Organization of the endogenous proviruses of chickens: implications for origin and expression

Eleven of the endogenous proviruses of white leghorn chickens have been mapped with restriction endonucleases and specific nucleic acid hybridization reagents. The restriction maps of these endogenous proviruses have been compared with restriction maps of avian sarcoma virus (ASV) and Rous-associated virus O (RAV-O), an endogenous virus which is spontaneously released by cells from certain lines of chickens. Endogenous proviruses have the same basic structure as proviruses acquired by exogenous infection; the gene order is the same in the provirus as in viral RNA, and the ends of the provirus form a characteristic direct repeat which contains sequences derived from both ends of viral RNA. The endogenous proviruses can thus be described “cell DNA-3′5′-gag-pol-env-3′5′-cell DNA,” where 3′ and 5′ denote sequences homologous to the 3′ and 5′ ends of viral RNA. The endogenous proviruses of chickens are more closely related to RAV-O than to ASV, based on restriction maps and on hybridization with reagents specific for the 3′ ends of RAV-O and ASV. However, all but two of the endogenous proviruses lack at least one of the twoSstI sites in RAV-O DNA (see the preceding paper) and can thus be distinguished from RAV-O by digestion withSstI. One of the two exceptions,ev-2, is found in the DNA of line 7₂ and line 100 chickens and is genetically linked to the production of RAV-O. The only other provirus (which we call B) having both theSstI sites in RAV-O DNA was seen only once in a line 100 sample. Of the nine remaining elements, six had large deletions. Three proviruses (ev-4,ev-6, and an element we call A) are missing the left 3′5′ repeat and have sustained deletions extending varying distances intogag orpol. One of these,ev-6, is associated with the gs^?chf⁺ phenotype; the phenotype can be explained from the structure of the provirus.ev-3 has both terminal repeats intact but has sustained a deletion near thegag-pol boundary. This provirus is associated with the gs⁺chf⁺ phenotype and the structure of the provirus could account for the peculiar RNA and protein associated with this phenotype. We have also found two elements which apparently consist of sequences present only in the 3′5′ terminal repeat unit, with no other associated virus specific sequences. Such structures might arise by homologous recombination between the terminal repeats of a normal provirus.     

Mutants of Rous sarcoma virus with extensive deletions of the viral genome.

Deletion mutants of Rous sarcoma virus (RSV) have been isolated from a stock of Prague RSV which had been irradiated with ultraviolet light. Quail fibroblasts were infected with irradiated virus and transformed clones isolated by agar suspension culture. Three clones were obtained which did not release any virus particles. Analysis of DNA from these non-producer clones with restriction endonucleases and the Southern DNA transfer technique indicated that the clones carry defective proviruses with deletions of approximately 4 × 10⁶ daltons of proviral DNA. The defective proviruses, which retain the viral transformation (src) gene, contain only 1.7–2.0 × 10⁶ daltons of DNA. Multiple species of viral RNA containing the sequences of the src gene were detected in these clones; some of these RNAs may contain both viral and cellular sequences. The protein product of the src gene, p60^src (Brugge and Erikson, 1977), was also synthesized in the nonproducer clones. However these clones did not contain the products of the group-specific antigen (gag), DNA polymerase (pol), or envelope glycoprotein (env) genes, nor did they contain the 35 and 28 S RNA species which are believed to represent the messengers for these viral gene-products. The properties of these mutants indicate that expression of the src gene is sufficient to induce transformation. These clones may represent useful tools for the study of the expression of this region of the genome.     

Esh avian sarcoma virus codes for a gag-linked transformation-specific protein with an associated protein kinase activity

Esh sarcoma virus, initially isolated from a spontaneous tumor of a chicken, transforms fibroblasts in vitro and induces fibrosarcomas in vivo. It is defective for replication, and infectious viral stocks consist of a mixture of a sarcomagenic virus (ESV) and an a avian leukosis virus of subgroup A (EAV) which serves as helper. Cloned stocks of infectious ESV contain two RNA components of M_r, 3 and 1.5 × 10⁶, respectively, as determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The component of M_r 1.5 × 10⁶ appears to be the genome of defective ESV, since it is not detected in preparations of the helper virus EAV. The size of the ESV genome suggests major deletions of replicative genes, and ESV-transformed nonproducer cells fail to express functional translation products of the gag, pol, and env genes. ESV-transformed producer and nonproducer clones also do not express pp60^src but contain a gag-related protein of M_r 80,000 (p80). Two-dimensional analyses of the [³⁵S]methionine-labeled tryptic peptides of p80 indicate that this protein contains part of the sequences of gag-p19 covalently linked to additional sequences unrelated to gag, pol, and env gene products. These ESV-specific sequences are also unrelated to pp60^src and to gag-linked polyproteins found in cells transformed by defective avian sarcoma viruses PRCII and Fujinami or defective leukemia viruses AEV, MC29, and MH2. P80 is phosphorylated in vivo at two major sites, one involving phosphoserine and the other phosphotyrosine residues. Immunoprecipitates containing ESV-p80 are associated with a protein kinase activity that is specific for tyrosine residues of several acceptor molecules including p80 itself, rabbit immunoglobulin H chain of the immune complex and exogenously added α-casein. p80 is phosphorylated in vitro at the same tyrosine site as in vivo suggesting that the enzyme activity detected in vitro is of physiological significance. The p80-associated protein kinase activity is strictly dependent on the presence of Mg²⁺ or Mn²⁺ but was found independent of known effectors of cellular protein kinases Ca²⁺, cAMP, or cGMP.     

Isolation of three new avian sarcoma viruses: ASV 9, ASV 17, and ASV 25

The newly isolated avian sarcoma viruses, ASV 9, 17, and 25, cause fibrosarcomas in young chickens and induce foci of transformed cells in chick embryo fibroblast cultures. They are defective in replication and belong to envelope subgroup A. The sizes of their genomes are 6 kb (ASV 9), 5 kb (ASV 17), and 6 kb (ASV 25), respectively. All three contain long terminal repeat (LTR) and gag sequences but lack pol. env is absent from ASV 9 and ASV 25, but some env sequences are detectable in ASV 17. None of the defective viral genomes hybridized to selected onc probes representing src, fps, yes, myc, myb, and erb A. erb B appears absent from ASV 9 and ASV 17, but some hybridization between the erb B probe and the RNA of ASV 25 was detected. ASV 9 codes for a transformation-specific gag-linked protein of 130kDa. Multiple gag-linked transformation-specific proteins are seen in ASV 17 and 25; they require further study.     

A nonconditional replication-defective mutant of the Schmidt-Ruppin strain of Rous sarcoma virus.

A nonconditional mutant of the Schmidt-Ruppin strain of Rous sarcoma virus (subgroup A) that fails to synthesize infectious progeny virus is described. Nonproducing cells transformed by this mutant LA7365 have functional viral src and env genes. The gag gene of LA7365 is defective. There is no complementation with a gag ts mutant, and pulse-chase experiments show that the proteolytic processing of the gag precursor pr76 is strongly inhibited in mutant-infected cells. Whether there is an additional lesion in the pol gene of LA 7365 could not be determined from the available data.     

Size and genetic content of virus-specific RNA in myeloblasts transformed by Avian Myeloblastosis Virus (AMV)

Radiolabeled complementary DNA probes representing sequences specific for avian myeloblastosis virus (AMV) and sequences of different regions of avian leukosis virus (ALV) genome were used to analyze the gene content and size distribution of viral-specific RNA in AMV-transformed myeloblasts. The producer myeloblasts contained 8–10 times more copies per cell of virus-specific RNA than those of nonproducer (NP) myeloblasts. In the producer myeloblast, 35 S, 34 S, and 21 S virus-specific RNAs were detected. The 35 S RNA contained gag, pol, env, and c sequences; representing both the genomic and the messenger RNA of the helper virus. The 34 S RNA contained gag, pol, AMV, and c sequences, representing the genomic and the messenger RNA of AMV. The 21 S RNA of producer cells contained two different species: one contained env and c sequences and represented the glycoprotein messenger RNA of the helper virus; another contained AMV and c sequences and represented the subgenomic messenger RNA of AMV. In NP cells, two virus-specific RNAs were found; a 34 S RNA containing gag, pol, AMV, and c sequences and a 21 S RNA containing AMV and c sequences. Both RNAs hybridized to a probe specific for 5′ sequences of ALV. About 15% of pol sequences of virus-specific RNA in NP cells were found to be deleted.     

Genome structure of the defective avian sarcoma virus PRCIV

The genome of PRCIV, a defective avian sarcoma virus of class II, was studied by nucleic acid hybridization and oligonucleotide mapping. It has a length of about 6.1 kilobases (kb). A stretch of retrovirus replicative sequences encompassing part of the gag, all of the pol, and most of the env region, in all about 5 kb, is deleted. In its place a 2.9 kb substitution that is transformation-specific for PRCIV was found. Flanking the PRCIV-specific substitution are sequences shared with the helper virus, a stretch of 2.0 kb on the 5′-side, including part of the gag region, and a stretch of up to 1 kb on the 3′-side, including the C-region and possibly some env sequences. A PRCIV-specific cDNA probe representing the transformation-specific sequences was prepared. This probe hybridizes only with RNAs of other class II sarcoma viruses but fails to hybridize with either class I or class III sarcoma viruses.     

Restriction endonuclease mapping of the DNA of Rous-associated virus O reveals extensive homology in structure and sequence with avian sarcoma virus DNA

We have prepared a physical map of the DNA of Rous-associated virus O (RAV-O), an endogenous virus released by certain chicken lines, in order to examine the relationship between the genome of this virus and closely related proviruses endogenous to chickens. Nineteen recognition sties for 11 restriction endonucleases have been mapped on the unintegrated linear and circular forms of RAV-O DNA isolated from acutely infected quail cells.PvuI is the only enzyme tested which does not cleave RAV-O DNA. Most of the sites (18 of 19) occur at similar or identical positions in the DNA of avian sarcoma virus (ASV). Significant differences between the maps of corrseponding regions of RAV-O and ASV DNA are observed only with those endonucleases which recognize sites encoded near the 3′ terminus of ASV RNA (PvuI andEcoRI). Both ends of RAV-O linear DNA contain sequences copied from both the 3′ and the 5′ ends of viral RNA. Two species of closed circular DNA were found: one the same size as the terminally redundant linear DNA (5.0 × 10⁶M_r and the other lacking ca. 0.35 kb from an end of linear DNA. Thus the unintegrated forms of RAV-O DNA appear structurally similar to those of ASV DNA[Shank, P. R.,et al. (1978b).Cell15, 1383–1395;Hsu, W.,et al. (1978).J. Virol.28, 810–818]; presumably one copy of a ca. 0.35-kb terminal repeat unit is lost during formation of the smaller circle from linear DNA. The following paper illustrates the utility of the physical map for differentiating between endogenous proviruses which might or might not have sequence identity with the RAV-O genome.     

Detection and enumeration of transformation-defective strains of avian sarcoma virus with molecular hybridization.

Serial propagation of avian sarcoma viruses generates deletions in the viral gene responsible for cellular transformation (src). We have devised an assay for these deletion mutants which utilizes molecular hybridization and exploits the availability of DNA (cDNA_sarc) complementary to the nucleotide sequences affected by the deletion in src. Our procedure is also applicable to deletions in other viral genes and offers several advantages over conventional bioassays for the deletion mutants; moreover, it can be used to detect deletions in virus-specific intracellular nucleic acids. In order to illustrate the utility of the assay, we demonstrate that all 20 copies of the proviral DNA for avian sarcoma viruses in XC cells contain src, and we show that single avian cells can contain functioning proviruses for both avian sarcoma virus and a congenic deletion mutant. It should now be possible to use molecular hybridization to study the mechanism by which deletions in src are generated.     

Measurement of endogenous leukosis virus nucleotide sequences in the DNA of normal avian embryos by RNA-DNA hybridization

Rous associated virus type O (RAV-O) is a subgroup E avian leukosis virus selected as an example of “endogenous” leukosis viruses which are associated with normal avian embryo cells. DNA sequences complementary to RAV-O 60–70S RNA genome were detected and quantitated in DNA from normal avian embryos by the technique of RNA-DNA hybridization with DNA excess. DNA from “leukosis free” white leghorn chicken embryos, as well as from line 7 chicken embryos, contained sequences related to at least 70%, if not all, of RAV-O RNA sequences. A tentative estimate of gene frequency is low, in the range of two to four copies of RAV-O per cell genome. Some difficulties in precisely calculating gene frequency by this method are discussed. Embryos of ring-necked pheasant contain only about 10% of the RAV-O genome. These findings are not affected by the presence or absence of viral group specific (gs) antigen in chicken or pheasant embryos. Embryos of Japanese quail, a species which lacks a known leukosis virus and does not synthesize avian gs antigen, contained DNA sequences specifically related to no more than 4% of the RAV-O genome. The data document the specificity and sensitivity of this hybridization technique and provide additional firm support for the proposition that normal avian cells carry endogenous avian leukosis virus in the form of DNA proviruses.     

Recombinant avian oncoviruses. I. Alterations in the precursor to the internal structural proteins.

The synthesis of the gag precursor protein (Pr76) was studied in a number of recombinant avian oncoviruses, which were selected for recombination between the env and pol genes or the env and src genes. Such studies show that the electrophoretic mobility of the gag precursor protein of recombinant viruses (ΔPr76) was greater than that of the parental gene product (Pr76) in 16 of 24 cases. Viruses derived from recombination between endogenous (RAV-0) and exogenous viruses (RSV), as well as between two exogenous viruses, showed the ΔPr76 phenotype. In an mRNA-dependent rabbit reticulocyte translation system, 35 S RNA isolated from PR-RSV-C directed the synthesis of Pr76, while RNA isolated from a recombinant between PR-RSV-C and RAV-0 directed the synthesis of ΔPr76. These observations show that the synthesis of ΔPr76 is due to an alteration in the genome related to recombination. An analysis of the RNase T₁-resistant oligonucleotides demonstrated a crossover near the 5′ end of the genome (which may be within the gag gene) in two recombinant virus clones which synthesize ΔPr76 in infected cells; but no crossover was detected near the 5′ end of the genome in a third recombinant virus clone which synthesizes Pr76 in infected cells. Our data suggest that the synthesis of ΔPr76 is a consequence of recombination near the 5′ end of the genome.     

RNA tumor viruses of pheasants: Characterization of avian leukosis subgroups F and G

Endogenous leukosis-like viruses of ring-necked pheasants (Phasianus colchicus) and golden pheasants (Chrysolophus pictus) have been isolated and characterized. The majority of the normal pheasant embryo cultures contain helper activity for the defective Bryan high titer strain of Rous sarcoma virus. The Rous sarcoma pseudotypes produced with endogenous helper activity from ring-necked pheasants belong to subgroup F. The pseudotypes from golden pheasant cells constitute subgroup G. Subgroup F and G pseudotypes can infect all known genetic types of chicken fibroblasts as well as pheasant and Japanese quail cells, but do not plate on goose cells. Duck cells are resistant to subgroup G but not to F.The subgroup F and G helper viruses isolated from Rous sarcoma viral pseudotypes show interference with their homologous subgroup. RAV-61, a standard of subgroup F, interferes with pseudotypes produced with endogenous helper activity from ring-necked pheasant cells but not with subgroup G pseudotypes.Subgroups F and G do not cross-react with subgroup A to E in neutralization tests. Some normal ring-necked pheasant sera have anti-F activity.Subgroup F and probably also G leukosis-like viruses can undergo genetic recombination with nondefective avian sarcoma viruses.     

Avian oncovirus MH2 is defective in Gag, Pol, and Env.

The defectiveness of avian oncovirus MH2 is characterized further by genetic experiments and by tryptic peptide mapping. The results show that MH2 lacks full function of all three viral genes necessary for replication, namely, gag, env, and pol. The polyprotein MH2 p100 made in MH2 nonproducer cells contains the tryptic peptides of gag proteins p19 and p27.     

Translation of avian type C virus messenger RNA: evidence for multiple initiation sites.

The avian sarcoma virus genome has been shown to consist of four genes arranged in the following order: 5′-gag pol-env-src-3′. In the present study, the pol gene product was found to be expressed in avian tumor virus-infected cells at several-hundred-fold lower levels than the translational products of the gag and env genes. Moreover, a translational block or possible deletion involving the carboxy-terminal region of the gag gene in chicken embryo cells had no apparent influence on the level of expression of the env gene. These findings indicate the involvement of multiple initiation sites for translation of the avian type C viral genome.     

New endogenous proviruses in the genome of brown Leghorn chickens

Genetic differences and differences in localization of endogenous proviruses in chicken genomes were found in studies on endogenous provirus sequences in DNA of white and brown Leghorn chickens. In brown Leghorn DNA, proviruses both similar with Raus-associated virus (endogenous virus of white Leghorns) and differing from it in the pattern of hydrolysis by EcoRI restricting endonuclease were found, the latter occurring more frequently. Different sets of variously localized proviruses were found in DNA of individual chickens; none of the proviruses was found in DNA of all brown Leghorns examined. No single provirus common for chickens of the two species was found. The genetic diversity of endogenous proviruses observed in brown Leghorns indicates the possibility of coexistence in the chicken DNA of message on several significantly different endogenous viruses.     

New Simian β Retroviruses from Rhesus Monkeys (Macaca Mulatta) and Langurs (Semnopithecus Entellus) from Rajasthan, India

Natural infection of feral Indian rhesus monkeys (Macaca mulatta) by a new simian β retrovirus, provisionally called simian retrovirus-7 (SRV-7) is described. The virus is capable of in vitro replication in primary human peripheral blood lymphocytes (PBL) and B and T cell lines. We have earlier reported a novel SRV, SRV-6 from Indian langurs (Semnopithecus entellus). Additional sequence analyses from gp20 transmembrane (TM) env genes of SRV-6 and SRV-7 place them in a separate cluster, related to but distinct from known exogenous SRVs and also close to the simian endogenous β retrovirus, (SERV) from African baboon. Phylogenetic analyses of pol gene of SRV-7 place it closer to SERV when the stop codons of the SERV genes are removed. On the other hand, additional sequence data from gp70, surface glycoprotein (SU) region of the env gene of SRV-6 suggest it is more closely related to known exogenous SRVs, (SRV-1 to 3). It is also related to the endogenous langur virus, Po-1-Lu. We hypothesize that SRV-6 and SRV-7 probably originated from a progenitor exogenous SRV which recombined with an endogenous SERV in the TM env and pol genes during evolution, based on the phylogenetic analyses. Genbank Accession numbers of the new sequences: SRV-6 TM (gp20) env: AF401239, SRV-6 SU (gp70) env: AY598468, SRV-7 pol region: AY594212, SRV-7 TM env (gp20): AY594213.