Sequence of the Long Terminal Repeat and Adjacent Segments of the Endogenous Avian Virus Rous-Associated Virus 0

Rous-associated virus 0 (RAV-0), an endogenous chicken virus, does not cause disease when inoculated into susceptible domestic chickens. An infectious unintegrated circular RAV-0 DNA was molecularly cloned, and the sequence of the long terminal repeat (LTR) and adjacent segments was determined. The sequence of the LTR was found to be very similar to that of replication-defective endogenous virus EV-1. Like the EV-1 LTR, the RAV-0 LTR is smaller (278 base pairs instead of 330) than the LTRs of the oncogenic members of the avian sarcoma virus-avian leukosis virus group. There is, however, significant homology. The most striking differences are in the U(3) region of the LTR, and in this region there are a series of small segments present in the oncogenic viruses which are absent in RAV-0. These differences in the U(3) region of the LTR could account for the differences in the oncogenic potential of RAV-0 and the avian leukosis viruses. I also compared the regions adjacent to the RAV-0 LTR with the available avian sarcoma virus sequences. A segment of approximately 200 bases to the right of the LTR (toward gag) is almost identical in RAV-0 and the Prague C strain of Rous sarcoma virus. The segment of RAV-0 which lies between the end of the env gene and U(3) is approximately 190 bases in length. Essentially this entire segment is present between env and src in the Schmidt-Ruppin A strain of Rous sarcoma virus. Most of this segment is also present between env and src in Prague C; however, in Prague C there is an apparent deletion of 40 bases in the region adjacent to env. In Schmidt-Ruppin A, but not in Prague C, about half of this segment is also present between src and the LTR. This arrangement has implications for the mechanism by which src was acquired. The region which encoded the gp37 portion of env appears to be very similar in RAV-0 and the Rous sarcoma viruses. However, differences at the very end of env imply that the carboxy termini of RAV-0, Schmidt-Ruppin A, and Prague C gp37s are significantly different. The implications of these observations are considered.     

Structural protein markers in the avian oncoviruses.

The proteins of purified avian oncoviruses were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and isoelectric focusing. Certain members of the avian leukosis-sarcoma viruses (ALSV) had group-specific antigens with altered electrophoretic properties. (i) The p27 protein of Rous-associated virus 0 (RAV-0) had a lower electrophoretic mobility in SDS gels and a lower isoelectric point than the p27 of other ALSV. (ii) The p19 proteins of RAV-1, RAV-2, and the Bryan high-titer strain of Rous sarcoma virus had higher mobilities in SDS gels than did the corresponding protein of other viruses. This altered electrophoretic mobility was correlated with specific differences in the tryptic peptides of radioiodinated p19s. (iii) The p15 protein of RAV-7 had a lower mobility in SDS gels than did the p15 of other ALSV. These markers were used in a study of the structural proteins of subgroup E RAV-60 produced after infection of chicken embryo cells by exogenous ALSV. Although exogenous group-specific protein markers could often be identified in the subgroup E isolates, one RAV-60 had a p27 that comigrated with the p27 of RAV-0. The p19s of two other RAV-60 isolates had electrophoretic properties that were different than those of p19s from either RAV-0 or the exogenous viruses. These results support the hypothesis that RAV-60 is generated by recombination between endogenous and exogenous oncoviruses and indicate that at least the p27 encoded by RAV-0 is closely related to a protein specified by endogenous viral information in chicken cells.     

Chicken leukosis virus genome sequences in DNA from normal chick cells and virus-induced bursal lymphomas.

Genome sequences of two recent field isolates of avian leukosis viruses in the DNA of normal and neoplastic chicken cells were studied by DNA-RNA hybridization under conditions of DNA excess. Comparisons were made between 60-70S RNA from these viruses and that of a chicken endogenous type C virus (RAV-0), and of a series of "laboratory" leukosis and sarcoma viruses, by competitive hybridization analysis. A minimum of 18% of the genome sequences of both ALV isolates detected in DNA from lymphomas they induced were not detected in normal chicken DNA. The vast majority of the fraction of RNA sequences from ALV which do form hybrids with normal chick DNA appear to be reacting with the endogenous provirus of RAV-0. The genomic representation of a variety of avian leukosis and sarcoma viruses in normal chicken cells could not be distinguished by these methods (except that 13% of the RAV-0 genome was not shared with any of the other viruses). In contrast, the portion of the ALV genome exogenous to the normal chicken geome showed significant divergence from that of two sarcoma viruses (Pr RSV-C and B-77). The increased hybridization of ALV RNA with lymphoma DNA was used to detect the appearance of ALV specific sequences in the bursa of Fabricius following infection.increased hybridization was correlated with both the time after infection and the extent of replacement of the bursa by lymphoma. About one half of the increase in hybridization preceded histologic evidence of transformation.     

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.     

Embryonic infection with the endogenous avian leukosis virus Rous-associated virus-0 alters responses to exogenous avian leukosis virus infection.

We inoculated susceptible chicken embryos with the endogenous avian leukosis virus Rous-associated virus-0 (RAV-0) on day 6 of incubation. At 1 week after hatching, RAV-0-infected and control chickens were inoculated with either RAV-1 or RAV-2, exogenous viruses belonging to subgroups A and B, respectively. The chickens injected with RAV-0 as embryos remained viremic with exogenous virus longer and either failed to develop type-specific humoral immunity to exogenous virus or developed it later than the control chickens not inoculated with RAV-0. The RAV-0-injected chickens also developed neoplasms at a much higher frequency than did the control chickens. We suggest that the lower immune responses of the RAV-0-injected chickens were due to an immunological tolerance to envelope group-specific glycoproteins shared among endogenous and exogenous viruses.     

Genetic determinants of neoplastic diseases induced by a subgroup F avian leukosis virus.

Two subgroup F avian leukosis viruses, ring-necked pheasant virus (RPV) and RAV-61, were previously shown to induce a high incidence of a fatal proliferative disorder in the lungs of infected chickens. These lung lesions, termed angiosarcomas, appear rapidly (4 to 5 weeks after infection), show no evidence of proto-oncogene activation by proviral integration, and are not induced by avian leukosis viruses belonging to other subgroups. To identify the viral sequences responsible for induction of these tumors, we constructed recombinant viruses by exchanging genomic segments of molecularly cloned RPV with those of a subgroup A leukosis virus, UR2AV. The ability to induce rapid lung tumors segregated only with the env sequences of RPV; the long terminal repeat of RPV was not required. However, recombinants carrying both env and long terminal repeat sequences of RPV induced lung tumors with a shorter latency. In several cases, recombinant viruses exhibited pathogenic properties differing from those of either parental virus. Recombinants carrying the gag-pol region of RPV and the env gene of UR2AV induced a high incidence of a muscle lesion termed infiltrative intramuscular fibromatosis. One recombinant, EU-8, which carries the gag-pol and LTR sequences of RPV, and the env gene of UR2AV, induced lymphoid leukosis after an unusually short latent period. The median time of death from lymphoid leukosis was 6 to 7 weeks after infection with EU-8 compared with approximately 5 months for UR2AV.     

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.     

Component of Strain MC29 Avian Leukosis Virus with the Property of Defectiveness

Three clones of morphologically altered cells (L(-)MC29) of singular properties were isolated from MC29 (subgroup A) leukosis virus-infected chick embryo cells. Supernatant fluids from cultures of the cloned cells produced no transforming or interfering activity on chick embryo cells susceptible to known avian leukosis-sarcoma viruses. No virus associated with the cells was demonstrable by fluorescent-antibody staining or by electron microscopy. All L(-)MC29 clone cells were activated, however, by four strains of Rous-associated viruses (RAV) representative of A, B, C, and D subgroup avian leukosis viruses and by two strains of MC29 virus. Virus L(-)MC29 cells activated by superinfection with RAV-1 and RAV-2 was characterized by helper-dependent and helper-independent properties. These findings suggest that the strain MC29 leukosis virus, or a component thereof, possesses properties of defectiveness similar to those of the Bryan high-titer Rous sarcoma virus.     

Sequences outside of the long terminal repeat determine the lymphomogenic potential of Rous-associated virus type 1.

Recombinant avian leukosis viruses have been constructed from the molecularly cloned DNAs of Rous-associated virus type 1 (RAV-1) and Rous-associated virus type 0(RAV-0). Virus encoded by the cloned RAV-1 DNA induced a high incidence of B-cell lymphoma and a moderate incidence of a variety of other neoplasms. Virus encoded by the cloned RAV-0 DNA did not cause disease. Virus recovered from DNA constructions that encoded the gag, pol, and 5' env sequences of RAV-0 and the 3' env and long terminal repeat sequences of RAV-1 did not cause a high incidence of lymphoma. Rather, these constructed viruses induced a low incidence of a variety of neoplasms. Virus recovered from reconstructed pRAV-1 DNA had the same disease potential as did virus recovered from the parental pRAV-1 DNA. These results indicate that the long terminal repeat sequences of RAV-1 do not confer the potential to induce a high incidence of B-cell lymphoma.     

Ciliary Extracellular Vesicles: Txt Msg Organelles

Cilia are sensory organelles that protrude from cell surfaces to monitor the surrounding environment. In addition to its role as sensory receiver, the cilium also releases extracellular vesicles (EVs). The release of sub-micron sized EVs is a conserved form of intercellular communication used by all three kingdoms of life. These extracellular organelles play important roles in both short and long range signaling between donor and target cells and may coordinate systemic responses within an organism in normal and diseased states. EV shedding from ciliated cells and EV–cilia interactions are evolutionarily conserved phenomena, yet remarkably little is known about the relationship between the cilia and EVs and the fundamental biology of EVs. Studies in the model organisms Chlamydomonas and Caenorhabditis elegans have begun to shed light on ciliary EVs. Chlamydomonas EVs are shed from tips of flagella and are bioactive. Caenorhabditis elegans EVs are shed and released by ciliated sensory neurons in an intraflagellar transport-dependent manner. Caenorhabditis elegans EVs play a role in modulating animal-to-animal communication, and this EV bioactivity is dependent on EV cargo content. Some ciliary pathologies, or ciliopathies, are associated with abnormal EV shedding or with abnormal cilia–EV interactions. Until the 21st century, both cilia and EVs were ignored as vestigial or cellular junk. As research interest in these two organelles continues to gain momentum, we envision a new field of cell biology emerging. Here, we propose that the cilium is a dedicated organelle for EV biogenesis and EV reception. We will also discuss possible mechanisms by which EVs exert bioactivity and explain how what is learned in model organisms regarding EV biogenesis and function may provide insight to human ciliopathies.     

ART-CH, a new chicken retroviruslike element.

A 3' region of a previously unknown retroviruslike element named ART-CH (avian retrotransposon from chicken genome) was obtained in the course of polymerase chain reaction-mediated cloning of avian leukosis virus long terminal repeats (LTRs) from DNAs of infected chicken cells. About 50 copies of ART-CH are present in the genome of chickens of different breeds. ART-CH is not found in DNA of quails, ducks, turkeys, or several other birds tested. The ART-CH element is about 3 kb in size, including 388 bp LTRs. The major class of ART-CH-specific RNA, also 3 kb in size, is detected in various organs of chickens. An ART-CH polypurine tract, a tRNA(Trp)-binding site, regions around the TATA box and polyadenylation signal, and the beginning of the putative gag gene strongly resemble the corresponding regions of avian leukosis viruses and EAV, the two described classes of chicken retroviruses. An open reading frame capable of encoding a polypeptide with a putative transmembrane domain is located upstream of the right ART-CH LTR. This sequence, as well as the U3 and U5 regions of the ART-CH LTR, has no obvious similarities with the corresponding parts of other known vertebrate retroviruses and retrotransposons. A short sequence upstream of the right LTR of ART-CH is very similar to sequences which flank the 3' ends of the oncogenes v-src, v-myc, v-fps, and v-crk in four different recombinant avian retroviruses and which are absent from the genomes of other studied avian retroviruses. Thus, ART-CH is a new endogenous chicken provirus that may participate in the formation of recombinant oncogenic retroviruses.     

Recombination between endogenous and exogenous RNA tumor virus genes as analyzed by nucleic acid hybridization.

Certain chicken cells that do not spontaneously release virus particles have been shown to produce a subgroup E avian RNA tumor virus, Rous-associated virus 60 (RAV-60), after infection with viruses of other subgroups. The nucleic acids of RAV-60 were analyzed for sequence homologies with the viral nucleic acids contained in the uninfected cell and with those of RAV-2, the exogenous virus used for the preparation of this particular RAV-60 isolate. In addition, these nucleic acids were compared with those of RAV-0, an endogenous virus spontaneously released from line 100 chicken cells. RAV-60 appears to be intermediate between RAV-0 and RAV-2 in its genetic composition, based on the pattern of hybridization obtained with the nucleic acids of these viruses and on the melting profiles of the various hybrid combinations. Of the three viruses tested, RAV-0 appears to have the greatest sequence homology with the viral nucleic acids of the uninfected cell. Hybridization between RAV-60 3-H-labeled complementary DNA and either DNA or RNA from the uninfected cell indicates that RAV-60 contains some nucleic acid sequences which are not present in the cell. In addition, some RAV-60 sequences which hybridize with the cell nucleic acid contain significant amounts of mismatching, as indicated by the lower thermal stability of these hybrid duplexes. Hybrid formation between these partially homologous sequences was excluded under stringent annealing conditions. The data indicate that RAV-60 is a recombinant between exogenous and endogenous viral genes.     

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.     

Transmission of avian influenza A viruses among species in an artificial barnyard

Waterfowl and shorebirds harbor and shed all hemagglutinin and neuraminidase subtypes of influenza A viruses and interact in nature with a broad range of other avian and mammalian species to which they might transmit such viruses. Estimating the efficiency and importance of such cross-species transmission using epidemiological approaches is difficult. We therefore addressed this question by studying transmission of low pathogenic H5 and H7 viruses from infected ducks to other common animals in a quasi-natural laboratory environment designed to mimic a common barnyard. Mallards (Anas platyrhynchos) recently infected with H5N2 or H7N3 viruses were introduced into a room housing other mallards plus chickens, blackbirds, rats and pigeons, and transmission was assessed by monitoring virus shedding (ducks) or seroconversion (other species) over the following 4 weeks. Additional animals of each species were directly inoculated with virus to characterize the effect of a known exposure. In both barnyard experiments, virus accumulated to high titers in the shared water pool. The H5N2 virus was transmitted from infected ducks to other ducks and chickens in the room either directly or through environmental contamination, but not to rats or blackbirds. Ducks infected with the H7N2 virus transmitted directly or indirectly to all other species present. Chickens and blackbirds directly inoculated with these viruses shed significant amounts of virus and seroconverted; rats and pigeons developed antiviral antibodies, but, except for one pigeon, failed to shed virus.     

Intracellular restriction on the growth of induced subgroup E avian type C viruses in chicken cells.

Subgroup E avian type C viruses produced by bromodeoxyuridine-treated 100 X 7, line 7, or line C chicken cells were restricted in their intracellular growth on K28 chicken cells but not on line 15 chicken cells. Cells from embryos of line 15 chickens bred with K28 chickens did not restrict the growth of the subgroup E induced leukosis viruses (ILVs). This result indicates that the phenotype for the intracellular restriction of the growth of subgroup E ILVs found in K28 cells is recessive. Long-term growth of the subgroup E ILVs in K28 cells resulted in the appearance of subgroup E virus that grew well on K28 cells. No change in growth characteristics was observed for subgroup E ILVs grown in line 15 cells indicating that appearance of nonrestricted virus occurred only during growth of the subgrouo E ILVs on a restrictive host. RAV-0, a subgroup E virus closely related to the ilvs, had the same growth characteristics as the subgroup E ILVs. RAV-60, a subgroup E virus formed by recombination of exogenous avian leukosis virus with endogenous subgroup E virus coat information, grew well on both line 15 and K28 cells.     

Ultrastructure of the Surfaces of Cells Infected with Avian Leukosis-Sarcoma Viruses

When stained with ruthenium red (RR), chick embryo cells infected with various strains of Rous sarcoma virus (RSV) and with avian leukosis viruses RAV-1 and RAV-3 showed an increase in the layer of acid mucopolysaccharides (AMPS) at their surfaces as compared with uninfected cells. This increase was most prominent in cells infected with the Fujinami strain of RSV. The layer was resistant to digestion with neuraminidase or trypsin but was readily removed by exposure to hyaluronidase. The thickness of this AMPS layer was not correlated with the varying degree of loss of contact inhibition exhibited by cells infected with the different strains of virus. The staining of the cell envelope with a solution of phosphotungstic and chromic acids (PTA-CR) suggested the presence of glycoproteins. The outer surface of the virions showed the same staining as the cell surface with RR and PTA-CR, and the budding virus particle was seen to incorporate the RR layer of the cell into its structure. The RR layers of cells and virions appeared to fuse, as did those between virus particles, suggesting that these layers play a role in the aggregation of virus particles and in their adherence to the surface of the cell.