Rous-associated Virus Research Articles

Cooperation of v-jun and v-erbB oncogenes in embryo fibroblast transformation in vitro and in vivo

Retroviral vectors carrying either the v-jun and v-erbB sequences or the v-jun gene linked to the neomycin resistance gene were constructed on the basis of the structural genome organization of avian erythroblastosis virus (AEV). These viruses, called JB and JN, respectively, were rescued as Rous-associated virus-1 pseudotypes, and they were shown to successfully transform chicken embryo fibroblasts in vitro. However, in agar, colonies developed from JB-infected fibroblasts were three to five times larger than those obtained after infection with JN or with AEV Pst124 carrying only a functional v-erbB gene. In vivo, on chorioallantoic membrane (CAM) assays, JB produced fibrosarcomas that were more rapidly growing and much larger than those induced by JN or AEV Pst124. Moreover, in chickens infected in ovo with JB, multiple fibrosarcomas arose in different organs a few days after birth, whereas no tumor could be detected in parallel experiments in either JN- or AEV Pst124-infected animals. These results demonstrate that in embryo fibroblast cells, v-jun and v-erbB can act synergistically to enhance the transformation potential of either oncogene alone both in vitro and in vivo.

A transformation-competent recombinant between v-src and Rous-associated virus RAV-1

The LTR, v-src, LTR provirus, which arose by the reverse transcription and integration of src mRNA in the H-19 hamster tumor, has been successfully rescued by fusion with chicken fibroblasts infected with Rous-associated virus RAV-1. One rescued virus, E6, acquired 1 kilobase of the 5' end of the gag gene structure. Recombination took place in the region of 15-nucleotide homology exactly between v-src exon (position 7054) and gag (position 1417). This recombination resulted in the alteration of src splice acceptor site sequences, but this site is maintained as a functional splice acceptor site. The nucleotide structure of the long terminal repeat of recombinant E6 virus suggests that it arose by the intermolecular jump of reverse transcription from RAV-1 to src mRNA and then the switch of templates between already depicted regions of homology. The second jump of reverse transcription was apparently an intramolecular event. The acquisition of 1 kilobase of the 5' gag by E6 resulted in maintaining the balance of unspliced and spliced E6 RNAs and assured the replication advantage of rescued E6 virus over rescued F6 virus, the genome of which corresponds to that present in ancestral H-19 cells.

A new avian leukosis virus-based packaging cell line that uses two separate transcomplementing helper genomes

An avian leukosis virus-based packaging cell line was constructed from the genome of the Rous-associated virus type 1. The gag, pol, and env genes were separated on two different plasmids; the packaging signal and the 3' long terminal repeat were removed. On a plasmid expressing the gag and pol genes, the env gene was replaced by the hygromycin resistance gene. The phleomycin resistance gene was inserted in the place of the gag-pol genes on a plasmid expressing the env gene. The plasmid containing the gag, pol, and Hygror genes was transfected into QT6 cells. Clones that produced high levels of p27gag were transfected with the plasmid containing the Phleor and env genes. Clones that produced high levels of env protein (as measured by an interference assay) were tested for their ability to package NeoR-expressing replication-defective vectors (TXN3'). One of the clones (Isolde) was able to transfer the Neo+ phenotype to recipient cells at a titer of 10(5) resistance focus-forming units per ml. Titers of supernatants of cells infected with Rous-associated virus type 1 prior to transfection by Neor vectors were similar. Tests for recombination events that might result in intact helper virus showed no evidence for the generation of replication-competent virus. The use of selectable genes inserted next to the viral genes to generate high-producer packaging cell lines is discussed.

Molecular and biological properties of c-mil transducing retroviruses generated during passage of Rous-associated virus type 1 in chicken neuroretina cells

IC1, IC2, and IC3 are novel c-mil transducing retroviruses generated during serial passaging of Rous-associated virus type 1 (RAV-1) in chicken embryo neuroretina cells. They were isolated by their ability to induce proliferation of these nondividing cells. IC2 and IC3 were generated during early passages of RAV-1 in neuroretina cells, whereas IC1 was isolated after six consecutive passages of virus supernatants. We sequenced the transduced genes and the mil-RAV-1 junctions of the three viruses. The 5' RAV-1-mil junction of IC2 and IC3 was formed by a splicing process between the RAV-1 leader sequence and exon 8 of the c-mil gene. The 5' end of IC1 resulted from homologous recombination between gag and mil sequences. Reconstitution experiments showed that serial passaging of IC2 in neuroretina cells also led to the formation of a gag-mil-containing retrovirus. Therefore, constitution of a U5-leader-delta c-mil-delta RAV-1-U3 virus represents early steps in c-mil transduction by RAV-1. This virus further recombined with RAV-1 to generate a gag-mil-containing virus. The three IC viruses transduced the serine/threonine kinase domain of the cellular gene. Hence, amino-terminal truncation is sufficient to activate the mitogenic property of c-mil. Comparison of the transforming properties of IC2 and IC1 showed that the transduced mil gene, expressed as a unique protein independent of gag sequences, was weakly transforming in avian cells. Acquisition of gag sequences by IC1 not only increased the rate of virus replication but also enhanced the transforming capacity of the virus.

Intrinsic properties of reverse transcriptase in reverse transcription : Associated RNase H is essentially regarded as an endonuclease

The intrinsic properties of reverse transcriptase in reverse transcription were studied using a synthetic, partial ovalbumin mRNA with a synthetic DNA oligonucleotide annealed to the 3′-end of the RNA as a model substrate (see Fig. 1). With or without concomitant cDNA synthesis, the RNase H activity of avian myeloblastosis virus (AMV)-reverse transcriptase cleaved the substrate at a site which would leave a hybrid of between 7 and 14 base pairs between the 3′ termini of the RNA and DNA oligonucleotide. Variability in the exact size of the hybrid probably reflects some weak base preference for cleavage by the enzyme. These short hybrids can be recognized as substrates by Escherichia coli RNase H and can be utilized by reverse transcriptase as sites for continuation of cDNA synthesis. Substrates with 5′-triphosphorylated termini, 3′-OH, 3′-phosphate, 3′-end hairpin structures and 20 base pair hybrids on the middle region of long RNA more than 300 bases or on circular RNA were all cleaved by AMV-reverse transcriptase-associated RNase H, indicating that the RNase H activity is essentially regarded as an endonuclease degrading RNA moiety in RNA-DNA hybrid. The modes of action of reverse transcriptase from murine leukemia virus and Rous-associated virus 2 were the same as that of AMV-reverse transcriptase, except that the size of the remaining hybrid and the specificity for cleavage depended on the reverse transcriptase. We propose a possible model to explain the mode of action of RNase H and RNA-dependent DNA polymerase activities in reverse transcription.

Rescue of infectious virus from nonproducer Rous cells by chick cellular DNA.

The rescue of infectious virus from nonproducer BH RSV(-) cells by chick cellular DNA was attempted in order to investigate the functional state of endogenous and exogenous retroviral genes integrated within the cellular DNA. No infectious virus was rescued by transfection with DNAs of chick helper factor (chf)-negative chick embryo cells (CEC), chf-positive CEC or uninfected CEC producing endogenous Rous associated virus (RAV-0). On the other hand, infectious Rous viruses with the phenotype of RAV-0 and RAV-1 were rescued by transfection with DNAs of CEC which had been infected with RAV-0 and RAV-1. From these results, it seems that exogenous retroviral genes integrated in the cellular DNA are expressed rather easily by transfection while those present endogenously are not.

Cytocidal effect caused by the envelope glycoprotein of a newly isolated avian hemangioma-inducing retrovirus

We isolated a field strain of avian hemangioma retrovirus (AHV) which induces a cytopathic effect (CPE) on cultured avian and mammalian cells shortly after infection. The kinetics of cell killing were dependent on the multiplicity of infection. The CPE on avian and mammalian cells was independent of virus replication, because UV-irradiated virus led to cell death as well. Biochemical and genetic experiments indicated that AHV env gene products were responsible for the CPE. Partially purified AHV envelope glycoproteins (gp85), but not those of the Rous sarcoma virus Prague C strain, induced a CPE. Rous-associated virus type 1, in which the env region was replaced by the AHV gp85 region, induced a CPE on avian and mammalian cultured cells. Therefore, we suggest that CPE is induced by AHV via interaction between viral gp85 and the cell membrane. This mode of CPE is unique among avian sarcoma-leukemia viruses.

Inhibition of reverse transcriptases by flavonoids

Selected naturally occurring flavonoids were shown to inhibit three reverse transcriptases (RT): avian myeloblastosis (AMV) RT, Rous-associated virus-2 (RAV-2) RT and Maloney murine leukemia virus (MMLV) RT when poly (rA)oligo(dT) 12–18 or rabbit globin mRNA were used as template. Amentoflavone, scutellarein and quercetin were the most active compounds and their effect was concentration-dependent. The enzymes exhibited differential sensitivity to the inhibitory effects of the flavonoids. The compounds also inhibited rabbit globin mRNA-directed, MMLV RT-catalyzed DNA synthesis. Amentoflavone and scutellarein inhibited ongoing new DNA synthesis catalyzed by RAV-2 RT. Kinetic studies were performed in an attempt to elucidate the mechanism of action of amentoflavone and scutellarein. A model is proposed for the mechanism of action of the flavonoids on RT activity.

Polyadenylation at correct sites in genome RNA is not required for retrovirus replication or genome encapsidation

RNA transcripts polyadenylated at sites derived from flanking cellular DNA (readthrough transcripts) make up about 15% of the viral RNA in cells infected with avian leukosis virus. To test the functionality of such transcripts, a virus was created by introducing two mutations into the AAUAAA polyadenylation signal of Rous-associated virus 1, converting it to AAGGAA. The replication of this virus was not greatly affected at any level. However, less than 1% of viral transcripts produced during mutant virus replication were cleaved and polyadenylated at the correct site within viral long terminal repeat-related sequence. These results imply that readthrough transcripts, which are produced during normal viral replication, are polyadenylated and packaged into virions as normal transcripts and can serve as RNA genomes in the next round of replication. These results show that polyadenylation within virus-related sequences is not a necessary requirement for virus replication and that readthrough transcripts have the necessary properties to be intermediates in the process of transduction of cellular sequences.

Influence of avian leukosis viral sequences on transmission to the egg and embryo

Rous associated virus type-0 (RAV-0), a subgroup E replication-competent endogenous virus of chickens, is associated with a low efficiency of virus shedding into the egg albumen and failure to establish congenital transmission. In contrast, RAV-1, a subgroup A virus of exogenous origin, is efficiently shed into the albumen and readily infects the embryo. Among a series of in vitro constructed recombinants between RAV-0 and RAV-1, we have identified subgroup E recombinants that efficiently shed virus into the egg albumen but do not undergo efficient congenital transmission. The LTR region, subgroup-determining sequences in env, and sequences within a 375 by Sacl- Xhol fragment at the 5′ end of the genome each influenced the efficiency of virus shedding into the albumen. Egg inoculations with viruses differing only in env were used to confirm the low rate of congenital transmission of subgroup E viruses. These studies revealed that subgroup A envelope antigens are at least 100-fold more effective for the establishment of embryonic infection than subgroup E.

Avian retroviral long terminal repeats bind CCAAT/enhancer-binding protein.

DNA-protein interactions involving enhancer and promoter sequences within the U3 regions of several avian retroviral long terminal repeats (LTRs) were studied by DNase I footprinting. The rat CCAAT/enhancer-binding protein, C/EBP, bound to all four viral LTRs examined. The Rous sarcoma virus binding site corresponded closely to the 5' limit of the LTR enhancer; nucleotides -225 to -188 were protected as a pair of adjacent binding domains. The Fujinami sarcoma virus LTR bound C/EBP at a single site at nucleotides -213 to -195. C/EBP also bound to the promoter region of the enhancerless Rous-associated virus-0 LTR at nucleotides -77 to -57. The avian myeloblastosis virus LTR bound C/EBP at three sites: nucleotides -262 to -246, -154 to -134, and -55 to -39. We have previously observed binding of C/EBP to an enhancer in the gag gene of avian retroviruses. A heat-treated nuclear extract from chicken liver bound to all of the same retroviral sequences as did C/EBP. Alignment of the avian retroviral binding sequences with the published binding sites for C/EBP in two CCAAT boxes and in the simian virus 40, polyoma, and murine sarcoma virus enhancers suggested TTGNNGCTAATG as a consensus sequence for binding of C/EBP. When two bases of this consensus sequence were altered by site-specific mutagenesis of the Rous sarcoma virus LTR, binding of the heat-stable chicken protein was eliminated.

Avian retroviral long terminal repeats bind CCAAT/enhancer-binding protein.

DNA-protein interactions involving enhancer and promoter sequences within the U3 regions of several avian retroviral long terminal repeats (LTRs) were studied by DNase I footprinting. The rat CCAAT/enhancer-binding protein, C/EBP, bound to all four viral LTRs examined. The Rous sarcoma virus binding site corresponded closely to the 5' limit of the LTR enhancer; nucleotides -225 to -188 were protected as a pair of adjacent binding domains. The Fujinami sarcoma virus LTR bound C/EBP at a single site at nucleotides -213 to -195. C/EBP also bound to the promoter region of the enhancerless Rous-associated virus-0 LTR at nucleotides -77 to -57. The avian myeloblastosis virus LTR bound C/EBP at three sites: nucleotides -262 to -246, -154 to -134, and -55 to -39. We have previously observed binding of C/EBP to an enhancer in the gag gene of avian retroviruses. A heat-treated nuclear extract from chicken liver bound to all of the same retroviral sequences as did C/EBP. Alignment of the avian retroviral binding sequences with the published binding sites for C/EBP in two CCAAT boxes and in the simian virus 40, polyoma, and murine sarcoma virus enhancers suggested TTGNNGCTAATG as a consensus sequence for binding of C/EBP. When two bases of this consensus sequence were altered by site-specific mutagenesis of the Rous sarcoma virus LTR, binding of the heat-stable chicken protein was eliminated.

Generation of a helper cell line for packaging avian leukosis virus-based vectors

We constructed an avian leukosis virus-based packaging cell line, pHF-g, containing Rous-associated virus DNA with several alterations to abolish RNA packaging. One of them is a 52-base-pair deletion encompassing the putative encapsidation signal in the leader region. The 3' long terminal repeat was also removed and replaced by the polyadenylation sequence from the herpes simplex virus thymidine kinase gene. When pHF-g cells were transfected by an avian leukosis virus-based vector, they produced replication-defective virus at high titer but they did not release any replication-competent particles. Proviral DNA was shown to be correctly integrated as well as correctly expressed. Viral RNAs were shown to be correctly translated into gag-related polypeptides.

Helper-independent retrovirus vectors with Rous-associated virus type O long terminal repeats

We have constructed nonpermuted replication-competent avian retrovirus vectors that derive from Rous sarcoma virus (S. H. Hughes, J. J. Greenhouse, C. J. Petropoulos, and P. Sutrave, J. Virol. 61:3004-3012, 1987). We describe here the construction and properties of corresponding vectors in which the long terminal repeats (LTRs) of the parental virus have been replaced by the LTRs of the endogenous chicken virus Rous-associated virus type O. The Rous-associated virus type O LTR vectors replicated approximately 1/10 as well as the parental vectors and expressed a test gene, chloramphenicol acetyltransferase, approximately 1/30 to 1/50 as well.

Activation and transduction of c-mil sequences in chicken neuroretina cells induced to proliferate by infection with avian lymphomatosis virus.

We report that nondividing neuroretina cells from chicken embryos can be induced to proliferate following infection with Rous-associated virus type 1 (RAV-1), an avian lymphomatosis retrovirus lacking transforming genes. Multiplication of RAV-1-infected neuroretina cells is observed after a long latency period and takes place initially in a small number of cells. We also show that serial virus passaging onto fresh neuroretina cultures leads to the generation of novel mitogenic viruses containing the mil oncogene. DNA analysis indicated that RAV-1 is the only provirus detected in cells infected at virus passage 1, whereas neuroretina cells infected at subsequent virus passages harbor mil-containing proviruses. Three viruses, designated IC1, IC2, and IC3, were molecularly cloned. Restriction mapping indicated that in each virus, truncated c-mil sequences were inserted within different portions of the RAV-1 genome. In addition, IC1 and IC2 viruses have transduced novel sequences that belong to the 3' noncoding portion of the c-mil locus. All three viruses induce neuroretina cell multiplication and direct the synthesis of mil-specific proteins. Proliferation of neuroretina cells infected at passage 1 of RAV-1 was not associated with any detectable rearrangement of c-mil, when a v-mil probe was used. However, these cells expressed high levels of an aberrant 2.8-kilobase mRNA hybridizing to mil but not to a long terminal repeat probe. Therefore, transcriptional activation of a portion of c-mil could represent the initial events induced by RAV-1 infection and lead to retroviral transduction of activated c-mil sequences.

A novel oncogene related to c-mil is transduced in chicken neuroretina cells induced to proliferate by infection with an avian lymphomatosis virus.

Non-dividing neuroretina cells from chicken embryos are induced to proliferate after a long latency, following infection with Rous associated virus type 1, an avian retrovirus which does not carry a transforming gene. We have isolated from these proliferating cells an acutely mitogenic retrovirus, designated IC10, which contains a novel oncogene. Nucleotide sequencing showed that the IC10 virus has transduced 1101 nucleotides of cellular origin inserted between the gag and env genes of RAV-1. This oncogene, designated v-Rmil, is 70.1% homologous to v-mil. v-Rmil encodes a protein of 40,976 daltons sharing 83.8% homology with the catalytic domain of the v-mil protein. Divergence with the v-mil gene product is observed at the NH2- and COOH-terminal portions of the v-Rmil protein. Restriction analysis of normal chicken DNA indicated that v-Rmil is derived from a cellular gene distinct from c-mil. The c-Rmil gene is transcribed through a major mRNA, greater than 10 kb in length, that is detected at much higher levels in neuroretinas, as compared to other embryonic tissues.

Long terminal repeat (LTR) sequences, env, and a region near the 5' LTR influence the pathogenic potential of recombinants between Rous-associated virus types 0 and 1.

A series of recombinants between Rous-associated virus type 0 (RAV-0), RAV-1, and a replication-competent avian leukosis virus vector (RCAN) have been tested for disease potential in day-old inoculated K28 chicks. RAV-0 is a benign virus, whereas RAV-1 and RCAN induce lymphoma and a low incidence of a variety of other neoplasms. The results of the oncogenicity tests indicate that (i) the long terminal repeat regions of RAV-1 and RCAN play a major role in disease potential, (ii) subgroup A envelope glycoproteins are associated with a two- to fourfold higher incidence of lymphoma than subgroup E glycoproteins, and (iii) certain combinations of 5' viral and env sequences cause osteopetrosis in a highly context-dependent manner. Long terminal repeat and env sequences appeared to influence lymphomogenic potential by determining the extent of bursal infection within the first 2 to 3 weeks of life. This would suggest that bursal but not postbursal stem cells are targets for avian leukosis virus-induced lymphomogenesis. The induction of neutralizing antibody had no obvious influence on the incidence of lymphoma.

Isolation of a variant endogenous avian leukosis virus: non-productive exogenous infection with endogenous viruses containing p27 and p27(0).

Ten replication-competent endogenous avian leukosis viruses (ALVs) with subgroup E specificity were isolated from six commercial chicken lines of mixed breed. All the ALVs isolated belonged to a variant class of endogenous viruses with a major group-specific antigen, p27, identical in size to that of exogenous viruses and different from the p27 degrees of endogenous viruses isolated from White Leghorn chickens. It was possible to infect chickens exogenously with endogenous viruses (containing p27) and with Rous-associated virus type 0 (containing p27 degrees) but infection was non-productive. Congenital transmission and shedding did not occur in chickens infected with endogenous viruses containing p27 degrees or in chickens infected with endogenous viruses containing p27. Restriction of endogenous virus replication in some tissues (heart, spleen and meconia) of viraemic chickens was observed suggesting that exogenous infection of chickens with endogenous viruses differs from an infection with exogenous ALVs.

Association of the Slow Feathering (K) and an Endogenous Viral (ev 21) Gene on the Z Chromosome of Chickens

A dominant sex-linked gene, K, regulates slow feathering (SF), whereas a recessive allele, k+, determines rapid feathering (RF) in chickens. This trait provides a convenient and inexpensive approach to gender identification of White Leghorn (WL) chicks at hatch, i.e., in a sex-linked mating using k+/k+ males mated with K/– females, the K/k+ male chicks are SF, and the k+/– females are RF. However, in many WL strains, female progeny of SF dams produce fewer eggs and have higher mortality than progeny of RF dams. This loss in productivity has been attributed to higher infection and shedding rates for leukosis viruses (ALV) in SF than in RF dam lines. Because infectious endogenous viruses (EV) can induce immunological tolerance to ALV, we examined the expression and distribution of ev genes in SF and RF siblings from heterozygous K/k+ sires and k+/– dams.Infectious ALV and EV were detected by cocultivation of frozen heparinized blood cells on selected chick embryo fibroblasts and culture supernatants were tested for viral antigen by enzyme-linked immunosorbent assay tests. Specific ev genes were identified as restriction fragment length polymorphisms after hybridization with a recombinant plasmid containing the complete genome of a Rous-associated virus. It was concluded that ev 21 and K genes are tightly linked because, in different WL crosses, all SF chicks inherited ev21 but RF siblings uniformly lacked ev 21. Alternatively, the K gene in WL may be a mutation resulting from the insertion of ev 21 in the k+ gene. The SF chicks which harbor ev 21 expressed infectious EV21; evidence that EV21 may influence susceptibility to ALV is presented.

Role of RAV-0 genes in the permissive replication of subgroup E avian leukosis viruses on line 15 Bev1 CEF

Rous associated virus type-0 (RAV-0) is a replication-competent endogenous virus of chickens which grows more efficiently on chick embryo fibroblasts (CEFs) from line 15 B chickens than on CEFs from line K28. Differences in viral growth on these two sources of cells have been attributed to an early event in the retrovirus life-cycle, at or before viral DNA synthesis. Five in vitro constructed avian leukosis viruses (ALVs) as well as RAV-0 (a subgroup E ALV), RAV-1 (subgroup A), and RAV-2 (subgroup B) have been assessed for their relative growth on 15 B ev1 and K28 CEFs. More efficient replication on 15 B ev1 CEFs than on K28 CEFs was determined by subgroup E-encoding sequences in env. Subgroup A and B envelope sequences as well as viral LTR, gag, and pol sequences did not obviously bias relative rates of viral replication on the two cell types. We suggest that the unusually permissive replication of subgroup E viruses on 15 B CEFs is a receptor-mediated phenomenon and that the line 15 B receptor for subgroup E ALVs is more efficient than that of line K28.