Antiviral Activity and Adaptive Evolution of Avian Tetherins.

Tetherin/BST-2 is an antiviral protein that blocks the release of enveloped viral particles by linking them to the membrane of producing cells. At first, BST-2 genes were described only in humans and other mammals. Recent work identified BST-2 orthologs in nonmammalian vertebrates, including birds. Here, we identify the BST-2 sequence in domestic chicken (Gallus gallus) for the first time and demonstrate its activity against avian sarcoma and leukosis virus (ASLV). We generated a BST-2 knockout in chicken cells and showed that BST-2 is a major determinant of an interferon-induced block of ASLV release. Ectopic expression of chicken BST-2 blocks the release of ASLV in chicken cells and of human immunodeficiency virus type 1 (HIV-1) in human cells. Using metabolic labeling and pulse-chase analysis of HIV-1 Gag proteins, we verified that chicken BST-2 blocks the virus at the release stage. Furthermore, we describe BST-2 orthologs in multiple avian species from 12 avian orders. Previously, some of these species were reported to lack BST-2, highlighting the difficulty of identifying sequences of this extremely variable gene. We analyzed BST-2 genes in the avian orders Galliformes and Passeriformes and showed that they evolve under positive selection. This indicates that avian BST-2 is involved in host-virus evolutionary arms races and suggests that BST-2 antagonists exist in some avian viruses. In summary, we show that chicken BST-2 has the potential to act as a restriction factor against ASLV. Characterizing the interaction of avian BST-2 with avian viruses is important in understanding innate antiviral defenses in birds.IMPORTANCE Birds are important hosts of viruses that have the potential to cause zoonotic infections in humans. However, only a few antiviral genes (called viral restriction factors) have been described in birds, mostly because birds lack counterparts of highly studied mammalian restriction factors. Tetherin/BST-2 is a restriction factor, originally described in humans, that blocks the release of newly formed virus particles from infected cells. Recent work identified BST-2 in nonmammalian vertebrate species, including birds. Here, we report the BST-2 sequence in domestic chicken and describe its antiviral activity against a prototypical avian retrovirus, avian sarcoma and leukosis virus (ASLV). We also identify BST-2 genes in multiple avian species and show that they evolve rapidly in birds, which is an important indication of their relevance for antiviral defense. Analysis of avian BST-2 genes will shed light on defense mechanisms against avian viral pathogens.

The interferon-inducible antiviral protein Daxx is not essential for interferon-mediated protection against avian sarcoma virus.

BACKGROUND: The antiviral protein Daxx acts as a restriction factor of avian sarcoma virus (ASV; Retroviridae) in mammalian cells by promoting epigenetic silencing of integrated proviral DNA. Although Daxx is encoded by a type I (α/ß) interferon-stimulated gene, the requirement for Daxx in the interferon anti-retroviral response has not been elucidated. In this report, we describe the results of experiments designed to investigate the role of Daxx in the type I interferon-induced anti-ASV response. FINDINGS: Using an ASV reporter system, we show that type I interferons are potent inhibitors of ASV replication. We demonstrate that, while Daxx is necessary to silence ASV gene expression in the absence of interferons, type I interferons are fully-capable of inducing an antiviral state in the absence of Daxx. CONCLUSIONS: These results provide evidence that Daxx is not essential for the anti-ASV interferon response in mammalian cells, and that interferons deploy multiple, redundant antiviral mechanisms to protect cells from ASV.

[Detection of fps tumor antigen with mono-specific anti-fps serum in tumors induced by acute transforming ALV].

OBJECTIVE: To prepare anti-fps mono-specific serum, and detect the fps antigen in tumors induced by acute transforming avian leukosis/sarcoma virus containing v-fps oncogene. METHODS: Two part of v-fps gene was amplified by RT-PCR using the Fu-J viral RNA as the template. Mono-specific serum was prepared by immuning Kunming white mouse with both two recombinant infusion proteins expressed by the prokaryotic expression system. Indirect immunofluorescent assay was used to detect fps antigen in tumor tissue suspension cells and CEF infected by sarcoma supernatant. Immunohistochemical method was used to detect fps antigen in tumor tissue. RESULTS: The mouse mono-specific serum was specific as it had no cross reaction with classical ALV-J strains. The result reveals that the tumor tissue suspension cells, the CEF infected by sarcoma supernatant, and the slice immunohistochemistry of the sarcoma showed positive results. CONCLUSION: The anti-fps mono-specific serum was prepared, and the detection method was established, which laid the foundation for the study of viral biological characteristics and mechanism of tumourgenesis of acute transforming avian leukosis/sarcoma virus containing v-fps oncogene.

The interferon-induced gene ISG15 blocks retrovirus release from cells late in the budding process.

The release of retroviruses from cells requires ubiquitination of Gag and recruitment of cellular proteins involved in endosome sorting, including the ESCRT-III proteins and the Vps4 ATPase. In response to infection, cells have evolved an interferon-induced mechanism to block virus replication through expression of the interferon-stimulated gene 15 (ISG15), a dimer homologue of ubiquitin, which interferes with ubiquitin pathways in cells. Previously, it has been reported that ISG15 expression inhibited the E3 ubiquitin ligase, Nedd4, and prevented association of the ESCRT-I protein Tsg101 with human immunodeficiency virus type 1 (HIV-1) Gag. The budding of avian sarcoma leukosis virus and HIV-1 Gag virus-like particles containing L-domain mutations can be rescued by fusion to ESCRT proteins, which cause entry into the budding pathway beyond these early steps. The release of these fusions from cells was susceptible to inhibition by ISG15, indicating that there was a block late in the budding process. We now demonstrate that the Vps4 protein does not associate with the avian sarcoma leukosis virus or the HIV-1 budding complexes when ISG15 is expressed. This is caused by a loss in interaction between Vps4 with its coactivator protein LIP5 needed to promote the formation of the ESCRT-III-Vps4 double-hexamer complex required for membrane scission and virus release. The inability of LIP5 to interact with Vps4 is the probable result of ISG15 conjugation to the ESCRT-III protein, CHMP5, which regulates the availability of LIP5. Thus, there appear to be multiple levels of ISG15-induced inhibition acting at different stages of the virus release process.

Ultrastructural distribution of cell surface antigens in avian tumor virus-infected chick embryo fibroblasts.

The distribution of neoantigens in the surface membrane of avian tumor virus-infected chicken embryo fibroblasts was examined on carbon replicas of cell cultures using hemocyanin-labeled antibody. New determinants appearing on the cell surface of virally infected but not transformed cells are thought to be common with components of the viral envelope. These antigens were found to exist in a diffuse, random array on the dorsal cell surface, with a denser accumulation along the cell processes. In living cells, surface antigens are capable of several types of redistribution when activated by reaction with antibody. Leukosis virus-infected (non-transformed) cells showed two apparently independent modes of redistribution: a relocation of some antibody-related sites to the cell margin; or an involvement of essentially all sites in randomly dispersed aggregates. Viral antigenic sites on sarcoma virus-infected (transformed) cells, reacted with antibody, were able to produce weak marginal relocation; but revealed a more striking tendency to migrate to some central location. The centripetal coalescence thus formed resembles the "cap" noted in other systems. Prior aggregation into "patches" may not be a prerequisite for such cap formation. Tumor-specific surface antigen detection and mapping was attempted by this technique, but results were equivocal. An antigen possibly characteristic of rapidly dividing cells occurred in a sparse, diffuse fashion over the surface of morphologically distinct "round" cells.

Neural differentiation, NCAM-mediated adhesion, and gap junctional communication in neuroectoderm. A study in vitro.

We studied the development of NCAM and gap junctional communication, and their mutual relationship in chick neuroectoderm in vitro. Expression of NCAM, as detected by monoclonal and polyclonal antibodies, and development of junctional communication, as detected by extensive cell-to-cell transfer of 400-500-D fluorescent tracers, occurred in cultures from stage-2 embryos onward. Both expressions presumably required primary induction. The differentiating cells formed discrete fields of expression on the second to third day in culture, with the NCAM fields coinciding with the junctional communication fields delineated by the tracers. Other neural differentiations developed in the following order: tetanus toxin receptors, neurofilament protein, and neurite outgrowth. Chronic treatment with antibody Fab fragments against NCAM interfered with the development of communication, suggesting that NCAM-mediated adhesion promotes formation of cell-to-cell channels. Temperature-sensitive mutant Rous sarcoma virus blocked (reversibly) communication and the subsequent development of neurofilament protein and neurites, but expression of NCAM continued.

Complement-fixing antigens in hamster tumors induced by the Bryan strain of Rous sarcoma virus.

Hamster tumors transplanted subcutaneously from primary intracranial tumors which developed after inoculation of the Bryan strain of Rous sarcoma virus, contained virusspecific tumor antigens indistinguishable from those induced by the Schmidt-Ruppin strain.

Rous sarcomas in chickens: enhanced growth coexisting with concomitant immunity.

Chickens bearing Rous sarcoma virus-induced tumors in one wing did not develop new tumors when subsequently inoculated with Rous sarcoma virus in the other wing. However, the second inoculation of Rous sarcoma virus caused accelerated growth of the established tumors. This phenomenon was found to be bursa-dependent. Paradoxically, established tumors in bursectomized chickens grew at a diminished rate if the chickens were reinoculated with Rous sarcoma virus.

Absence of polymerase protein in virions of alpha-type rous sarcoma virus.

Noninfectious particles of a mutant of Rous sarcoma virus failed to exhibit DNA polymerase activity even with the use of the most sensitive synthetic template-primer complexes. A neutralization blocking test against antibody to DNA polymerase revealed that these mutants did not contain protein immunologically related to the DNA polymerase.

Recombinant human polyclonal antibodies: A new class of therapeutic antibodies against viral infections.

The mammalian immune system eliminates pathogens by generating a specific antibody response. Polyclonality is a key feature of this immune response: the immune system produces antibodies which bind to different structures on a given pathogen thereby increasing the likelihood of its elimination. The vast majority of current recombinant antibody drugs rely on monospecific monoclonal antibodies. Inherently, such antibodies do not represent the benefits of polyclonality utilized by a natural immune system and this has impeded the identification of efficacious antibody drugs against infectious agents, including viruses. The development of novel technologies has allowed the identification and manufacturing of antigen-specific recombinant polyclonal human antibodies, so-called symphobodies. This review describes the rationale for designing drugs based on symphobodies against pathogenic viruses, including HIV, vaccinia and smallpox virus, and respiratory syncytial virus.

Low oncogenic potential of avian endogenous RNA tumor virus infection or expression.

Of chickens either spontaneously producing or exogenously infected in ovo with Rous-associated virus, type O (RAV-O), an endogenous virus of the chicken, only 1 died with lymphoid leukosis (LL), the most common neoplasm associated with the leukosis-sarcoma virus group. Because the chickens were not kept in strict isolation, it could not be assumed that the one LL was induced by RAV-O. In contrast, RAV-1-infected chickens from the same lines had a high incidence of LL and other neoplasms. Over 800 chickens of several inbred lines were maintained in plastic isolators free of exogenous avian leukosis-sarcoma virus infection for from 500 to nearly 1,000 days of age. No LL was observed, even though some lines are known to produce RAV-O spontaneously or to express inherited gs antigen. Three neoplasms of unknown etiology were observed, but none generally associated with leukosis virus infection. We concluded that avian endogenous virus expression had little, if any, oncogenic potential, and that exogenous avian leukosis viruses were responsible for most naturally occurring neoplasms.

Failure to confirm evidence for a nonvirion tumor-specific surface antigen in avian retrovirus-transformed cells.

Triton X-100 or Nonidet P40-deoxycholate extracts of [3H]fucose-labeled Rous sarcoma virus-transformed chick embryo fibroblasts were examined by indirect immunoprecipitation for the presence of a tumor-specific neoantigen of 100,000 daltons. Extracts were incubated with immune IgG from Rous tumor-sensitized chickens, and the resultant antigen-antibody complexes were precipitated with rabbit antichicken IgG and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactivity appeared between the migration positions of proteins having molecular weights of 65,000 and 95,000 daltons and at about 30,000 daltons. These antigens were group-specific, and thei precipitation could be inhibited by competition with extracts from cultured fibroblasts that had been infected with a nontransforming avian leukosis virus. They were not precipitated with IgG from unimmunized chickens or chickens immunized with the culture supernatants of uninfected chick embryo fibroblasts. In contrast to results reported recently, the present results could not confirm by immunoprecipitation, the existence of a tumor neoantigen different from that associated with viral components. However, the tumor-specific antigen possibly existed on the cell surface but was not preserved in these detergent extracts.

Tumor antigen induction by the envelope-defective Bryan strain of Rous sarcoma viruses.

Chicken, quail, and turkey cells were infected and/or transformed by various avian leukosis or sarcoma oncovirus (ALSV) strains as well as by the envelope-defective Bryan high-titer strain of Rous sarcoma virus [BH-RSV(-)]. All fibroblast-transforming avian sarcoma viruses (ASV), including BH-RSV(-), were able to induce the expression of the previously described avian tumor-specific cell-surface antigen(s) (TSSA). Thus TSSA was group-specific for all tested transforming ASV. Since BH-RSV(-) is a stable deletion mutant lacking the capacity to code for the 85 000d major virus envelope glycoprotein (gp85), it seemed unlikely that the group-specific antigen determinants of gp85 that were recently detected on ALSV-infected cells could account for TSSA expression.

No expression of a Rous sarcoma virus-induced tumor antigen in mammalian cells infected with retroviruses transducing other oncogenes of the src gene family.

Immunization with mouse and rat cells transformed by Rous sarcoma virus (RSV) or by B77 avian sarcoma virus (ASV) induced complete transplantation resistance against an RSV-induced mouse tumor (CSA1M) in syngeneic hosts. In contrast, most of the mice immunized with a Fujinami sarcoma virus-transformed rat fibroblast line (FSV-3Y1), a feline sarcoma virus-transformed cat fibroblast line (FeSV-FEF), an Abelson leukemia virus-infected Balb/3T3 cell line (AbLV-3T3), or an uninfected 3Y1 cell line could not reject the CSA1M. Serologic analysis with the use of a complement-dependent cytotoxicity assay supported the results of transplantation studies. The mouse and rat cells transformed by RSV or B77 ASV expressed a common tumor-specific cell surface antigen (TSSA) detected by syngeneic antiserum against the CSA1M, whereas none of the FSV-3Y1, FeSV-FEF, and AbLV-3T3 cells expressed the TSSA. These results suggest that the common TSSA in the mouse and rat cells transformed by RSV or B77 ASV containing src gene is not shared with mammalian cells infected with retroviruses transducing other oncogenes of the src gene family (i.e., fps, fes, and abl).

Integration and expression of provirus in human cells transformed by avian sarcoma virus.

Previously, human diploid fibroblasts from some donors infected in vitro by avian sarcoma virus (ASV) were transformed and found, by electron microscopy, to produce small numbers of virus particles that were infectious by bioassay; also, a line of human osteosarcoma cells infected with ASV developed additional characteristics of transformation and released a small number of infectious virus particles. In this study the complete proviral sequence was shown to be integrated in the genome of these cells. The env-related proteins gp85 and gp37 and the gag-related proteins pr76, pr60, and p19 can be detected in cytoplasmic extracts of ASV-infected human cells. Comparable amounts of pp60v-src were found in human and avian cells infected with ASV. The associated kinase activity in infected human cells was dramatically increased as compared to that of uninfected controls; the enzyme had the same cation and substrate requirements as those from ASV-transformed avian cells. Replicating particles from infected human cells were purified and were significantly modified compared to those from avian hosts as shown by a) higher specific gravity, b) the presence of RSV gag-related but not env-related antigens, and c) the fact that the virus-associated reverse transcriptase preferred the divalent cations Mn2+ and Fe2+ over Mg2+.

Cellular immunity in rats with primary brain tumors: inhibition of macrophage migration by soluble extracts of avian sarcoma virus-induced tumors.

Rats bearing primary tumors of the brain induced by avian sarcoma virus (ASV) were studied with the migration-inhibition factor (MIF) assay for the presence of cell-mediated immunity to tumor-associated antigens. Astrocytomas and sarcomas of the brain were induced in 34 neonatal F344 rats by the intracerebral inoculation of Bratislava-77 ASV. At weekly intervals from 4 to 9 weeks after the inoculation with virus, peritoneal exudate cells (PEC) from rats bearing brain tumors were tested an an MIF assay against soluble and KCl-treated extracts of a syngeneic, ASV-induced sarcoma. Incubation of the PEC with a soluble extract of syngeneic liver or with a KCl extract of a syngeneic, chemically induced tumor served as controls. Of 14 rats tested against the soluble tumor extract, 6 (43%) had statistically significant inhibition of migration (P less than or equal to 0.05). Of 23 animals tested against the KCl extract, 16 (70%) had significant inhibition. Immunity to the KCl extract was significant in most rats at each period. Ten rats were tested against a KCl extract of a hamster ASV-induced tumor; 7 gad significant inhibition of migration. None of 3 tested against a soluble extract of a syngeneic, chemically induced tumor had significant inhibition. Rats bearing ASV-induced brain tumors displayed cell-mediated immunity to tumor-associated antigen or antigens of ASV-induced tumors, which could be solubilized.

Relationship between Rous sarcoma virus-induced expression of membrane antigen and phenotypic transformation.

Rous sarcoma virus-transformed hamster BHK fibroblasts express a virus-induced cell surface antigen undetectable in cells either transformed by unrelated viruses or infected by transformation-defective strains of Rous sarcoma virus. To clarify whether this antigen plays any role in the process of malignant transformation or is expressed at the cell surface only as a consequence of the acquisition of the transformed phenotype, we have investigated its expression at the cell surface of Rous sarcoma virus-transformed BHK cells treated with dibutyryl cyclic adenosine 3':5'-monophosphate and at the surface of parental BHK cells transiently transformed by the tumor promoter phorbol myristate acetate. In the dibutyryl cyclic adenosine 3':5'-monophosphate-treated cells, in which most of the parameters of the transformed phenotype are reverted to normality, but the product of the transforming gene is still present, virus-induced cell surface antigen is expressed. In the mirror experiment, this antigen is not expressed by phenotypically transformed but genetically normal phorbol myristate acetate-treated cells. It is concluded that the tumor membrane antigen studied is intimately associated with the expression of the function(s) controlled by the transforming gene.

Association of polyoma virus middle T antigen and pp60src with cytoskeletal elements.

Polyoma virus middle T antigen (mT) as well as pp60v-src, the oncogene product of Rous sarcoma virus, associate with membranes and cytoskeletal structures in virus-infected cells. Furthermore, mT has been shown to be tightly bound to pp60c-src, the cellular homolog of pp60v-src. While the presence of pp60v-src in focal contacts has been demonstrated, the localization of mT and pp60c-src is less clear. We show here that mT is associated with focal contacts and that the phosphorylated protein can be immunoprecipitated with anti-vinculin serum. We suggest that the mT/pp60c-src complex and pp60v-src are tightly bound to vinculin. This interaction may be relevant in the process of cell transformation since disorganization of the actin filament network seems to play an important role in the deregulation of cell growth observed in tumor cells.

A glial fibrillary acidic protein-expressing and tumorigenic cell line derived from an avian sarcoma virus-induced rat astrocytoma.

A permanent cell line, S635c15, was derived from an anaplastic astrocytoma induced by the Schmidt-Ruppin strain of avian sarcoma virus (ASV) in a female F-344 rat. Persistent expression of the astrocytic differentiation protein, glial fibrillary acidic protein (GFAP), was detected both in cultured cells after 100 passages in vitro and in transplanted tumors. Subcutaneous and intracerebral transplantation of S635c15 cells in syngeneic rats resulted in a 100% tumor incidence and a reproducible mortality distribution. S635c15 cells formed discrete masses after subcutaneous injection but grew intracranially as infiltrative lesions. Tumor blood flow and blood-to-tissue transport studies yield comparable values to other rat glioma models; S635c15 intracranial tumors proved to be a homogeneous model with little variation within and between tumors with respect to morphology, GFAP expression, blood flow, and permeability. This cell line provides a GFAP-expressing brain tumor model that extends the use of autochthonous ASV-induced astrocytomas by allowing in vitro and in vivo studies. It may be useful for further studies in neurobiology and brain tumor biology, diagnosis, and therapy.