 × 10(7) IU/mL is considered appropriate. Importantly, the virus reverse transcriptase is error prone; errors will accumulate in the virus produced over time. Therefore, virus producer cells should not be cultured for >4-6 wk before being replaced with fresh producer cells. Low passage virus producer cells may be frozen and stored at -80°C; thawed cells will not display a reduction in virus titer. Virus can be collected regularly, concentrated, and stored at -80°C for long-term use; thawed viral stocks typically show a 10-fold decrease in titer.

A threshold level of gag expression is required for particle formation in rat cells transformed by avian retroviruses.

To test the hypothesis that the block of polyprotein precursor processing and particle formation in RSV-transformed mammalian cells is due to a low level of pr76gag expression, rat tumor cell lines with different amounts of precursor molecules were used. The wild-type forms of pr76gag have been expressed at a high level by use of SV40-based vector and thirty-two stable transfected cell clones were isolated. The gag protein expression was detected in the cell lysate by immunoblotting. Untransfected cells released no proteins that could be detected by immunoprecipitation with anti-RSV serum. Membrane-enclosed gag precursor-polyprotein molecules and infectious virus particles from different stably transfected clones have been found in the medium. Both immature and mature virions of type C morphology were directly detected by transmission electron microscopy. Surprisingly, virus-like particles of morphology similar to mature type C retroviruses were found enclosed within intracellular membranes in a stably transfected nonproducing clone.

High prevalence of "Simkania Z," a novel Chlamydia-like bacterium, in infants with acute bronchiolitis.

The newly described microorganism "Simkania Z" ("Z"), an obligate intracellular, penicillin-resistant microorganism most closely related to the chlamydiae, has been associated with adult community-acquired pneumonia. The possible involvement of "Z" in bronchiolitis in infants was examined in a prospective study of 239 infants with bronchiolitis and 78 controls. Other potential etiologic agents sought were respiratory syncytial virus (RSV), adenovirus, and cytomegalovirus. Evidence for the presence of "Z" in nasopharyngeal wash specimens (polymerase chain reaction and/or culture) was found in 25% of infants with bronchiolitis, while controls were all negative (P < .001). A serum IgA response to "Z" infection was detected by immunoperoxidase assay in 15% of infants with bronchiolitis versus 1.3% of controls (P < .001). Clinical findings were not different for infants with bronchiolitis associated with RSV alone, "Z" alone, or RSV and "Z" together. The high prevalence of "Z" in infants with bronchiolitis, often accompanied by an immune response, suggests a possible etiologic role of this agent in the disease.

Selection of an avian retrovirus mutant with extended receptor usage.

Receptor recognition by avian retroviruses is thought to involve the interaction of two regions of the SU protein, hr1 and hr2, with the host cell surface receptor. These regions exhibit considerable variation, concordant with differences in receptor usage among the many avian leukosis virus subgroups. We hypothesize that some retroviruses have altered receptor usage in response to selective pressures imposed by receptor polymorphisms in their hosts. To test this hypothesis, we passaged td-Pr-RSV-B on cocultured permissive chicken (C/E) and nonpermissive quail (QT6/BD) cells. A variant virus with an expanded host range was identified at passage 29 and ultimately shown to be identical in sequence to td-Pr-RSV-B, except for changes at codons 155 and 156 of SU amino acid corresponding to two amino acid changes within hr1. Superinfection resistance studies suggest that the variant virus recognizes the subgroup B receptor on chicken cells and the subgroup E receptor on quail cells. These findings indicate that altered receptor usage can be conferred by small changes in env and may point to a key region for receptor interaction. Further, they demonstrate the evolutionary potential of retroviral env genes to alter receptor usage in response to appropriate selective pressure.

Injury models of the vascular endothelium: apoptosis and loss of thromboresistance induced by a viral protein.

Endothelial injury caused by viruses usually involves viral replication or transformation. We report a novel mechanism of endothelial damage by a toxic viral protein. We have isolated a new retrovirus from hemangiosarcomas which appeared among layer hens. The isolated avian hemangiosarcoma virus (AHV) is capable of inducing hemangiomas in hens in-vivo and causes a cytopathic effect (CPE) and loss of thromboresistance in cultured bovine aortic endothelial cells (BAEC). These effects do not require viral replication and can be induced by purified AHV envelop glycoprotein (gp85). AHV causes CPE in BAEC through a typical programmed cell death (apoptosis). Quiescent G0/G1-BAEC are much more sensitive to AHV induced apoptosis than actively dividing cells. These experiments demonstrate the capacity of viral proteins to affect the integrity and functionality of vascular endothelial cells.

Characterization of a small (25-kilodalton) derivative of the Rous sarcoma virus Gag protein competent for particle release.

Retroviral Gag proteins have the ability to induce budding and particle release from the plasma membrane when expressed in the absence of all of the other virus-encoded components; however, the locations of the functional domains within the Gag protein that are important for this process are poorly understood. It was shown previously that the protease sequence of the Rous sarcoma virus (RSV) Gag protein can be replaced with a foreign polypeptide, iso-1-cytochrome c from a yeast, without disrupting particle assembly (R. A. Weldon, Jr., C. R. Erdie, M. G. Oliver, and J. W. Wills, J. Virol. 64:4169-4179, 1990). An unexpected product of the chimeric gag gene is a small, Gag-related protein named p25C. This product was of interest because of its high efficiency of packaging into particles. The goal of the experiments described here was to determine the mechanism by which p25C is synthesized and packaged into particles. The results demonstrate that it is not the product of proteolytic processing of the Gag-cytochrome precursor but is derived from an unusual spliced mRNA. cDNA clones of the spliced mRNA were obtained, and each expressed a product of approximately 25 kDa, designated p25M1, which was released into the growth medium in membrane-enclosed particles that were much lighter than authentic retrovirions as measured in sucrose density gradients. DNA sequencing revealed that the clones encode the first 180 of the 701 amino acids of the RSV Gag protein and no residues from iso-1-cytochrome c. This suggested that a domain in the carboxy-terminal half of Gag is important for the packaging of Gag proteins into dense arrays within the particles. In support of this hypothesis, particles of the correct density were obtained when a small segment from the carboxy terminus of the RSV Gag protein (residues 417 to 584) was included on the end of p25.

Retroviral integrase functions as a multimer and can turn over catalytically.

A number of studies have demonstrated that the retroviral protein integrase (IN) alone is sufficient to carry out two discrete steps required for retroviral integration: the endonucleolytic processing of viral DNA ends and the cleavage and joining of host DNA to the processed viral DNA termini. Little is known about the biochemical and biophysical mechanisms involved in these reactions. Here, we employ in vitro assays of Rous sarcoma virus IN to demonstrate for the first time that IN is capable of multiple turnover in both the processing and joining reactions. The turnover number calculated for the processing reaction is 0.26 cleavages/min/mol of IN. Our steady state kinetic studies indicate that both the processing and joining activities require a multimeric form of IN. Ultracentrifugation analyses reveal a substrate-independent reversible equilibrium among the monomeric, dimeric, and tetrameric forms of this protein. From these results we conclude that the minimal functional unit for both the processing and joining of each viral DNA end is an IN dimer.

Multiple complex families of endogenous retroviruses are highly conserved in the genus Gallus.

We have analyzed the genome of the domestic chicken for the presence of genetic sequences related to the envelope protein-encoding genes of avian sarcoma/leukosis retroviruses to determine the organization, structure, potential functionality, and distribution of such sequences. We have previously identified in the genus Gallus an extensive group of endogenous avian retroviruses termed EAV-0. Southern blot and sequence analysis presented here of EAV-0 elements revealed that the majority of the EAV-0 elements in the domestic chicken genome have large deletions in their env genes. Screening of a line 0 chicken genomic DNA library for potential full-length env gene-containing endogenous elements yielded three provirus clones of a previously unrecognized group of endogenous retroviruses. These three clones, E13, E33, and E51, are more closely related to each other (80% or more sequence identity) than to other avian retroviruses (70% or less sequence identity). The E13 element has a large deletion in env, but the E51 element has full-length and highly divergent SU- and TM-coding domains. Complete sequence analysis of the E51 env gene region revealed a defective SU-coding domain and an intact TM-coding domain. Sequence analysis of the E51, E33, and E13 3' termini revealed highly distinctive long terminal repeats of approximately 360 bp which appear to be the products, in part, of long terminal repeat domain shuffling. Hybridization analysis with E51 and E33 env gene probes indicated that they are members of an extensive group of elements present in all Gallus species, and at least one element, E51, could be shown by polymerase chain reaction amplification and direct sequencing to have integrated prior to Gallus speciation.

Phosphorylation of integrin in differentiating ts-Rous sarcoma virus-infected myogenic cells.

The differentiation of primary myogenic cultures requires the attachment of the cells to an extracellular matrix substrate using an integrin family receptor. These integrin receptors can be phosphorylated on both their alpha and beta chains, and it has been postulated that phosphorylation regulates the receptor function. Quail myogenic clones transformed with ts-LA24A differentiated into mature myotubes following a temperature shift to nonpermissive temperature which inactivates the viral src kinas. Phosphorylation of integrin beta-1 chain and of at least one alpha chain was detected on both serine and tyrosine. An additional alpha chain(s) with a mobility similar to alpha 5 was not phosphorylated at either temperature. Following the induction of differentiation by a temperature shift, there was a marked decrease in integrin phosphorylation of both alpha and beta integrin chains. This decrease was more prominent for serine than for tyrosine, suggesting that src could not be the only kinase involved. The drop in integrin phosphorylation correlated with the initiation of differentiation, suggesting that integrin phosphorylation could be at least part of the mechanism by which myogenic differentiation is blocked by v-src.

[The detection of the chicken sarcoma virus D6 in a tumor induced by 7,12-dimethylbenz(a)anthracene]. / O vyiavlenii virusa sarkomy kur D6 v opukholi, indutsirovannoi 7,12-dimetilbenz(a)antratsenom.

A tumor induced by 7.12-dimethylbenz (a) anthracene in the hen is described from which a virus was isolated and classified as C type of Rous sarcoma virus. The virus is described in brief. It does not belong to any known strain of Rous sarcoma virus.

A variant Schmidt-Ruppin strain of Rous sarcoma virus with increased affinity for mammalian cells.

SR-RSV-D(H), a variant virus with extremely high tropism for mammalian cells, was isolated by passage of the Schmidt-Ruppin strain of Rous sarcoma virus of subgroup D (SR-RSV-D) through hamster cells. This variant virus has acquired an altered envelope glycoprotein, encoded by the env gene, that has high affinity for receptors on the surface of mammalian cells. The variant virus transforms rat cells at about 100 times the efficiency of the parental virus, SR-RSV-D(S), as assayed by focus formation. Addition of amphotericin B (Fungizone) to the medium at a concentration of 0.2 micrograms/ml completely inhibited rat cell transformation by SR-RSV-D(H), possibly by blocking virus penetration into the cells, whereas the drug showed no inhibitory effect on transformation of chick embryo fibroblast (CEF) cells by the variant virus or on transformation of rat cells by the parental virus. The efficiency of transformation of rat cells by the variant virus was much less than its efficiency of transformation of CEF cells. Analysis of infection of rat cells suggested that the virus can infect rat cells as efficiently as CEF cells but that rat cells were not transformed by the virus as fully as CEF cells because of inefficiency of some post-penetrational step involved in viral gene expression. The finding that E1AY cells, rat cells expressing adenovirus E1A gene, were transformed by SR-RSV-D(H) as efficiently as CEF cells supports this conclusion and suggests that expression of the E1A gene in rat cells may overcome the defect in the transforming step(s) in rat cells.

A newly isolated avian sarcoma virus, ASV-1, carries the crk oncogene.

Avian sarcoma virus 1 (ASV-1) was isolated from a spontaneous sarcoma of an adult chicken. It is a replication-defective virus that induces fibrosarcomas in young chickens and oncogenic transformation in cultured chick embryo fibroblasts. The ASV-1 genome has been cloned in the lambda gt WES.lambda B vector from closed circular viral DNA extracted from infected cells. The nucleotide sequence of the oncogene insert in the ASV-1 genome has been determined. It is virtually identical to the sequence of the oncogene crk recently discovered in the avian sarcoma virus CT10. ASV-1 and CT10 are two independent retrovirus isolates carrying the crk oncogene.

Evaluation of the cocarcinogenic effect of wounding in Rous sarcoma virus tumorigenesis.

Chickens given injections of Rous sarcoma virus form sarcomas at the site of inoculation (primary tumor) and at the site of experimentally introduced wounds (wound tumor). This latter finding provides a model system to study systematically the mechanisms underlying the cocarcinogenic effects of wounding. Our experiments show the following. (a) Chickens inoculated with a Rous sarcoma virus-derived, replication-defective virus construct fail to elaborate wound tumors in spite of aggressively growing primary tumors. We thus rule out metastasis as a mechanism and conclude that infectious virus is required for wound tumor formation; (b) using bromodeoxyuridine incorporation and immunofluorescence on frozen sections we demonstrate proliferation in the unwounded wing in cell types which are normally targets for Rous sarcoma virus infection and transformation and conclude that proliferation per se is not sufficient to induce wound tumors; (c) using immunohistochemistry for the viral protein p19gag we show that wounding induces virus expression in fibroblasts of newly forming granulation tissue 2 days after injury. We also demonstrate expression of viral mRNA in wound tumors by in situ hybridization with a v-src probe. We discuss the possibility of activation of integrated, silent virus or the preferential infection of a special target cell population as a result of wounding as well as the potential role of wound factors in transformation.

Cellular immunity in chicks expressing fibrosarcomatous liver tumor following embryo infection with subgroup A Rous sarcoma virus.

Bryan standard strain of Rous sarcoma virus (BS-RSV) of subgroup A was inoculated into heavy and light breeds of chicken embryos via chorioallantoic membrane (CAM) to ascertain cell-mediated immune response, as measured by a leukocyte migration inhibition (LMI) test. Chicks hatched from eggs with pock-positive CAMs were more likely to develop liver tumors than those hatched from eggs with pock-negative CAMs. Chicks that developed tumors usually had a positive cell-mediated immune response, and those that were negative for liver tumor were negative, based on the LMI test.

Rescue of infectious virus from nonproducer Rous cells by chick cellular DNA.

The rescue of infectious virus from nonproducer BH RSV(-) cells by chick cellular DNA was attempted in order to investigate the functional state of endogenous and exogenous retroviral genes integrated within the cellular DNA. No infectious virus was rescued by transfection with DNAs of chick helper factor (chf)-negative chick embryo cells (CEC), chf-positive CEC or uninfected CEC producing endogenous Rous associated virus (RAV-0). On the other hand, infectious Rous viruses with the phenotype of RAV-0 and RAV-1 were rescued by transfection with DNAs of CEC which had been infected with RAV-0 and RAV-1. From these results, it seems that exogenous retroviral genes integrated in the cellular DNA are expressed rather easily by transfection while those present endogenously are not.

[Occurrence of viral particles of avian sarcoma viruses in sarcoma tissue]. / Výskyt vírusových castíc vtácieho sarkómového vírusu v sarkómovom tkanive.

An electron-microscopic examination was performed of chicken fibrosarcoma caused by avian sarcoma virus (ASV), strain B 77, to investigate virus budding and release through the cytoplasmic membrane. The virus particles of type C- were 90-100 nm in size, the electron-optically denser nucleoids being clearly differentiated from the outer membrane.

The LTR, v-src, LTR provirus generated in the mammalian genome by src mRNA reverse transcription and integration.

Different types of altered proviruses of Rous sarcoma virus (RSV) have been detected in mammalian tumor cell lines. We cloned and sequenced one of these altered proviruses with the structure LTR, v-src, LTR. The presence of an intact viral splice junction, as well as duplications of the chromosomal sequence GCGGGG flanking the two 2-base-pair-deleted LTRs, demonstrated reverse transcription and normal retroviral integration of src mRNA in mammalian cells. In addition, a 1-nucleotide deletion 2 bases upstream from the AAUAAA polyadenylation signal is suspected to be responsible for the absence of a poly(A) track in the src mRNA present in virions of rescued viruses.

Transduction of the cellular src gene and 3' adjacent sequences in avian sarcoma virus PR2257.

When injected into chickens, a transformation-defective mutant of the Prague C strain of Rous sarcoma virus induced tumors at low incidence and after a long latency. One such tumor released a replication-defective virus designated PR2257. We molecularly cloned and sequenced the proviral DNA from quail fibroblasts transformed by PR2257. Comparison of PR2257 sequence with that of Prague C, cellular src, and 3' adjacent cellular DNA showed that the spliced version of the c-src gene and about 950 base pairs (bp) of 3'-flanking cellular DNA were transduced into PR2257. This transduction eliminated nearly all replicative genes, since the gag gene splice donor site was linked to the splice acceptor site of the src gene and, on the 3' side, recombination occurred in the end of env gene. Insertion of two extra cytosines 23 bp before and 19 bp after the c-src stop codon resulted in an extension of the coding portion up to 587 amino acids, divergence of sequences after Pro-525 and replacement of Tyr-527 by a valine residue. In addition, it appears that the 5' and 3' untranslated regions of PR2257 result from multiple recombinations between exogenous and endogenous virus genomes. Limited digestion of p66src encoded by PR2257 with Staphylococcus aureus V8 protease yielded a V2 peptide (C-terminal moiety) with an apparent molecular mass of 31 kilodaltons, consistent with the 5.7-kilodalton increase expected from the DNA sequence. The structure of PR2257 suggests that the first step in the capture of c-src gene by avian lymphomatosis viruses is the trans splicing of the viral leader mRNA to exon 1 of c-src.