Transfection of molecularly cloned Friend murine leukemia virus DNA yields a highly leukemogenic helper-independent type C virus.

Unintegrated viral DNA was isolated via the Hirt procedure from mouse fibroblasts newly infected with Friend murine leukemia virus (F-MuLV) clone 201, a biologically cloned helper virus isolated from stocks of F-MuLV complex. A physical map of the unintegrated in vivo linear viral DNA was generated for several restriction endonucleases. The supercoiled viral DNA was digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized viral DNA was then inserted into lambda gtWES.lambda B at the EcoRI site and cloned in an approved EK2 host. Eight independent lambda-mouse recombinants were identified as containing F-MuLV DNA inserts by hybridization with F-MuLV 32P-labeled complementary DNA. One of the F-MuLV DNA inserts was 9.1 kilobases (kb) and had the same restriction enzyme sites as the unintegrated linear F-MuLV DNA. Six inserts were 8.5 kb; each lacked a single copy of the terminally redundant sequences of the unintegrated linear viral DNA. One insert was 8.2 kb and contained a 0.9-kb deletion. After digestion with EcoRI, one recombinant DNA preparation containing an 8.5-kb insert was infectious for NIH 3T3 cells. Undigested recombinant DNA was not infectious. The infectivity of the EcoRI-digested DNA followed multihit kinetics, indicating that more than one molecule was required to register as an infectious unit. The virus isolated from this transfection (F-MuLV-57) was NB-ecotropic, helper-independent, and formed XC plaques. Inoculation of this virus into newborn NIH Swiss mice induced leukemia and splenomegaly in greater than 90% of animals within 3 to 4 weeks. The gross and microscopic abnormalities induced by F-MuLV clone 57 were identical to those seen with the original parent stocks of F-MuLV clone 201. These results indicate that this helper-independent F-MuLV can induce a rapid nonthymic leukemia in the absence of the spleen focus-forming virus.     

Transplacental transmission of BK virus in human.

The prevalance and distribution of BK virus antibody in women during pregnancy and the occurrence of transplacental transmission of BK virus was determined by measurement of IgM antibody in the serum. Sera were collected from 63 nonpregnant women, 71 women who had experienced spontaneous abortion, 80 in the first trimester of pregnancy and the same 80 at delivery. Umbilical cord blood was also taken at delivery. Hemagglutination inhibition (HI) tests for BK virus used the micromethod of Gardner. Results indicate that a significant level of HI antibody was present in 70% of sera from all 4 experimental groups. This showed that BK virus infection was not limited to cases of spontaneous abortion. Of the 80 pregnant mothers, 6 showed a 4-fold or greater HI antibody seroconversion to BK virus after delivery. Of these 6 seroconversion patients, sensitive antibody was detected in 3 umbilical cords. Umbilical cords of those without seroconversion had no sensitive antibody. As evidenced by 2-NE-sensitive antibody, BK virus infections were also recognized in 6 of 71 women who aborted, 4 of 80 in the first trimester of pregnancy and 2 collected after delivery. The 2-ME-sensitive antibody was not found in any of 63 samples from nonpregnant women. Data indicate that 2-ME-sensitive antibody was present only in sera of women during pregnancy and after abortion. It may be possible that BK virus persists in a latent form in many healthy women and becomes activated during pregnancy.     

Prenatal transmission and pathogenicity of endogenous ecotropic murine leukemia virus Akv.

OBJECTIVE: Mouse strains carrying endogenous ecotropic murine leukemia viruses (MuLV) are capable of expressing infective virus throughout life. Risk of transplacental transmission of MuLV raises concerns of embryo infection and induction of pathogenic effects, and postnatal MuLV infection may lead to tumorigenesis. METHODS: Endogenous ecotropic MuLV-negative SWR/J embryos were implanted into Akv-infected viremic SWR/J mice, into spontaneously provirus-expressing AKR/J mice, and into noninfected SWR/J control mice; virus integration and virus expression were investigated at 14 days' gestation. Tumor development was monitored over 18 months. RESULTS: Of 111 embryos, 20 (18%) recovered from Akv-infected SWR/J mice, which had developed normally, were infected. New proviruses were detected in 10 of 111 (9%) embryos from Akv-infected SWR/J mice, and in 2 of 60 (3%) embryos from AKR/J mice; none expressed viral protein. Of 127 embryos recovered from Akv-infected SWR/J mice, 16 (13%) were dead; 4 of 5 (80%) were infected and expressed viral protein. Of 71 embryos from AKR/J mice, 11 (15%) were dead, and 2 of 2 had virus integration; virus expression was not detected. Numbers of dead embryos recovered from experimentally infected, viremic SWR/J mice and from spontaneously endogenous MuLV-expressing AKR/J mice were significantly higher, compared with numbers from nonviremic SWR/J control mice, and embryo lethality was significantly associated with prenatal provirus expression. Postnatal inoculation of Akv induced lymphoblastic lymphomas in 15 of 24 (61%) SWR/J mice within mean +/- SD latency of 14 +/- 2.4 months. Only 3 of 39 (8%) control mice developed lymphomas (P < 0.005). CONCLUSION: Embryos in MuLV-viremic dams are readily infected, and inappropriate prenatal expression of leukemogenic endogenous retroviruses may play a critical role in embryo lethality and decreased breeding performance in ecotropic provirus-positive mouse strains.     

Introduction of a cis-acting mutation in the capsid-coding gene of moloney murine leukemia virus extends its leukemogenic properties

Inoculation of newborn mice with the retrovirus Moloney murine leukemia virus (MuLV) results in the exclusive development of T lymphomas with gross thymic enlargement. The T-cell leukemogenic property of Moloney MuLV has been mapped to the U3 enhancer region of the viral promoter. However, we now describe a mutant Moloney MuLV which can induce the rapid development of a uniquely broad panel of leukemic cell types. This mutant Moloney MuLV with synonymous differences (MSD1) was obtained by introduction of nucleotide substitutions at positions 1598, 1599, and 1601 in the capsid gene which maintained the wild-type (WT) coding potential. Leukemias were observed in all MSD1-inoculated animals after a latency period that was shorter than or similar to that of WT Moloney MuLV. Importantly, though, only 56% of MSD1-induced leukemias demonstrated the characteristic thymoma phenotype observed in all WT Moloney MuLV leukemias. The remainder of MSD1-inoculated animals presented either with bona fide clonal erythroid or myelomonocytic leukemias or, alternatively, with other severe erythroid and unidentified disorders. Amplification and sequencing of U3 and capsid-coding regions showed that the inoculated parental MSD1 sequences were conserved in the leukemic spleens. This is the first report of a replication-competent MuLV lacking oncogenes which can rapidly lead to the development of such a broad range of leukemic cell types. Moreover, the ability of MSD1 to transform erythroid and myelomonocytic lineages is not due to changes in the U3 viral enhancer region but rather is the result of a cis-acting effect of the capsid-coding gag sequence.     

Genetic analysis of myeloproliferative leukemia virus, a novel acute leukemogenic replication-defective retrovirus.

The myeloproliferative leukemia virus (MPLV) is a new acute leukemogenic, nonsarcomatogenic retroviral complex that is generated during the in vivo passage of a molecularly cloned Friend ecotropic helper virus. Examination of viral RNA expression in MPLV-producing cells revealed the presence of two distinct molecular species that hybridized with a long terminal repeat or an ecotropic env-specific probe but not with a xenotropic mink cell focus-forming virus env-specific probe derived from a spleen focus-forming virus: an 8.2-kilobase species corresponding to a full-length Friend murine leukemia virus (F-MuLV) and a deleted species with a genomic size of 7.4 kilobases. This deleted virus was biologically cloned by limiting dilutions and single cell cloning in Mus dunni fibroblasts. Three nonproducer clones with normal morphologies and containing one single integrated copy of the deleted virus were superinfected with F-MuLV, Moloney murine leukemia virus, Gross murine leukemia virus, mink cell focus-forming virus (HIX), or the amphotropic 1504 murine leukemia virus. All pseudotypes caused macroscopic and microscopic abnormalities in mice that were similar to those seen in the parental stock. A comparison of the physical maps of F-MuLV and MPLV, which was deduced from the restriction enzyme digests of unintegrated proviral DNAs, indicated that the MPLV-defective genome (i) is probably derived from F-MuLV, (ii) has conserved the F-MuLV gag and pol regions, and (iii) is deleted and rearranged in the env region in a manner that is clearly distinct from that of Friend or Rauscher spleen focus-forming viruses.     

The leukemogenic potential of an enhancer variant of Moloney murine leukemia virus varies with the route of inoculation.

We previously showed that the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV) is poorly leukemogenic when inoculated subcutaneously (s.c.) into neonatal mice. We recently found that intraperitoneal (i.p.) inoculation of neonatal mice with the same virus significantly enhanced its leukemogenicity. In this study, infections of neonatal mice by the two different routes of inoculation were compared. We studied replication of the virus in vivo to identify critical preleukemic events. These would be observed in mice inoculated i.p. by Mo+PyF101 M-MuLV but not when inoculation was s.c. Infectious center assays indicated that regardless of the route of inoculation, Mo+PyF101 M-MuLV showed delayed infection of the thymus compared with wild-type M-MuLV. On the other hand, i.p.-inoculated mice showed more rapid appearance of infectious centers in the bone marrow than did s.c.-inoculated animals. Thus, the enhanced leukemogenicity of i.p. inoculation correlated with efficient early infection of the bone marrow and not with early infection of the thymus. These results suggest a role for bone marrow infection for efficient leukemogenesis in Mo+PyF101 M-MuLV-infected mice. Consistent with this notion, if bone marrow infection was decreased by injecting 10- to 12-day-old animals i.p., leukemogenicity resembled that of s.c. inoculation. Thus, two cell types that are critical for the induction of efficient leukemia were implicated. One cell delivers virus from the site of s.c. inoculation (the skin) to the bone marrow and is apparently restricted for Mo+PyF101 M-MuLV replication. The second cell is in the bone marrow, and its early infection is required for efficient leukemogenesis.     

Immunogenic determinants of a neuropathogenic murine leukemia virus.

Previous studies of Cas-Br-M murine leukemia virus (MuLV) (Cas-MuLV) infection demonstrated that cytotoxic T cells (CTL) of the CD8+ phenotype play a role in resistance to the neuropathogenic effects of the virus in NFS/N mice. In the current study, we sought to identify the Cas-MuLV epitopes that are immunogenic for the CTL response. Infection of adult NFS/N mice with a well-characterized neuropathogenic variant of Friend MuLV, PVC-211 MuLV (PVC-MuLV), was not immunogenic for MuLV-specific CTL. Therefore, we constructed chimeric viruses between Cas-MuLV and PVC-MuLV. Infectious chimeras contained the Cas-MuLV env gene on a PVC-MuLV background (PVC-CasenvMuLV) and the PVC-MuLV env gene on a Cas-MuLV background (Cas-PVCenvMuLV). Cas-MuLV-specific CTL were found following inoculation of both the chimeric viruses and the parental Cas-MuLV but not the parental PVC-MuLV, despite evidence of antibody responses to both parental and chimeric MuLV. CTL generated in response to infection with PVC-CasenvMuLV and Cas-PVCenvMuLV were exclusively of the CD8+ phenotype. These results indicate that both the env and gag-pol regions of Cas-MuLV express epitopes that are immunogenic for CTL.     

Molecular cloning of viral DNA from leukemogenic Gross passage A murine leukemia virus and nucleotide sequence of its long terminal repeat.

The viral DNA genome of the leukemogenic Gross passage A virus was cloned in phage Charon 21A as an infectious molecule. The virus recovered by transfection with this infectious DNA was ecotropic, N-tropic, fibrotropic, and XC+. It was leukemogenic when reinjected into newborn SIM mice, indicating that ecotropic murine leukemia virus (MuLV) from an AKR mouse thymoma can harbor leukemogenic sequences. Its restriction map was similar to that of nonleukemogenic AKR MuLV, its putative parent, but differed at the 3' end and in the long terminal repeat (LTR). The nucleotide sequence of the Gross A virus LTR was identical to the AKR MuLV LTR sequence (Van Beveren et al., J. Virol. 41:542-556, 1982) in U5, R, and part of U3. All differences between both LTRs were found in U3. Only one copy of the U3 tandem direct repeat was conserved in the Gross A virus LTR, and it was rearranged by the insertion of a 36-base-pair sequence and by five point mutations. Only one additional point mutation common to several oncogenic MuLVs was present in U3. These structural changes in the U3 LTR and at the 3' end of the genome may be related to the leukemogenicity of this virus.     

Characterization of recombinant murine leukemia virus integrase.

Retroviral integration involves two DNA substrates that play different roles. The viral DNA substrate is recognized by virtue of specific nucleotide sequences near the end of a double-stranded DNA molecule. The target DNA substrate is recognized at internal sites with little sequence preference; nucleosomal DNA appears to be preferred for this role. Despite this apparent asymmetry in the sequence, structure, and roles of the DNA substrates in the integration reaction, the existence of distinct binding sites for viral and target DNA substrates has been controversial. In this report, we describe the expression in Escherichia coli and purification of Moloney murine leukemia virus integrase as a fusion protein with glutathione S-transferase, characterization of its activity by using several model DNA substrates, and the initial kinetic characterization of its interactions with a model viral DNA substrate. We provide evidence for functionally and kinetically distinct binding sites for viral and target DNA substrates and describe a cross-linking assay for DNA binding at a site whose specificity is consistent with the target DNA binding site.     

Monoclonal proliferation of Friend murine leukemia virus-transformed myeloblastic cells occurs early in the leukemogenic process.

Integrated Friend murine leukemia virus copies were analyzed by the Southern blotting procedure in myeloblastic cell lines obtained after in vitro infection of long-term mouse bone marrow cultures. Several steps leading to the generation of malignant myeloblastic cells after a long latency period were observed in the evolution of infected cultures. Shortly after infection, a random distribution of integrated provirus copies was observed in the DNA of normally differentiating myeloid cells. In contrast, a distinct pattern of integrated Friend murine leukemia virus copies was evident in the first non-differentiating immature myeloblastic cells appearing in cultures, suggesting a monoclonal origin of these cells. For each cell line, characteristic hybridizing fragments were conserved during the 1-year culture period necessary for the acquisition of tumorigenic properties and were also observed in tumors grafted in vivo. We can conclude that monoclonality is effective very early in the myeloid transformation process, as soon as the precursor cells are blocked in their differentiation.     

New class of leukemogenic ecotropic recombinant murine leukemia virus isolated from radiation-induced thymomas of C57BL/6 mice

We previously reported the establishment of several lymphoid cell lines from X-ray-induced thymomas of C57BL/Ka mice, and all, except one, produce retroviruses (P. Sankar-Mistry and P. Jolicoeur, J. Virol.35:270-275, 1980). Biological characterization of five of these new primary radiation leukemia viruses (RadLVs) indicated that they had a B-tropic, fibrotropic, and ecotropic host range and were leukemogenic when reinjected into C57BL/Ka newborn mice. The leukemogenic potential of one isolate (G(6)T(2)) was further assessed and shown to be retained after prolonged passaging on fibroblasts in vitro. Restriction endonuclease analysis of the DNA of four of our new RadLV isolates (G(6)T(2), Ti-7, Ti-8, and Ti-9) revealed that G(6)T(2) and Ti-7 murine leukemia virus (MuLV) genomes had identical restriction maps, whereas Ti-8 and Ti-9 genomes were different from each other and from the G(6)T(2) and Ti-7 genomes. The physical maps of these genomes were similar to that of known ecotropic MuLV genomes (including the C57BL/Ka endogenous ecotropic MuLV) within their long terminal repeats, env, the right portion of pol, and the left portion of gag. However, a region covering the end of gag and the beginning of pol was different and showed several similarities with xenotropic MuLV genomes of BALB/c, AKR, and C58 mice previously mapped. Our results suggest that these primary RadLV genomes are recombinants between the parental ecotropic MuLV genome and a nonecotropic (xenotropic) sequence. This nonecotropic gag-pol region might be important in conferring the leukemogenic potential to these isolates. Therefore, these RadLVs appear to form a new class of leukemogenic recombinant MuLVs recovered from leukemic tissues of mice. They appear to be distinct from the recombinant AKR mink cell focus-inducing MuLVs which have a dual-tropic host range and harbor xenotropic env sequences. To further study the leukemogenic potential of these RadLVs, the genome of one of them (G(6)T(2)) was cloned in Charon 21A as an infectious molecule.     

Immunofluorescent antibody studies of a murine leukemia virus

Brown, Eric R. (Northwestern University Medical School, Chicago, Ill.), Peter Buinauskas, and Steven O. Schwartz. Immunofluorescent antibody studies of a murine leukemia virus. J. Bacteriol. 92:978-982. 1966.-Correlation was close between in vitro complement fixation, immunodiffusion, and passive cutaneous anaphylaxis tests with the S-63 and GC murine leukemia viruses and immunofluorescence reactions with these viruses. When fluorescein-isothiocyanate-conjugated convalescent sera obtained from mice initially infected with S-63 leukemia virus were used, the reactive site was within the cytoplasm of the infected cell. By electron microscopic examination, virus particles were demonstrated in the same areas within the cells that exhibited specific fluorescence with the conjugates. Spleen and mesenteric nodes contained the most virus, whereas kidney tissues contained the least amount. Fluorescence was not observed within the nuclei of infected cells.     

Structure of the Abelson murine leukemia virus genome.

Virions produced from cells transformed by A-MuLV contain a 30S, 5.6 kb RNA that can be translated in a cell-free system to form the characteristic A-MuLV protein. This RNA was mapped by heteroduplex methods using DNA probes from M-MuLV, the presumed parent of A-MuLV. The overall organization of the RNA was determined by using full-length M-MuLV reverse transcribed DNA and visualizing the heteroduplexes in the electron microscope. This showed that A-MuLV and M-MuLV have homologous sequences at both ends of their RNAs but that the central portion of the A-MuLV genome is not homologous to sequences in M-MuLV RNA. A precise measure of the lengths of the shared regions was obtained by using S1 nuclease to digest hybrids between 32P-labeled M-MuLV DNA and A-MuLV RNA; the resulting fragments were analyzed for their length by electrophoresis. The regions of homology were shown to be 1320 nucleotides long at the 5' end and 730 nucleotides long at the 3' end. Thus approximately 6200 nucleotides of the approximately 8300 in M-MuLV RNA were deleted when the A-MuLV genome was formed, but an insert of 3600 nucleotides, presumably derived from the normal murine genome, was inserted in place of the deleted region.     

Sialylatin of glycoproteins of murine mammary tumor virus, murine leukemia virus, and Mason-Pfizer monkey virus.

Neuraminidase treatment of mouse mammary tumor virus, Rauscher murine leukemia virus, and Mason-Pfizer monkey virus resulted in loss of their capacity to inhibit hemagglutination of influenza virus. Hemagglutination-inhibition activity of these RNA tumor viruses could be restored by in vitro resialylation catalyzed by sialyl transferase. The major glycoprotein in the intact envelope of desialylated and, to some extent, native virions could be specificallly labeled in vitro with CMP-(14C) sialic acid. These studies further characterize the individual glycoproteins of mouse mammary tumor virus, Rauscher murine leukemia virus, and Mason-Pfizer monkey virus.     

Thymocyte subsets transformed by Abelson murine leukemia virus.

The infectious complex of Abelson murine leukemia virus was altered by replacing its usual helper virus, Moloney leukemia virus, with radiation leukemia virus (RadLV). After intrathymic injection of the Abelson-RadLV complex, thymomas arose rapidly, as described previously for injection of the Abelson-Moloney complex. Cell lines were derived from thymomas induced by each Abelson virus complex and were classified according to normal thymus cell phenotypes. Each virus complex induced some cell lines which were like a 0.7% subpopulation of murine thymocytes in that they failed to express the Thy-1 cell-surface antigen. These lines are thus far indistinguishable from some Abelson-derived bone marrow transformants classified as pre-B cells. However, the Abelson-Moloney complex induced some cell lines which expressed low levels of Thy-1 and which shared most markers with immature blast cells of the thymic medulla, whereas the Abelson-RadLV complex induced some lines which were clearly like thymic cortex blast cells. Thus, Abelson virus can induce thymoma cell lines of at least two, and possibly three, distinct phenotypes corresponding to normal thymocyte blast subsets, the determination of which can be influenced by helper virus sequences.     

Generation of a uniform 3'' end RNA of murine leukemia virus.

Using the S1 nuclease mapping technique, we demonstrated that the majority of Moloney murine leukemia RNA molecules, isolated either from the nucleus or cytoplasm of infected mouse cells, share a uniform 3' end located at the border of the R and U-5 regions of the long terminal repeat. When the long terminal repeat sequences were inserted in the pSV plasmid downstream of the simian virus 40 late promoter, the 3' end of the viral RNA was also generated close to the R region of the long terminal repeat. These results demonstrate that the long terminal repeat signals the generation of an authentic 3' end when situated downstream of an actively transcribed region.