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

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

env Gene of Chicken RNA Tumor Viruses: Extent of Conservation in Cellular and Viral Genomes

The env gene of avian sarcoma-leukosis viruses codes for envelope glycoproteins that determine viral host range, antigenic specificity, and interference patterns. We used molecular hybridization to analyze the natural distribution and possible origins of the nucleotide sequences that encode env; our work exploited the availability of radioactive DNA (cDNA(gp)) complementary to most or all of env. env sequences were detectable in the DNAs of chickens which synthesized an env gene product (chick helper factor positive) encoded by an endogenous viral gene and also in the DNAs of chickens which synthesized little or no env gene product (chick helper factor negative). env sequences were not detectable in DNAs from Japanese quail, ring-necked pheasant, golden pheasant, duck, squab, salmon sperm, or calf thymus. The detection of sequences closely related to viral env only in chicken DNA contrasts sharply with the demonstration that the transforming gene (src) of avian sarcoma viruses has readily detectable homologues in the DNAs of all avian species tested [D. Stehelin, H. E. Varmus, J. M. Bishop, and P. K. Vogt, Nature (London) 260: 170-173, 1976] and in the DNAs of other vertebrates (D. Spector, personal communication). Thermal denaturation studies on duplexes formed between cDNA(gp) and chicken DNA and also between cDNA(gp) and RNAs of subgroup A to E viruses derived from chickens indicated that these duplexes were well matched. In contrast, cDNA(gp) did not form stable hybrids with RNAs of viruses which were isolated from ring-necked and golden pheasants. We conclude that substantial portions of nucleotide sequences within the env genes of viruses of subgroups A to E are closely related and that these genes probably have a common, perhaps cellular, evolutionary origin.     

Inversion of a chicken ets-1 proto-oncogene segment in avian leukemia virus E26.

The segment of the avian leukemia virus E26 genome near the termination of the p135gag-myb-ets open reading frame contains an inversion of the chicken ets-1 sequence. The inversion contains at least 41 bp and may be as large as 46 bp. This results in the replacement of 13 amino acids of chicken ets-1, with 16 amino acids derived from reverse complement of the normal ets-1 coding strand or read-through into E26 env sequences. At least 13 of these codons are specified by the inverted ets sequences. This represents the first reported occurrence of inverted oncogene sequences in a natural retrovirus. The inverted ets sequences are immediately followed by sequences homologous to the Rous sarcoma virus Prague B env gene. Since the E26 env sequence is more closely related to subgroup B avian retroviruses than to avian retroviruses from subgroups A, C, D, or E, the progenitor of E26 was a virus belonging to avian retrovirus subgroup B.     

The Unique Envelope Gene of the Subgroup J Avian Leukosis Virus Derives from ev/J Proviruses, a Novel Family of Avian Endogenous Viruses

A new subgroup of avian leukosis virus (ALV), designated subgroup J, was identified recently. Viruses of this subgroup do not cross-interfere with viruses of the avian A, B, C, D, and E subgroups, are not neutralized by antisera raised against the other virus subgroups, and have a broader host range than the A to E subgroups. Sequence comparisons reveal that while the subgroup J envelope gene includes some regions that are related to those found in env genes of the A to E subgroups, the majority of the subgroup J gene is composed of sequences either that are more similar to those of a member (E51) of the ancient endogenous avian virus (EAV) family of proviruses or that appear unique to subgroup J viruses. These data led to the suggestion that the ALV-J env gene might have arisen by multiple recombination events between one or more endogenous and exogenous viruses. We initiated studies to investigate the origin of the subgroup J envelope gene and in particular to determine the identity of endogenous sequences that may have contributed to its generation. Here we report the identification of a novel family of avian endogenous viruses that include env coding sequences that are over 95% identical to both the gp85 and gp37 coding regions of subgroup J viruses. We call these viruses the ev/J family. We also report the isolation of ev/J-encoded cDNAs, indicating that at least some members of this family are expressed. These data support the hypothesis that the subgroup J envelope gene was acquired by recombination with expressed endogenous sequences and are consistent with acquisition of this gene by only one recombination event.     

Vertebrate DNAs contain nucleotide sequences related to the transforming gene of avian myeloblastosis virus.

Avian myeloblastosis virus contains a continuous sequence of approximately 1,000 nucleotides which may represent a gene (amv) responsible for acute myeloblastic leukemia in chickens. This sequence appears to have been acquired from chicken DNA and to be substituted for the envelope gene in the viral genome. We used hybridization probes enriched for the amv sequences and conditions that facilitate annealing of partially homologous nucleotide sequences to show that cellular sequences related to amv are present in the genomes of all vertebrates ranging from amphibians to humans but were not detected in fish, sea urchins, or Escherichia coli. In contrast to the preceding findings, nontransforming endogenous proviral nucleotide sequences closely related to the remainder of the avian myeloblastosis virus genome and to the entire myeloblastosis-associated helper virus are present only in chicken DNA. The amv-related cellular sequences appear to be highly conserved during evolution and to be contained at only one or a few locations in the genome of vertebrates. Within closely related species, they appear to share common evolutionary genetic loci. These findings and similar ones obtained with other highly oncogenic retroviruses containing a transforming gene suggest a general mechanism for acquisition of viral oncogenic sequences and an essential role for these sequences in the normal cellular state.     

Detection and Molecular Characterization of J Subgroup Avian Leukosis Virus in Wild Ducks in China

To assess the status of avian leukosis virus subgroup J (ALV-J) in wild ducks in China, we examined samples from 528 wild ducks, representing 17 species, which were collected in China over the past 3 years. Virus isolation and PCR showed that 7 ALV-J strains were isolated from wild ducks. The env genes and the 3′UTRs from these isolates were cloned and sequenced. The env genes of all 7 wild duck isolates were significantly different from those in the prototype strain HPRS-103, American strains, broiler ALV-J isolates and Chinese local chicken isolates, but showed close homology with those found in some layer chicken ALV-J isolates and belonged to the same group. The 3′UTRs of 7 ALV-J wild ducks isolates showed close homology with the prototype strain HPRS-103 and no obvious deletion was found in the 3′UTR except for a 1 bp deletion in the E element that introduced a binding site for c-Ets-1. Our study demonstrated the presence of ALV-J in wild ducks and investigated the molecular characterization of ALV-J in wild ducks isolates.     

Production of avian oncoviral subgroups after multiple infection.

The number of different oncoviral env genes that can be expressed by a single chicken embryo fibroblast was investigated. Fibroblasts were infected with one to three subgroups of Rous-associated virus, which is a nontransforming avian oncovirus, then superinfected with a transforming virus, Rous sarcoma virus, of a different subgroup. The subgroups of viruses released by the resulting clones were analyzed. When two viral subgroups were used for preinfection, all the resulting clones produced transforming virus particles having the subgroup of the superinfecting virus, and most clones produced transforming virus particles of all the infecting viral subgroups. However, when cells were preinfected with three viral subgroups, many of the resulting clones did not produce transforming virus particles having the subgroup of the superinfecting virus, and only 1 of 23 clones produced transforming particles of all the infecting viral subgroups. DNA annealing experiments showed that cells infected with three or four viral subgroups had an additional 8 to 20 copies of proviral DNA per cell. Finally, most clones resulting from cells simultaneously infected with three or four viral subgroups were able to produce virus of all infecting subgroups. It appears that the number of exogenous oncoviral env genes that can be expressed by a single cell is limited, and in the range of 4 to 8-20 per cell.     

A myristylated form of the sea oncoprotein can transform chicken embryo fibroblasts

The transforming gene product of the S13 avian erythroblastosis virus, v-sea, is a member of the growth factor receptor class of tyrosine kinases. In the virus genome, the sea sequences are fused in frame to the virus env gene, thereby generating an abnormally large envelope protein because of the presence of a cytoplasmic tyrosine kinase domain. To determine what role these envelope sequences play in v-sea transformation, we generated a myristylated form of v-sea which contains no envelope sequences. In this report, we show that this myristylated sea-encoded protein retained the ability to transform chicken embryo fibroblasts, indicating that envelope sequences are not essential for transformation by the v-sea tyrosine kinase.     

Induction of neoplasms by subgroup E recombinants of exogenous and endogenous avian retroviruses (Rous-associated virus type 60).

Chickens susceptible to infection with subgroup E viruses were inoculated with four independent isolates of Rous-associated virus type 60 (RAV-60) that are subgroup e recombinants of endogenous and exogenous virus. Neoplasms developed in each inoculated group. Therefore, nontransforming viruses of subgroup E can induce lymphoid leukosis at a moderate rate compared with RAV-0, a subgroup E endogenous virus, suggesting that oncogenicity is not a viral envelope (env)-related characteristic. Since the common (c) regions of the RAV-60s examined were of exogenous origin, we suggest that the c region rather than env is important for a high rate of induction of lymphoid leukosis and related neoplasms.     

Purification of DNA complementary to the env gene of avian sarcoma virus and analysis of relationships among the env genes of avian leukosis-sarcoma viruses.

The env gene of avian leukosis-sarcoma viruses encodes a glycoprotein that determines the host range and surface antigenicitiy of virions. We have purified radioactive DNA (cDNAgp) complementary to at least a portion of the env gene for viral subgroups A and C; complementary DNA was synthesized with purified virions of wild-type avian sarcoma virus, and RNA from a mutant with a deletion in env was used to select DNA specific to env by molecular hybridization. The genetic complexity of cDNAgp for subgroup A (ca. 2,000 nucleotides) was sufficient to represent the entire deletion and most or all of the env cistron. The deletions in env in two independently isolated strains of virus (Bryan and rdNY8SR) overlap, and cDNAgp represents nucleotide sequences common to both deletions. By contrast, we could detect no overlap between deletions in env and deletions in the adjacent viral gene src. Laboratory stocks of viral subgroups A, B, C, D and E do not contain detectable amounts of env deletions when tested by molecular hybridization; hence, segregation of deletions in env is a less frequent event that the segregation of deletions in the viral transforming gene src (Vogt, 1971). We found extensive homology among the nucleotide sequences encoding the env genes of virus strains indigenous to chickens (subgroups A, B, C, D, and E) although subgorups B, D and E appear to differ slightly from subgroups A and C at the env locus. By contrast, viruses obtained from pheasant cells (subgroups F and G) have env genes with little or no relationship to env genes of chikcen viruses. According to available data, viruses of subgroup F arose by recombination between an avarian sarcoma virus and viral genes in the genome of ring-necked pheasants, whereas subgroup G viruses may be entirely endogenous to golden pheasants.     

Transfection by DNAs of avian erythroblastosis virus and avian myelocytomatosis virus strain MC29.

Chicken embryo fibroblasts and NIH 3T3 mouse cells were transformable by DNAs of chicken cells infected with avian myelocytomatosis virus strain MC29 or with avian erythroblastosis virus. Transfection of chicken cells appeared to require replication of MC29 or avian erythroblastosis virus in the presence of a nontransforming helper virus. In contrast, NIH 3T3 cells transformed by MC29 or avian erythroblastosis virus DNA contained only replication-defective transforming virus genomes.     

Influence of env and long terminal repeat sequences on the tissue tropism of avian leukosis viruses.

Adsorption and penetration of retroviruses into eucaryotic cells is mediated by retroviral envelope glycoproteins interacting with host receptors. Recombinant avian leukosis viruses (ALVs) differing only in envelope determinants that interact with host receptors for subgroup A or E ALVs have been found to have unexpectedly distinctive patterns of tissue-specific replication. Recombinants of both subgroups were highly expressed in bursal lymphocytes as well as in cultured chicken embryo fibroblasts. In contrast, the subgroup A but not subgroup E host range allowed high levels of expression in skeletal muscle, while subgroup E but not subgroup A envelope glycoproteins permitted efficient replication in the thymus. A subgroup B virus (RAV-2), like the subgroup E viruses, demonstrated a distinct bursal and thymic tropism, further supporting the theory that genes encoding receptors for subgroup B and E viruses are allelic. The source of long terminal repeats (LTRs) or adjacent sequences also influenced tissue-specific replication, with the LTRs from endogenous virus RAV-0 supporting efficient replication in the bursa and thymus but not in skeletal muscle. These results indicate that ALV env and LTR regions are responsible for unexpectedly distinctive tissue tropisms.     

禽白血病病毒J亚群env基因的克隆和序列分析

应用多聚酶链反应（PCR）的方法增出ADOL－4817毒株的囊膜蛋白env基因,并克隆进大肠杆菌。经核酸序列分析证明,env基因的大小为1746bp,其中gp85和gp37mh 1554bp组成,可翻译成517个氨基酸,分子量为57.7kD。根据糖基化位点N－X－S／T的特点,发现ADOL－4817的env蛋白有15个潜在的糖基化位点。同源性分析证明,ADOL－4817的env基因与其它ALV－J的env基因序列同源性为88.8％－92.4%,而与外源性ALVs的相应序列的同源性仅为40.5％－51.4％,然而,与内源性的EAV－HP毒株的类env基因的同源性高达91.2％;另外,ADOL－4817毒株的gp37d C末端多了13个氨基酸,这些结果提示,ALV－J的env基因存在广泛的变异性,env基因可能来源于内源性和外源性ALVs的重组。     

Molecular cloning and characterization of gag-, pol-, and env-related gene sequences in the ev- chicken.

Using less stringent hybridization conditions and cloned viral DNA probes representing the avian sarcoma virus gag, pol, env, and long terminal repeat (LTR) gene sequences, we detected related sequences in two avian species purportedly lacking all endogenous avian leukosis viruses, the ev- chicken and the Japanese quail. The blot hybridization patterns obtained with the various probes suggest the presence of between 40 and 100 copies of retrovirus-related sequences in the genomes of these two species. An ev- chicken genomic DNA library was prepared and screened with gag-specific and pol-specific DNA probes. Several different clones were obtained from this library and characterized. Analysis of these clones revealed that the retrovirus-related gene sequences are linked in the order LTR-gag-pol-env-LTR, a structure indicative of a complete provirus. These data indicate the presence of previously unidentified endogenous retrovirus species in avian cells, suggesting that under the appropriate conditions of hybridization additional, more distantly evolved families of endogenous retrovirus genes may be identified in vertebrate species.     

Genome structure and expression of the ev/J family of avian endogenous viruses.

We recently reported the identification of sequences in the chicken genome that show over 95% identity to the novel envelope gene of the subgroup J avian leukosis virus (S. J. Benson, B. L. Ruis, A. M. Fadly, and K. F. Conklin, J. Virol. 72:10157-10164, 1998). Based on the fact that the endogenous subgroup J-related env genes were associated with long terminal repeats (LTRs), we concluded that these LTR-env sequences defined a new family of avian endogenous viruses that we designated the ev/J family. In this report, we have further characterized the content and expression of the ev/J proviruses. The data obtained indicate that there are between 6 and 11 copies of ev/J proviruses in all chicken cells examined and that these proviruses fall into six classes. Of the 18 proviruses examined, all share a high degree of sequence identity and all contain an internal deletion that removes all of the pol gene and various amounts of gag and env gene sequences. Sequencing of the gag genes, LTRs, and untranslated regions of several ev/J proviruses revealed a high level of identity between isolates, indicating that they have not undergone significant sequence variation since their introduction into the avian germ line. Although the ev/J gag gene showed a relatively weak relationship (46% identity and 61% similarity at the amino acid level) to that of the avian leukosis-sarcoma virus family, it retains several sequences of demonstrated importance for virus assembly, budding, and/or infectivity. Finally, evidence was obtained that at least some members of the ev/J family are expressed and, if translated, could encode Gag- and Env-related polypeptides.     

Expression of Endogenous Retrovirus ev/J gp85 Gene and Analysis of Its Immunoreactivity in Comparison with Exogenous Viral Protein

The envelope gene gp85 of ev/J,a new family of endogenous avian retroviral sequences identified recently, has the most extensive nucleotide sequence identity ever described with ALV-J avian ieukosis virus. This report described expression of ev/J envelope gene gp85 derived from commercial meat-type chicken using the Invitrogen Bac-to-Bac baculovirus expression system. The antigenicity and immunoreactivity of the recombinant endogenous gp85 gene product (SU) were analyzed by indirect immunofluorescence, Western blot, indirect and blocking Enzyme-Linked ImmunoSorbent Assay (ELISA) using JE9 monoclonal antibody (MAb) against the envelope protein of ALV-J (ADOL-4817), positive mouse antiserum against the ev/J gp85 SU and sera from chicken naturally infected with ALV-J. The results showed that the ev/J gp85 SU can bind specifically to JE9 MAb and antiserum from chicken naturally infected with ALV-J, and the binding reactivity between exogenous ALV-J gp85 SU and natural positive chicken serum against exogenous ALV-J can be blocked by positive mouse serum against the ev/J gp85 SU. It is concluded that recombinant endogenous gp85 gene product (SU) has close immunological relatedness to the envelope protein of exogenous ALV-J (ADOL-4817 and IMC<,10200> strain).