Two different molecular defects in the Tva receptor gene explain the resistance of two tvar lines of chickens to infection by subgroup A avian sarcoma and leukosis viruses

The subgroup A to E avian sarcoma and leukosis viruses (ASLVs) are highly related and are thought to have evolved from a common ancestor. These viruses use distinct cell surface proteins as receptors to gain entry into avian cells. Chickens have evolved resistance to infection by the ASLVs. We have identified the mutations responsible for the block to virus entry in chicken lines resistant to infection by subgroup A ASLVs [ASLV(A)]. The tva genetic locus determines the susceptibility of chicken cells to ASLV(A) viruses. In quail, the ASLV(A) susceptibility allele tva(s) encodes two forms of the Tva receptor; these proteins are translated from alternatively spliced mRNAs. The normal cellular function of the Tva receptor is unknown; however, the extracellular domain contains a 40-amino-acid, cysteine-rich region that is homologous to the ligand binding region of the low-density lipoprotein receptor (LDLR) proteins. The chicken tva(s) cDNAs had not yet been fully characterized; we cloned the chicken tva cDNAs from two lines of subgroup A-susceptible chickens, line H6 and line 0. Two types of chicken tva(s) cDNAs were obtained. These cDNAs encode a longer and shorter form of the Tva receptor homologous to the Tva forms in quail. Two different defects were identified in cDNAs cloned from two different ASLV(A)-resistant inbred chickens, line C and line 7(2). Line C tva(r) contains a single base pair substitution, resulting in a cysteine-to-tryptophan change in the LDLR-like region of Tva. This mutation drastically reduces the binding affinity of Tva(R) for the ASLV(A) envelope glycoproteins. Line 7(2) tva(r2) contains a 4-bp insertion in exon 1 that causes a change in the reading frame, which blocks expression of the Tva receptor.     

The receptor for the subgroup C avian sarcoma and leukosis viruses, Tvc, is related to mammalian butyrophilins, members of the immunoglobulin superfamily

The five highly related envelope subgroups of the avian sarcoma and leukosis viruses (ASLVs), subgroup A [ASLV(A)] to ASLV(E), are thought to have evolved from an ancestral envelope glycoprotein yet utilize different cellular proteins as receptors. Alleles encoding the subgroup A ASLV receptors (Tva), members of the low-density lipoprotein receptor family, and the subgroup B, D, and E ASLV receptors (Tvb), members of the tumor necrosis factor receptor family, have been identified and cloned. However, alleles encoding the subgroup C ASLV receptors (Tvc) have not been cloned. Previously, we established a genetic linkage between tvc and several other nearby genetic markers on chicken chromosome 28, including tva. In this study, we used this information to clone the tvc gene and identify the Tvc receptor. A bacterial artificial chromosome containing a portion of chicken chromosome 28 that conferred susceptibility to ASLV(C) infection was identified. The tvc gene was identified on this genomic DNA fragment and encodes a 488-amino-acid protein most closely related to mammalian butyrophilins, members of the immunoglobulin protein family. We subsequently cloned cDNAs encoding Tvc that confer susceptibility to infection by subgroup C viruses in chicken cells resistant to ASLV(C) infection and in mammalian cells that do not normally express functional ASLV receptors. In addition, normally susceptible chicken DT40 cells were resistant to ASLV(C) infection after both tvc alleles were disrupted by homologous recombination. Tvc binds the ASLV(C) envelope glycoproteins with low-nanomolar affinity, an affinity similar to that of binding of Tva and Tvb with their respective envelope glycoproteins. We have also identified a mutation in the tvc gene in line L15 chickens that explains why this line is resistant to ASLV(C) infection.     

The CAR1 Gene Encoding a Cellular Receptor Specific for Subgroup B and D Avian Leukosis Viruses Maps to the Chicken tvb Locus

Host susceptibility to subgroup B, D, and E avian leukosis viruses (ALV) is determined by specific alleles of the chicken tvb locus. Recently, a chicken gene that encodes a cellular receptor, designated CAR1, specific for subgroups B and D ALV was cloned, and it was proposed that this gene was the s3 allele of tvb (J. Brojatsch, J. Naughton, M. M. Rolls, K. Zingler, and J. A. T. Young, Cell 87:845–855, 1996). We now report that in a backcross derived from an F₁ (Jungle Fowl × White Leghorn [WL]) male mated with inbred WL females, the cloned ALV receptor gene cosegregated with two markers linked to tvb. The two markers used were a tvb^s1-specific antigen recognized by the chicken R2 alloantiserum and restriction fragment length polymorphisms associated with the expressed sequence tag com152e. With all three markers, no crossovers were observed among 52 backcross progeny tested and LOD linkage scores of 15.7 were obtained. These data demonstrate that CAR1 is the subgroup B and D ALV susceptibility gene located at tvb^s3.     

Intronic deletions that disrupt mRNA splicing of the tva receptor gene result in decreased susceptibility to infection by avian sarcoma and leukosis virus subgroup A

The group of closely related avian sarcoma and leukosis viruses (ASLVs) evolved from a common ancestor into multiple subgroups, A to J, with differential host range among galliform species and chicken lines. These subgroups differ in variable parts of their envelope glycoproteins, the major determinants of virus interaction with specific receptor molecules. Three genetic loci, tva, tvb, and tvc, code for single membrane-spanning receptors from diverse protein families that confer susceptibility to the ASLV subgroups. The host range expansion of the ancestral virus might have been driven by gradual evolution of resistance in host cells, and the resistance alleles in all three receptor loci have been identified. Here, we characterized two alleles of the tva receptor gene with similar intronic deletions comprising the deduced branch-point signal within the first intron and leading to inefficient splicing of tva mRNA. As a result, we observed decreased susceptibility to subgroup A ASLV in vitro and in vivo. These alleles were independently found in a close-bred line of domestic chicken and Indian red jungle fowl (Gallus gallus murghi), suggesting that their prevalence might be much wider in outbred chicken breeds. We identified defective splicing to be a mechanism of resistance to ASLV and conclude that such a type of mutation could play an important role in virus-host coevolution.     

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.     

Cospeciation and horizontal transmission of avian sarcoma and leukosis virus gag genes in galliform birds

In a study of the evolution and distribution of avian retroviruses, we found avian sarcoma and leukosis virus (ASLV) gag genes in 26 species of galliform birds from North America, Central America, eastern Europe, Asia, and Africa. Nineteen of the 26 host species from whom ASLVs were sequenced were not previously known to contain ASLVs. We assessed congruence between ASLV phylogenies based on a total of 110 gag gene sequences and ASLV-host phylogenies based on mitochondrial 12S ribosomal DNA and ND2 sequences to infer coevolutionary history for ASLVs and their hosts. Widespread distribution of ASLVs among diverse, endemic galliform host species suggests an ancient association. Congruent ASLV and host phylogenies for two species of Perdix, two species of Gallus, and Lagopus lagopus and L. mutus also indicate an old association with vertical transmission and cospeciation for these ASLVs and hosts. An inference of horizontal transmission of ASLVs among some members of the Tetraoninae subfamily (grouse and ptarmigan) is supported by ASLV monophyletic groups reflecting geographic distribution and proximity of hosts rather than host species phylogeny. We provide a preliminary phylogenetic taxonomy for the new ASLVs, in which named taxa denote monophyletic groups.     

Genetic Mapping of the Cloned Subgroup A Avian Sarcoma and Leukosis Virus Receptor Gene to the TVA Locus

A chicken gene conferring susceptibility to subgroup A avian sarcoma and leukosis viruses (ASLV-A) was recently identified by a gene transfer strategy. Classical genetic approaches had previously identified a locus, TVA, that controls susceptibility to ASLV-A. Using restriction fragment length polymorphism (RFLP) mapping in inbred susceptible (TVA*S) and resistant (TVA*R) chicken lines, we demonstrate that in 93 F₂ progeny an RFLP for the cloned receptor gene segregates with TVA. From these analyses we calculate that the cloned receptor gene lies within 5 centimorgans of TVA, making it highly probable that the cloned gene is the previously identified locus TVA. The polymorphism that distinguishes the two alleles of TVA in these inbred lines affects the encoded amino acid sequence of the region of Tva that encompasses the viral binding domain. However, analysis of the genomic sequence encoding this region of Tva in randomly bred chickens suggests that the altered virus binding domain is not the basis for genetic resistance in the chicken lines analyzed.Avian sarcoma and leukosis viruses (ASLV) may be classified into several subgroups based on properties of the viral envelope proteins. Five major subgroups of ASLV (A to E) have been defined based on immunological reactivity of the viral envelope proteins, host range, and infection interference patterns (reviewed in reference 18). The subgroup specificity of the virus maps to the env gene, and distinct hypervariable regions within env have been shown to determine the viral subgroup (2, 3, 7, 8).Patterns of susceptibility of inbred chicken lines to infection with the various ASLV subgroups suggest that three loci control viral entry for the five major ASLV subgroups (11, 12, 15, 17). Dominant alleles at the TVA and TVC (TVA*S and TVC*S) loci confer susceptibility to subgroups A and C viruses, respectively. The TVB locus is more complex, with various alleles determining infection by subgroup B, D, and E viruses. TVA, TVB, and TVC appear to act at the level of viral entry into the cell; therefore, it has been assumed that the TV loci encode or control expression of the cellular receptors for ASLV (5, 13).Recently, a gene that confers susceptibility to subgroup A ASLV (ASLV-A) was cloned by a gene transfer strategy (1, 19). Molecular clones containing coding sequences for this gene relieve the block to viral entry when expressed in a number of mammalian cell lines (1, 19). The gene encodes a surface glycoprotein that has been shown to bind directly and specifically to the ASLV-A envelope protein (4, 9). In addition, binding of the receptor protein to ASLV-A envelope protein induces conformational changes in the viral glycoproteins that appear to be associated with viral entry (10). Finally, an antibody to the receptor protein specifically blocks infection of chicken cells by subgroup A viruses, suggesting that this gene encodes the normal ASLV-A receptor on chicken cells (1). Taken together, these results demonstrate that the cloned gene encodes a subgroup A virus receptor.Since it is clear that the cloned gene encodes an ASLV-A receptor, the protein supposed to be encoded by the classical TVA locus, we asked whether the identified receptor gene mapped to the TVA locus. In this paper we show that a restriction fragment length polymorphism (RFLP) of DNA from inbred chickens can be used to demonstrate that the cloned ASLV-A receptor gene maps to within 5 centimorgans (cM) of the TVA locus as defined by susceptibility to ASLV-A, making it highly probable that the cloned gene is TVA. In addition, analysis of the genomic sequences encoding the viral interaction domain of the receptor in both inbred and randomly bred chickens demonstrates that alterations in this region of the receptor are not responsible for the resistance phenotype.

TVA segregation analysis.

Inbred chicken lines homozygous for TVA alleles encoding either sensitivity (TVA*S) or resistance (TVA*R) were used to generate birds in which the TVA segregation pattern could be studied. Regional Poultry Research Lab lines 6₃ (TVA*S/*S TVB*S/*S) and 7₂ (TVA*R/*R TVB*R/*R) (6) were crossed, and the resulting heterozygous F₁ progeny were mated to generate 93 F₂ chicks. The susceptibility phenotype of the F₂ chicks was determined on the basis of tumor formation following subcutaneous injection of 500 focus-forming units (FFU) of subgroup A Bryan high-titer Rous sarcoma virus (RSV) (RAV-1) and 500 FFU of subgroup B Bryan high-titer RSV (RAV-2) into the right and left wing webs, respectively, of 4-week-old chicks. At 2, 3, and 4 weeks postinjection the wings were palpated to check for tumor development. Chicks were scored as positive for susceptibility (e.g., TVA*S/*S or TVA*S/*R) if a tumor of any size formed in the appropriate wing web.Table ​Table11 summarizes the distribution of susceptibility to subgroups A and B RSV in the F₂ chicks. Segregation of TVB yielded roughly the predicted frequency of susceptible (67 observed versus 70 expected) and resistant (26 observed versus 23 expected) progeny for a dominant locus (P > 0.05). In contrast, the distribution of subgroup A-susceptible and -resistant birds deviated significantly from the expected 3:1 ratio, with an excessive number of subgroup A-resistant chicks observed and a ratio of 2:1 (χ² = 3.4) (Table ​(Table1).1). Previous analysis of the segregation of TVA using the same chicken lines did not reveal an altered distribution of sensitive and resistant birds (6), suggesting that the bias seen here may be a chance deviation from the expected ratio. Segregation of two endogenous virus loci, ALVE2 (previously designated ev2) (carried by line 7₂) and ALVE3 (previously designated ev3) (carried by line 6₃), also gave roughly the expected 3:1 ratio in these F₂ chicks (data not shown). When the segregation bias in the ASLV-A-susceptible birds was accounted for, then susceptibility to subgroup A and B RSV segregated independently, as was expected since the TVA and TVB genes have been reported to be unlinked (6). Recent mapping studies also confirmed that TVA and TVB are found on different linkage groups (3a).

TABLE 1

Segregation of resistance and susceptibility to subgroup A and B viruses in F₂ progeny obtained by crossing lines 6₃ and 7₂

Close linkage of genes encoding receptors for subgroups A and C of avian sarcoma/leucosis virus on chicken chromosome 28

Avian sarcoma and leucosis viruses (ASLV) are classified into six major subgroups (A to E and J) according to the properties of the viral envelope proteins and the usage of cellular receptors for virus entry. Subgroup A and B receptors are identified molecularly and their genomic positions TVA and TVB are mapped. The subgroup C receptor is unknown, its genomic locus TVC is reported to be genetically linked to TVA, which resides on chicken chromosome 28. In this study, we used two chicken inbred lines that carry different alleles coding for resistance (TVC(R) and sensitivity (TVC(S)) to infection by subgroup C viruses. A backross population of these lines was tested for susceptibility to subgroup C infection and genotyped for markers from chicken chromosome 28. We confirmed the close linkage between TVA and TVC loci. Further, we have described the position of TVC on chromosome 28 relative to markers from the consensus map of the chicken genome.     

[125I]LSD binding to serotonin and dopamine receptors in bovine caudate membranes

[¹²⁵I]LSD (labeled at the 2 position) has been introduced as the first ¹²⁵I-labeled ligand for serotonin 5-HT₂ (S2) receptors. In the present study we examined the binding of [¹²⁵I]LSD and its non-radioactive homologue, 2I-LSD, to bovine caudate homogenates. The binding of [¹²⁵I]LSD is saturable, reversible, stereospecific and is destroyed by boiling the membranes. The specific to total binding ratio in this tissue is 75–80% and Scatchard plots of the binding data reveal K_d = 1.1 nM, B_max = 9.6 fmol/mg wet weight tissue. The association and dissociation rate constants are highly temperature dependent. At 0°C the net dissociation is less than 5% after 1 h and the association rate is proportionately slow. IC₅₀ values for a variety of compounds show a clear 5-HT₂ (S2) serotonergic pattern at this [¹²⁵I]LSD site. Blockage of this primary 5-HT₂ (S2) caudate binding site by 0.3 μM mianserin reveals the presence of a weaker [¹²⁵I]LSD binding site with a K_d = 9.1 nM, B_max = 7.6 fmol/mg tissue. This secondary site is a D3 dopaminergic receptor site, as shown by the relative abilities of various displacers to inhibit this binding. Binding studies with nonradioactive 2I-LSD reveal a clear preference for D2 over D3 dopamine receptor sites. [¹²⁵I]LSD is a sensitive and selective label for 5-HT₂ (S2) serotonin receptor sites in both rat frontal cortex and bovine caudate membranes. Blockage of the primary bovine caudate [¹²⁵I]LSD binding site with mianserin allows the high sensitivity of [¹²⁵I]LSD to be applied to D2 dopamine receptor studies as well.     

Role of basic residues in the subgroup-determining region of the subgroup A avian sarcoma and leukosis virus envelope in receptor binding and infection.

Receptor specificity in avian sarcoma and leukosis viruses (ASLV) maps to the central region of the envelope surface protein, SU. Two hypervariable regions, hr1 and hr2, within this region of SU are the principal determinants of receptor specificity. The cellular receptor for subgroup A ASLV, Tva, utilizes a 40-residue, acidic, cysteine-rich sequence for viral binding and entry. This domain in Tva is closely related to the ligand-binding domain of the low-density lipoprotein receptor (LDLR). Ligands bind to LDLR via the interaction of clustered basic residues in the ligand with the acidic cysteine-rich domains of the receptor. Analysis of the ASLV envelope sequences revealed a cluster of basic residues within hr2 that is unique to the subgroup A viruses, suggesting a possible role for these residues in receptor recognition. Therefore, the effects of altering these basic residues on subgroup A envelope expression, receptor binding, and infectivity were examined. Most of the mutant proteins were transported to the cell surface and processed normally. Receptor binding was diminished approximately 50% by alanine substitution at amino acid R213 or K227, whereas substitution by alanine at R210, R223, or R224 had no effect. However, when coupled with mutations at R213 or K227, changes at R223,R224 reduced envelope binding by 90%. Mutation of all five basic residues abrogated receptor binding. The effect of the hr2 mutations on ASLV envelope-mediated infection did not parallel the effect on receptor binding. Residues 210, 213, 223, and 224 were important for efficient infection, while mutations at residue 227 had little effect on infectivity. These results demonstrate that the basic residues in the ASLV envelope have roles in both receptor recognition and post-receptor binding events during viral entry.     

H-2-linked control of resistance to Ectromelia virus infection in 1310 congenic mice

Several B 10 strains of mice, recombinant at theH-2 locus, have been shown to differ in their resistance to infection with ectromelia virus, a natural mouse pathogen. Of 10 strains, 1310, B 10.A(2R), B10.A(4R) and B10.D2 were the most resistant, while B10.G and B 10.A(5R) were the most susceptible. Other strains were intermediate between these extremes. Several genes conferring resistance have been mapped toD ^b in B10.A(2R),K ^k I-A ^k I-B ^k in B10.A,I-J ^b in B10.A(2R) and toD ^d in B 10.T(6R). In general, death among susceptible strains was not a consequence of acute liver necrosis as in other non-B10 strains, and occurred randomly from 8–14 days after infection. The exact cause of death is unknown but is characterized by persisting high titers of virus in the spleen and sometimes the liver, despite an ongoing immune response indicated by strong cytotoxic T-cell activity detectable in the spleens of all mice. The most resistant B10 and B10.A(2R) strains cleared virus from the spleen and liver by 8 days after infection. Analysis of infection in chimeric mice indicates thatH-2 genes, which determine susceptibility to virus persistence in the spleen, operate via radiosensitive cells of the lymphomyeloid system. This evidence, together with several examples ofH-2-linked differences in cytotoxic T-cell responsiveness between resistant and susceptible strains, is consistent with the hypothesis that the mechanism by whichH-2 genes control resistance to ectromelia virus in B10 strain mice is by their influence on the effectiveness of a cell-mediated immune response.     

Studies on construction of a recombinant Eimeria tenella SO7 gene expressing Escherichia coli and its protective efficacy against homologous infection

Eimeria spp. are the causative agents of coccidiosis, a major disease affecting the poultry industry. A recombinant non-antibiotic Escherichia coli that expresses the Eimeria tenella SO7 gene was constructed and its protective efficacy against homologous infection in chickens was determined. The three-day-old chickens were orally immunized with the recombinant non-antibiotic SO7 gene expressing E. coli and boosted two weeks later. Four weeks after the second immunization, the chickens were challenged with 5 × 10⁴ homologous sporulated oocysts. The protective effects of the recombinant non-antibiotic E. coli were determined by measuring body weight change, mortality, histopathology, lesion scores, oocyst counts, the specific antibody response and the frequency of CD4⁺ and CD8⁺ lymphocytes in peripheral blood. The results showed that immunization with SO7 expressing E. coli resulted in significantly improved body weight gain, reduced lesion scores and oocyst shedding in immunized chickens compared to controls. Furthermore, administration of recombinant SO7 expressing E. coli leads to a significant increase in serum antibody, CD4⁺ and CD8⁺ T cells in peripheral blood of chickens. These results, therefore, suggest that the recombinant non-antibiotic E. coli that expresses the SO7 gene is able to effectively stimulate host protective immunity as evidenced by the induction of development of both humoral and cell-mediated immune responses against homologous challenge in chickens.     

Effect of repeatedly sensitized bursal extract on the resistance of WLH chickens to experimental infection of Ancylostoma caninum larvae

Goyal P. K. and Johri G. N. 1982. Effect of repeatedly sensitized bursal extract on the resistance of WLH chickens to experimental infection of Ancylostoma caninum larvae. International Journal for Parasitology12: 245–249. Bursal extract from chickens, repeatedly sensitized with Ancylostoma caninum larvae and from normal nonsensitized donor WLH chickens were injected separately into experimental and control groups of isologous recipients of same age and weight. Extract from donors infected with repeatedly high dose (250 + 250 + 500) and low dose (125 + 125 + 250) of A. caninum larvae produced significantly greater acquired immunity in recipients compared to those which received nonsensitized (normal) extract. However, no significant difference was observed between the two experimental groups.     

A Charged Second-Site Mutation in the Fusion Peptide Rescues Replication of a Mutant Avian Sarcoma and Leukosis Virus Lacking Critical Cysteine Residues Flanking the Internal Fusion Domain

The entry process of the avian sarcoma and leukosis virus (ASLV) family of retroviruses requires first a specific interaction between the viral surface (SU) glycoproteins and a receptor on the cell surface at a neutral pH, triggering conformational changes in the viral SU and transmembrane (TM) glycoproteins, followed by exposure to low pH to complete fusion. The ASLV TM glycoprotein has been proposed to adopt a structure similar to that of the Ebola virus GP2 protein: each contains an internal fusion peptide flanked by cysteine residues predicted to be in a disulfide bond. In a previous study, we concluded that the cysteines flanking the internal fusion peptide in ASLV TM are critical for efficient function of the ASLV viral glycoproteins in mediating entry. In this study, replication-competent ASLV mutant subgroup A [ASLV(A)] variants with these cysteine residues mutated were constructed and genetically selected for improved replication capacity in chicken fibroblasts. Viruses with single cysteine-to-serine mutations reverted to the wild-type sequence. However, viruses with both C9S and C45S (C9,45S) mutations retained both mutations and acquired a second-site mutation that significantly improved the infectivity of the genetically selected virus population. A charged-amino-acid second-site substitution in the TM internal fusion peptide at position 30 is preferred to rescue the C9,45S mutant ASLV(A). ASLV(A) envelope glycoproteins that contain the C9,45S and G30R mutations bind the Tva receptor at wild-type levels and have improved abilities to trigger conformational changes and to form stable TM oligomers compared to those of the C9,45S mutant glycoprotein.All retroviruses have envelope glycoproteins that interact with a receptor protein on the cell surface to initiate entry (18, 36). The viral glycoprotein is synthesized as a precursor polyprotein consisting of the surface (SU) glycoprotein, which contains the domains that bind with the cellular receptor, and the transmembrane (TM) glycoprotein, which tethers the protein to the viral surface and contains the domains responsible for fusion of the viral and cellular membranes (32). After synthesis, the precursor viral glycoproteins form trimers through the interaction of the TM domains. The SU and TM domains are then cleaved by a cellular protease, forming a mature, metastable complex capable of mediating viral entry. A specific receptor protein interaction with the SU domain of the mature Env is required to initiate a conformational change in the trimer, separating the globular SU domains to allow the TM glycoproteins to form a structure that projects the fusion peptide toward the target membrane. Two domains in TM, the N-terminal heptad repeat and the C-terminal heptad repeat, are critical for the formation of the extended structure (13, 31)}. The fusion peptide is thought to interact with a target membrane irreversibly, forming an extended prehairpin TM oligomer structure anchored in both the viral and target membranes (35). The cooperation of several of these extended prehairpin TM oligomer structures is most likely required to complete fusion. The viral and target membranes are brought into close proximity when the C-terminal heptad repeats fold back into grooves formed by the N-terminal heptad repeats, forming presumably the most stable TM structure, the six-helix bundle (6HB). Fusion of the membranes proceeds through the initial mixing of the outer lipid leaflets, hemifusion, followed by initial fusion pore formation, pore widening, and the completion of fusion. The 6HB may undergo some additional structural rearrangement in order to bring the fusion peptide and membrane-spanning domain of TM into close proximity to form the final trimeric hairpin structure (22, 24, 33).Until recently, the triggering of class I virus fusion proteins was thought to occur by one of two mechanisms (13, 35, 36). In one mechanism, the viral glycoproteins interact with receptors on the cell surface, resulting in the trafficking of the virion into an endocytic compartment, followed by the triggering of structural rearrangements in the viral glycoproteins to initiate fusion by exposure to low pH (e.g., influenza virus hemagglutinin [HA]). In a second entry mechanism, the interaction of the viral glycoproteins with receptors on the cell surface in a neutral pH environment triggers the structural rearrangements in the viral glycoproteins directly, initiating viral entry. Retroviruses predominately employ the second entry mechanism, although two cellular protein receptors may be required to complete the conformational changes in the viral glycoproteins necessary to complete entry (e.g., human immunodeficiency virus type 1). However, the entry process of the avian sarcoma and leukosis virus (ASLV) family of retroviruses demonstrates a third entry mechanism for the action of class I virus fusion proteins (25). ASLV entry requires both a specific interaction between the viral glycoproteins and receptors at the cell surface at neutral pH, triggering initial conformational changes in the viral glycoproteins, and a subsequent exposure to low pH to complete fusion (2, 3, 22-24).The fusion peptides of ASLVs are not at the N terminus of the cleaved TM, as in all other retroviral TM proteins, but in a proposed internal loop (TM residues 22 to 37) flanked by two cysteine residues (residues C9 and C45) (Fig. ​(Fig.1).1). The ASLV TM glycoprotein has been proposed to adopt a structure similar to that of the Ebola virus GP2 protein: both contain an internal fusion peptide flanked by cysteine residues predicted to be in a disulfide bond (10). Other viruses contain internal fusion peptides also predicted to be in looped structures (35). In a study to determine if the cysteines that flank the ASLV fusion peptide are required for function, mutant ASLV Env proteins were constructed with one or both of these cysteines changed to serine (C9S, C45S, or C9S C45S [C9,45S]) (8). The mutant subgroup A ASLV [ASLV(A)] Env proteins were expressed, processed, and incorporated into virions at levels similar to those of wild-type (WT) ASLV(A) Env. The mutant and WT ASLV(A) Env proteins bound the Tva receptor with similar affinities. However, murine leukemia virus (MLV) virions pseudotyped with the mutant Envs were ∼500-fold less infectious (titer, ∼2 × 10³ inclusion-forming units [IFU]/ml) than MLV virions pseudotyped with WT ASLV(A) Env (titer, ∼1 × 10⁶ IFU/ml). The ability of the mutant Envs to mediate cell fusion was also greatly impaired compared to that of WT ASLV(A) Env in a cell-cell fusion assay. We concluded that the cysteines flanking the internal fusion peptide in ASLV TM are critical for efficient function of the ASLV viral glycoproteins in mediating entry. In a recent study, the cysteines flanking the fusion peptide region were shown to be critical for the lipid mixing stage of fusion (6).Open in a separate windowFIG. 1.Schematic representations of the ASLV-based RCASBP retroviral vector and the major domains of the envelope glycoproteins. The RCASBP(A)AP replication-competent vector contains a subgroup A env and a reporter gene coding for heat-stable AP. The hypervariable domains (vr1, vr2, hr1, hr2, and vr3) of the SU glycoprotein, the proteolytic cleavage site, the putative fusion peptide region (shaded box), and the membrane-spanning domain (MSD) of the TM glycoprotein are shown schematically. The first 45 residues of the TM glycoprotein are shown for wild-type subgroup A Env (WT) and for the three mutants tested in this study, with either a substitution of serine for the cysteine at position 9 in TM (C9S), a substitution of serine for the cysteine at position 45 in TM (C45S), or both substitutions (C9,45S). The complete sequence of the ASLV(A) WT TM glycoprotein is shown, with the fusion peptide region, N-terminal and C-terminal heptad repeat regions (N-alpha-helix; C-alpha helix), and membrane-spanning domain indicated.Very little is known about the structures of fusion peptides in the context of full-length, trimeric, viral glycoproteins upon interaction with target membranes. Also, natural membrane targets contain a variety of lipid and protein compositions in an asymmetrical organization that is difficult to reproduce experimentally (27). In addition, little is known about how fusion proteins with internal fusion peptide regions interact with target membranes or the possible conformational changes that might be required to complete the fusion process (19, 20). In this study, replication-competent ASLV(A) viruses containing the C9S, C45S, or C9,45S mutations were constructed and genetically selected for improved replication in chicken fibroblasts in order to further explore the importance of these cysteines for proper TM function. Viruses with single cysteine-to-serine mutations reverted to the WT sequence. However, viruses with both the C9S and the C45S mutation retained both mutations and acquired a second-site mutation that significantly enhanced the infectivity of the genetically selected virus population. Unexpectedly, the selected second-site mutation was a charged residue located in the middle of the hydrophobic fusion peptide within TM.     

Gene Expression Profiling of the Local Cecal Response of Genetic Chicken Lines That Differ in Their Susceptibility to Campylobacter jejuni Colonization

Campylobacter jejuni (C. jejuni) is one of the most common causes of human bacterial enteritis worldwide primarily due to contaminated poultry products. Previously, we found a significant difference in C. jejuni colonization in the ceca between two genetically distinct broiler lines (Line A (resistant) has less colony than line B (susceptible) on day 7 post inoculation). We hypothesize that different mechanisms between these two genetic lines may affect their ability to resist C. jejuni colonization in chickens. The molecular mechanisms of the local host response to C. jejuni colonization in chickens have not been well understood. In the present study, to profile the cecal gene expression in the response to C. jejuni colonization and to compare differences between two lines at the molecular level, RNA of ceca from two genetic lines of chickens (A and B) were applied to a chicken whole genome microarray for a pair-comparison between inoculated (I) and non-inoculated (N) chickens within each line and between lines. Our results demonstrated that metabolism process and insulin receptor signaling pathways are key contributors to the different response to C. jejuni colonization between lines A and B. With C. jejuni inoculation, lymphocyte activation and lymphoid organ development functions are important for line A host defenses, while cell differentiation, communication and signaling pathways are important for line B. Interestingly, circadian rhythm appears play a critical role in host response of the more resistant A line to C. jejuni colonization. A dramatic differential host response was observed between these two lines of chickens. The more susceptible line B chickens responded to C. jejuni inoculation with a dramatic up-regulation in lipid, glucose, and amino acid metabolism, which is undoubtedly for use in the response to the colonization with little or no change in immune host defenses. However, in more resistant line A birds the host defense responses were characterized by an up-regulation lymphocyte activation, probably by regulatory T cells and an increased expression of the NLR recognition receptor NALP1. To our knowledge, this is the first time each of these responses has been observed in the avian response to an intestinal bacterial pathogen.     

Heterophils are associated with resistance to systemic Salmonella enteritidis infections in genetically distinct chicken lines

Heterophils mediate acute protection against Salmonella in young poultry. We evaluated susceptibility of genetically distinct lines of broilers to systemic Salmonella enteritidis (SE) infections. SE was administered into the abdomen of day-old chickens (parental lines [A and B]; F1 reciprocal crosses [C and D]) to assess modulation of leukocytes and survivability of chickens. Line A was more resistant to SE than line B; likewise cross D was more resistant than cross C. Significantly more heterophils migrated to the abdominal cavity post-infection in the resistant lines. These data indicate that increased heterophil influx to the infection site contributes to increased resistance against systemic SE infections in neonatal chickens.     

Genetic targeting of Card19 is linked to disrupted NINJ1 expression,impaired cell lysis,and increased susceptibility to Yersinia infection

Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1β release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation. However, the factors and mechanisms that regulate terminal cell lysis downstream of GSDMD cleavage remain poorly understood. In the course of studies to define regulation of pyroptosis during Yersinia infection, we identified a line of Card19-deficient mice (Card19^lxcn) whose macrophages were protected from cell lysis and showed reduced apoptosis and pyroptosis, yet had wild-type levels of caspase activation, IL-1 secretion, and GSDMD cleavage. Unexpectedly, CARD19, a mitochondrial CARD-containing protein, was not directly responsible for this, as an independently-generated CRISPR/Cas9 Card19 knockout mouse line (Card19^Null) showed no defect in macrophage cell lysis. Notably, Card19 is located on chromosome 13, immediately adjacent to Ninj1, which was recently found to regulate cell lysis downstream of GSDMD activation. RNA-seq and western blotting revealed that Card19^lxcn BMDMs have significantly reduced NINJ1 expression, and reconstitution of Ninj1 in Card19^lxcn immortalized BMDMs restored their ability to undergo cell lysis in response to caspase-dependent cell death stimuli. Card19^lxcn mice exhibited increased susceptibility to Yersinia infection, whereas independently-generated Card19^Null mice did not, demonstrating that cell lysis itself plays a key role in protection against bacterial infection, and that the increased infection susceptibility of Card19^lxcn mice is attributable to loss of NINJ1. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent gene Ninj1, disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during Yersinia infection.     

Partial purification of the nuclear estrogen receptor from chicken liver by affinity chromatography

The crude nuclear extract from the liver of estrogenized chickens contains 0.3–1 pmol/g tissue of the estrogen receptor. The receptor has been partially purified by ammonium sulphate precipitation and affinity chromatography on 17β-estradiol-17-hemisuccinyl-ovalbumin-Sepharose 4B. A 12% pure receptor preparation (2700-fold purification) with a yield of 17% could be obtained. The partially purified receptor has retained most properties which it displayed in cruder preparations, e.g. the dissociation constant of 10^?9?10^?10 M, the hormone specificity and the sedimentation coefficient of 3.9 S. The size (Stokes radius, 2.9 nm; molecular weight, 49 000) and the asymetry (f/f₀ = 1.10) of the receptor molecule, however, appear slightly reduced after the purification.     

Infectivity of Steinernema carpocapsae and S. feltiae to Larvae and Adults of the Hazelnut Weevil,Curculio nucum: Differential Virulence and Entry Routes

We investigated the existing susceptibility differences of the hazelnut weevil, Curculio nucum L. (Coleoptera:, Curculionidae) to entomopathogenic nematodes by assessing the main route of entry of the nematodes, Steinernema carpocapsae strain B14 and S. feltiae strain D114, into larvae and adult insects, as well as host immune response. Our results suggested that S. carpocapsae B14 and S. feltiae D114 primarily entered adult insects and larvae through the anus. Larvae were more susceptible to S. feltiae D114 than S. carpocapsae B14 and adults were highly susceptible to S. carpocapsae B14 but displayed low susceptibility to S. feltiae D114. Penetration rate correlated with nematode virulence. We observed little evidence that hazelnut weevils mounted any cellular immune response toward S. carpocapsae B14 or S. feltiae D114. We conclude the differential susceptibility of hazelnut weevil larvae and adults to S. carpocapsae B14 and S. feltiae D114 primarily reflected differences in the ability of these two nematodes to penetrate the host.