Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors

We have used vectors derived from avian leukosis viruses to transduce exogenous genes into early somatic stem cells of chicken embryos. The ecotropic helper cell line, Isolde, was used to generate stocks of NL-B vector carrying theNeo ^r selectable marker and theEscherichia coli lacZ gene. Microinjection of the NL-B vector directly beneath unincubated chicken embryo blastoderms resulted in infection of germline stem cells. One of the 16 male birds hatched (6.25%) from the injected embryos contained vector DNA sequences in its semen. Vector sequences were transmitted to G₁ progeny at a frequency of 2.7%.Neo ^r andlacZ genes were transcribedin vitro in chicken embryo fibroblast cultures from transgenic embryos of the G₂ progeny.     

Structure and membrane interaction of the internal fusion peptide of avian sarcoma leukosis virus.

The structure and membrane interaction of the internal fusion peptide (IFP) fragment of the avian sarcoma and leucosis virus (ASLV) envelope glycoprotein was studied by an array of biophysical methods. The peptide was found to induce lipid mixing of vesicles more strongly than the fusion peptide derived from the N-terminal fusion peptide of influenza virus (HA2-FP). It was observed that the helical structure was enhanced in association with the model membranes, particularly in the N-terminal portion of the peptide. According to the infrared study, the peptide inserted into the membrane in an oblique orientation, but less deeply than the influenza HA2-FP. Analysis of NMR data in sodium dodecyl sulfate micelle suspension revealed that Pro13 of the peptide was located near the micelle-water interface. A type II beta-turn was deduced from NMR data for the peptide in aqueous medium, demonstrating a conformational flexibility of the IFP in analogy to the N-terminal FP such as that of gp41. A loose and multimodal self-assembly was deduced from the rhodamine fluorescence self-quenching experiments for the peptide bound to the membrane bilayer. Oligomerization of the peptide and its variants can also be observed in the electrophoretic experiments, suggesting a property in common with other N-terminal FP of class I fusion proteins.     

Mutational analysis of the subgroup A avian sarcoma and leukosis virus putative fusion peptide domain

Short hydrophobic regions referred to as fusion peptide domains (FPDs) at or near the amino terminus of the membrane-anchoring subunit of viral glycoproteins are believed to insert into the host membrane during the initial stage of enveloped viral entry. Avian sarcoma and leukosis viruses (ASLV) are unusual among retroviruses in that the region in the envelope glycoprotein (EnvA) proposed to be the FPD is internal and contains a centrally located proline residue. To begin analyzing the function of this region of EnvA, 20 substitution mutations were introduced into the putative FPD. The mutant envelope glycoproteins were evaluated for effects on virion incorporation, receptor binding, and infection. Interestingly, most of the single-substitution mutations had little effect on any of these processes. In contrast, a bulky hydrophobic substitution for the central proline reduced viral titers 15-fold without affecting virion incorporation or receptor binding, whereas substitution of glycine for the proline had only a nominal effect on EnvA function. Similar to other viral FPDs, the putative ASLV FPD has been modeled as an amphipathic helix where most of the bulky hydrophobic residues form a patch on one face of the helix. A series of alanine insertion mutations designed to interrupt the hydrophobic patch on the helix had differential effects on infectivity, and the results of that analysis together with the results observed with the substitution mutations suggest no correlation between maintenance of the hydrophobic patch and glycoprotein function.     

Proper processing of avian sarcoma/leukosis virus capsid proteins is required for infectivity

The formation of the mature carboxyl terminus of CA in avian sarcoma/leukemia virus is the result of a sequence of cleavage events at three PR sites that lie between CA and NC in the Gag polyprotein. The initial cleavage forms the amino terminus of the NC protein and releases an immature CA, named CA1, with a spacer peptide at its carboxyl terminus. Cleavage of either 9 or 12 amino acids from the carboxyl terminus creates two mature CA species, named CA2 and CA3, that can be detected in avian sarcoma/leukemia virus (R. B. Pepinsky, I. A. Papayannopoulos, E. P. Chow, N. K. Krishna, R. C. Craven, and V. M. Vogt, J. Virol. 69:6430-6438, 1995). To study the importance of each of the three CA proteins, we introduced amino acid substitutions into each CA cleavage junction and studied their effects on CA processing as well as virus assembly and infectivity. Preventing cleavage at any of the three sites produced noninfectious virus. In contrast, a mutant in which cleavage at site 1 was enhanced so that particles contained CA2 and CA3 but little detectable CA1 was infectious. These results support the idea that infectivity of the virus is closely linked to proper processing of the carboxyl terminus to form two mature CA proteins.     

Characteristics of two-step RNA dimerization in avian sarcoma and leukosis viruses

Dimerization of two genomic RNA copies is essential for the assembly of retrovirus particles. This process has been studied in detail, and a two-step mechanism has been proposed for the human immunodeficiency virus type 1 (HIV-1). A similar model can be assumed for avian sarcoma and leukosis viruses (ASLV), despite the lack of homology between the dimerization initiation site (DIS) of ASLV and that of HIV-1. The structural features of the ASLV DIS were studied with the examples of the avian leukosis virus HPRS-103 and the avian sarcoma virus CT-10. The rate of spontaneous transition from loose to tight dimers at a higher temperature was studied as dependent on the stem length in the DIS hairpin. Dimers of both types were formed by the selected RNA fragments of the two viruses. The conditions of loose dimer formation differed considerably, although the two viruses had identical sequences (5-A-CUGCAG-3) of the hairpin loop. Dimerization of CT-10 RNA fragments required an RNA concentration at least an order of magnitude higher than in the case of HPRS-103. The difference was explained by deletion of an adenine from the hairpin stem of C-10.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 147–154.Original Russian Text Copyright © 2005 by Beniaminov, Samokhin, Ulyanov, Minyat.     

Development of avian sarcoma and leukosis virus-based vector-packaging cell lines.

We have constructed an avian leukosis virus derivative with a 5' deletion extending from within the tRNA primer binding site to a SacI site in the leader region. Our aim was to remove cis-acting replicative and/or encapsidation sequences and to use this derivative, RAV-1 psi-, to develop vector-packaging cell lines. We show that RAV-1 psi- can be stably expressed in the quail cell line QT6 and chicken embryo fibroblasts and that it is completely replication deficient in both cell types. Moreover, we have demonstrated that QT6-derived lines expressing RAV-1 psi- can efficiently package four structurally different replication-defective v-src expression vectors into infectious virus, with very low or undetectable helper virus release. These RAV-1 psi--expressing cell lines comprise the first prototype avian sarcoma and leukosis virus-based vector-packaging system. The construction of our vectors has also shown us that a sequence present within gag, thought to facilitate virus packaging, is not necessary for efficient vector expression and high virus production. We show that quantitation and characterization of replication-defective viruses can be achieved with a sensitive immunocytochemical procedure, presenting an alternative to internal selectable vector markers.     

Characteristic of the two-step RNA dimerization in avian sarcoma and leukosis viruses

Dimerization of two copies of genomic RNA is a necessary step of retroviral replication. In the case of human immunodeficiency virus type 1 (HIV-1) the process is explored in many details. It is proved that conserved stem-loop structure is an essential element in RNA dimerization. Similar model of two-step dimerization mechanism can be considered for avian sarcoma and leukosis virus group (ASLV) in spite of the absence of homology between dimer initiation site (DIS) of ASLV and that of HIV-1. In this paper, short RNA fragments of two viruses: avian sarcoma virus CT-10 and avian leukosis virus HPRS-103 have been chosen in order to investigate the structural requirements of dimerization process and compare them to that of HIV-1. The rate of spontaneous transition from loose to tight dimer was studied as a function of stem length and temperature. Although both types of dimers were observed for both avian retroviruses chosen, fragments of CT-10 requires much higher RNA concentration to form loose dimer. In spite of identical sequence of the loops (5'-A-CUGCAG-3') avian sarcoma virus CT-10 RNA fragments dimerization was greatly impaired. The differences can be explained by deletion of adenine 271 in avian sarcoma virus CT-10 in the stem and by resulting shortening of the self-complementary loop.     

The follicle promotes the evolution of variants of avian leukosis virus subgroup J in vertical transmission

Avian leukosis virus (ALV) is one of the main causative agent of tumor development, which brings enormous economic losses to the poultry industry worldwide. ALV can be transmitted horizontally and vertically, and the latter often give rise to more adverse pathogenicity. However, the propagation and evolution of ALV underlying vertical transmission remain not-well understood. Herein, an animal model for the evolution of variants of ALV subgroup J (ALV-J) in the vertical transmission was built and different organs from infected hens and plasma from their ALV-positive progenies were collected, and then three segments in the hypervariable regions of ALV (gp85-A, gp85-B, LTR-C) were amplified and sequenced using conventional Sanger sequencing and MiSeq high-throughput sequencing, respectively. The results showed that the genomic diversity of ALV-J occurred in different organs from ALV-J infected hen, and that the dominant variants in different organs of parental hens, especially in follicle, changed significantly compared with original inoculum strain. Notably, the dominant variants in progenies exhibited higher homologies with variants in parental hens’ follicle (88.9%–98.9%) than other organs (85.6%–91.1%), and most consistent mutations in the variants were observed between the progenies and parental hen’s follicle. Furthermore, HyPhy analysis indicated that the global selection pressure value (ω) in the follicle is significantly higher than those in other organs. In summary, an animal model for vertical transmission was built and our findings revealed the evolution of variants of ALV in the process of vertical transmission, moreover, the variants were most likely to be taken to the next generation via follicle, which may be related to the higher selection pressure follicle underwent.     

The avian retrovirus avian sarcoma/leukosis virus subtype A reaches the lipid mixing stage of fusion at neutral pH

We previously showed that the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at >or=22 degrees C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and >or=22 degrees C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate avian retroviral Envs: receptor binding at neutral pH, followed by exposure to low pH (W. Mothes et al., Cell 103:679-689, 2000). Therefore, we evaluated the requirements for intact ASLV-A particles to bind to target bilayers and fuse with cells. We found that ASLV-A particles bind stably to liposomes in a receptor- and temperature-dependent manner at neutral pH. Using ASLV-A particles biosynthetically labeled with pyrene, we found that ASLV-A mixes its lipid envelope with cells within 5 to 10 min at 37 degrees C. Lipid mixing was neither inhibited nor enhanced by incubation at low pH. Lipid mixing of ASLV-A was inhibited by a peptide designed to prevent six-helix bundle formation in EnvA; the same peptide inhibits virus infection and EnvA-mediated cell-cell fusion (at both neutral and low pHs). Bafilomycin and dominant-negative dynamin inhibited lipid mixing of Sindbis virus (which requires low pH for fusion), but not of ASLV-A, with host cells. Finally, we found that, although EnvA-induced cell-cell fusion is enhanced at low pH, a mutant EnvA that is severely compromised in its ability to support infection still induced massive syncytia at low pH. Our results indicate that receptor binding at neutral pH is sufficient to activate EnvA, such that ASLV-A particles bind hydrophobically to and merge their membranes with target cells. Possible roles for low pH at subsequent stages of viral entry are discussed.     

A study of low pH-induced refolding of Env of avian sarcoma and leukosis virus into a six-helix bundle

The fusion protein of avian sarcoma and leukosis virus is likely to fold into a six-helix bundle as part of its final configuration. A peptide, R99, inhibits fusion, probably by binding into the grooves of the triple-stranded coiled coil that becomes the central core of the six-helix bundle. The stages at which the envelope protein (Env) of avian sarcoma and leukosis virus subgroup A folds into a bundle during low pH-induced fusion were determined. Effector cells expressing Env were bound to target cells expressing the cognate receptor Tva, and intermediates of fusion were created. R99 was added and the extent of fusion inhibition was used to distinguish between a prebundle state with exposed grooves and a state in which the grooves were no longer exposed. The native conformation of Env was not sensitive to R99. But adding a soluble form of Tva to effector cells conferred sensitivity. Acidic pH applied at low temperature created an intermediate state of local hemifusion. Surprisingly, R99 caused these locally hemifused membranes to separate. This indicates that the grooves of Env were still exposed, that prebundle configurations of Env stabilized hemifused states, and that binding of R99 altered the conformation of Env. In the presence of an inhibitory lipid that blocks fusion before hemifusion, applying low pH at 37 degrees C created an intermediate in which R99 was without effect. This suggests that the six-helix bundle can form before hemifusion and that subsequent conformational changes, such as formation of the trimeric hairpin, are responsible for pore formation and/or growth.     

Phylogeny of Tetraoninae and other galliform birds using mitochondrial 12S and ND2 genes

The avian subfamily Tetraoninae (grouse and ptarmigan) is a Holarctic group in the order Galliformes distinguished by morphological adaptations to cold environments and behavioral traits associated with elaborate courtship. Here we investigate the relationships of 17 tetraonines and 12 other galliform species using mitochondrial 12S and ND2 sequence data. We found support for the recent phylogenetic classification that separates the genus Dendragapus into two genera, Falcipennis and Dendragapus. In addition, we found support for a tetraonine clade in which the first divergence is between Bonasa umbellus and all others, followed by divergence between a Bonasa bonasia/Bonasa sewerzowi clade and the remaining tetraonines. Falcipennis canadensis is sister to a clade with four Tetrao species, and the genus Centrocercus is sister to a Dendragapus obscurus/Tympanuchus clade. Our data indicate a basal position for Cracidae and Megapodiidae among the five recognized galliform families. We also found strong support for the monophyly of Phasianidae, although the relative positions of Numididae and Odontiphoridae remains unresolved. We use a maximum likelihood approach to infer ages of 37mya for divergence of Numididae and Phasianidae and 28mya for the divergence of Tetraoninae and Meleagris gallopavo. These estimates must be viewed as tentative as they depend on tests of rates of molecular evolution and accurate fossil dates.     

Selective shedding and congenital transmission of endogenous avian leukosis viruses.

Shedding and congenital transmission of endogenous avian leukosis viruses were studied in viremic White Leghorn hens exogenously infected with viruses with endogenous long terminal repeats (LTRs) and in four semicongenic lines of hens that naturally express infectious endogenous viruses (EVs). Relatively high titers of infectious virus EV7 (encoded at locus ev7), Rous-associated virus-0 (RAV-0), and recombinant 882/-16 RAV-0 were detected in blood cells and sera from exogenously infected hens, but marked differences were noted in the incidence of congenitally infected progeny. In enzyme immunoassays that detect viral group-specific antigen, little or no p27 was detected in albumens from dams infected with RAV-0. However, hatchmates infected with either EV7 or recombinant 882/-16 RAV-0, which was constructed with an RAV-0 LTR, shed high titers of p27. Similarly, semicongenic hens that expressed RAV-0 (EV2) (encoded at locus ev2) shed little or no p27 into albumens, but hens that harbored ev10, ev11, and ev12 shed high titers of p27. A slower electrophoretic mobility of p27, considered to be characteristic of EVs that are restricted in congenital transmission, was not associated with low levels of shedding or congenital transmission; p27 from other EVs and p27 from an avian leukosis virus field strain, all of which are shed at high levels, had mobilities identical to that of p27 from RAV-0. Although shedding and congenital transmission appear to be controlled by the viral genome, there was no correlation between low efficiency of shedding or congenital transmission and endogenous LTR or p27 sequences.     

Novel monoclonal antibody directed at the receptor binding site on the avian sarcoma and leukosis virus Env complex

We report here on the generation of a mouse monoclonal antibody directed against Rous sarcoma virus (RSV) subgroup A Env that will be useful in functional and structural analysis of RSV Env, as well as in approaches employing the RCAS/Tva system for gene targeting. BALB/c mice were primed and given boosters twice with EnvA-expressing NIH 3T3 cells. Resulting hybridomas were tested by enzyme-linked immunosorbent assay against RCANBP virions and SU-A-immunoglobulin G immunoadhesin. One highly reactive hybridoma clone, mc8C5, was subcloned and tested in immunofluorescence, immunoprecipitation (IP), and Western blotting assays. In all three assays, mc8C5-4 subgroup-specifically recognizes SR-A Env, through the SU domain, expressed from different vectors in both avian and mammalian cells. This multifunctionality is notable for a mouse monoclonal. We furthermore observed a preference for binding to terminally glycosylated Env over core-glycosylated Env precursor in IPs, suggesting that the epitope is at least partially conformational and dependent on glycosylation. Most importantly, we found mc8C5-4 inhibited Env function: in vitro, the monoclonal not only interferes with binding of the EnvA receptor, Tva, but it also blocks the Tva-induced conformational change required for activation of the fusion peptide, without inducing that change itself. Infection of Tva-expressing avian or mammalian cells by avian sarcoma and leukosis virus (ASLV) or EnvA-pseudotyped murine leukemia virus, respectively, is efficiently inhibited by mc8C5-4. The apparent interference of the monoclonal with the EnvA-Tva complex formation suggests that the epitope seen by mc8C5 overlaps with the receptor binding site. This is supported by the observation that mutations of basic residues in hr2 or of the downstream glycosylation site, which both impair Tva-binding to EnvA, have similar effects on the binding of mc8C5. Thus, anti-ASLV-SU-A mc8C5-4 proves to be a unique new immunoreagent that targets the receptor-binding site on a prototypical retroviral envelope.     

Role of calcium in protein folding and function of Tva, the receptor of subgroup A avian sarcoma and leukosis virus

Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue cysteine-rich motif called the LDL-A module. In this study, we expressed and purified the wild-type (wt) Tva LDL-A module as well as several mutants and examined their in vitro folding properties. We found that, as for other LDL-A modules, correct folding and structure of the Tva LDL-A module is Ca2+ dependent. When calcium was present during in vitro protein folding, the wt module was eluted as a single peak by reverse-phase high-pressure liquid chromatography. Furthermore, two-dimensional nuclear magnetic resonance (NMR) spectroscopy gave well-dispersed spectra in the presence of calcium. In contrast, the same protein folded in vitro in the absence of calcium was eluted as multiple broad peaks and gave a poorly dispersed NMR spectrum in the presence of calcium. The calcium affinity (Kd) of the Tva LDL-A module, determined by isothermal titration calorimetry, is approximately 40 microM. Characterization of several Tva mutants provided further evidence that calcium is important in protein folding and function of Tva. Mutations of the Ca2+-binding residues (D46A and E47A) completely abrogated the Ca2+-binding ability of Tva, and the proteins were not correctly folded. Interestingly, mutations of two non-calcium-binding residues (W48A and L34A) also exerted adverse effect on Ca2+-dependent folding, albeit to a much less extent. Our results provide new insights regarding the structure and function of Tva in ASLV-A entry.     

Critical role for the cysteines flanking the internal fusion peptide of avian sarcoma/leukosis virus envelope glycoprotein

The transmembrane subunit (TM) of the envelope glycoprotein (Env) of the oncovirus avian sarcoma/leukosis virus (ASLV) contains an internal fusion peptide flanked by two cysteines (C9 and C45). These cysteines, as well as an analogous pair in the Ebola virus GP glycoprotein, are predicted to be joined by a disulfide bond. To examine the importance of these cysteines, we mutated C9 and C45 in the ASLV subtype A Env (EnvA), individually and together, to serine. All of the mutant EnvAs formed trimers that were composed of the proteolytically processed surface (SU) and TM subunits. All mutant EnvAs were incorporated into murine leukemia virus pseudotyped virions and bound receptor with wild-type affinity. Nonetheless, all mutant EnvAs were significantly impaired ( approximately 1,000-fold) in their ability to support infectivity. They were also significantly impaired in their ability to mediate cell-cell fusion. Our data are consistent with a model in which the internal fusion peptide of ASLV-A EnvA exists as a loop that is stabilized by a disulfide bond at its base and in which this stabilized loop serves an important function during virus-cell fusion. The fusion peptide of the Ebola virus GP glycoprotein may conform to a similar structure.