Cytolytic T lymphocyte-defined retroviral antigens on normal cells: Encoding by the Akv-1 proviral locus

We previously described the generation and specificity of H-2-restricted cytolytic lymphocytes (CTL) directed against tumors induced by AKR/Gross murine leukemia viruses (MuLV). Such anti-AKR/Gross virus CTL demonstrated type specificity; only tumors induced by endogenous MuLV that expressed the Gross cell-surface antigen were lysed. These CTL and their precursor also recognized normal spleen cells from AKR-H-2 ^b , but not AKR-H-2 ^b , Fv-1 ^b mice, however, suggesting that N-ecotropic, retrovirus-associated antigens were primarily involved. Here, expression of these CTL-defined retroviral antigens by H-2^b-positive AKR × C57L recombinant inbred strains was examined by using normal spleen cells as stimulators in the generation of specific anti-AKR/Gross virus CTL. Analysis of the strain distribution pattern of stimulation indicated that a single proviral locus, Akv-1, was primarily, if not entirely, responsible for CTL-defined retroviral antigen expression. The lack of correlation with two other well-defined proviral loci was interesting. Whereas Akv-3 is known to encode a defective virus, Akv-4 has been shown to code for an infectious virus thought to be very similar or identical to that of Akv-1. Although quantitative differences cannot be formally excluded, dose response experiments argued against this possibility and suggested that Akv-1 and Akv-4 may exhibit qualitative differences germane to antiviral CTL recognition.     

T1 oligonucleotide maps of N-, B-, and B leads to NB-tropic murine leukemia viruses derived from BALB/c.

We previously described and characterized RNase T1 RNA fingerprints of an N-, a B-, and five B leads to NB-tropic murine leukemia viruses derived from BALB/c mice (Faller and Hopkins, J. Virol. 23:188-195, 1977, and J. Virol. 24:609-617, 1977). These viruses share the majority of their large RNase T1-resistant oligonucleotides, but each possesses some "unique" oligonucleotides relative to the others. We have ordered the large T1-resistant oligonucleotides of the N-, the B-, and one NB-tropic virus relative to the 3' end of their genomes to obtain oligonucleotide maps. These maps indicate that (i) the large T1 oligonucleotides shared by the N-, B-, and NB-tropic viruses probably occupy the same relative positions on their genomes; (ii) the 14 T1 oligonucleotides that differ between the N- and B-tropic viruses are derived from regions scattered along the genomes; and (iii) an oligonucleotide that is present in five NB-tropic viruses but not in their B-tropic virus progenitors lies toward the 5' end of the NB-tropic virus oligonucleotide map.     

Emv-13 (Akv-3): a noninducible endogenous ecotropic provirus of AKR/J mice

All AKR/J mice carry at least three endogenous ecotropic viral loci which have been designated Emv-11 (Akv-1), Emv-13 (Akv-3), and Emv-14 (Akv-4) (Jenkins et al., J. Virol. 43:26-36, 1982.) Using two independent AKR/J-derived sets of recombinant inbred mouse strains, AKXL (AKR/J x C57L/J) and AKXD (AKR/J x DBA/2J), as well as the HP/EiTy strain (an Emv-13-carrying inbred strain partially related to AKR/J mice) (Taylor et al., J. Virol. 23:106-109, 1977), we have examined the association of these endogenous viral loci with virus expression. Strains which transmit Emv-11 or Emv-14 or both were found to produce virus spontaneously, whereas strains that transmit Emv-13 alone were negative for virus expression. Restriction endonuclease digestion and hybridization with an ecotropic virus-specific hybridization probe of DNAs from strains which transmit only Emv-13 yielded enzyme cleavage patterns identical to those observed with DNAs from strains transmitting Emv-11 or Emv-14 or both. These findings indicate the absence of any gross rearrangement of Emv-13 proviral sequences. Cell cultures derived from recombinant inbred strains that carry only Emv-13 failed to express detectable infectious virus, viral proteins, or cytoplasmic ecotropic virus-specific RNA even after treatment with 5-iodo-2-deoxyuridine or 5-azacytidine, an inhibitor of DNA methylation. Our results indicate that a mechanism(s) other than methylation of Emv-13 proviral DNA is responsible for inhibition of Emv-13 expression.     

Linkage of the Igl-1 structural and regulatory genes to Akv-2 on chromosome 16

Evidence is presented here for a close linkage between Akv-2, an ecotropic provirus found uniquely on chromosome 16 of AKR/N mice, and the immunoglobulin ₁ light chain locus, Igl-1. No recombinants between the Igl-1 locus and Akv-2 were found by Southern blot analysis of DNA obtained from progeny of the backcross of (AKR/N × SJL/J)F₁ to SJL/J, indicating that these genes map within 5.9 cM of each other. A probe specific for the flanking sequence of Akv-2 was used to detect the provirus, while one specific for the IgI-1 constant region was used to determine which allele of the structural gene was expressed in the backcross mice. The constant region of Igl-1 differs between AKR/N and SJL/J with respect to a site for the restriction endonuclease KpnI. This backcross was also used to seek recombinants between the regulatory, Igl-1r, and structural, Igl-1, loci of the immunoglobulin light chain locus, since the existence of such recombinants would prove that these loci are distinct. Since only parental types were recovered in the offspring, the structural and regulatory loci are no more than 2.3 cM apart, and the implications of this finding are discussed.Deceased.     

Phenotypic alteration in retroviral gene expression by leukemia-resistant thymocytes differentiating in leukemia-susceptible recipients

(AKR x NZB)F1 mice possess the dominant genes, Akv-1, Akv-2, Nzv-1a and Nzv-2a, which determine the expression of ecotropic and xenotropic viruses. Nevertheless, their thymic lymphocytes fail to produce these agents, and these mice are resistant to leukemia. We investigated the mechanism of this cell-specific restriction in radiation chimeras. (AKR x NZB)F1 thymocytes that had differentiated in lethally irradiated AKR recipients produced high levels of ecotropic and xenotropic viruses and showed marked amplification of MuLV antigen expression. Polytropic viruses could also be isolated from such thymocytes. These virological changes in chimeric thymocytes were donor- and host-specific and occurred only when (AKR x NZB)F1 bone marrow cells were inoculated into AKR recipients. This inductive capacity of the host environment could be detected in irradiated AKR recipients as early as age 2 months. The phenotypic changes brought about in leukemia-resistant (AKR x NZB)F1 thymocytes by the leukemia-susceptible AKR thymic microenvironment may be the result of a three-component inductive system.     

Genetic alterations of RNA leukemia viruses associated with the development of spontaneous thymic leukemia in AKR/J mice.

T1-oligonucleotide fingerprinting and mapping were used to study the expression of RNA leukemia viruses in leukemic and preleukemic AKR/J mice, with techniques designed to minimize the loss or inadvertent selection of viruses in vitro before biochemical analysis. In leukemic animals, complex mixtures of ecotropic and mink-tropic viruses were expressed. Unique but similar polytropic virus-like genomes were present in each tumor isolate. In preleukemic mice, viral isolates from the thymus that were grown on NIH3T3 fibroblasts contained genomes with non-Akv polytropic virus-related oligonucleotides. This phenomenon was not evident in fingerprints of viruses from the spleen and bone marrow of the same animals. Remarkably, the non-Akv oligonucleotides located in the 3' portion of the P15E gene, the U3 noncoding region, and the 5' part of the gp70 gene were often expressed independently. Our results suggest the following. (i) Recombinant viruses can be detected in the thymuses of young preleukemic AKR mice and increase in relative abundance with age. (ii) During in vivo generation of the recombinant leukemogenic viruses, the selection of polytropic virus-related sequences in the 3' part of p15E and the U3 region and the 5' portion of gp70 occurs independently. (iii) Independent biological properties encoded in the gp70 and p15E regions of env of the recombinant viruses may mediate viral selection or leukemogenicity. (iv) The leukemogenic polytropic viruses of AKR/J mice arise via genetic recombination involving at least three endogenous viral sequences.     

RNase T1-resistant oligonucleotides of an N- and a B-tropic murine leukemia virus of BALB/c: evidence for recombination between these viruses.

We used two-dimensional gel electrophoresis to obtain fingerprints of 32P-labeled RNase T1-resistant oligonucleotides derived from the genomes of an N- and a B-tropic murine leukemia virus of BALB/c. These viruses share approximately 30 large T1-resistant oligonucleotides. In addition, there are eight large oligonucleotides unique to the N-tropic virus, and there are six B-trophic virus-specific oligonucleotides. Viruses, designated XLP-N, which appear by biological criteria and analysis of virion proteins to be recombinants between these N- and B-tropic viruses, possess some but not all of the N or B virus-specific oligonucleotides.     

Chromosomal assignment of two endogenous ecotropic murine leukemia virus proviruses of the AKR/J mouse strain.

The AKR/J mouse strain is genetically fixed for three different ecotropic murine leukemia virus genomes, designated Akv-1, Akv-3, and Akv-4 (Emv-11, Emv-13, and Emv-14). With recombinant inbred strains and crosses with linkage-testing stocks, Akv-3 and Akv-4 were placed on the mouse chromosome map. Akv-3, which encodes a replication-defective provirus, maps near the agouti coat color locus, a, on chromosome 2. Akv-4, which is replication competent, maps near the neurological mutant gene locus trembler, Tr, on chromosome 11. Akv-1 and Akv-2 (Emv-12), an ecotropic provirus carried by AKR/N but not AKR/J, have previously been mapped to chromosome 7 and 16, respectively. Thus, the four Akv proviruses mapped to date are on four different chromosomes. Akv-3 is the second ecotropic murine leukemia virus provirus to be mapped near the agouti locus. The results are discussed in relation to possible nonrandomness of viral integration.     

RNase T1-resistant oligonucleotides of B-tropic murine leukemia virus from BALB/c and five of its NB-tropic derivatives.

We used two-dimensional gel electrophoresis to obtain fingerprints of RNase T1-resistant oligonucleotides of a B-tropic murine leukemia virus from BALB/c and five NB-tropic viruses independently derived from this B virus by passage through NIH Swiss mouse embryo cells in vitro. The fingerprints of the B- and NB-tropic viruses were very similar: approximately 33 of 35 large T1-resistant oligonucleotides appeared to be shared by these viruses. However, the five NB-tropic viruses possessed an apparently common alteration relative to their B virus progenitor. This change involved the acquisition of one oligonucleotide and, tentatively, the loss of one oligonucleotide. We do not know whether these changes represent an alteration responsible for the change from B- to NB-tropism. Fingerprints of B- and NB-tropic viruses were not affected when the viruses were grown in cells of different Fv-1 type.     

Comparative analysis of the genomes of feline leukemia viruses.

The genomes of several strains of feline leukemia virus (FeLV) were compared by two-dimensional polyacrylamide gel electrophoresis of the large RNase T1-resistant oligonucleotides of the 70S RNA. Differences between each strain of FeLV tested were detected by this method. We estimate that the degree of sequence identity between the viruses is: FeLV A (Glasgow-1) to FeLV B (Snyder-Theilen), 52%; FeLV A (Glasgow-1) to FeLV C(Sarma), 66%; FeLV B(Snyder-Theilen) to FeLV C (Sarma), 37%. The fingerprints of two independent isolates of FeLV strains of subgroup A (Glasgow-1 and Rickard) were detectably different. We conclude that the RNase T1 oligonucleotide fingerprint pattern provides a useful tool for identification of FeLV strains.     

Characterization of genome structure of amphotropic and ecotropic wild mouse retroviruses.

We studied the RNA genomes of several wild mouse type C retroviruses by using RNase T1-oligonucleotide fingerprinting. The amphotropic and ecotropic viruses of field strain 1504 produced very similar oligonucleotide fingerprints, but each also had several unique oligonucleotides. All of these unique oligonucleotides were located in the env gene region and were probably responsible for the host range differences between these viruses, as well as the lymphomagenic and paralytogenic properties of the viruses. We obtained similar results with the amphotropic and ecotropic viruses of another field strain (4070), which was isolated from a mouse from a different trapping area. The amphotropic viruses of several field strains (strains 1504, 4070, and 1313) were more closely related than the ecotropic viruses of different strains (strains 1504, 4070, and 4996). These findings suggested that the genetic sequences of the amphotropic viruses are more conserved than those of ecotropic viruses isolated from the same wild mice.     

Structure of retroviral RNAs produced by cell lines derived from spontaneous lymphomas of AKR mice.

The retrovirus expression of eight independent lymphoid cell lines derived from spontaneous thymomas of AKR mice was investigated. The RNase T1 fingerprints of viral 70S RNA produced by these cell lines were compared with genome structures of the non-leukemogenic Akv virus and with two types of cloned leukemogenic viruses derived from one of the thymoma cell lines. Viral RNAs from three cell lines, SL3, 4, and 7, were indistinguishable from one another. The fingerprint patterns indicated that these cell lines produce equal amounts of two prototype, leukomogenic SL viruses that were previously isolated from the SL3 cell line. Viral RNA produced by the SL1 and SL2 cell lines contained similar components, but at a different ratio. Two other cell lines (SL5 and SL11) produced viral RNAs that resemble those of AKR mink cell focus-forming viruses. One additional line, SL9, produced viral RNA of a novel structure. The complex pattern of viral RNA expression observed for these lymphoid cell lines can be interpreted in terms of recombination among three types of endogenous viral sequences: the Akv virus, a xenotropic virus, and an SL (for spontaneous leukemia) virus.     

Relationship of retroviruses isolated from human leukemia tissues to the woolly monkey-gibbon ape leukemia viruses.

Retroviruses have been isolated from the tissues of human leukemia patients. Previous studies have shown that these isolates share some antigenic determinants with the family of viruses isolated from the woolly monkey and gibbon ape and that they exhibit partial nuclei acid homology with this same group of viruses. We have compared the RNAs of the viruses by two-dimensional polyacrylamide gel electrophoresis of the large RNase T1-resistant oligonucleotides. The degree of sequence identity between the RNAs was determined by the similarity of their RNase T1-resistant oligonucleotide pattern on gels, fingerprints, and in some cases by partial sequence analysis of individual oligonucleotides. This technique permits us to determine the degree of sequence identity among related RNA species. From our studies we conclude that viruses isolated from the tissues of two human leukemia patients, A1476 and SKA 21-3, as well as some subcultures of a virus isolated from the leukemic tissues of a third patient, HL23V, are closely related to the wooly monkey virus. However, the fingerprints of other HL23 viral isolates are very similar to that of GaLVSF, a gibbon ape leukemia virus isolated from a lymphosarcoma.     

Induction of anti-AKR/gross virus cytolytic T lymphocytes in AKR.H-2b:Fv-1b congenic mice: age-dependent conversion to a nonresponder phenotype

Previously, we reported that the generation of cytolytic T lymphocytes (CTL) specific for syngeneic tumors induced by AKR/Gross leukemia viruses was under multi-gene control. Thus, although carrying the required immune response gene(s) encoded by the H-2b haplotype and characteristic of responder strains such as C57BL/6, AKR.H-2b congenic mice failed to mount antiviral CTL responses. Young adult AKR.H-2b:Fv-1b "doubly congenic" mice, however, were able to generate specific anti-AKR/Gross virus CTL activity. These results demonstrated that the positive effect of MHC-encoded immune response gene control could be overcome by the action of the Fv-1n allele. The responder status of the B6.Fv-1n congenic, however, indicated that this Fv-1n-mediated inhibition was dependent on the interaction of Fv-1n with another gene(s) encoded by the AKR background. The results of experiments performed with AKXL recombinant inbred mice further suggested that a single additional genetic locus, encoding the Akv-1 provirus, was necessary along with Fv-1n to cause inhibition of antiviral CTL generation. Here we show that the responsiveness of AKR.H-2b:Fv-1b mice is dependent on their age. Thus, with moderate aging these doubly congenic mice converted to a nonresponder status with respect to anti-AKR/Gross virus CTL production: 85% of mice less than or equal to 9 wk of age responded compared with 0% of mice greater than 9 wk old. As with nonresponder AKR.H-2b mice, an inverse correlation was observed between CTL responsiveness and the expression of CTL-defined viral antigens by normal cells. Namely, spleen cells from young AKR.H-2b:Fv-1b mice showed little or no expression of such viral antigens, whereas with moderate aging there was a steady increase in their display. These results are discussed with reference to possible mechanisms of unresponsiveness of AKR.H-2b vs moderately aged AKR.H-2b:Fv-1b mice, and with respect to the utility of this system as a model for naturally occurring retrovirus infections and the interactions of retroviruses with the immune system.     

"Fingerprinting" high molecular weight RNA by two-dimensional gel electrophoresis: application to poliovirus RNA.

Conditions are described under which complete RNase T1 digests of high molecular weight RNA can be separated into numerous components by two-dimensional gel electrophoresis. Small and large oligonucleotides (n = 1 - 2c0) can be resolved without losses. The procedure yields fingerprints which are diagnostic for a particular species of RNA and an index of its purity as will be shown for the genomes of poliovirus type 1 and 2.     

Class II polytropic murine leukemia viruses (MuLVs) of AKR/J mice: possible role in the generation of class I oncogenic polytropic MuLVs

We examined the frequency of occurrence of polytropic murine leukemia viruses (MuLVs) in the spleens and thymuses of preleukemic AKR/J mice from 1 week to 6 months of age and analyzed the genomic RNAs of several polytropic isolates by RNase T1 oligonucleotide fingerprinting. Polytropic MuLVs were first detected in the spleens of 3-week-old mice and preceded the appearance of polytropic MuLVs in the thymus by over 1 month. At 4 months of age and older, nearly all mice expressed polytropic MuLVs in both organs. In contrast to previous studies which have identified class I polytropic MuLVs in AKR/J mice, fingerprint analysis of polytropic MuLVs from both young (3- to 4-week-old) and older (5- to 6-month-old) preleukemic mice indicated that a large proportion of viruses at both ages were class II polytropic MuLVs. All polytropic viruses (five isolates) analyzed from 3- to 4-week-old mice were recovered from spleen cells and were class II polytropic MuLVs. In older preleukemic mice, five of seven isolates were class II polytropic MuLVs and two were class I polytropic viruses. Class I and class II polytropic MuLVs were recovered from both the spleens and thymuses of older preleukemic mice. A detailed comparison of the class I and class II polytropic MuLVs from 5- to 6-month-old mice revealed that the nonecotropic gp70 sequences of most of the class I and class II MuLVs were identical, consistent with a common origin for these sequences. In contrast, the nonecotropic p15E sequences of class I MuLVs were clearly derived from different endogenous sequences than the nonecotropic p15E sequences of the class II MuLVs. The in vitro host ranges of class I and class II polytropic viruses were clearly distinguishable. Examination of the in vitro host range of several isolates suggested that the predominant polytropic viruses initially identified in the thymus (2 to 3 months of age) were class II polytropic viruses. The order of appearance of the class I and class II polytropic MuLVs and the identity of the gp70 oligonucleotides of these MuLVs suggested a model for the stepwise generation of class I polytropic MuLVs involving a class II polytropic MuLV intermediate.     

Genetic control of CTL responses to AKR/Gross virus: effect of inheritance of Akv proviruses

Our earlier observations suggested that the AKR/Gross leukemia virus-specific C57BL/6 cytolytic T lymphocyte (CTL) response was directed to Akv-1, but not Akv-3 or Akv-4, provirus-associated determinants. Based on these data, the present experiments were performed with various AKXL RI mouse strains of the responder H-2 ^b haplotype which had inherited different combinations of the Akv-1, –3, and –4 proviruses, to determine whether these strains were able to mount specific antiviral CTL responses. In a comparison with control responder C57BL/6 mice, a clear pattern emerged. Akv-negative mice of the AKXL-29 strain were fully responsive, but five other AKXL strains which had inherited the Akv-1 provirus failed to mount significant antiviral CTL responses ( 10% of control). In contrast, an Akv-1-negative but Akv-4-positive strain (AKXL-5) was partially responsive (approximately 24 % of the C57BL/6 control). These results were consistent with a direct relationship between the Akv-1 provirus and the nominal antigens recognized by antiviral CTL, and with an inverse correlation between in vivo expression of viral antigens by normal cells and the ability to generate antiviral CTL. The possible mechanisms accounting for this unresponsiveness are discussed along with the utility of this system for investigating the interactions of retroviruses with the immune system.     

Oligonucleotide fingerprints of RNA species obtained from rhabdoviruses belonging to the vesicular stomatitis virus subgroup.

The relationships among the genomes of various rhabdoviruses belonging to the vesicular stomatitis virus subgroup were analyzed by an oligonucleotide fingerprinting technique. Of 10 vesicular stomatitis viruses, Indiana serotype (VSV Indiana), obtained from various sources, either no, few, or many differences were observed in the oligonucleotide fingerprints of the 42S RNA species extracted from standard B virions. Analyses of the oligonucleotides obtained from RNA extracted from three separate preparations of VSV Indiana defective T particles showed that their RNAs contain fewer oligonucleotides than the corresponding B particle RNA species. The fingerprints of RNA obtained from five VSV New Jersey serotype viruses were easily distinguished from those of the VSV Indiana isolates. Three of the VSV New Jersey RNA fingerprints were similar to each other but quite different from those of the other two viruses. The RNA fingerprints of two Chandipura virus isolates (one obtained from India and one from Nigeria) were also unique, whereas the fingerprint of Cocal virus RNA was unlike that of the serologically related VSV Indiana.     

Genomic complexities of murine leukemia and sarcoma, reticuloendotheliosis, and visna viruses.

The genetic complexities of several ribodeoxyviruses were measured by quantitative analysis of unique RNase T1-resistant oligonucleotides from 60-70S viral RNAs. Moloney murine leukemia virus was found to have an RNA complexity of 3.5 x 10(6) daltons, whereas Moloney murine sarcoma virus had a significantly smaller genome size of 2.3 x 10(6). Reticuleondotheliosis and visna virus RNAs had complexities of 3.9 x 10(6), respectively. Analysis of RNase A-resistant oligonucleotides of Rous sarcoma virus RNA gave a complexity of 3.6 x 10(6), similar to that previously obtained with RNase T1-resistant oligonucleotides. Since each of these viruses was found to have a unique sequence genomic complexity near the molecular weight of a single 30-40S viral RNA subunit, it was concluded that ribodeoxyvirus genomes are at least largely polyploid.     

Physical map of the Kirsten sarcoma virus genome as determined by fingerprinting RNase T1-resistant oligonucleotides.

From analysis of the large RNase T1-resistant oligonucleotides of Kirsten sarcoma virus (Ki-SV), a physical map of the virus genome was deduced. Kirsten murine leukemia virus (Ki-MuLV) sequences were detected in T1 oligonucleotides closest to the 3' end of the viral RNA and extended approximately 1,000 nucleotides into the genome. The rat genetic sequences started at this point and extended all the way to the very 5' end of the RNA molecules, where a small stretch of Ki-MuLV sequence was detected. By comparison of the fingerprints of Ki-SV RNA and the RNA of the endogenous rat src genetic sequences, it was found that more than 50% of the T1 oligonucleotides were similar between Ki-SV and the endogenous rat src RNA, suggesting an identical primary nucleotide sequence in over 50% of the viral genomes. The results indicate that Ki-SV arose by recombination between the 5' and 3' ends of Ki-MuLV and a large portion of the homologous sequences of the endogenous rat src RNA.