Endogenous Proviruses Research Articles

Origins of enhancer sequences of recombinant murine leukemia viruses from spontaneous B- and T-cell lymphomas of CWD mice.

Recombinant murine leukemia viruses from the highly leukemic mouse strains AKR, HRS, and C58 usually acquire pathogenic U3 region sequences fro the endogenous xenotropic virus, Bxv-1. However, the majority of tumors from another highly leukemic strain, CWD, contained recombinant viruses that lacked Bxv-1-specific sequences. The nucleotide sequence of the U3 regions of two such CWD recombinants was nearly identical to that of the endogenous ecotropic virus parent Emv-1, but they shared three nucleotide substitutions immediately 3' of the enhancer core. These substitutions were found in recombinant proviruses from about one-third of spontaneous CWD lymphomas as determined by an oligonucleotide hybridization assay of proviral fragments that had been nucleotide substitutions in the CWD viruses were inherited from an endogenous polytropic provirus that is absent in the other highly leukemic strains. On the basis of the results of these and previous studies, we propose that CWD recombinants acquire pathogenic U3 region sequences through recombination with an endogenous polytropic virus or Bxv-1 and that the pathogenicity of these sequences may be related to a sequence motif that is known to bind members of the basic helix-loop-helix class of transcription factors.

The induction of peripheral tolerance by the chronic activation and deletion of CD4+V beta 5+ cells.

In C57BL/6 mice transgenic for a rearranged gene encoding a V beta 5+ beta-chain of the TCR, transgene expression among CD4+ cells decreases with age, such that approximately 40% of CD4+ cells express an endogenous beta-chain gene in 8-mo-old mice. A similar deletion of V beta 5+ cells is observed among CD4+ cells from nontransgenic littermates. V beta 5+ T cells are deleted intrathymically in I-E+Mtv-9+ strains of mice, but this chronic deletion occurs in the lymphoid periphery, in the absence of I-E. We now demonstrate the increased expression of the activation markers CD44 and VLA-4 among CD4+V beta 5+ cells, in the absence of either an increase in size or IL-2 receptor expression. Functional as well as phenotypic differences distinguish CD4+ from CD8+ cells in older V beta 5+ transgenic mice. Relative to their CD8+ counterparts, CD4+V beta 5+ cells are hyporesponsive to plate-bound anti-V beta 5 Abs, and this anergy is partially reversible by the addition of exogenous IL-2. These data suggest the deletion of CD4+V beta 5+ cells is the result of a process that includes their activation, loss of function, and their eventual removal. To investigate the involvement of the principal V beta 5 superantigen Mtv-9 in this chronic deletion, we have derived several lines of V beta 5+I-E-Mtv-9- mice. Transgene expression also declines with age in CD4+ T cells in these mice, clearly demonstrating that the chronic deletion of CD4+V beta 5+ cells does not require Mtv-9. There is considerable variation in the kinetics and efficiency of CD4+V beta 5+ deletion between lines of Mtv-9- transgenic mice that is not from differences in the profiles of endogenous mammary tumor proviruses nor readily explained by environmental differences that influence proviral expression. These results suggest the existence of genetic factors other than mammary tumor proviruses that influence the deletion of CD4+V beta 5+ cells in the absence of I-E.

Distribution of mouse mammary tumor virus in Asian wild mice.

Several groups of wild mice (Mus musculus) were captured from eight different locations in Asia and bred for several generations in a facility free of any laboratory strains of mice carrying mouse mammary tumor virus (MMTV). The distribution of endogenous MMTV proviral sequences in the liver tissues of these mice was investigated by using Southern blot hybridizations. Four categories of mice were identified. Mice originating from Bogor, Indonesia (Cas-Bgr); He-mei, Taiwan (Cas-Hmi/1); and Malaysia (Cas-Mal) were found to carry an endogenous MMTV provirus consisting of the env, gag-pol, and long terminal repeat sequences. Mice captured from Kojuri, Republic of Korea (Sub-Kjr); Nagoya, Japan (Mol-nag); and three Chinese provinces, Shanghai (Sub-Shh), Beijing (Sub-Bjn), and Jiayuguang (Sub-Jyg/1), appeared to carry defective proviruses. Some mice originating from He-mei (Cas-Hmi/2) and Jiayuguang (Sub-Jyg/2) were found to be completely free of endogenous MMTV. Interestingly, however, the Sub-Jyg/2 mice, after several generations of inbreeding, were found, unlike all of the other subspecies that we examined in the present study, to develop mammary tumors at a high incidence (80 to 90%) with a short period of latency. Electron microscopic examination of the mammary glands and mammary tumors of these mice revealed the presence of numerous intracytoplasmic A, immature, budding, and mature B particles. Furthermore, the mammary tumors were found to contain MMTV proviral sequences. It seems, therefore, that Sub-Jyg/2 mice carry an exogenous MMTV which contributes to their developing mammary tumors.

Mutational Analysis of the Human Endogenous ERV9 Proviruses Promoter Region

ERV9 is a low repeated family of human endogenous retrovital elements whose expression is mainly detectable in undifferentiated embryonal carcinoma NT2/D1 cells. To define all the elements required for the correct transcription activity of the ERV9 promoter and to establish a precise correlation between the elements important for basal transcription, we have systematically analyzed the in vivo and in vitro transcriptional activity of many different ERV9 promoter mutants, including a series of linker-scanning mutations across the promoter region. We report here that the ERV9 promoter contains two elements controlling the selection of the correct start sites, a TATA box and an Inr-like region; the concerted action of both elements is necessary for faithful transcription. Finally, using a series of GAL4 protein fusion constructs in contransfection experiments, we demonstrated that various transcription factors can synergistically induce a high level of transcription when bound to an ERV9 DNA promoter.

Defective endogenous proviruses are expressed in feline lymphoid cells: evidence for a role in natural resistance to subgroup B feline leukemia viruses.

Endogenous feline leukemia virus (FeLV)-related sequences (enFeLV) are a family of proviral elements found in domestic cats and their close relatives. These elements can recombine with exogenous, infectious FeLVs of subgroup A (FeLV-A), giving rise to host range variants of FeLV-B. We found that a subset of defective enFeLV proviruses is highly expressed in lymphoma cell lines and in a variety of primary tissues, including lymphoid tissues from healthy specific-pathogen-free cats. At least two RNA species were detected, a 4.5-kb RNA containing gag, env, and long terminal repeat sequences and a 2-kb RNA containing env and long terminal repeat sequences. Cloning of enFeLV cDNA from two FeLV-free lymphoma cell lines (3201 and MCC) revealed a long open reading frame (ORF) encoding a truncated env gene product corresponding to the N-terminal portion of gp70env. Interestingly, all of three natural FeLV-B isolates include 3' env sequences which are missing from the highly transcribed subset and hence must be derived from other enFeLV elements. The enFeLV env ORF cDNA clones were closely similar to a previously characterized enFeLV provirus, CFE-16, but were polymorphic at a site corresponding to an exogenous FeLV neutralization epitope. Site-specific antiserum raised to a C-terminal 30-amino-acid peptide of the enFeLV env ORF detected an intracellular product of 35 kDa which was also shed from cells in stable form. Expression of the 35-kDa protein correlated with enFeLV RNA levels and was negatively correlated with susceptibility to infection with FeLV-B. Cell culture supernatant containing the 35-kDa protein specifically blocked infection of permissive fibroblast cells with FeLV-B isolates. We suggest that the truncated env protein mediates resistance by receptor blockade and that this form of enFeLV expression mediates the natural resistance of cats to infection with FeLV-B in the absence of FeLV-A.

Determination of avian endogenous provirus ‐ cellular junction sequences using inverse polymerase chain reactions

Abstract The inverse polymerase chain reaction (invPCR), based on using sets of oligonucleotide primers oriented in the reverse direction of the usual PCR, was used to amplify cell sequences that flank chicken endogenous virus (ev) genes. Inverse PCR products flanking the 5’ region of ev7 and ev12 were cloned and cell nucleotide sequences were determined. Subsequent PCRs were conducted using primers based on cell sequences flanking ev7, ev12, and the proviral long terminal repeat of ev1. In a survey of experimental and commercial lines and breeds, ev12 was found among three broiler lines. This approach facilitates the identification of ev genes in breeding stocks without conducting prior conventional progeny testing. Moreover, specific ev genes may be detected in individuals harboring a variety of other ev genes.

Retroviruses in invertebrates: the gypsy retrotransposon is apparently an infectious retrovirus of Drosophila melanogaster.

Retroviruses are commonly considered to be restricted to vertebrates. However, the genome of many eukaryotes contains mobile sequences known as retrotransposons with long terminal repeats (LTR retrotransposons) or viral retrotransposons, showing similarities with integrated proviruses of retroviruses, such as Ty elements in Saccharomyces cerevisiae, copia-like elements in Drosophila, and endogenous proviruses in vertebrates. The gypsy element of Drosophila melanogaster has LTRs and contains three open reading frames, one of which encodes potential products similar to gag-specific protease, reverse transcriptase, and endonuclease. It is more similar to typical retroviruses than to LTR retrotransposons. We report here experiments showing that gypsy can be transmitted by microinjecting egg plasma from embryos of a strain containing actively transposing gypsy elements into embryos of a strain originally devoid of transposing elements. Horizontal transfer is also observed when individuals of the "empty" stock are raised on medium containing ground pupae of the stock possessing transposing elements. These results suggest that gypsy is an infectious retrovirus and provide evidence that retroviruses also occur in invertebrates.

Association of Xmv-10 and the non-agouti (a) mutation explained by close linkage instead of causality

In a previous survey of endogenous proviruses among inbred mouse strains, the Xmv-10 provirus was found only in strains that carried the non-agouti (a) mutation (Frankel et al. J. Virol. 63: 1763-1774, 1989). To determine whether insertion of Xmv-10 caused the a mutation, we cloned a portion of Xmv-10 and its insertion site. Using a fragment of flanking cellular DNA as a Southern hybridization probe, we found that the Xmv-10 provirus was still present in revertant alleles of a to a(t) or AW. A restriction fragment length variant (RFLV) in cellular DNA at the Xmv-10 insertion site was found to be correlated with the presence or absence of the provirus among inbred strains of laboratory mice regardless of their agouti allele. This correlation did not extend to wild mice, however, in which none of the samples contained Xmv-10, yet one, Mus domesticus poschiavinus, contained the insertion site RFLV correlated with Xmv-10 in laboratory mice. Analysis of an intersubspecific backcross with RFLVs at the insertion sites of Xmv-10 and Emv-15 (an endogenous provirus associated with Ay) revealed the following genetic map information: cen-A-0.31 +/- 0.31 cM-Emv-15-0.62 +/- 0.27 cM-Xmv-10-tel. Haplotype analysis of inbred strains in which a was not associated with Xmv-10 and in which Ay was not associated with Emv-15 demonstrated that these "exceptions" were explained most simply by a single recombination that disturbed the linkage relationships evident in most inbred strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Major and Minor Kb-Restricted Epitopes Encoded by the Endogenous Ecotropic Murine Leukemia Virus AKR623 That Are Recognized by Anti-AKR/Gross MuLV CTL

C57BL/6 mice can generate a type-specific and class IH-2Kb-restricted CTL response against histocompatible AKR/Gross murine leukemia virus (MuLV) cell surface antigen positive (GCSA+) tumor cells. These anti-AKR/Gross MuLV CTL are also known to lyse SC.Kb/623 target cells expressing the molecular MuLV clone AKR623 (derived from the endogenous ecotropic MuLV provirus emv-11). To help identify AKR623 viral epitopes recognized by these CTL, four chimeric proviruses were constructed from two parental plasmids, pAKR623 and pAK7. It has been shown that SC.Kb/7 fibroblast targets expressing the emv-14-derived molecular clone AK7 are only poorly lysed by anti-AKR/Gross MuLV CTL. Data from experiments employing SC.Kb cells infected with the chimeras as targets against anti-AKR/Gross MuLV CTL supported the location of a previously identified immunodominant epitope located within the viral p15E transmembrane envelope protein, peptide TM134-141 (KSP-WFTTL). Furthermore, the use of Kb-motif-defined AKR623 encoded peptides together with data obtained using the chimeric viruses allowed the identification of three additional anti-AKR/Gross MuLV CTL epitopes. Peptides representing these epitopes, MA125-132 (RSALY-PAL), RT142-149 (SHRWYTVL), and RT456-463 (RMTHYQAM), are characterized herein with respect to their ability to confer lysis upon EMV- target cells and to stimulate tumor primed splenocytes in vitro. The identification and characterization of these additional epitopes allow for a better understanding of both the CTL response against GCSA+ tumor cells and the dysfunctional CTL response against EMV-14 and AK7.

Mouse mammary tumor virus-induced tumorigenesis in sag transgenic mice: a laboratory model of natural selection.

Transgenic mice that expressed the superantigen protein encoded in the C3H exogenous mouse mammary tumor virus long terminal repeat deleted their V beta 14+ T cells during the shaping of their immune repertoire and showed no evidence of virus production in their mammary glands after infection by milk-borne C3H exogenous virus. However, they developed mammary gland tumors that had newly integrated copies of C3H exogenous virus, although the latency of tumor formation was much longer than in their nontransgenic littermates that retained their V beta 14+ T cells. After four generations, infectious C3H virus was completely eliminated from the transgenic mouse pedigree. These data support the hypothesis that endogenous mouse mammary tumor proviruses are retained in the genome as protection against exogenous virus infection and subsequent tumorigenesis and show that there may be natural selection against the virus in vivo.

The endogenous ecotropic murine retroviruses Emv-16 and Emv-17 are both capable of producing new proviral insertions in the mouse genome

New germ line proviral insertions are acquired at a high frequency by the progeny of SWR/J-RF/J hybrid female mice that carry the endogenous ecotropic murine leukemia proviruses Emv-16 and Emv-17. The tight linkage of these RF/J strain proviral loci has prevented genetic segregation of the retroviral genomes. Hence, it is not known whether both of these proviruses are capable of giving rise to new proviral insertions. We have molecularly cloned Emv-16 and Emv-17 and have characterized them in vitro and in vivo. Restriction enzyme analysis of the recombinant clones revealed that the proviral genomes are very similar to each other and closely resemble the wild-type AKR virus. A comparison of the flanking cellular DNA suggests that the Emv-16 and Emv-17 loci did not arise by simple duplication of a viral insertion site within the RF/J genome but most likely are independent integration events. Both proviruses produce infectious virus when transfected into NIH 3T3 cells, indicating that they are nondefective retroviruses. Exogenous infection of SWR/J mice with either Emv-16 or Emv-17 leads to viremia in the host animals, and in both cases, progeny of viremic females acquire new proviral insertions. The ability of these retroviruses to generate novel retroviral integration sites in the mouse genome provides a simple method for inducing insertional mutations in mice.

Sequence Similarity between the Long Terminal Repeat Coding Regions of Mammary-Tumorigenic BALB/cV and Renal-Tumorigenic C3H-K Strains of Mouse Mammary Tumor Virus

The long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) encodes a protein which functions as a superantigen. The BALB/cV strain differs from other exogenous MMTVs antigenically, biochemically, on the basis of restriction fragment analysis, and by the specificity of its superantigen for Vβ2 +T cells. In order to elucidate the origin of the BALB/cV virus and to better understand the interaction of its superantigen with the T cell receptor, we have determined the nucleotide sequence of the BALB/cV LTR open reading frame, including 93 bases downstream of the translation termination site. The encoded protein's C-terminal portion, thought to control superantigenic specificity, is identical to the C3H-K strain of MMTV, isolated from a rare kidney adenocarcinoma. The remainder of the coding sequence is highly related to many MMTV strains. Like other MMTV strains, the BALB/cV LTR maintains intact an 18 base pair sequence, located downstream of the translational termination site, which is lacking in the C3H-K LTR. Sequence comparison between the BALB/cV LTR and other MMTV strains indicates that the most likely origin for the BALB/cV open reading frame sequence is a recombination event involving the endogenous provirus mtv-6.

Identification of human endogenous retroviruses with complex mRNA expression and particle formation.

Retroviruses comprise strains with considerable disease potential in animals and humans. In addition to exogenous strains transmitted horizontally, endogenous proviruses are transmitted through the germ line. Some of these endogenous retroviruses can be pathogenic in mice and possibly in other animal species. They may also be considered as mobile genetic elements with the potential to produce mutations. In humans, genomic DNA contains numerous endogenous retroviral sequences detected by their partial relatedness to animal retroviruses. However, all proviruses sequenced so far have been found to be defective. In this communication, we describe the expression of a family of human endogenous retrovirus sequences (HERV-K) in GH cells, a teratocarcinoma cell line producing the human teratocarcinoma-derived retrovirus (HTDV) particles previously described by us. Four viral mRNA species could be identified, including a full-length mRNA. The other three subgenomic mRNAs are generated by single or double splicing events. This expression pattern is reminiscent of the more complex control of virus gene regulation observed, for example, with lenti- or spumavirus strains, although HERV-K shows no sequence homology to human T-lymphotropic virus or human immunodeficiency virus. Sequence analysis of expressed HERV-K genomes revealed non-defective gag genes, a prerequisite for particle formation. Open reading frames were also observed in pol and env. Antisera raised against recombinant gag proteins of HERV-K stained HTDV particles in immunoelectron microscopy, linking them to the HERV-K family.

Aberrant induction of T cell tolerance in B cell suppressed mice.

Self-tolerance is the process by which the T cell repertoire develops without expressing self-reactive specificities. The mechanisms which functionally eliminate self-reactive T cells are clonal deletion and clonal inactivation, and both of these phenomena have been studied in T cell populations reactive to endogenous superantigens that are encoded by endogenous mouse mammary tumor proviruses (Mtv). The studies described here demonstrate that the kinetics of Etc-1 (encoded by the Mtv-9 open reading frame gene)-mediated deletion are much slower than that seen for Mls 1a (encoded by the Mtv-7 open reading frame), and that Etc-1-reactive T cells are present in the periphery up to 2 wk after birth. The deletion of peripheral Etc-1-reactive T cells late in ontogeny indicates an efficient mechanism of peripheral clonal deletion in these animals. The clonal deletion of Etc-1-reactive cells is abrogated in B cell-suppressed animals; however, clonal elimination of peripheral V beta 5+ or V beta 11+ (Etc-1 reactive) T cells can be induced when these mice are allowed to recover their B cell population after cessation of anti-mu treatment. Finally, we establish that peripheral Etc-1-reactive V beta 11+/CD4+ T cells remaining in B cell-suppressed and recovering animals are markedly less responsive to stimulation through the TCR than are control T cells. These data support the idea that peripheral self-reactive T cells can be rendered tolerant by two mechanisms which may be temporally related. This model suggests that clonal hyporesponsiveness may be followed by clonal deletion.

Phylogenetic and Structural Analyses of MMTV LTR ORF Sequences of Exogenous and Endogenous Origins

The long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) harbors an open reading frame (ORF) that encodes a glycoprotein and is present in all exogenous and endogenous MMTV proviruses. The ORF protein has been reported to interact with the immune system of mice to cause deletion of specific Vβ-bearing subsets of T cells, Twenty-two MMTV LTR ORF sequences were analyzed. Although highly conserved, the MMTV ORF sequences are not identical, with ≈35% of the total variation clustered at the carboxy terminus. Statistical analysis revealed the presence of two conserved regions in the protein, one of which contained a transmembrane-like domain (residues 45-63). Two potential nuclear localization signals were recognized. Many ORF sequences shared polymorphisms. To analyze relationships, phylogenetic trees were constructed on the basis of alignments of LTR ORF sequences. A tree generated from the carboxy-terminal 35 residues clustered the sequences into three divergent families. The topology of the tree based on the amino-terminal 288 residues differed significantly, with some MMTV sequences rearranged relative to their carboxy-terminal families. A continuum of exogenous-like to endogenous-like character was suggested by the amino-terminal tree. The discordance between the topologies of the two trees suggests that some type of genetic exchange has occurred in the MMTV LTR gene. Mechanisms and implications of such genetic exchange are discussed.

Genetic control of retroviral disease in aging wild mice.

Different populations of wild mice (Mus musculus domesticus) in Los Angeles and Ventura Counties were observed over their lifespan in captivity for expression of infectious murine leukemia virus (MuLV) and murine mammary tumor virus (MMTV) and for the occurrence of cancer and other diseases. In most populations of feral mice these indigenous retroviruses were infrequently expressed and cancer seldom occurred until later in life (> 2 years old). MMTV was found in the milk of about 50% of wild mice, but was associated with only a low incidence (> 1%) of breast cancer after one year of age. By contrast, in several populations, most notably at a squab farm near Lake Casitas (LC), infectious MuLV acquired at birth via milk was highly prevalent, and the infected mice were prone to leukemia and a lower motor neuron paralytic disease after one year of age. These two diseases were both caused by the same infectious (ecotropic) strain of MuLV and were the principal cause of premature death in these aging LC mice. A dominant gene called FV-4R restricting the infection with ecotropic MuLV was found segregating in LC mice. Mice inheriting this FV-4R allele were resistant to the ecotropic MuLV associated lymphoma and paralysis. The FV-4R allele represents a defective endogenous MuLV provirus DNA segment that expresses an ecotropic MuLV envelope-related glycoprotein (gp70) on the cell surface. This FV-4R encoded gp70 presumably occupies the receptor for ecotropic MuLV and blocks entry of the virus. The FV-4R gene was probably acquired by the naturally occurring crossbreeding of LC feral mice with another species of feral mice (Mus castaneus) from Southeast Asia. The FV-4R gp70 does not block entry of the amphotropic MuLV that uses a separate cell surface receptor. Therefore LC mice continued to be susceptible to the highly prevalent but weakly lymphogenic and nonparalytogenic amphotropic strain of MuLV. The study points out the potential of feral populations to reveal genes associated with specific disease resistance.

T-cell receptor b-V repertoire expression in the absence of an endogenous mouse mammary tumor provirus.

T-cell receptor b-V repertoire expression in the absence of an endogenous mouse mammary tumor provirus.

Carbon tetrachloride induction of rapid changes in liver nuclear protein factors capable of sequence-specific binding to regulatory elements in the long terminal repeat of polytropic-class endogenous murine leukemia virus-related proviruses.

Treatment of mice with hepatic carcinogens, including CCl4, has been shown to rapidly enhance the transcription of endogenous murine leukemia virus-related proviral sequences in the liver. To understand the mechanism for this transcriptional stimulation, we used nuclear protein preparations from mouse livers to perform DNase I protection analyses and identified nuclear protein binding on approximately 20 individual sequences within the regulatory regions of the long terminal repeat (LTR) of a polytropic-class endogenous provirus clone. From 3 to 144 h after treatment with CCl4, the livers of FVB/N mice were analyzed for specific nuclear protein binding to the LTR DNA. Three to nine hours after CCl4 treatment, decreased protection was seen at potential regulatory cis-elements throughout the LTR, including specific sites within the putative negative regulatory element (located 5' of the consensus enhancer sequences) and the 3' terminal portion of the polytropic class-specific enhancer-like inserted sequence element and around the CCAA(C/T) box in the promoter region. In addition, by 3-6 h after treatment, a transient increase in protection activity for the transcription initiation site occurred. The loss of cis-element protection expanded to other binding sites and became most marked by 48 h after treatment. As the regenerating liver recovered, the nuclear protein binding activities for these LTR sequences also recovered, but protection at the TATAA and transcription initiation sites remained deprotected at 144 h after treatment. Nuclear protein protection of other sites, particularly in the conserved LTR enhancer sequences, was minimally affected by CCl4 treatment. Three nuclear protein binding sites that showed rapid CCl4-induced kinetic changes were homologous to the consensus sequence for the binding of the transcription factor families MEF-2, HNF-1, and C/EBP. The complex kinetic changes in factors that may contribute to the rapid and transient induction of endogenous retroviral gene expression in the liver after CCl4 exposure are discussed.

Beta cell expression of endogenous xenotropic retrovirus distinguishes diabetes-susceptible NOD/Lt from resistant NON/Lt mice.

Endogeneous retroviral expression in beta cells is a feature of prediabetes in nonobese diabetic (NOD) mice. The purpose of this study was to characterize the class-specific pattern of retroviral gene expression in NOD/Lt beta cells versus a related, but diabetes-resistant strain, NON/Lt. Electron microscopic comparison of beta cells from both strains indicated low constitutive expression of the intracisternal type A (IAP) retroviral class. However, NOD beta cells, in contrast to NON beta cells, expressed an additional intracisternal retroviral form resembling a type C particle. Antibodies against both IAP and type C were detected in NOD, with the humoral response to type C, but not IAP, preceding decline in beta cell function. RNA was extracted from freshly isolated islets from NOD and NON males. Comparative Northern blot analysis of total type C retroviral gene expression using a gag-pol DNA probe corroborated expression of endogenous type C proviruses in both NOD and NON islet cells and thymus. Use of class-specific retroviral probes identified the class of expressed endogenous retrovirus distinguishing the two inbred strains. The single ecotropic provirus present in both the NOD and NON genome (Emv-30) was not expressed in islets or thymus of either strain. Comparison of endogenous xenotropic provirus content by Southern blot analysis revealed two unique xenotropic loci (Xmv-65, -66) in NOD; 8.4 and 3.0 kb xenotropic envelope (env) RNA transcripts were detected in NOD, but not NON islets and thymus. NON contained three xenotropic loci common to other inbred strains (Xmv-21, -25, and -28). Both strains were partially characterized for content of recombinant (polytropic and modified polytropic) proviruses. IAP RNA expression was common to both NOD and NON islets and hence could not be specifically associated with the unique intracisternal type C particle found in NOD, but not NON beta cells. In conclusion, this study shows that expression of xenotropic type C but not IAP distinguishes retroviral activity in NOD/Lt versus NON/Lt beta cells. The potential pathogenic role of retroviral gene expression in NOD beta cells is discussed.

Biologically selected recombinants between Feline Leukemia Virus (FeLV) subgroup a and an endogenous FeLV element

In efforts to elucidate the proximal leukemogens that might be produced during a feline leukemia virus (FeLV) infection of cats, homologous recombinations between molecularly cloned exogenous and endogenous FeLV proviruses of known sequences were examined in cell cultures in vitro. A plasmid containing an infectious member of the most commonly occurring FeLV subgroup (FeLV subgroup A or FeLV-A) was coexpressed with noninfectious constructs containing the envelope ( env) gene of an endogenously inherited FeLV-like feline genomic element in transfected feline fibroblasts. The viruses generated were selected for their ability to propagate in human cells which are resistant to infection by the parental ecotropic FeLV-A or the noninfectious endogenous constructs. An analysis of the recombinants thus derived identified a limited number of sites in the env gene which were preferentially utilized in the generation of recombinant FeLVs under the selection conditions used. These sites were clustered in the surface glycoprotein (SU) moiety of the env gene, and it appeared that most, but not all, of the SU gene product of FeLV-A, beginning from the N-terminus, can be replaced by sequences from an endogenous element, still allowing the virus to be biologically viable. In fact, these substitutions in the env gene expanded infectivity of the parental FeLV-A from ecotropic to polytropic cell tropism. Additionally, substitutions in the SU region yielded many recombinants in which a primary neutralizing pentapeptide epitope of FeLV-A was altered because of its variance in the endogenous element. In several of the recombinants, this sequence was also found to be frequently mutated. Consistent with the changes identified in this antibody-binding domain, the recombinant viruses were only weakly inhibited by a monoclonal antibody directed against this epitope, while FeLV-A was highly sensitive to neutralization.