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.

Antibody binding in proximity to the receptor/glycoprotein complex leads to a basal level of virus neutralization.

Hypothetically, antibodies may neutralize enveloped viruses by diverse mechanisms, such as disruption of receptor binding, interference with conformational changes required for virus entry, steric hindrance, or virus aggregation. Here, we demonstrate that retroviral infection mediated by the avian sarcoma-leukosis virus (ASLV-A) envelope glycoproteins can be neutralized by an antibody directed against a functionally unimportant component of a chimeric receptor protein. Thus, the binding of an antibody in proximity to the retroviral envelope glycoprotein-receptor complex, without binding to the entry machinery itself, results in neutralization. This finding provides additional support for the hypothesis that steric hindrance is sufficient for antibody-mediated neutralization of retroviruses.

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.

Dual requirement for the Ig alpha immunoreceptor tyrosine-based activation motif (ITAM) and a conserved non-Ig alpha ITAM tyrosine in supporting Ig alpha beta-mediated B cell development.

Surface Ig (sIg) expression is a critical checkpoint during avian B cell development. Only cells that express sIg colonize bursal follicles, clonally expand, and undergo Ig diversification by gene conversion. Expression of a heterodimer, in which the extracellular and transmembrane domains of murine CD8alpha or CD8beta are fused to the cytoplasmic domains of chicken Igalpha (chIgalpha) or Igbeta, respectively (murine CD8alpha (mCD8alpha):chIgalpha + mCD8beta:chIgbeta), or an mCD8alpha:chIgalpha homodimer supported bursal B cell development as efficiently as endogenous sIg. In this study we demonstrate that B cell development, in the absence of chIgbeta, requires both the Igalpha ITAM and a conserved non-ITAM Igalpha tyrosine (Y3) that has been associated with binding to B cell linker protein (BLNK). When associated with the cytoplasmic domain of Igbeta, the Igalpha ITAM is not required for the induction of strong calcium mobilization or BLNK phosphorylation, but is still necessary to support B cell development. In contrast, mutation of the Igalpha Y3 severely compromised calcium mobilization when expressed as either a homodimer or a heterodimer with the cytoplasmic domain of Igbeta. However, coexpression of the cytoplasmic domain of Igbeta partially complemented the Igalpha Y3 mutation, rescuing higher levels of BLNK phosphorylation and, more strikingly, supporting B cell development.

Major histocompatibility (B) complex control of responses against Rous sarcomas.

The chicken major histocompatibility (B) complex (MHC) affects disease outcome significantly. One of the best characterized systems of MHC control is the response to the oncogenic retrovirus, Rous sarcoma virus (RSV). Genetic selection altered the tumor growth pattern, either regressively or progressively, with the data suggesting control by one or a few loci. Particular MHC genotypes determine RSV tumor regression or progression indicating the crucial B complex role in Rous sarcoma outcome. Analysis of inbred lines, their crosses, congenic lines, and noninbred populations has revealed the anti-RSV response of many B complex haplotypes. Tumor growth disparity among lines identical at the MHC but differing in their background genes suggested a non-MHC gene contribution to tumor fate. Genetic complementation in tumor growth has also been demonstrated for MHC and non-MHC genes. RSV tumor expansion reflects both tumor cell proliferation and viral replication generating new tumor cells. In addition, the B complex controls tumor growth induced by a subviral DNA construct encoding only the RSV v-src oncogene. Immunity to subsequent tumors and metastasis also exhibit MHC control. Genotypes that regressed either RSV or v-src DNA primary tumors had enhanced protection against subsequent homologous challenge. Regressor B genotypes had lower tumor metastasis compared with progressor types. Together, the data indicate that B complex control of RSV tumor fate is strongly defined by the response to a v-src-determined function. Differential RSV tumor outcomes among various B genotypes may include immune recognition of a tumor-specific antigen or immune system influences on viral replication.

Genetic analysis of a divergent selection for resistance to Rous sarcomas in chickens.

Selection for disease resistance related traits is a tool of choice for evidencing and exploring genetic variability and studying underlying resistance mechanisms. In this framework, chickens originating from a base population, homozygote for the B19 major histocompatibility complex (MHC) were divergently selected for either progression or regression of tumors induced at 4 weeks of age by a SR-D strain of Rous sarcoma virus (RSV). The first generation of selection was based on a progeny test and subsequent selections were performed on full-sibs. Data of 18 generations including a total of 2010 birds measured were analyzed for the tumor profile index (TPI), a synthetic criterion of resistance derived from recording the volume of the tumors and mortality. Response to selection and heritability of TPI were estimated using a restricted maximum likelihood method with an animal model. Significant progress was shown in both directions: the lines differing significantly for TPI and mortality becoming null in the "regressor" line. Heritability of TPI was estimated as 0.49 +/- 0.05 and 0.53 +/- 0.06 within the progressor and regressor lines respectively, and 0.46 +/- 0.03 when estimated over lines. Preliminary results showed within the progressor line a possible association between one Rfp-Y type and the growth of tumors.

Resistance to infection by subgroups B, D, and E avian sarcoma and leukosis viruses is explained by a premature stop codon within a resistance allele of the tvb receptor gene.

Here we present the first molecular characterization of the defect associated with an avian sarcoma and leukosis virus (ASLV) receptor resistance allele, tvb(r). We show that resistance to infection by subgroups B, D, and E ASLV is explained by the presence of a single base pair mutation that distinguishes this allele from tvb(s1), an allele which encodes a receptor for all three viral subgroups. This mutation generates an in-frame stop codon that is predicted to lead to the production of a severely truncated protein.

DNA vaccination against v-src oncogene-induced tumours in congenic chickens.

DNA vaccination is particularly efficient for induction of cytotoxic T-lymphocyte (CTL) response. In our experiments, we used MHC(B) congenic chicken lines CB and CC (regressors and progressors of v-src-induced tumours, respectively) and a mutated, non-oncogenic v-src gene construct as the DNA vaccine. A high degree of vaccine protection against oncogenic v-src challenge was achieved in the CB line chickens. CTL response was demonstrated in vitro and by adoptive transfer of immune cells to the syngeneic host and to the CC line chickens rendered tolerant to CB cells. In the CC line chickens we observed tumour growth retardation after a low-dose DNA vaccination administered to immature recipients while higher amounts of DNA vaccine in immunocompetent chickens exerted an enhancing effect.

Nonmajor histocompatibility complex alloantigen effects on the fate of Rous sarcomas.

Rous sarcoma virus-induced tumor outcome is controlled by the MHC (B). Additional data, using controlled segregation in families, has indicated non-MHC effects as well, but few studies have focused on blood groups other than the B complex. Segregating combinations of genes encoding erythrocyte (Ea) alloantigen systems A, C, D, E, H, I, P, and L in B2B5 and B5B5 MHC (B) backgrounds were examined for their effects on Rous sarcomas. Six-week-old chickens were inoculated in the wing-web with 30 pfu of Rous sarcoma virus (RSV). Tumors were scored six times over a 10-wk period. A tumor profile index (TPI) was assigned to each chicken based on the six tumor size scores. Response was evaluated using tumor size at each measurement period, TPI, and mortality. The genotypes of Ea systems A, C, D, E, H, I, and P had no significant effect on any parameter in either B complex population. The Ea-L system had an effect on Rous sarcomas in the B2B5 intermediate responders and B5B5 progressors. Tumor size, TPI, and mortality were all significantly lower in B2B5 L1L1 chickens than in B2B5 L1L2 chickens. Mortality was lower in the B5B5 L1L1 birds than in B5B5 L1L2 chickens. It appears that the Ea-L system, or one closely linked, is acting in a manner independent of the B complex in response to RSV challenge.

The genetics of mortality and survival of broiler chicks infected as embryos by subgroup A Rous sarcoma virus.

A study using two high-performance broiler lines, a synthetic male line (SML) and a synthetic dam line (SDL), was undertaken to investigate the pattern of mortality due to induced liver tumours (LT) and the immune response to subgroup A virus inoculated via the chorioallantoic membrane (CAM) route. The distribution patterns of the four possible phenotypes were similar in both sire and dam lines. The occurrence of conversely associated phenotypes was about 30S, in both the lines. The percentages of CAM(+) LT(-) and CAM(-) LT(+) were 14.26%, and 14.46% in the dam line and 20.0% and 9.57% in the male line. The LT mortality was 30-50% in the birds with low pock counts, whereas it was 80-93% in the birds with high pock counts. The group specific antigen shedding status did not influence death due to LT. In birds in the high pock count group, 98% of deaths due to LT were completed by the sixth week, whereas in those in the low pock count group, death due to LT was spread over 24 weeks. The SDL birds survived better than SML birds in the high pock count groups. In both lines, about 20% of deaths occurred owing to non-specific causes. The average survival time after hatching before death from LT was 26 days, whereas that for non-specific death was 81 days.

Genes of chicken MHC regulate the adherence activity of blood monocytes in Rous sarcomas progressing and regressing lines.

The influence of the chicken major histocompatibility (B) complex (MHC) on the adherence potential of monocyte-derived macrophages was examined using the congenic chicken lines CB and CC. These lines represent well-defined genetic models for the study of resistance (CB) or susceptibility (CC) to the progressive growth of Rous sarcomas. Using a monoclonal antibody specific for chicken monocytes/macrophages, CB and CC chickens were shown by flow cytometry analyses to have similar proportions of peripheral blood monocytes. However, when the glass-adherence potential of these cells was compared during incubation in tissue culture medium over 24, 48 and 72 h at 40 degrees C, significant differences were seen between cells from these two inbred lines. After 24 and 48 h, glass-adherence by CB cells was 2-3 fold higher than that of CC cells. After 72 h this difference decreased to 1.5 fold. At 24 and 48 h, the adherent CB macrophages also appeared about 1.5 times larger than those of CC chickens. Genetic analysis using F1 hybrids (CBxCC) showed that this trait is regulated by a dominant gene that segregates with the B12 haplotype in the backcross generation F1xCC. From the results obtained with the recombinant congenic lines CB.R1 and CC.R1, we conclude that the gene regulating adherence potential is localized within the B-F/L region of the chicken MHC. About 50% of adherent cells were able to phagocytose opsonised FITC-labelled Zymosan particles. The level of nitric oxide production in vitro by CB and CC macrophages was equal. The importance of cells of the mononuclear phagocyte system for the response to Rous sarcoma virus (RSV) infection was studied in CB chickens using the anti-macrophage agents silica, carrageenan, and C12MDP, encapsulated in liposomes. In those chickens treated with silica and carrageenan, we observed progressive growth of RSV-induced tumors. The graft-versus-host reactivity of peripheral blood lymphocytes (PBL) of treated chickens was comparable to controls. In vitro nitric oxide production by macrophages from silica-treated chickens was higher than by macrophages from untreated controls.

The genetic control of susceptibility to subgroup D RNA tumour virus infection in commercial breeds of chickens.

The genetic control of resistance to RSV (RAV-50) in chick embryos inoculated via the chorioallantoic membrane route was studied in three breeds of chickens. The relative susceptibility to subgroup D infection was 0.01, 0.02 and 0.003 in broilers, White Leghorns and Bantam x Australorp crosses, respectively. The distribution pattern of putative dr gene frequencies were 0.80, 0.68 and 0.90, respectively, in the same three breeds. Liver tumours resulting from the virus infection were observed in a very few birds about 28 days after hatching.

Highly specific antibody to Rous sarcoma virus src gene product recognizes nuclear and nucleolar antigens in human cells.

An antiserum to the Rous sarcoma virus-transforming protein pp60v-src, raised in rabbits immunized with the bacterially produced protein alpha p60 serum (M. D. Resh and R. L. Erikson, J. Cell Biol. 100:409-417, 1985) previously reported to detect very specifically a novel population of pp60v-src and pp60c-src molecules associated with juxtareticular nuclear membranes in normal and Rous sarcoma virus-infected cells of avian and mammalian origin, was used here to investigate by immunofluorescence microscopy localization patterns of Src molecules in human cell lines, either normal or derived from spontaneous tumors. We found that the alpha p60 serum reveals nuclear and nucleolar concentrations of antigens in all the human cell lines tested and in two rat and mouse hepatoma cell lines derived from adult tumorous tissues but not in any established rat and mouse cell lines either untransformed or transformed by the src and ras oncogenes. Both the nuclear and nucleolar stainings can be totally extinguished by preincubation of the serum with highly purified chicken c-Src. We show also that the partitioning of the alpha p60-reactive proteins among the whole nucleus and the nucleolus depends mostly on two different parameters: the position in the cell cycle and the degree of cell confluency. Our observations raise the attractive possibility that, in differentiated cells, pp60c-src and related proteins might be involved not only in mediating the transduction of mitogenic signals at the plasma membrane level but also in controlling progression through the cell cycle and entry in mitosis by interacting with cell division cycle regulatory components at the nuclear level.

Transport of a lysosomally targeted Rous sarcoma virus envelope glycoprotein involves transient expression on the cell surface.

The details of intracellular transport pathways for glycosylated proteins remain incompletely described. We previously described a mutant Rous sarcoma virus envelope glycoprotein (gp), mu 26, with an altered membrane-spanning domain that was targeted to lysosomes after traversing the trans-Golgi. This mutant protein was not detectable on the cell surface by immunofluorescence, but its pathway for degradation remained unclear. To investigate this we have employed a second env mutation, S19, that results in a protein which is defective for normal cleavage/activation by intracellular enzymes, but remains susceptible to cleavage by extracellular proteases. Cleavage/activation of the double mutant by trypsin, which could only occur if it was exposed on the cell surface, was observed, indicating that the plasma membrane is an intermediate destination in the transport of this mutant protein. To substantiate these results, cells expressing the mu 26 glycoprotein were incubated with an antibody specific for the native protein in the presence of chloroquine. The specific accumulation of this antibody/gp complex in vesicles, as detected by internal immunofluorescence, confirmed the trypsin cleavage results. We conclude that this rapidly degraded mutant protein is transported from the trans-Golgi to the cell surface, where it is only transiently exposed, and then rapidly endocytosed and lysosomally degraded. The relevance of these results to the targeting of lysosomal proteins is discussed.

Detection of antibodies to avian leukosis/sarcoma viruses (ALSV) and reticuloendotheliosis viruses (REV) in humans by ELISA.

We used a modified commercial ELISA kit to test for antibodies to avian leukosis/sarcoma and reticuloendotheliosis viral antigens in the sera of 45 poultry workers and their matched controls. We found that 42% of sera from poultry workers had anti-avian leukosis Sarcoma viruses (anti-ALSV) and 20% had anti-reticuloendotheliosis viruses (anti-REV), antibody titers that were higher than the highest titer recorded in control subjects, and hence were regarded as positive. To determine the specificity of these reactions, selected sera were absorbed with ALSV or REV antigens alone, or with chick embryo fibroblasts (CEF) alone, or with both CEF and ALSV/REV, and then retested. In each case, absorption resulted in a statistically significant reduction in absorbance, which was greatest for the combined CEF and ALSV/REV absorption, thus suggesting that the reactions involved viral as well as chicken antigens. However, definitive tests such as Western Blot analyses are needed to confirm whether indeed antibodies to these viruses were specifically elicited in human sera.

Vaccination with a low-oncogenic strain of Rous sarcoma virus prevents visceral tumors in chickens.

The oncogenic potential of different strains of Rous sarcoma virus (RSV) varies significantly in two Mhc(B) congenic chicken lines CB and CC and in their F1 hybrids with the unrelated inbred line IA. The Bryan high-titer pseudotype of RSV of antigenic subgroups D and B (BH-D, and BH-B) was highly oncogenic, eliciting mostly fatal, progressively growing tumors. Visceral tumor formation and erythroblastosis were seen after challenge with BH-B in CB and CC chickens, but not in their F1 hybrids with IA. Tumor regression induced by a low-oncogenic RSV strain PR-C elicited a protective immunity capable of preventing not only the progressive growth of challenge tumors at the site of inoculation, but also visceral tumor formation and erythroblastosis.