Rescue and Transmission of a Replication-Defective Variant of Moloney Murine Leukemia Virus

We have described a clone of mouse cells, termed "8A," which appears to be infected with a replication-defective variant of Moloney murine leukemia virus (MuLV) (Rein et al., J. Virol. 25:146-156, 1978). Clone 8A cells release virus particles which do not form plaques in the standard XC test. However, approximately 10(2) particles per ml of clone 8A supernatant do form plaques in a modified XC test (the "complementation plaque assay"), in which the assay cells are coinfected with the XC-negative, nondefective amphotropic MuLV as well as the test virus. Superinfection of clone 8A cells themselves with amphotropic MuLV results in the production of approximately 10(5), rather than approximately 10(2), particles per ml which register in the complementation plaque assay. This increase is due to the rescue of replication-defective ecotropic MuLV from clone 8A cells by amphotropic MuLV since (i) this ecotropic MuLV can only form XC plaques in cells which are coinfected with amphotropic MuLV; and (ii) it is possible to transmit this defective variant, rescued from superinfected clone 8A cells, to a fresh clone of normal mouse cells. The time course of production of the rescued MuLV particles by superinfected clone 8A cells is virtually identical to that of rescue from these cells of murine sarcoma virus. Amphotropic MuLV superinfection of "NP-N" cells, which contain a "non-plaque-forming" variant of N-tropic MuLV (Hopkins and Jolicoeur, J. Virol. 16:991-999, 1975), also increases the titer of particles registering in the complementation plaque assay; thus, NP-N cells, like clone 8A cells, contain a rescuable defective variant of ecotropic MuLV.     

Oncogenicity of AKR endogenous leukemia viruses.

Four biologically distinct groups of endogenous murine leukemia virus (MuLV) have been isolated from AKR mice. These viruses included (i) ecotopic XC+ MuLV that occur in high titer in normal tissues and serum of AKR mice throughout their life span, (ii) ecotropic XC- MuLV that are produced in high titers by leukemia cells, (iii) xenotropic MuLV that are readily demonstrable only in aged mice, and (iv) polytropic MuLV thatarise in the thymuses of aged mice as a consequence of genetic recombination between ecotropic and xenotropic MuLV. Virus of each of these biological classes were assayed in AKR mice for their ability to accelerate the occurrence of spontaneous leukemia. Certain isolates of ecotropic XC- MuLV and polytropic MuLV were found to have high oncogenic activity. These viruses induced 100% leukemias within 90 days of inoculation. In contrast, ecotropic XC+ MuLV that were obtained from AKR embryo fibroblasts and xenotropic MuLV that were obtained from the lymphoid tissues of aged AKR mice did not demonstrate oncogenic activity. These findings demonstrate fundamental differences between XC- and XC+ ecotropic MuLV that are found in leukemic and normal tissues, respectively. Furthermore, these findings point to the role of ecotropic XC- and polytropic MuLV in the spontaneous leukemogenesis of AKR mice.     

Cell fusion induced by the murine leukemia virus envelope glycoprotein.

To determine whether ecotropic murine leukemia virus (MuLV) envelope glycoproteins are sufficient to cause cell-to-cell fusion when expressed in the absence of virus production, we used an ecotropic MuLV, AKV, to construct env expression vectors that lack the gag and pol genes. The rat cell line XC, which undergoes cell-to-cell fusion upon infection with ecotropic MuLV, was transfected with wild-type env expression vectors, and high levels of syncytium formation resulted. Transfection of the murine cell line NIH 3T3 with expression vectors containing the wild-type or mutated env region did not result in syncytium formation. Immunoprecipitation analysis of the envelope glycoproteins expressed in NIH 3T3 and XC cells showed that the mature surface glycoprotein expressed in XC cells was of a much lower apparent molecular weight than that expressed in NIH 3T3 cells. Further characterization showed that most if not all of this difference was the result of differences in glycosylation. Finally, site-directed mutagenesis was used to introduce several conservative and nonconservative changes into the amino-terminal region of the transmembrane protein. Analysis of the effect of these mutations confirmed that this region is a fusion domain.     

Analysis of the Fusion of XC Cells Induced by Homogenates of Murine Leukemia Virus-Infected Cells and by Purified Murine Leukemia Virus

The fusion of XC cells induced by murine leukemia virus (MuLV)-infected cells is also induced by homogenates prepared from the infected cells and by purified MuLV. The fusion-inducing factor appears to contain a heat-labile lipoprotein. No synthesis of specific macromolecules by the XC cells is necessary to obtain fusion. The results suggest that specific components of the viral particle are the activators for the fusion process and they may also be present in the membranes of infected cells.     

Mechanism of restriction of ecotropic and xenotropic murine leukemia viruses and formation of pseudotypes between the two viruses.

Ecotropic and xenotropic murine leukemia viruses (MuLV's) constitute separate interference groups; within each group there is cross-interference, but between the groups there is no detectable interference. Interference is manifest against pseudotypes in which the vesicular stomatitis virus genome is contained within the coat of one of the murine leukemia viruses. The pseudotypes display the cell specificity of the leukemia viruses: pseudotypes with an ecotropic MuLV coat infect mouse cells but not rabbit or mink cells; pseudotypes with a xenotropic MuLV coat infect rabbit or mink cells well but mouse cells very poorly. Efficient pseudotype formation also occurs between the two MuLV classes, and both the interference patterns and the cell specificity of these pseudotypes are entirely determined by their envelope. Using these pseudotypes, ecotropic MuLV infection could be established in xenogeneic cells, and the resulting progeny could be scored by using a conventional XC cell assay. Also, xenotropic MuLV infection could be established in a mouse cell, showing that no absolute intracellular barrier against xenotropic virus growth exists in murine cells. The major barriers against both xenotropic and ecotropic MuLV therefore are cell surface barriers. Xenogeneic cells probably lack receptors for ecotropic MuLV, but murine cells may either lack receptors for xenotropic MuLV or have receptors that are blocked by endogenous expression of the glycoprotein of endogenous xenotropic MuLV.     

Induction of syncytia by Moloney murine leukemia virus in myoblasts defective in differentiation.

fu-1 cells, a nonfusing variant of the L8 line of rat myoblasts, form syncytia upon infection with murine leukemia virus (MuLV) or upon cocultivation with MuLV-infected cells; L8 cells do not form these syncytia, but do fuse into multinucleate myotubes. Syncytia of fu-1 cells form within 1 h after infection. The number of syncytia formed is proportional to the multiplicity of virus within a range of 4 to 16 and is maximum when the cell density is subconfluent. When either XC or fu-1 cells are productively infected with MuLV, they become resistant to syncytia formation by passage 3. The rapid formation of syncytia in fu-1 cells was found amenable for selection of temperature-sensitive mutants of MuLV and for screening additional variants of the L8 line.     

Molecular cloning of infectious viral DNA from ecotropic neurotropic wild mouse retrovirus.

Among a mixture of amphotropic and ecotropic murine leukemia viruses (MuLVs) isolated from paralyzed wild mice, only N-tropic ecotropic MuLV, cloned by cell culture techniques, has been shown to induce paralysis after reinjection into susceptible mice (M. B. Gardner, Curr. Top. Microbiol. Immunol. 79:215-239, 1978). The viral DNA genome of one of these neurotropic MuLVs (Cas-Br-E) has been cloned in Charon 21A at the SalI site. One clone, designated NE-8, was studied in more detail. A restriction endonuclease map of this cloned DNA was derived. Cloned viral DNA microinjected into NIH 3T3 cells produced infectious MuLV which was characterized as XC+, ecotropic, and N-tropic. The virus that was recovered after the microinjection of NE-8 DNA was also injected into susceptible SIM.S and NIH Swiss mice and was found to induce lower limb paralysis in these animals. These results make it highly unlikely that other agents (which might have escaped detection and separation from ecotropic MuLV by the techniques previously used) play a role in the etiology of this disease and clearly indicate that the ecotropic MuLV genome harbors sequences responsible for this paralysis. The availability of this clone DNA would now allow us to map these sequences on the genome.     

Virological properties and nucleotide sequences of Cas-E-type endogenous ecotropic murine leukemia viruses in South Asian wild mice, Mus musculus castaneus

Two types of endogenous ecotropic murine leukemia viruses (MuLVs), termed AKV- and Cas-E-type MuLVs, differ in nucleotide sequence and distribution in wild mouse subspecies. In contrast to AKV-type MuLV, Cas-E-type MuLV is not carried by common laboratory mice. Wild mice of Mus musculus (M. m.) castaneus carry multiple copies of Cas-E-type endogenous MuLV, including the Fv-4(r) gene that is a truncated form of integrated MuLV and functions as a host's resistance gene against ecotropic MuLV infection. Our genetic cross experiments showed that only the Fv-4(r) gene was associated with resistance to ecotropic F-MuLV infection. Because the spontaneous expression of infectious virus was not detected in M. m. castaneus, we generated mice that did not carry the Fv-4(r) gene but did carry a single or a few endogenous MuLV loci. In mice not carrying the Fv-4(r) gene, infectious MuLVs were isolated in association with three of six Cas-E-type endogenous MuLV loci. The isolated viruses showed a weak syncytium-forming activity for XC cells, an interfering property of ecotropic MuLV, and a slight antigenic variation. Two genomic DNAs containing endogenous Cas-E-type MuLV were cloned and partially sequenced. All of the Cas-E-type endogenous MuLVs were closely related, hybrid-type viruses with an ecotropic env gene and a xenotropic long terminal repeat. Duplications and a deletion were found in a restricted region of the hypervariable proline-rich region of Env glycoprotein.     

Mutations in the cytoplasmic tail of murine leukemia virus envelope protein suppress fusion inhibition by R peptide

During viral maturation, the cytoplasmic tail of the murine leukemia virus (MuLV) envelope (Env) protein undergoes proteolytic cleavage by the viral protease to release the 16-amino-acid R peptide, and this cleavage event activates the Env protein's fusion activity. We introduced Gly and/or Ser residues at different positions upstream of the R peptide in the cytoplasmic tail of the Friend MuLV Env protein and investigated their effects on fusion activity. Expression in HeLa T4 cells of a mutant Env protein with a single Gly insertion after I619, five amino acids upstream from the R peptide, induced syncytium formation with overlaid XC cells. Env proteins containing single or double Gly-Ser insertions after F614, 10 amino acids upstream from the R peptide, induced syncytium formation, and mutant proteins with multiple Gly insertions induced various levels of syncytium formation between HeLa T4 and XC cells. Immunoprecipitation and surface biotinylation assays showed that most of the mutants had surface expression levels comparable to those of the wild-type or R peptide-truncated Env proteins. Fluorescence dye redistribution assays also showed no hemifusion in the Env proteins which did not induce fusion. Our results indicate that insertion mutations in the cytoplasmic tail of the MuLV Env protein can suppress the inhibitory effect of the R peptide on membrane fusion and that there are differences in the effects of insertions in two regions in the cytoplasmic tail upstream of the R peptide.     

Role of carbohydrate in biological functions of Friend murine leukemia virus gp71.

Purified gp71 of Friend murine leukemia virus (FLV) can interfere with virus infection, absorb neutralizing antibody, and in the presence of group-specific anti-gp71 antibody, hemagglutinate sheep erythrocytes. Interference by FLV gp71 with several murine leukemia viruses (MuLV) was tested in the XC and S + L- assay systems. Treatment of gp71 with trypsin or Pronase eliminated its interfering capacity. However, treatment with neuraminidase or a mixture of glycosidase enzymes, which left the major serological properties of gp71 intact, did not reduce the interference potential of gp71 for FLV or AKR MuLV. The capacity of gp71 to absorb type- or group-specific virus-neutralizing antibodies was similarly affected by the various enzyme treatments. In contrast, indirect hemagglutination by gp71 was abolished not only by proteases but also by treatment with glycosidase enzymes, although neuraminidase had no effect. Preliminary data indicate that infectivity of FLV or xenotropic MuLV was not affected by short treatment with glycosidase enzymes.     

Rescue of Murine Sarcoma Virus from a Sarcoma-Positive Leukemia-Negative Cell Line: Requirement for Replicating Leukemia Virus

The nature of murine sarcoma virus (MSV) "defectiveness" was investigated by employing an MSV-transformed mouse 3T3 cell line which releases noninfectious virus-like particles. Rescue kinetics of MSV, observed after murine leukemia virus (MuLV) superinfection of these "sarcoma-positive leukemia-negative (S + L -)" mouse 3T3 cells, consisted of a 9- to 12-hr eclipse period followed by simultaneous release of both MSV and MuLV with no evidence for release of infectious MSV prior to the production of progeny MuLV. Addition of thymidine to the growth medium of MuLV-superinfected S + L - cells at a concentration suppressing deoxyribonucleic acid synthesis inhibited the replication of MuLV and the rescue of MSV. MSV production closely paralleled MuLV replication under a variety of experimental conditions. These results suggest that replication of MuLV is required for the rescue of infectious MSV from S + L - cells and that one (or more) factor, produced late in the MuLV replicative cycle, is utilized by both viruses during virion assembly. During the course of these experiments, virus stocks were recovered which contained infectious MSV in apparent excess over MuLV. These stocks were used for generating new S + L - cell lines by simple end point dilution procedures.     

Sequence analysis of amphotropic and 10A1 murine leukemia viruses: close relationship to mink cell focus-inducing viruses.

Viral interference studies have demonstrated the existence of four distinct murine leukemia virus (MuLV) receptors on NIH 3T3 mouse cells. The four viral interference groups are ecotropic MuLV; mink cell focus inducing virus (MCF); amphotropic MuLV; and 10A1, a recombinant derivative of amphotropic MuLV that uses a unique receptor but also retains affinity for the amphotropic MuLV receptor. We report here that 10A1 infects rat and hamster cells, unlike its amphotropic parent. We isolated an infectious molecular clone of 10A1 and present here the sequences of the env genes and enhancer regions of amphotropic MuLV and 10A1. The deduced amino acid sequences of amphotropic MuLV and 10A1 gp70su are remarkably similar to those of MCF and xenotropic MuLV (for which mouse cells lack receptors), with 64% amino acids identical in the four groups. We generated a consensus from these comparisons. Further, the differences are largely localized to a few discrete regions: (i) amphotropic MuLV has two short insertions relative to MCF, at residues 87 to 92 and 163 to 169, and (ii) amphotropic MuLV and MCF are totally different in a hypervariable region, which is greater than 30% proline, at residues approximately 253 to 304. 10A1 closely resembles amphotropic MuLV in its N terminus but contains an MCF-type hypervariable region. These results suggest the possibility that receptor specificity is localized in these short variable regions and further that the unique receptor specificity of 10A1 is due to the novel combination of amphotropic MuLV and MCF sequences rather than to the presence of any novel sequences. The Env proteins of ecotropic MuLV are far more distantly related to those of the other four groups than the latter are to each other. We also found that the enhancer regions of amphotropic MuLV and 10A1 are nearly identical, although 10A1 is far more leukemogenic than amphotropic MuLV.     

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.     

Cleavage of the Murine Leukemia Virus Transmembrane Env Protein by Human Immunodeficiency Virus Type 1 Protease: Transdominant Inhibition by Matrix Mutations

We have identified mutations in the human immunodeficiency virus type 1 (HIV-1) matrix protein (MA) which block infectivity of virions pseudotyped with murine leukemia virus (MuLV) envelope (Env) glycoproteins without affecting infectivity conferred by HIV-1 Env or vesicular stomatitis virus G glycoproteins. This inhibition is very potent and displays a strong transdominant effect; infectivity is reduced more than 100-fold when wild-type and mutant molecular clones are cotransfected at a 1:1 ratio. This phenomenon is observed with both ecotropic and amphotropic MuLV Env. The MA mutations do not affect the incorporation of MuLV Env into virions. We demonstrate that in HIV-1 virions pseudotyped with MuLV Env, the HIV-1 protease (PR) efficiently catalyzes the cleavage of the p15(E) transmembrane (TM) protein to p12(E). Immunoprecipitation analysis of pseudotyped virions reveals that the mutant MA blocks this HIV-1 PR-mediated cleavage of MuLV TM. Furthermore, the transdominant inhibition exerted by the mutant MA on wild-type infectivity correlates with the relative level of p15(E) cleavage. Consistent with the hypothesis that abrogation of infectivity imposed by the mutant MA is due to inhibition of p15(E) cleavage, mutant virions are significantly more infectious when pseudotyped with a truncated p12(E) form of MuLV Env. These results indicate that HIV-1 Gag sequences can influence the viral PR-mediated processing of the MuLV TM Env protein p15(E). These findings have implications for the development of HIV-1-based retroviral vectors pseudotyped with MuLV Env, since p15(E) cleavage is essential for activating membrane fusion and virus infectivity.     

HIV-1 predisposed to acquiring resistance to maraviroc (MVC) and other CCR5 antagonists in vitro has an inherent, low-level ability to utilize MVC-bound CCR5 for entry

In 2009, a newly discovered human retrovirus, xenotropic murine leukemia virus (MuLV)-related virus (XMRV), was reported by Lombardi et al. in 67% of persons from the US with chronic fatigue syndrome (CFS) by PCR detection of gag sequences. Although six subsequent studies have been negative for XMRV, CFS was defined more broadly using only the CDC or Oxford criteria and samples from the US were limited in geographic diversity, both potentially reducing the chances of identifying XMRV positive CFS cases. A seventh study recently found polytropic MuLV sequences, but not XMRV, in a high proportion of persons with CFS. Here we tested blood specimens from 45 CFS cases and 42 persons without CFS from over 20 states in the United States for both XMRV and MuLV. The CFS patients all had a minimum of 6 months of post-exertional malaise and a high degree of disability, the same key symptoms described in the Lombardi et al. study. Using highly sensitive and generic DNA and RNA PCR tests, and a new Western blot assay employing purified whole XMRV as antigen, we found no evidence of XMRV or MuLV in all 45 CFS cases and in the 42 persons without CFS. Our findings, together with previous negative reports, do not suggest an association of XMRV or MuLV in the majority of CFS cases.     

Infection of bone marrow cells in vitro with FLV: Effects on stem cell proliferation,differentiation and leukemogenic capacity

Long-term cultures of proliferating hematopoietic stem cells derived from bone marrow permit the study of the interaction between murine leukemia virus (MuLV) infection and the proliferation and differentiation of stem cells. We have used this system to analyze the replication of different biological variants of MuLV in bone marrow cells; the effect of MuLV infection upon pluripotent stem cell (CFU-S) proliferation; and the effect of MuLV on differentiation of CFU-S along different hematopoietic pathways. Two MuLV variants were studied in detail: the Moloney strain of lymphatic leukemia virus (Mol-MuLV) and the erythroleukemic Friend virus complex (FLV) consisting of the lymphoid leukemia helper virus and the defective spleen focus-forming virus (SFFV). Mol-MuLV and its sarcoma virus pseudotype, MSV(Mol-MuLV), replicate efficiently in the bone marrow cultures; however, CFU-S are lost more readily than in uninfected cultures, and the cultures are soon represented by a majority population of mononuclear macrophages. On the other hand, infection with FLV produces a prolonged survival of the spleen colony-forming cells, CFU-S, and CFU-C (the committed granulocytic precursor cells). Production of erythroleukemogenic SFFV is maintained in these cultures for more than 40 weeks. No erythroblastic differentiation was observed in vitro, however, neither erythroblast precursor cells (CFU-E) nor hemoglobin-producing cells could be detected. This suggests that the target cell for FLV is an earlier precursor cell.     

Naturally occurring murine leukemia viruses in wild mice: characterization of a new "amphotropic" class.

A new class of murine leukemia viruses, isolated from wild Mus musculus trapped in California, is described. These viruses, designated "amphotropic," replicate in mouse, rabbit, mink, human, guinea pig, and rat cells, but not in hamster, quail, or duck cells. They show N-tropism for mouse cells, and do not trigger the XC cell response. They are distinct by interference and virus neutralization testing from the previously recognized mouse-tropic and xenotropic MuLV classes. Mouse-tropic viruses occuring along with three of the four amphotropic isolates were found to be distinguishable by virus neutralization from other mouse-tropic murine leukemia virus strains of laboratory mouse origin.     

Substitution of the U3 long terminal repeat region of the neurotropic Cas-Br-E retrovirus affects its disease-inducing potential.

The Cas-Br-E and ts-Mo BA-1 murine leukemia viruses (MuLV) induce a spongiform neurodegenerative disease with different clinical manifestations, namely, either hind limb paralysis (Cas-Br-E) or tremors, spasticity, and hind limb weakness (ts-Mo Ba-1). We constructed the chimeric NEBA-1 MuLV by replacing the long terminal repeat of Cas-Br-E MuLV with that of ts-Mo BA-1 MuLV. In SWR/J or CFW/D mice, NEBA-1 MuLV induced an ataxic neurological disease characterized by clinical signs different from those induced by both parents. Although NEBA-1 MuLV did not induce lesions in novel brain areas, the spongiform lesions were more severe in deep cerebellar nuclei and in the spinal cord than those found in paralyzed mice inoculated with Cas-Br-E MuLV. By in situ hybridization, we found that the distribution of the spongiform lesions closely correlated with the distribution of the infected central nervous system cells. In the spinal cord, a close correlation was found between the number of infected cells and the severity of the spongiform degeneration. Sequencing of the substituted ts-BA-1 MuLV fragment and comparison with homologous sequences of Cas-Br-E and Moloney MuLV showed differences mainly in the U3 tandem direct repeats. Our results show that a few modifications within the U3 long terminal repeat allow the virus to cause more severe lesions in some central nervous system regions and that the severity of the spongiform degeneration correlates with the level of viral replication.