Endogenous Avian Retrovirus Research Articles

Detection of long terminal repeat loci derived from endogenous retrovirus in junglefowl using whole-genome sequencing

Endogenous retroviruses (ERVs) are genetic elements present in the genome that retain traces of past viral infections. Characterization of ERVs can provide crucial insights into avian evolution. This study aimed to identify novel long terminal repeat (LTR) loci derived from ERVs (ERV-LTRs) absent in the reference genome using whole-genome sequencing data of red junglefowl, gray junglefowl, Ceylon junglefowl, and green junglefowl. In total, 835 ERV-LTR loci were identified across the four Gallus species. The numbers of ERV-LTRs loci detected in red junglefowl and its subspecies gray junglefowl, Ceylon junglefowl, and green junglefowl were 362, 216, 193, and 128, respectively. The phylogenetic tree was congruent with previously reported trees, suggesting the potential for inferring relationships among past junglefowl populations from the identified ERV-LTR loci. Of the detected loci, 306 ERV-LTRs were identified near or within the genes, and some were associated with cell adhesion. The detected ERV-LTR sequences were classified as endogenous avian retrovirus family, avian leukosis virus subgroup E, Ovex-1, and murine leukemia virus-related ERVs. In addition, the sequence of the EAV family was divided into four patterns by combining the U3, R, and U5 regions. These findings contribute to a more comprehensive understanding of the characteristics of junglefowl ERVs.

Prototype endogenous avian retroviruses of the genus Gallus.

Ancient endogenous retroviruses (ERVs), designated endogenous avian retrovirus (EAVs), are present in all Gallus spp. including the chicken, and resemble the modern avian sarcoma and leukosis viruses (ASLVs). The EAVs comprise several distinct retroviruses, including EAV-0, EAV-E51 and EAV-HP, as well as a putative member previously named the avian retrotransposon of chickens (ART-CH). Thus far, only the EAV-HP elements have been well characterized. Here, we determined sequences of representative EAV-0 and EAV-E51 proviruses by cloning and data mining of the 2011 assembly of the Gallus gallus genome. Although the EAV-0 elements are primarily deleted in the env region, we identified two complete EAV-0 env genes within the G. gallus genome and prototype elements sharing identity with an EAV-E51-related clone previously designated EAV-E33. Prototype EAV-0, EAV-E51 and EAV-E33 gag, pol and env gene sequences used for phylogenetic analysis of deduced proteins showed that the EAVs formed three distinct clades, with EAV-0 sharing the last common ancestor with the ASLVs. The EAV-E51 clade showed the greatest level of divergence compared with other EAVs or ASLVs, suggesting that these ERVs represented exogenous retroviruses that evolved and integrated into the germline over a long period of time. Moreover, the degree of divergence between the chicken and red jungle fowl EAV-E51 sequences suggested that they were more ancient than the other EAVs and may have diverged through mutations that accumulated post-integration. Finally, we showed that the ART-CH elements were chimeric defective ERVs comprising portions of EAV-E51 and EAV-HP rather than authentic retrotransposons.

Identification and characterization of avian retroviruses in chicken embryo-derived yellow fever vaccines: investigation of transmission to vaccine recipients.

All currently licensed yellow fever (YF) vaccines are propagated in chicken embryos. Recent studies of chick cell-derived measles and mumps vaccines show evidence of two types of retrovirus particles, the endogenous avian retrovirus (EAV) and the endogenous avian leukosis virus (ALV-E), which originate from the chicken embryonic fibroblast substrates. In this study, we investigated substrate-derived avian retrovirus contamination in YF vaccines currently produced by three manufacturers (YF-vax [Connaught Laboratories], Stamaril [Aventis], and YF-FIOCRUZ [FIOCRUZ-Bio-Manguinhos]). Testing for reverse transcriptase (RT) activity was not possible because of assay inhibition. However, Western blot analysis of virus pellets with anti-ALV RT antiserum detected three distinct RT proteins in all vaccines, indicating that more than one source is responsible for the RTs present in the vaccines. PCR analysis of both chicken substrate DNA and particle-associated RNA from the YF vaccines showed no evidence of the long terminal repeat sequences of exogenous ALV subgroups A to D in any of the vaccines. In contrast, both ALV-E and EAV particle-associated RNA were detected at equivalent titers in each vaccine by RT-PCR. Quantitative real-time RT-PCR revealed 61,600, 348,000, and 1,665,000 ALV-E RNA copies per dose of Stamaril, YF-FIOCRUZ, and YF-vax vaccines, respectively. ev locus-specific PCR testing of the vaccine-associated chicken substrate DNA was positive both for the nondefective ev-12 locus in two vaccines and for the defective ev-1 locus in all three vaccines. Both intact and ev-1 pol sequences were also identified in the particle-associated RNA. To investigate the risks of transmission, serum samples from 43 YF vaccine recipients were studied. None of the samples were seropositive by an ALV-E-based Western blot assay or had detectable EAV or ALV-E RNA sequences by RT-PCR. YF vaccines produced by the three manufacturers all have particles containing EAV genomes and various levels of defective or nondefective ALV-E sequences. The absence of evidence of infection with ALV-E or EAV in 43 YF vaccine recipients suggests low risks for transmission of these viruses, further supporting the safety of these vaccines.

Segregation of EAV-HP ancient endogenous retroviruses within the chicken population.

Avian leukosis virus subgroup J (ALV-J), an exogenous avian retrovirus, is thought to have evolved by recombination with the highly identical env gene of the endogenous avian retrovirus EAV-HP. Embryonic expression of EAV-HP env has been suggested to be associated with the induction of immunological tolerance, a feature observed in a significant proportion of meat-type chickens infected with ALV-J. In support of this hypothesis, we demonstrate that EAV-HP loci, some of which could be associated with tolerance, are still segregating within the chicken population.

Lack of evidence of endogenous avian leukosis virus and endogenous avian retrovirus transmission to measles, mumps, and rubella vaccine recipients.

The identification of endogenous avian leukosis virus (ALV) and endogenous avian retrovirus (EAV) in chick cell-derived measles and mumps vaccines in current use has raised concern about transmission of these retroviruses to vaccine recipients. We used serologic and molecular methods to analyze specimens from 206 recipients of measles, mumps, and rubella (MMR) vaccine for evidence of infection with ALV and EAV. A Western blot assay for detecting antibodies to endogenous ALV was developed and validated. All serum samples were negative for antibodies to endogenous ALV by Western blot analysis. Peripheral blood lymphocyte samples from 100 vaccinees were further tested by polymerase chain reaction for both ALV and EAV proviral sequences; all were negative. Matching serum samples were tested by reverse transcriptase polymerase chain reaction for ALV and EAV RNA, and all 100 samples were negative, providing no evidence of viremia. These findings do not indicate the presence of either ALV or EAV infection in MMR vaccine recipients and provide support for current immunization policies.

Lack of Evidence of Endogenous Avian Leukosis Virus and Endogenous Avian Retrovirus Transmission to Measles Mumps Rubella Vaccine Recipients

Lack of Evidence of Endogenous Avian Leukosis Virus and Endogenous Avian Retrovirus Transmission to Measles Mumps Rubella Vaccine Recipients

Evidence of avian leukosis virus subgroup E and endogenous avian virus in measles and mumps vaccines derived from chicken cells: investigation of transmission to vaccine recipients.

Reverse transcriptase (RT) activity has been detected recently in all chicken cell-derived measles and mumps vaccines. A study of a vaccine manufactured in Europe indicated that the RT is associated with particles containing endogenous avian retrovirus (EAV-0) RNA and originates from the chicken embryonic fibroblasts (CEF) used as a substrate for propagation of the vaccine. We investigated the origin of RT in measles and mumps vaccines from a U.S. manufacturer and confirm the presence of RT and EAV RNA. Additionally, we provide new evidence for the presence of avian leukosis virus (ALV) in both CEF supernatants and vaccines. ALV pol sequences were first identified in particle-associated RNA by amplification with degenerate retroviral pol primers. ALV RNA sequences from both the gag and env regions were also detected. Analysis of hypervariable region 2 of env revealed a subgroup E sequence, an endogenous-type ALV. Both CEF- and vaccine-derived RT activity could be blocked by antibodies to ALV RT. Release of ALV-like virus particles from uninoculated CEF was also documented by electron microscopy. Nonetheless, infectivity studies on susceptible 15B1 chicken cells gave no evidence of infectious ALV, which is consistent with the phenotypes of the ev loci identified in the CEF. PCR analysis of ALV and EAV proviral sequences in peripheral blood mononuclear cells from 33 children after measles and mumps vaccination yielded negative results. Our data indicate that the sources of RT activity in all RT-positive measles and mumps vaccines may not be similar and depend on the particular endogenous retroviral loci present in the chicken cell substrate used. The present data do not support transmission of either ALV or EAV to recipients of the U.S.-made vaccine and provide reassurance for current immunization policies.

Novel endogenous retroviral sequences in the chicken genome closely related to HPRS-103 (subgroup J) avian leukosis virus.

HPRS-103, the prototype of avian leukosis virus (ALV) subgroup J, is a recently identified retrovirus associated with myeloid leukosis in meat-type chickens. Although this virus shows high sequence identity to other ALV subgroups within the gag and pol genes, its env gene is highly diverged (with only about 40% sequence identity) from other ALV subgroups. On the other hand, the sequence of the env gene of HPRS-103 was 75% identical to that of E51, a member of the EAV family of endogenous avian retroviruses. It is reported here that the chicken genome also contains another EAV-related element, EAV-HP, showing much greater sequence identity (over 97%) to the HPRS-103 env gene. Southern blotting analysis showed that EAV-HP-related sequences were distinct from EAV-O and were present in all lines of chicken examined and in grey jungle fowl, but were absent from several other avian species. The potential role of these endogenous sequences in the evolution of ALV subgroup J viruses is discussed.

Retrovirus-induced disease in poultry

Three species of avian retrovirus cause disease in poultry: the avian leukosis/sarcoma virus (ALSV), reticuloendotheliosis virus (REV), and lymphoproliferative disease virus (LPDV) of turkeys. The ALSV can be classified as slowly transforming viruses, which lack a viral oncogene, and acutely transforming viruses, which possess a viral oncogene. Slowly transforming viruses induce late onset leukoses of the B cell lymphoid, erythroid, and myeloid cell lineages, and other tumors, by viral promoter insertion into the genome of a host cell and activation of a cellular protooncogene. The various acutely transforming leukemia and sarcoma viruses induce leukotic or other tumors rapidly and carry one or anther (sometimes two) viral oncogenes, of which some 15 have been identified. The ALSV fall into six envelope subgroups, A through E, and the recently recognized J subgroup, which induces myeloid leukosis. With the exception of Subgroup E viruses, these viruses spread vertically and horizontally as infectious virions, and are termed exogenous viruses. Subgroup E viruses are usually spread genetically as DNA proviruses (often defective) in host germ cell genome, and are termed endogenous viruses. Several other families of endogenous viruses also exist, one of which, endogenous avian retrovirus (EAV), is related to Subgroup J ALV. Exogenous viruses, and sometimes endogenous viruses, can have detrimental effects on commercially important production traits. Exogenous viruses are currently controlled by virus eradication schemes. Reticuloendotheliosis virus, which lacks a viral oncogene, causes chronic B cell and T-cell lymphomas in chickens, and also chronic lymphomas in turkeys and other species of birds. An acutely transforming variant of REV, Strain T, carries a viral oncogene, and induces reticuloendotheliosis within a few days. In chickens and turkeys, REV spreads vertically and horizontally. No commercial control schemes are operated. In turkeys, LPDV infection has occurred in several countries, where it caused a lymphoproliferative disease of uncertain nature.

The reverse transcriptase activity in cell-free medium of chicken embryo fibroblast cultures is not associated with a replication-competent retrovirus.

Reverse transcriptase (RT) activity has previously been reported in concentrated medium of primary chicken embryo cell cultures using the traditional RT assay. Recently, using the newly-developed and highly-sensitive product-enhanced reverse transcriptase (PERT) assay, RT activity has been detected in live, attenuated vaccines grown in chicken cell substrates. Furthermore, this activity has been associated with particles that contain RNA related to an ancient, endogenous avian retrovirus family designated as EAV-0. To investigate whether the RT activity present in vaccines produced in specific pathogen-free chicken cell substrates is associated with an infectious retrovirus that can replicate in human cells. The kinetics of RT activity produced by 10-day-old chicken embryo fibroblast (CEF) cultures was determined by analyzing cell-free medium in a PCR-based RT (PBRT) assay. Material containing the peak PBRT activity was used as the inoculum to infect various human cell lines and peripheral blood mononuclear cells. Filtered supernatants from control and test cultures were analyzed for the presence of replication-competent retroviruses by the PBRT assay. The cells were monitored for other adventitious agents by routine observation for cytopathic effect (CPE) and by transmission electron microscopy (TEM) at culture termination. The PBRT activity did not increase above the background level in the human target cells through at least five cell passages, thus indicating the absence of a replicating retrovirus. No other adventitious agents were detected based upon TEM analysis and the absence of CPE. The RT activity produced by chicken primary cell cultures is not associated with a retrovirus that can replicate in human cells.

Antigenic variants of J subgroup avian leukosis virus: sequence analysis reveals multiple changes in the env gene.

HPRS-103, the prototype of avian leukosis virus (ALV) subgroup J, was isolated in 1989 from meat-type chickens from commercial flocks where it induces myelocytic myeloid leukosis (ML). The HPRS-103 env gene differs considerably from other ALV subgroups but shows high identity (75-97%) to env-like sequences of the different members of the EAV family of endogenous avian retroviruses. Recently, we have isolated several viruses related to HPRS-103 from cases of ML. Although these isolates showed properties of ALV subgroup J, the majority of them resisted neutralization by HPRS-103-specific serum, suggesting antigenic variation. The nucleotide sequence of the env gene of the variant viruses showed several substitutions resulting in amino acid changes especially clustered in the variable regions hr1, hr2 and vr3. Analysis of the data suggests that selection pressure, probably from the immune response, is driving the antigenic variation among the isolates. Phylogenetic analysis of the sequences showed the evolutionary relationships of the isolates with HPRS-103 and the EAV family of endogenous avian retroviruses. The epidemiological significance of the antigenic variation and the emergence of variant viruses are discussed.

Reverse transcriptase activity in chicken embryo fibroblast culture supernatants is associated with particles containing endogenous avian retrovirus EAV-0 RNA.

We have recently shown that live attenuated virus vaccines produced on chicken-derived cells contain low levels of particle-associated reverse transcriptase (RT). In both virus and corresponding control harvests produced on chicken embryo fibroblasts, these activities were present at significantly higher concentrations than in the vaccines. In order to identify the putative retrovirus sequence responsible for this activity, a novel method for the selective PCR amplification of particle-associated retrovirus RNA that uses DNA primers complementary to the primer binding sites of the known exogenous retroviruses in combination with an anchor primer was applied. A product of the endogenous avian retrovirus family EAV-0, termed EAV-0(B1), was reproducibly generated with a tRNA(Trp)-derived primer from the RT peak fraction of a sucrose density gradient run with a harvest of a live attenuated measles vaccine. In contrast, no products were detected with primers derived from tRNA(Pro), tRNA(Lys)1,2 or tRNA(Lys)3. In the same fraction, genomic RNA of EAV-0(B1) was demonstrated by long PCR. Analysis of several sucrose density gradients from different harvests of various manufacturers demonstrated accumulation of, and colocalization with, RT activity for the EAV-0(B1) RNA but not for a chicken cellular mRNA. Synthesis of cDNA from EAV-0(B1) RNA was shown by endogenous RT reaction. Furthermore, complexes of naturally primed EAV-0(B1) RNA with RT were demonstrated. Taken together, these data strongly suggest that EAV-0 is able to produce virus-like particles with an active RT.

Structural and functional characterization of the unusually short long terminal repeats and their adjacent regions of a novel endogenous avian retrovirus

We have cloned the long terminal repeats and their flanking regions from four different proviruses belonging to a large, highly conserved, novel family of avian endogenous retroviruses. This family, termed the endogenous avian retrovirus (EAV) family, is distinct from the previously characterized avian endogenous and exogenous retroviruses. We have analyzed the sequences of the long terminal repeats and their adjacent noncoding viral sequences, including the gag leader region and the 3′ noncoding region, of several different members of the EAV family and have found that the regulatory region of these novel viruses contains several unique features. The LTRs of the EAV proviruses are extremely short (243 by long) but contain all of the essential regulatory features of longer avian retrovirus LTRs. The gag leader region and the 3′ noncoding region of the novel EAVs are only weakly related to those of other avian retroviruses. Northern blot hybridization analysis of RNA from Line-0 chicken embryos reveals several transcripts derived from the EAV proviruses. Primer extension analysis indicates that all transcripts initiated from 5′ proviral LTRs are initiated at the predicted +1 position within the EAV LTRs. The relative shortness, sequence divergence from other known LTRs, and the retention of the transcriptional integrity of the EAV LTRs make these LTRs an interesting model system for LTR function and for study of the potential involvement of such highly conserved retroviral elements in development.

Region coding for subgroup specificity of envelope of avian retroviruses does not determine lymphomagenicity

The purpose of this study was to test whether the region coding for subgroup envelope specificity was a determinant of oncogenic potential. An avian retrovirus recombinant was constructed to contain the envelope gene ( enu) determinant for subgroup A of an avian RNA tumor virus in one of the non-oncogenic, endogenous avian retroviruses, RAV-0. The results show that such a recombinant virus does not induce lymphoma when injected into susceptible, newborn chicks. Thus, it is concluded that the envelope determinant for subgroup of avian RNA tumor viruses does not determine malignant potential.

Replication of endogenous avian retrovirus in permissive and nonpermissive chicken embryo fibroblasts.

Clones of chicken embryo fibroblasts exogenously infected with the endogenous avian retrovirus were analyzed to examine the replication of this virus in permissive (Gr+) and nonpermissive (Gr-) cells. The results demonstrate that the endogenous virus was capable of infecting both Gr+ and Gr- cells with equal efficiency. Infected clones of Gr+ and Gr- cells differed, however, in two significant ways. At the time of their initial characterization, the Gr+ clones produced 100- to 1,000-fold more virus than the Gr- clones. Further, the amount of virus produced by Gr+ clones did not change significantly during serial passage of the cells. In contrast, continued passage of the infected Gr- clones resulted in a gradual increase in the amount of virus produced. Individual clones of infected Gr- cells produced infectious virus at rates that, initially, differed by a factor of more than 10(4). The large differences in the production of virus by these clones could not be explained by equally large differences in the number of infected cells within the clonal populations. Greater than 80% of the clonal populations examined ultimately produced virus at rates that were not significantly different from the rates observed in infected Gr+ cells. Virus produced by these infected Gr- cells exhibited the same restricted replication upon establishing a new infection in nonpermissive cells. Analysis of the appearance of free and integrated viral DNA sequences during endogenous virus infection of Gr+ and Gr- cells demonstrated that, after an initial delay in the synthesis of free viral DNA in Gr- cells, the nonpermissive cells ultimately acquired as many integrated viral DNA sequences as were found in infected Gr+ cells. These results indicate that a majority of the infectious particles of the endogenous virus are capable of establishing infection in a Gr- cell and, ultimately, of producing virus at a rate that is not significantly different from that produced by infected Gr+ cells. The virus produced from the Gr- cells is not a stable genetic variant of the original endogenous virus that is capable of unrestricted replication in nonpermissive cells. The reduced efficiency with which the endogenous virus initially replicates in nonpermissive cells and the increased length of time required for infected Gr- cells to produce maximal virus titers suggest that the endogenous virus may utilize a different mechanism of replication in Gr+ and Gr- fibroblasts.

Characterization of endogenous viral Loci in five lines of white leghorn chickens

Five lines of chickens have been examined for the presence of DNA sequences related to the endogenous avian retrovirus. Five new loci have been identified, based upon analysis with the restriction endonucleases SacI and BamHI. One locus has been associated with the production of infectious endogenous virus. Restriction endonuclease mapping suggested a limited similarity between the flanking cellular sequences of two of these loci, ev-17 and ev-18, and several endogenous loci, including ev-1, already characterized. The data suggested that these two loci might have been generated by chromosomal duplication. Hybridization analysis with a probe containing the cellular sequences that flank ev-1, however, revealed that these flanking sequences shared no detectable homology with the cellular sequences that surround ev-17, ev-18, or nine other endogenous loci that were examined. These results are consistent with the hypothesis that several of the endogenous viral loci resulted either from independent infections of the germ line or from virus transpositions.

Independent segregation of ev 10 and ev 11, genetic loci for spontaneous production of endogenous avian retroviruses

F 1 males produced by crossing Regional Poultry Research Laboratory line 15I 4 that carries endogenous viral ( ev) genes 1, 6, and 10 and a partially defined gene, ev 11, with line 15 B that carries ev 1 and 7 were backerossed to line 15 B females. The DNA of 72 progeny embryos was digested by BamHI and Kpn and hybridization of resulting fragments with 32P-labeled viral RNA showed that ev 6, 10, and 11 were autosomal genes and not closely linked, and confirmed that ev 7 was sex linked. ev 10 and 11 were consistently associated with complete virus production, confirming that the newly defined ev 11 codes for a complete endogenous retrovirus.

Endogenous avian retroviruses contain deficient promoter and leader sequences.

A sensitive and quantitative biological assay has been utilized to measure the ability of the exogenous and endogenous avian retroviral long terminal repeats (LTR) to promote gene expression in avian cells. This assay has revealed that the exogenous virus RAV-2 LTR is approximately equal to 10-fold more active than the LTRs of endogenous viruses RAV-0, ev-1, and ev-2. The endogenous viral LTRs show approximately equal activity. Upstream flanking cellular or viral sequences have no significant modulating effect on gene expression in our assay. Unexpectedly, we have detected and localized an additional defect outside of the LTR in the 5' noncoding leader sequence of ev-1 that further decreases gene expression relative to RAV-0 by approximately equal to 10-fold.

Analysis of endogenous avian retrovirus DNA and RNA: Viral and cellular determinants of retrovirus gene expression

We have characterized endogenous avian retrovirus DNAs and RNAs in uninfected white leghorn chicken embryos. The embryos we studied include representations of four phenotypic classifications of chickens (gs − chf − , gs + chf + , gs − chf +, and V +), and contained the endogenous proviruses evl, 2, 3, 4, 5, 6, 8, 9, and 15. We confirmed the previously described phenotypes and restriction maps of evl, 3, 4, 5, 6, 8, and 15, and we confirmed the phenotype for ev9 and derived a restriction map for this locus. Evaluation of the structure of the individual proviruses and characterization of the nuclear and cytoplasmic viral RNA isolated from embryos of defined genotype yielded the following major conclusions. (i) Viral RNA could not be detected for ev4, ev5, ev8, or ev15. (ii) The amount of stable RNA produced from evl, ev3, ev6, or ev9 varied over a range of at least 100- to 300-fold and was at least 10- to 1000-fold lower than the amounts found in cells productively infected with exogenous avian retroviruses. (iii) A deletion in the provirus at ev3 can account for three abnormalities: the production of a viral protein that results from fusion of internal regions of the gag and pol genes, the abnormal structure of a subgenomic viral RNA, and failure of the abnormal RNA to appear in the cytoplasm. (iv) A deletion in the provirus at ev6 gives rise to at least two major anomalies: the initiation of transcription at an upstream cellular promoter, and the abnormal metabolism of an ev6 viral RNA. (v) ev9 has an ostensibly normal structure; nevertheless, the metabolism of an RNA arising from this locus is aberrant. We conclude that the expression of endogenous retrovirus genes in chickens is subject to control by both viral and cellular determinants.

Clonal analysis of the integration and expression of endogenous avian retroviral DNA acquired by exogenous viral infection.

Rous-associated virus-0 is one of several endogenous avian retroviruses that are transmitted vertically and that can be isolated from different inbred lines of chickens. These viruses, referred to here as induced-leukosis viruses bearing a subgroup E glycoprotein (ILV-E), are all closely related. Clonal populations of fibroblasts from line 15B and line 100 inbred chickens have been examined for the presence and expression of exogenously acquired ILV-E sequences. Restriction enzyme analysis of uniform populations of line 15B fibroblasts, prepared by cloning cells either before or after infection with ILV-E, indicates that viral sequences were inserted at multiple sites within the cell genome. Analysis of 49 clonal populations of line 100 fibroblasts containing between one and five copies of exogenous ILV-E sequences demonstrated that each clone was characterized by a unique set of viral DNA insertions within the cell genome. The expression of the exogenous ILV-E sequences within these fibroblast clones was examined by using reverse transcriptase activity as a measure of virus production. Some clones produced an amount of virus equivalent to that produced by an equal number of the uncloned ILV-E-infected parental fibroblasts. Other clones produced 5- to 10-fold less virus. Still other clones produced no detectable virus at all. Among nine clones derived from cells containing a single copy of the ILV-E provirus, the level of virus production differed more than 100-fold. DNA from these clones was analyzed with several different restriction endonucleases to characterize the location and arrangement of the ILV-E sequences. All nine clones consisted of cells that appeared to contain a complete provirus inserted (i) in a different site within the cellular DNA and (ii) in an orientation that was colinear with the viral genomic RNA. It was observed that several cleavage sites potentially affected by methylation were equally available for cleavage in all clones regardless of the level of viral production.