Abrogation of Fv-1 restriction by genome-deficient virions produced by a retrovirus packaging cell line.

The Fv-1b-mediated restriction of N-tropic retrovirus vector infection of BALB/3T3 cells was partially abrogated by prior infection with N-tropic murine leukemia virus. Likewise, abrogation of the Fv-1b restriction of N-tropic murine leukemia virus replication was accomplished by prior infection with genome-deficient virions produced by an N-tropic murine leukemia virus packaging cell line. The latter observation suggests that the Fv-1 target in genome-deficient virions abrogates Fv-1 restriction in the absence of any viral genome-directed processes.     

Effect of the Fv-1 locus on the titration of murine leukemia viruses.

Titration of N- and B-tropic murine leukemia viruses on sensitive and resistant cell lines has been studied by direct XC plaque assay and infective center assay. The titration of cloned B-tropic virus by infective center assay on BALB/3T3 (Fv-1b/b) and NIH/3T3 (Fv-1n/n) cells gave one-hit patterns, with 100-fold less infected NIH/3T3 cells than BALB/3T3 cells. The titration of B-tropic virus on DBA/2 cells (Fv-1n/n) was also a one-hit. The titration of a one-hit curve, and there were about 100-fold less infected BALB/3T3 cells than NIH/3T3 cells. Comparable results were obtained by titrating the cloned N-tropic virus on congenic SIM (Fv-1n/n) and SIM.R (Fv-1b/b) cells or the Gross N-tropic virus on BALB/3T3 cells. Therefore, our data indicate that the multiple-hit phenomenon described previously may not be an essential part of the Fv-1 gene restriction.     

Physical mapping of the Fv-1 tropism host range determinant of BALB/c murine leukemia viruses

The murine leukemia viruses (MuLVs) have different host ranges and were originally designated N-tropic and B-tropic if they replicated preferentially in vitro on NIH and BALB/c fibroblasts, respectively. It was later found that N-tropic MuLVs were in fact restricted in BALB/c cells, that B-tropic MuLVs were restricted in NIH cells, and that both viruses were restricted in (BALB X NIH) F1 cells. A single gene, Fv-1, with two alleles, Fv-1b and Fv-1n, determines this dominant restriction. A virus-encoded protein seems to carry the viral host range determinant which is recognized by the Fv-1 gene product. To map the viral DNA sequences encoding this determinant, we constructed viral DNA recombinants in vitro between the cloned infectious viral DNA genomes from BALB/c N-tropic and B-tropic MuLVs. Infectious recombinant MuLVs were recovered by microinjecting these recombinant DNAs into murine Fv-1- SC-1 cells and were subsequently tested in vitro for their host ranges (N- or B-tropic). We found that a short 302-base pair 5'-end fragment was necessary and sufficient to confer a specific host range to a recombinant. Our sequencing data revealed that this fragment codes for amino acid sequences in gag p30. They also showed that only two consecutive amino acid differences, Gln-ArgN- and Thr-GluB-, in p30 are responsible for the N- and B-tropic host ranges of the BALB/c MuLVs, respectively. Therefore, it appears that the Fv-1b and Fv-1n gene products can discriminate between these two p30 amino acid sequences.     

Retrovirus-induced murine acquired immunodeficiency syndrome: natural history of infection and differing susceptibility of inbred mouse strains.

C57BL mice (Fv-1b) develop a severe immunodeficiency disease following inoculation as adults with LP-BM5 murine leukemia virus (MuLV), a derivative of Duplan-Laterjet virus which contains B-tropic ecotropic and mink cell focus-inducing MuLVs and a putative defective genome which may be the proximal cause of disease. The stages of development of this disease were defined for C57BL mice on the basis of lymphadenopathy and splenomegaly; histopathological changes consistent with B-cell activation; and alterations in expression of cell surface antigens affected by proliferation of T cells, B cells, and macrophages. By using this disease profile as a standard, the response of adult mice of various inbred strains and selected F1 hybrids was compared. We show that although the strains which are highly sensitive are of the Fv-1b genotype (i.e., permissive for B-tropic MuLVs), certain Fv-1b strains, e.g., BALB/c and A/J, are resistant to murine acquired immunodeficiency syndrome, whereas certain Fv-1n strains (permissive for N-tropic MuLVs but restrictive for B-tropic MuLVs), notably P/N, BDP, and AKR, show moderate sensitivity and (C57BL/6 x CBA/N)F1 mice (Fv-1n/b and thus dually restrictive) are of relatively high susceptibility. The results of virus recovery tests suggest that apparently anomalous sensitivity, based on predicted Fv-1 restriction, may reflect MuLV induction and/or mutation to provide a helper virus for which the host is permissive.     

Control of RFM strain endogenous retrovirus in RFM mouse cells

RFM/Un mice express an endogenous type C retrovirus throughout their life span in many tissues; primary or established embryo fibroblast cell cultures do not express a virus but can be induced by exposure to 5-iodo-2'-deoxyuridine. All of our sources yielded a single ecotropic virus (RFV) which appeared to be related more closely to the endogenous N-tropic virus (WN1802N) of BALB/c mice than to Gross leukemia virus on the basis of two-dimensional gel electropherograms of virion proteins. No xenotropic or recombinant viruses were isolated by cocultivation techniques. RFV is N-tropic, and RFM/Un cells possess the Fv-1n allele, as indicated by restriction of B-tropic virus and susceptibility to Gross strain N-tropic virus. However, RFM cells are highly resistant to RFV and other endogenous N-tropic viruses. This resistance is expressed by two-hit titration kinetics and by inhibition of viral linear duplex DNA formation. This is similar to the effects of the Fv-1 locus, but preliminary work has shown no apparent genetic linkage between the two restrictions. The relative strength of the restriction, the presence of a single class of ecotropic virus, and the absence of recombinant viruses suggest that in RFM mice virus is expressed only in cells in which it is induced and not by cell-to-cell transmission.     

Reversal of Fv-1 host range by in vitro restriction endonuclease fragment exchange between molecular clones of N-tropic and B-tropic murine leukemia virus genomes.

We molecularly cloned unintegrated viral DNA of the BALB/c endogenous N-tropic and B-tropic murine leukemia retroviruses and in vitro passaged N-tropic Gross (passage A) murine leukemia retroviruses. Recombinant genomes were constructed in vitro by exchanging homologous restriction enzyme fragments from N- or B-tropic parents and subsequent recloning. Infectious virus was recovered after transfection of these recombinant genomes into NIH-3T3 cells and cocultivation with the Fv-1 nonrestrictive SC-1 cells. XC plaque assays of recombinant virus progeny on Fv-ln and Fv-lb cells indicated that the Fv-l host range was determined by sequences located between the BamHI site in the p30 region of the gag gene (1.6 kilobase pairs from the left end of the map) and the HindIII site located in the pol gene (2.9 kilobase pairs from the left end of the map).     

Effect of Fv-1 gene product on synthesis of linear and supercoiled viral DNA in cells infected with murine leukemia virus.

Levels of unintegrated viral DNA made in Fv-1b/b (SIM.R, JLS-V9) and Fv-1n/n (NIH/3T3) cell lines after infection with N- or B-tropic murine leukemia virus (MuLV) have been measured. Different forms of viral DNA were sedimented on neutral sucrose or ethidium bromide-cesium chloride density gradients and detected by hybridization with complementary DNA. It was found that the major viral DNA species made in Fv-1 permissive or resistant cells was sedimenting at 20S on neutral sucrose gradient. Levels of this 20S viral DNA species were not significantly different in both systems. However levels of closed circular (form I) viral DNA separated on ethidium bromide-cesium chloride gradients were found to be decreased in Fv-1 resistant cells. Various species of viral DNA were also analyzed by the agarose gel-DNA transfer procedure of Southern. The major viral DNA species was found to migrate as a double-stranded linear DNA of 5.7 x 10(6) daltons. The molecular weight of linear viral DNA molecules extracted from Fv-1 permissive or resistant cells appeared to be the same. Levels of this linear viral DNA were almost identical in both systems except in B-tropic MuLV-infected resistant NIH/3T3 cells in which a moderate decrease has been measured. Two closed circular viral DNA species were observed by this technique. Their levels were markedly decreased in Fv-1 resistant cells. Our results indicate that the Fv-1 restriction does not grossly affect the formation of linear double-stranded viral DNA, but prevents the accumulation of closed circular viral DNA. Therefore the Fv-1 gene product could allow the synthesis of a normal linear viral DNA but interfere with the formation of supercoiled viral DNA. Alternatively, it could promote the synthesis of a faulty linear viral DNA whose defect (yet undetected) would prevent its circularization. In any case, the Fv-1 restriction mechanism appears to occur before the integration event itself.     

Transfer of Fv-1 locus-specific resistance to murine N-tropic and B-tropic retroviruses by cytoplasmic RNA.

A standardized bioassay for transfer of Fv-1 gene-specific resistance to N-tropic and B-tropic murine retroviruses was developed using X plaque reduction in SC-1 (Fv-1-) cells inoculated with virus. Testing of subcellular fractions of restrictive cells showed that the resistance transfer activity was present in the cytoplasmic (microsomal and cytosol) fractions. The activity of the cytoplasmic extract was destroyed by treatment with ribonuclease, but not with deoxyribonuclease or proteases. RNA prepared by phenol-chloroform extraction of mouse tissues, including embryos and livers of weanling mice, transferred Fv-1 locus-specific resistance into DEAE-dextran-treated SC-1 cells. The activity of isolated RNA preparations against virus of the appropriate host-range type has been demonstrated to correspond to the Fv-1 genotypes of the cell sources. The specific transfer of resistance with cellular RNA was effective within a 5- to 6-h period from 2 h before to 4 to 5 after virus infection. Sucrose gradient centrifugation of the RNA showed that the activity sedimented as a broad peak, with an apparent maximum in the 22S region. Affinity chromatography of whole-cell RNA on polyuridylic acid-Sepharose tended to separate more activity into the polyadenylic acid RNA fraction than the non-polyadenylic acid RNA fraction. Except for the reciprocal inhibitory activity for the two host-range virus types, the RNAs of Fv-1n and Fv-1b specificities showed similar properties in all aspects studied.     

Inherited resistance to N- and B-tropic murine leukemia viruses in vitro: titration patterns in strains SIM and SIM.R congenic at the Fv-1 locus.

We have investigated the titration patterns of murine leukemia viruses on mouse embryo cultures derived from a pair of congenic strains differing at the Fv-1 locus. XC plaque and infectious center assays carried out with N- and B-tropic viruses on both SIM (Fv-1nn) and SIM.R(Fv-1bb) host cells yielded results that were best approximated by Poisson one-hit curves. Titration curves of N-tropic virus by direct XC plaque assay were linear and parallel on the different hosts, with titers 1.8 to 2.7 log10 lower on SIM.R and on (SIM X SIM.R)F1 than on SIM cells; similar linear and parallel curves were found for B-tropic virus, with titers 1.4 to 2.0 log10 lower on SIM and (SIM XSIM-R)F1 than on SIM-R cells. In the infectious center assays, the proportion of infected cells was linearly related to multiplicity of infection on both permissive (N- on SIM and B- on SIM.R) restrictive (B- on SIM and N- on SIM.R) genotypes at multiplicities of infection below 0.5; the line relating the variables was about 1 log10 lower in the restrictive than in the permissive situations. At multiplicities of infection where the proportion of infected cells reached a plateau, differences between the results on permissive and restrictive genotypes were considerably reduced. This appeared to be due to the action of non-Fv-1 factors in permissive host. We conclude that the major action of the restrictive allele at the Fv-1 locus in this system is to reduce the probability of successful murine leukemia virus infection without a change in hitness.     

Genetic studies of the Fv-1 locus of mice: linkage with Gpd-1 in recombinant inbred lines.

Multiple recombinant inbred lines, derived from crosses between strains permissive to N-tropic murine leukemia viruses (Fv-1n) and strains permissive to B-tropic murine leukemia viruses (Fv-1b), have been characterized as to Fv-1 genotype and other chromosome 4 markers, including the closely linked hexose-6-phosphate dehydrogenase isozyme locus (Gpd-1). Only one recombinant between Fv-1 and Gpd-1 was found among 45 lines tested. On this basis, the distance between Fv-1 and Gpd-1 is estimated to be 0.6 centimorgans. None of the lines was either resistant or susceptible to both N- and B-tropic viruses. Nineteen other inbred strains, previously untested, were characterized as either Fv-1n or Fv-1b.     

Similarity of the genomic structure between the two members in a new family of heparin-binding factors.

By differential hybridization of cDNA libraries from Fv-1 restrictive and non-restrictive somatic mutant mouse cells, we isolated a cDNA clone specifically present in the Fv-1 restrictive cells. Its sequence was found identical to that of heparin-binding growth-associated molecule or pleiotrophin (HB-GAM/PTN). We report here its genomic organization which has not been yet reported. Although the exon organization was similar to that of midkine, another member of the same family of heparin-binding factors, the sizes of introns were much larger and occupied more than a three-fold larger chromosomal region than midkine. As its introduction into Fv-1 non-restrictive cells failed to confer the Fv-1 restrictive character, HB-GAM/PTN is probably unrelated to Fv-1 restriction.     

Loss of Fv-1 restriction in Balb/3T3 cells following infection with a single N tropic murine leukemia virus particle.

The ability of various murine leukemia viruses (MuLVs) to replicate in mouse cells exhibiting Fv-1 restriction was analyzed by quantitative dose-response assays. In particular, the effect of infection with N, B, or NB tropic MuLVs on Fv-1b restriction in Balb/3T3 cells was measured with an infection center technique in which pseudotypes of murine sarcoma virus (MSV), which have been shown to exhibit Fv-1 dependence of expression, were used to quantitate the degree of restriction. The resulting dose-response curves indicate that productive infection of a single Balb/3T3 cell with N tropic MSV requires co-infection with two MuLV particles. These two MuLV particles are functionally distinguishable. One of them must be N tropic and must be added less than 18 hr after infection with N tropic MSV. The second MuLV particle, on the other hand, need not be N tropic and may be added at any time. Balb/3T3 cultures infected with sufficient N tropic MuLV become fully permissive to transformation by N tropic MSV and to productive infection by N tropic MuLV. This effect, termed "abrogation" of Fv-1 restriction, results from infection of a Balb/3T3 cell with a single N tropic MuLV particle, but apparently occurs without viral replication. It seems probable that a requirement for abrogation of Fv-1b restriction by a single infectious particle of N tropic MuLV, which does not itself replicate, is responsible for the two-hit dose-response relationship observed in infectivity titrations of N tropic MuLV in Balb/3T3 cells. The requirements that N tropic MuLV be added within a specified time period with regard to N tropic MSV in order for abrogation to occur suggests that in the absence of N tropic MuLV, the cellular Fv-1b restriction mechanism inactivates N tropic MSV by 9 hr after infection.     

Helper-Dependent Properties of Friend Spleen Focus-Forming Virus: Effect of the Fv-1 Gene on the Late Stages in Virus Synthesis

Co-infection of neonatal BALB/c mice with Friend virus (FV) complex (containing defective spleen focus-forming virus [SFFV] and endogenous N-tropic leukemia-inducing helper virus [LLV-F]) and B-tropic Tennant leukemia virus (TenLV) resulted in the inhibition of LLV-F by the Fv-1(b) gene and recovery of a TenLV pseudotype of SFFV, abbreviated SFFV(TenLV). The host range of this pseudotype was B-tropic, since SFFV(TenLV) was 10 to 100 times more infectious for B-type (Fv-1(bb)) than for N-type (Fv-1(nn)) mice. The similar patterns of neutralization of N-tropic and B-tropic SFFV by type-specific murine antisera suggested that the difference in infectivity between these two SFFV preparations did not reside in envelope determinants. Rather, helper control of SFFV's host range was only apparent and dependent upon the ability of associated virus to provide a helper function for late stages in SFFV synthesis. Early stages in SFFV's infectious cycle were shown to be helper independent. The Fv-1 gene did not act at the level of the cell membrane to effectively restrict SFFV infection, since SFFV-induced transformed cells could be detected in the absence of spleen focus formation and SFFV synthesis. Further, the generation of these transformed cells by SFFV followed a one-hit, dose-response pattern, suggesting that SFFV-induced cell transformation is helper independent. Finally, restriction of helper function by Fv-1 may be an intracellular event, because both SFFV and its associated LLV-F helper share common envelope determinants and presumably adsorb onto and penetrate target cells with equal efficiency.     

Genetic transmission of endogenous N- and B-tropic murine leukemia viruses in low-leukemic strain C57BL/6.

Spontaneous expression of endogenous N- and B-tropic murine leukemia viruses was stu1bb), DDD (Fuv-1nn), DDD-Fvr (fv-1nn), (DDD or DDD-Fvr times C57BL/6)F1, and 16 partially inbredlines with either the Fv-1nn or Fv-1bb genotype, which had been established from hybrids between C57BL/6 and DDD-Fvr. When tested at middle age, virus-positive mice were found in C57BL/6, F1 hybrids, and 9 out of 16 partially inbred lines. N-tropic viruses were isolated from Fv-1nn, Fv-1bb mice, whereas B-tropic viruses, except for one isolate, were from Fv-1bb mice only. C57BL/6 mice were positive for both N- and B-tropic viruses, whereas DDD-Fvr mice were negative. With respect to the Fv-1 genotype and the presence of endogenous murine leukemia viruses, the partially inbred lines were grouped into five types: (i) Fv-1bb, both N- and B-tropic virus positive, like C57BL/6; (ii) Fv-1nn, virus negative, like DDD-Fvr; (iii) Fv-1bb, virus negative; (iv) Fv-1nn, only N-tropic virus positive; and (v) less convincingly, Fv-1bb, only B-tropic virus positive. These findings indicate that the transmission of N- and B-tropic viruses in C57BL/6 is genetically controlled and that the expression of B-tropic virus, but not of N-tropic virus, is closely associated with the Fv-1 genotype.     

Thymic epithelium controls thymocyte expression of preleukemic phenotype and leukemogenic retroviruses

Congenitally athymic AKR-streaker (nustr/nustr) mice were grafted separately with syngeneic or allogeneic, irradiated (1200 R) thymic reticuloepithelial (TRE) elements (stroma) or nonirradiated whole thymus grafts (control group) from N-tropic murine leukemia virus (MuLV) infection-susceptible (Fv-1n/n) or N-tropic-MuLV-infection-resistant (Fv-1b/n) murine strains. From 3 to 13 mo after grafting, the mononuclear cells repopulating the thymus grafts were stained with fluorescent monoclonal antibodies to thymocyte differentiation antigens, peanut agglutinin, and an antibody to MuLV antigens and were then analyzed by flow cytometry. Irradiated TRE of the Fv-1n/n genotype, whether from high or low leukemia-incidence strains, contained lymphoid cells of host (nustr/nustr) origin with alterations in thymocyte differentiation and MuLV antigen expression consistent with preleukemic changes. In contrast, transplanted TRE of the low leukemia-incidence Fv-1b/n genotype restricted preleukemic changes in thymocyte differentiation and MuLV antigen expression by lymphoid cells derived from the nustr/nustr host. Thus, nustr/nustr lymphocytes must infect susceptible TRE (Fv-1n/n) with N-tropic-MuLV before preleukemic changes occur in the mustr/nustr lymphocytes that later migrate to the thymus. Therefore, it was the radiation-resistant cells in the thymus that amplified or suppressed expression of AKR MCF retroviruses and the preleukemic phenotype, not the thymic lymphocytes. Thy-1.1+ splenocytes of ungrafted nustr/nustr mice were comparable in percentage to nustr/+ but were deficient in the Lyt-1+2- subpopulation and unresponsive to mitogens or alloantigens in vitro. Analysis of splenocyte cell surface markers, mitogen, MLC, and CML responses of Fv-1n/n-thymus-grafted nustr/nustr mice showed restoration of Lyt-1+2- cells to levels comparable to nustr/+ and reconstitution of in vitro proliferative and cytotoxic responses.     

Fv-1 determinants in xenotropic murine leukemia viruses studied with biological assay systems: Isolation of xenotropic virus with N-tropic Fv-1 activity in the cryptic form.

By a biological assay system using phenotypically mixed ecotropic and xenotropic murine leukemia viruses, we investigated whether in the virions of a xenotropic virus there is N- or B-tropic Fv-1 determinant in active form. The existence of N-tropic Fv-1 determinant was demonstrated in SL-XT-1 xenotropic virus isolated from the spleen of a 3-month-old SL mouse, and the N-tropic Fv-1 tropism was confirmed by analysis of the phenotypically mixed viruses harvested from clonal SC-1 cells doubly infected with the SL-XT-1 and B-tropic ecotropic viruses. However, neither N- nor B-tropic Fv-1 determinant was demonstrated in any xenotropic viruses isolated from embryo cells of BALB/c, NZB, or DBA/2 mice, or Cas E #1-IU, and xenotropic-like virus isolated from a wild mouse.     

Influence of murine leukemia proviral integrations on development of N-methyl-N-nitrosourea-induced thymic lymphomas in AKR mice.

The AKR mouse strain is characterized by a high incidence of spontaneous thymic lymphoma that appears in older animals (greater than 6 months of age) and is associated with novel provirus integrations of ecotropic and recombinant murine leukemia viruses (MuLVs). Treatment of 4- to 6-week-old AKR/J mice with the carcinogen N-methyl-N-nitrosourea (MNU) results in thymic lymphomas that arise as early as 3 to 4 months of age and contain novel somatically acquired MuLV provirus integrations. The AKR/J strain develops MNU-induced lymphoma with a higher incidence and shorter latency than has been observed for other inbred mouse strains. To determine whether provirus integrations of endogenous MuLV account for the enhanced susceptibility of the AKR strain, the incidence and latency of MNU-induced lymphoma development was compared in AKR/J and AKR.Fv-1b mice. The restrictive b allele of the Fv-1 locus restricts integration and replication of endogenous N-tropic MuLV; therefore, AKR-Fv-1b mice have a very low incidence of spontaneous lymphoma. In contrast, AKR.Fv-1b mice develop MNU-induced lymphomas with an incidence and latency similar to those of the AKR/J strain. Furthermore, thymic lymphomas from both strains express an immature CD4-8+ phenotype, indicating neoplastic transformation of the same thymocyte subset. Southern blot analysis confirmed that lymphoma DNA from AKR.Fv-1b mice did not contain somatically acquired provirus integrations. These results demonstrate that provirus integration does not contribute to the predisposition of AKR mice to develop a high incidence of early MNU-induced lymphomas. Nevertheless, MNU treatment stimulated high-level expression of infectious ecotropic MuLV in AKR.Fv-1b as well as in AKR/J mice, suggesting that viral gene products might enhance lymphoma progression.