Characterization of a novel murine retrovirus mixture that facilitates hematopoiesis.

A new virus previously arose in BALB/c females mated repeatedly to C57BL/6 (B6) males and then injected with fixed, activated B6 male spleen cells (V. S. Ter-Grigorov, O. Krifuks, E. Liubashevsky, A. Nyska, Z. Trainin, and V. Toder, Nat. Med. 3:37-41, 1997). In the present study, BALB/cJ mice inoculated with virus-containing plasma from affected mice developed splenomegaly, which was caused by increased numbers of Sca-1(+) Lin(-) hematopoietic stem cells (HSC) and their differentiated progeny. Biological and molecular analyses of a new virus revealed a mixture of murine leukemia viruses (MuLVs). These MuLVs comprised ecotropic and mink lung cell focus-forming (MCF) virus classes and are termed Rauscher-like MuLVs because they bear numerous similarities to the ecotropic and MCF viruses of the Rauscher MuLV complex but do not include a spleen focus-forming virus. The ecotropic virus component alone transferred some disease characteristics, while MCF virus alone did not. Thus, we have described a novel virus mixture, termed Rauscher-like MuLV, that causes an increase in hematopoiesis due to activation of pluripotent HSC. Experiments using mice and a protocol that replicated the pregnancy and immunization strategy of the original experiment demonstrated that endogenous BALB/c mouse ecotropic and xenotropic MuLVs are activated by these treatments. Emv1 was expressed in the spleens of multiparous mice but not in those of virgin mice, and Bxv1Emv1-pseudotyped MuLVs were recovered following injection of fixed, activated B6 cells. Thus, multiple pregnancies and allostimuli appear to have provided the signals required for activation of and recombination among endogenous viruses and could have resulted in generation of the Rauscher-like MuLV mixture.

Retrovirally induced switch from production of IL-12 to IL-4 in dendritic cells.

Dendritic cells (DC) in HIV-1 infection show a reduced capacity to stimulate primary T cell proliferation. Exposure of bone marrow-derived DC to Rauscher leukemia virus (RLV) provides a mouse model for studying retrovirally induced reduction in stimulatory capacity for T cells. Treatment with IL-12, a cytokine that promotes the development of Th1 cells, has been postulated as a treatment for AIDS and is effective at restoring cell-mediated immunity in mice infected with mouse AIDS virus or with RLV (see Knight, S. C. and Patterson, S., Annu. Rev. Immunol. 1994. 15: 593-615 for references). Here we studied the direct effect of RLV and of IL-12 on bone marrow-derived DC. Normal DC produced IL-12 and IL-10 and stimulated primary allogeneic T cell proliferation. Exposure of DC to RLV caused reduced production of IL-12, production of IL-4 was seen in DC for the first time and T cell stimulation was inhibited. Addition of IL-12 reinstated and enhanced IL-12 synthesis in RLV-treated DC, abrogated production of IL-10 and IL-4 and restored stimulatory activity. Manipulation of cytokine production in DC could be a stratagem that has evolved in the retrovirus to avoid stimulation of cellular responses.

Interleukin-12 administration in retroviral infection of mice increases the potential to produce functional dendritic cells from bone marrow stem cells.

Rauscher leukaemia virus (RLV) infection in mice causes production of lymph node and skin dendritic cells (DC) that fail to stimulate a primary mixed leukocyte reaction (MLR). Treatment of mice with IL-12 around the time of infection results in DC with normal stimulatory function (N.J. Williams, J.J. Harvey, I. Duncan, R.F.G. Booth, S.C. Knight, Cell Immunol. 183 (1988) 121-130). Here we derived DC from mouse bone marrow by culture with granulocyte macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha) for 10-12 days; DC were generated from bone marrow cells taken from normal mice, from mice injected 15 days earlier with RLV or from those receiving RLV plus five daily doses of 100 ng of IL-12 starting 2 days before infection. Infection of the DC with RLV was assessed from nested PCR with doubling dilutions of DNA and the capacity of DC to stimulate a MLR was tested. DC derived from bone marrow of IL-12 treated animals showed at least twice the level of infection with RLV as those from non-treated animals although infection never exceeded 20% of the cells. DC derived from bone marrow of mice given RLV caused negligible stimulation of the MLR but those from mice additionally treated with IL-12 functioned normally. Thus, treatment of mice with IL-12 promoted the potential of stem cells taken 12 days after the last IL-12 injection to develop into functional DC despite increased infection with virus. Treatment of mice with IL-12 may have a long term effect on the potential growth of DC from stem cells which may contribute to the potency of this cytokine in promoting cell mediated immune responses.

Phylogenetic relationship of the complete Rauscher murine leukemia virus genome with other murine leukemia virus genomes.

We report the complete nucleotide sequence of the genome of Rauscher murine leukemia virus (R-MuLV), the replication-competent helper virus present in the Rauscher virus complex, and its phylogenetic relationship with other murine leukemia virus genomes. An overall sequence identity of 97.6% was found between R-MuLV and the Friend helper virus (F-MuLV), and the two viruses were closely related on the phylogenetic trees constructed from either gag, pol, or env sequences. Moloney murine leukemia virus (Mo-MuLV) was the next closest relative to R-MuLV and F-MuLV on all trees, followed by Akv and radiation leukemia virus (RadLV). The most distantly related helper virus was Hortulanus murine leukemia virus (Ho-MuLV). Interestingly, Cas-Br-E branched with Mo-MuLV on the gag and pol trees, whereas on the env tree, it revealed the highest degree of relatedness to Ho-MuLV, possibly due to an ancient recombination with an Ho-MuLV ancestor. In summary, a phylogenetic analysis involving various MuLVs has been performed, in which the postulated close relationship between R-MuLV and F-MuLV has been confirmed, consistent with the pathobiology of the two viruses.

Specificity of disease induced by M-MuLV chimeric retroviruses containing v-myc or v-src is not determined by the LTR.

The long terminal repeats (LTRs) of defective Moloney Murine Leukemia Virus (M-MuLV) containing the avian v-myc and v-src oncogenes were exchanged for LTRs from murine retroviruses inducing myeloid and erythroid disease, in an attempt to retarget disease specificity. Chimeric MuLVs containing either the Myeloproliferative sarcoma virus or the Rauscher mink cell focus-inducing virus LTRs induced the same disease as the parental viruses, suggesting that for these viruses the v-myc and v-src oncogenes are the major determinants in the disease specificity. However, substitution of the LTRs did affect the efficiency of tumorigenesis.

Peptide-induced conformational changes in class I heavy chains alter major histocompatibility complex recognition.

Small peptides, derived from endogenous proteins bind within the antigen binding groove created by the beta-pleated sheets and alpha helices of the alpha 1 and alpha 2 domains of the class I molecule of the major histocompatibility complex (MHC). However, the precise role of peptide in class I MHC conformation remains unclear. Here, we have shown that, in at least some instances, changes induced in the MHC molecule by the binding of distinct peptides can be identified as specific alterations in serological epitopes expressed on the class I protein. The nature of specific peptides expressed by class I-bearing cells may, therefore, have a dramatic influence on T cell development, self-tolerance, and alloreactivity.

Effects of ecotropic murine retroviruses on the dual-function cell surface receptor/basic amino acid transporter.

The widely expressed Na(+)-independent transporter for basic amino acids (system y+) is the cell surface receptor (ecoR) for ecotropic host-range mouse retroviruses (murine leukemia viruses (MuLVs)), a class of retroviruses that naturally infects only mice or rats. Accordingly, expression of mouse ecoR cDNA in mink CCL64 fibroblasts yields cells (CEN cells) that have y+ transporter activity above the endogenous background and that bind and are infected by ecotropic MuLVs. The effect of ecotropic MuLV infection on expression of y+ transporter was analyzed in mouse and in mink CEN fibroblasts. Chronic infection with ecotropic MuLVs caused 50-70% loss (down-modulation) of mouse y+ transporter in plasma membranes, detected as a reduced Vmax for uptake and outflow of L-[3H]arginine with no effect on Km values. Down-modulation was specific for mouse y+ and did not affect other transporters or the endogenous mink y+, suggesting that it results from specific interaction between mouse y+ and the viral envelope glycoprotein gp70 in the infected cells. Because this partial loss of mouse y+ from cell surfaces is insufficient to explain the complete interference to superinfection that occurs in cells chronically infected with ecotropic MuLVs, alternative explanations for interference are proposed. In contrast to the y+ down-modulation caused by chronic infection, binding of extracellular envelope glycoprotein gp70 at 37 degrees C resulted in noncompetitive inhibition of amino acid import by mouse y+ but had no effect on export through this same transporter or on any transporter properties of mink y+. The effects of gp70 on transport kinetics suggest that it slows the rate-limiting step of the amino acid import cycle, a conformational transition of the empty transporter in which the binding site moves from the inside back to the outside of the cell, and that gp70 has no effect on the rate-limiting step of the amino acid export cycle. Infected cells retain substantial y+ activity. Moreover, the virus binding site on ecoR is in a mobile region that changes conformation during the amino acid transport cycle.

Mapping the orientation of an antigenic peptide bound in the antigen binding groove of H-2Kb using a monoclonal antibody.

The major histocompatibility complex class I molecules are receptors for intracellular peptides, both of self and non-self origin. When non-self peptides (eg., pathogen derived) are bound to the class I molecules, they form ligands for T cell receptors resulting in antigen specific lysis of the infected cells by cytotoxic T lymphocytes. Therefore, an understanding of the process of antigen recognition requires the precise definition of the structural features of the bimolecular complex formed by a single well defined antigenic peptide bound to the class I molecule. A strategy using antibodies was developed to probe the structural features of the H-2Kb containing a defined peptide in the antigen cleft. We report that the binding surface area of a Kb specific monoclonal antibody (28-13-3s) includes residues in the alpha 1 (Gly56 and Glu58) and alpha 2 (Trp167) helices of Kb thus, binding across the antigen binding groove. When cells treated with the antigenic peptide of vesicular stomatitis virus, N52-59, and its alanine substituted analogs were tested for 28-13-3s binding, it was found that position 1 of the peptide also forms a part of the antibody binding site. This finding strongly supports the positioning of the N-terminus of N52-59 proximal to pocket A, thus, assuming an orientation parallel to the alpha 1 helix.

Peptide selection by MHC class I molecules.

Synthetic peptides have been used to sensitize target cells and thereby screen for epitopes recognized by T cells. Most epitopes of cytotoxic T lymphocytes can be mimicked by synthetic peptides of 12-15 amino acids. Although in specific cases, truncations of peptides improves sensitization of target cells, no optimum length for binding to major histocompatibility complex (MHC) class I molecules has been defined. We have now analysed synthetic peptide captured by empty MHC class I molecules of the mutant cell line RMA-S. We found that class I molecules preferentially bound short peptides (nine amino acids) and selectively bound these peptides even when they were a minor component in a mixture of longer peptides. These results may help to explain the difference in size restriction of T-cell epitopes between experiments with synthetic peptides and those with naturally processed peptides.

Inhibition of mouse retroviral disease by bioactive glutaminase-asparaginase.

Glutamine depletion strongly inhibits the replication of Rauscher murine leukaemia retrovirus (RLV) in vitro. Pseudomonas 7A glutaminase-asparaginase (PGA), capable of depleting glutamine and asparagine for prolonged periods, was used to determine the therapeutic effectiveness of glutamine depletion in mice infected with RLV or Friend virus. During PGA treatment of viraemic animals, serum reverse transcriptase activity fell to control levels and infected animals did not develop splenomegaly. The therapeutic results obtained with PGA compared favourably with those of azidothymidine given intraperitoneally at 30 mg/kg/day. Western blots performed on splenic tissue from control and treated animals indicated that glutamine depletion prevented readthrough of an amber codon at the gag-pol junction, stopping translation of viral mRNA at that point. Treatment of RLV-infected animals with PGA resulted in nearly a 200% increase in mean survival time even when therapy was initiated late in the course of the disease. To our knowledge, this is the first demonstration that a nutrient required for viral replication can be enzymically depleted in vivo to inhibit viral replication.

Retroviral nucleocapsid protein-metal ion interactions: folding and sequence variants.

Retroviral nucleocapsid proteins contain one or two proposed metal-binding sequences of the form Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys. Previously, we reported that an 18-amino acid peptide derived from the nucleocapsid protein of Rauscher murine leukemia virus (RMLV) binds metals such as Co2+ and Zn2+. We have now synthesized the entire nucleocapsid protein from RMLV. We report here that the protein also binds Co2+ and Zn2+ and does so with a higher affinity than does the peptide. Limited proteolysis and circular dichroism studies reveal that metal binding induces folding of the metal-binding domain and, perhaps, the regions adjacent to it but the remainder of the protein remains in a relatively unstructured state. In addition, we have synthesized sequence variants of the metal-binding domain that correspond to viral mutations reported in the literature. In many cases, the metal-binding properties of these peptides correlate with the observed biological activity, providing further evidence for the importance of metal binding to nucleocapsid function.

Spi-1 oncogene activation in Rauscher and Friend murine virus-induced acute erythroleukemias.

The Friend viruses, like the Rauscher virus, cause murine acute erythroleukemias which evolve in a similar multistep process. In previous studies it has been described that the late malignant proerythroblastic transformation induced by the polycythemia-inducing strain of Friend spleen focus-forming virus (SFFVP) is correlated with Spi-1 oncogene activation by insertional mutagenesis. In this paper we report that Spi-1 genomic rearrangements were also observed in 90% of tumors induced by the anemia-inducing strain of Friend spleen focus-forming virus (SFFVA) and in all Rauscher-induced tumors analyzed. SFFVA and Rauscher proviral insertions occurred in the viral integration cluster previously characterized in SFFVP-induced tumors. The Spi-1 1.4-Kb messenger RNA was found highly expressed in all SFFVA and Rauscher-induced malignant cells as compared to normal tissues. The nucleotide sequence of Spi-1 cDNA isolated from a library constructed from SFFVA-induced tumor cells revealed no difference between the Spi-1 gene transcripts expressed in both SFFVP and SFFVA-induced leukemic cells. These results indicate that Spi-1 gene activation is a general feature in the malignant proerythroblastic transformation which occurs in mice infected with Friend and Rauscher viruses.

Identification, using synthetic peptides, of the minimum amino acid sequence from the retroviral transmembrane protein p15E required for inhibition of lymphoproliferation and its similarity to gp21 of human T-lymphotropic virus types I and II.

Synthetic peptides containing portions of a highly conserved region of retroviral transmembrane proteins of human and animal retroviruses were tested for their ability to inhibit lymphoproliferation to determine the minimum amino acid sequence required. The previously reported immunosuppression mediated by the peptide CKS-17 was confirmed and further localized to a sequence of eight residues essentially identical to the sequence present in the transmembrane protein gp21 of human T-lymphotropic virus types I and II (HTLV-I and -II). To substantiate the physiological relevance of the inhibition of lymphoproliferation observed with the synthetic peptides and to relate this activity to the intact protein, we purified the Rauscher murine leukemia virus transmembrane protein p15E by immunoaffinity chromatography and report that this purified component presented in the form of protein micelles inhibited the interleukin-2-dependent proliferation of the murine T-cell line CTLL-2 in a dose-dependent manner, with a half-maximal inhibitory dose (ID50) of approximately 16 nM. In comparison, the ID50 concentration of a recombinant form of p15E required to inhibit lymphoproliferation was approximately 2.2 microM. The results reported here support the hypothesis that the transmembrane protein gp21 of HTLV-I and -II participates in the mechanism of immunosuppression previously reported for the transmembrane proteins of feline leukemia virus and other animal retroviruses. Thus, the transmembrane protein of HTLV-I, the etiological agent of adult T-cell leukemia-lymphoma, may be partially responsible for the immunocompromised clinical course of this disease that results in fatal opportunistic infections in a majority of cases.

Molecular cloning of the Rauscher murine leukemia virus. Disease spectrum and integration pattern.

After transfection of NIH 3T3 cells with DNA from molecularly cloned Rauscher MuLV, virus was isolated which showed a disease spectrum comparable to that of R-MuLV cloned biologically by endpoint dilution. In both cases sites of proviral integration vary from 2-5 per leukemic tissue and occur apparently at random.

Oncogene expression in Rauscher murine leukemia virus induced erythroid, myeloid and lymphoid cell lines.

A comparative study on the expression of nuclear and cytoplasmic oncogenes was carried out using the Northern blotting technique, in Rauscher virus induced primary leukemias and the more malignant transformed cell lines derived from them. The latter grow permanently in vitro. Hyperplastic spleens obtained from mice recovering from anemia were analysed as controls. In addition to the detection of mRNAs, Southern blotting was carried out to observe whether rearrangement or amplification of oncogenes had occurred. The results show that the nuclear oncogenes c-myc, c-myb and p53 are strongly expressed in leukemic tissue, whereas c-fos transcripts show a much weaker hybridization. The expression of two of these oncogenes, c-myc and c-myb was followed during differentiation in myeloid leukemic cells and showed a gradual decrease when compared with the actin gene, which is constitutively transcribed. A large number of cytoplasmic oncogenes is expressed in the leukemic cells lines, i.e. c-abl, c-fms, c-fes, c-src, c-ros, c-H-ras, c-K-ras and N-ras. Of these, transcripts coding for c-abl and c-src were absent in blast cells of acute erythroid leukemias. Transcripts coding for c-erb, c-mos and c-sis could also not be detected. A number of putative oncogenes which are reported to play a role in Moloney and Friend virus induced leukemias for instance pim-1, fis-1, fim-1 and fim-2 were also used for screening. Only expression of pim-1 in Rauscher virus induced myeloid leukemic cells and in primary acute erythroid leukemias could be observed. At the DNA level no rearrangement or amplification of any of the oncogenes investigated could be detected. The results show that a number of oncogenes are expressed simultaneously in the same leukemic tissue or cell lines. It therefore seems likely that the presence of transcripts of different oncogenes is associated with the progression of leukemia, but is not the primary cause of leukemogenesis or of the transformation of these cells into established cell lines.

Nucleotide sequence of the Rauscher murine leukaemia virus long terminal repeat.

The long terminal repeat (LTR) of Rauscher murine leukaemia virus (MuLV) has been sequenced. It differs in only three positions from the LTR of Rauscher spleen focus-forming virus (SFFV), and in four positions from the LTR of Rauscher mink cell focus-inducing virus (MCFV). It is unlikely that these differences account for differences in leukaemogenicity or tissue tropism of Rauscher MuLV, SFFV and MCFV. In contrast to the LTR of Friend MuLV, the Rauscher MuLV LTR contains only one copy of a tandem direct repeat. This repeat includes an enhancer core sequence.

Specific sequences of the env gene determine the host range of two XC-negative viruses of the Rauscher virus complex.

Two viruses which do not give rise to XC plaques in the standard XC assay (XC-negative) have been isolated from the Rauscher virus (RV) complex. These viruses differ in their host range. One, R-MCF-1, is dualtropic and will therefore infect both murine and non-murine cells. However, unlike other mink cell focus-inducing (MCF) viruses, it cannot infect NIH 3T3 cells. The other, R-XC-, is ecotropic. It will infect murine cells, including NIH 3T3 cells, but does not infect mink lung cells. Analysis of hybrid viruses, in which homologous regions of the genomes of R-MCF-1 and R-XC- virus were exchanged, indicated that the NH2-terminal portion of the gp70 is responsible for the particular host ranges of these viruses. The nucleotide sequence of the env gene of R-XC- virus was therefore determined and compared with the known env sequences of ecotropic MLVs and dualtropic MCF viruses of the Rauscher and Friend virus complexes. R-XC- virus was found to be a recombinant virus. Its env gene contained sequences derived from an endogenous env gene which were closely related to those of the MCF viruses but differed from any previously described sequences. The particular properties of R-MCF-1 and R-XC- virus suggest that the two viruses arose by recombination between R-MLV and two endogenous env sequences which differ from those of the known MCF viruses. If so, this suggests that the mouse genome contains at least five env sequences which can give rise to MCF-like viruses. In addition, since the host range and interference properties of R-XC- virus are very similar to those of the previously described ecotropic recombinant viruses, it may be that the ecotropic recombinant viruses arose by recombination with the same endogenous env sequences as did R-XC- virus.

[Molecular cloning of MuLV proviruses integrated into the genome of mouse erythroleukemia cells. I. Characteristics of endogenous proviruses]. / Molekuliarnoe klonirovanie provirusov MuLV, integrirovannykh v genom myshinykh éritroleikoznykh kletok. I. Kharakteristika provirusov éndogennogo proiskhozhdeniia.

The library of genes was obtained from erythroleukemic AKR cells (C-1), that were maintained as suspension culture. Thirty four clones that had homology with 60-70S RNA of Rauscher Leukemia virus (RLV) were separated from this library. The restriction mapping was carried out with 14 clones, that contained most extensive proviral sequences. One clone (107) contains proviral sequences that are derived from one of the components of the RLV complex. The other 13 clones contain sequences of endogenous xenotropic viruses. The endogenous retroviral sequences obtained differ in restrictive maps from proviruses of ecotropic and xenotropic infectious endogenous MuLV and, apparently, might be attributed as non-inducible infectious xenotropic MuLV of class III. Some of the cloned retroviral sequences had symmetrical structure, that is typical for integrated proviruses, i. e. these sequences were separated from flanking cellular ones by long terminal repeats. All investigated retroviral sequences are deletion mutants of MuLV proviruses. It was shown that the inner regions of proviruses diverged more than the long terminal repeats. The expression of the main inner MuLV polypeptide (p30) was detected in NIH 3T3 cells, transfected with DNA of some clones.

Envelope gene and long terminal repeat determine the different biological properties of Rauscher, Friend, and Moloney mink cell focus-inducing viruses.

The nucleotide sequence of the envelope (env) gene and the long terminal repeat (LTR) of an infectious clone of Rauscher mink cell focus-inducing (R-MCF) virus has been determined and compared with the published env gene and LTR sequences of Friend (F)- and Moloney (M)-MCF viruses. The sequence shows that R-MCF virus, like other MCF viruses, is a recombinant virus. Its env gene contains sequences which were acquired from an env gene in the mouse genome and which confer on the MCF virus its dualtropic host range. Unlike F-MCF and M-MCF viruses, R-MCF virus will not replicate in NIH 3T3 cells. The deduced amino acid sequence for the gp70 of R-MCF differs from that of F- and M-MCF viruses by 15 amino acids between residues 49 and 138 of gp70. These differences in amino acid sequences may be responsible for the inability of R-MCF virus to replicate in NIH 3T3 cells. The host range of two hybrid viruses constructed in vitro is consistent with this hypothesis. R-MCF virus and Friend murine leukemia virus (F-MLV) show 98% identity in their env gene 3' from the acquired env sequences. This contrasts with 82% identity between the env gene of R-MCF virus and M-MLV. The LTR of R-MCF shows 98% identity with the LTR of F-MCF as compared to 88% identity with the LTR of M-MCF. This striking similarity between the sequences of R-MCF, F-MCF, and F-MLV is surprising since the Rauscher virus and the Friend virus are thought to have originated independently. The high degree of similarity suggests that Rauscher and Friend viruses have a common origin. In contrast to M-MLV, which induces predominantly a lymphoid disease, R- and F-MCF viruses induce an erythroproliferative disease in NIH Swiss mice. A hybrid R-MCF virus with a genome derived primarily from R-MCF virus and a 3' end including the U3 region derived from M-MLV induces a lymphoid disease instead of an erythroid disease. This result indicates that it is the U3 region which determines the tissue specificity of the MCF virus-induced disease. It is suggested that the putative viral enhancers in the U3 region play two roles in the process of leukemogenesis: in the Friend and Rauscher disease, the viral enhancers act by increasing the transcription of the MCF env gene; in the thymic lymphoma, the enhancers activate mainly the expression of cellular genes.

[Molecular cloning of provirus sequences of Rauscher leukemia virus from mouse erythroleukemia cell genome]. / Molekuliarnoe klonirovanie provirusnykh posledovatel'nostei leikoza Raushera iz genoma myshinykh éritroleikoznykh kletok.

Provirus from a component of Rauscher leukaemia virus (RLV) has been cloned. The provirus (the size of 5000 b. p.) contains two LTR sequences and shares expressed sequence homology with Mo-MuLV. Restriction analysis and determination of the LTR nucleotide sequence and of the site from 3'-end of proviral genome have shown the cloned provirus to be the SFEV component of RLV. LTR from this cloned provirus contains all sites necessary for transcription: CAAT and TATA sequences, "cap" site and polyadenylation signal. The LTR of the cloned provirus from SFEV component of RLV has been shown to function as a promoter in E. coli cells.