Phenotypic mixing between N- and B-tropic murine leukemia viruses: infectious particles with dual sensitivity to Fv-1 restriction.

In effort to understand how N or B tropism is determined in murine leukemia virus (MuLV) particles, we analyzed the MuLV produced after dual infection of mouse cells by N- and B-tropic MuLV. The progeny MuLV from such a mixed infection are sensitive to Fv-1 restriction in both N- and B-type cells, but are still highly infectious for mouse cells which do not exhibit Fv-1 restriction. This dual sensitivity to Fv-1 restriction is a phenotypic property of MuLV produced by mixedly infected cells, since individual virus clones derived from this MuLV are either N- or B-tropic. In further experiments, we superinfected murine sarcoma virus (MSV)-transformed cells with mixtures of N- and B-tropic MuLVs. The rescued MSV is restricted in its ability to transforms both N- and B-type cells. The results suggest that N- and B-tropic MuLVs specify different determinants, which are incorporated into virions along with the viral genome and which are the recognition sites for Fv-1 restriction. The presence of a given determinant in a virion renders the virus sensitive to restriction in cells of the opposite Fv-1 type.     

Fv-1 N- and B-tropism-specific sequences in murine leukemia virus and related endogenous proviral genomes

Oligonucleotide probes specific for the Fv-1 N- and B-tropic host range determinants of the gag p30-coding sequence were used to analyze DNA clones of various murine leukemia virus (MuLV) and endogenous MuLV-related proviral genomes and chromosomal DNA from four mouse strains. The group of DNA clones consisted of ecotropic MuLVs of known Fv-1 host range, somatically acquired ecotropic MuLV proviruses, xenotropic MuLV isolates, and endogenous nonecotropic MuLV-related proviral sequences from mouse chromosomal DNA. As expected, the prototype N-tropism determinant is carried by N-tropic viruses of several different origins. All seven endogenous nonecotropic MuLV-related proviral sequence clones derived from RFM/Un mouse chromosomal DNA, although not recognized by the N probe, showed positive hybridization with the prototype B-tropism-specific probe. The two xenotropic MuLV clones derived from infectious virus (one of BALB:virus-2 and one of AKR xenotropic virus) failed to hybridize with the N- and B-tropic oligonucleotide probes tested and with one probe specific for NB-tropic Moloney MuLV. One of two endogenous xenotropic class proviruses derived from HRS/J mouse chromosomal DNA (J. P. Stoye and J. M. Coffin, J. Virol. 61:2659-2669, 1987) also failed to hybridize to the N- and B-tropic probes, whereas the other hybridized to the B-tropic probe. In addition, analysis of mouse chromosomal DNA from four strains indicates that hybridization with the N-tropic probe correlates with the presence or absence of endogenous ecotropic MuLV provirus, whereas the B-tropic probe detects abundant copies of endogenous nonecotropic MuLV-related proviral sequences. These results suggest that the B-tropism determinant in B-tropic ecotropic MuLV may arise from recombination between N-tropic ecotropic MuLV and members of the abundant endogenous nonecotropic MuLV-related classes including a subset of endogenous xenotropic proviruses.     

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.     

Unstable resistance of G mouse fibroblasts to ecotropic murine leukemia virus infection.

G mouse cells were resistant to N- and NB-tropic Friend leukemia viruses and to B-tropic WN 1802B. Though the cells were resistant to focus formation by the Moloney isolate of murine sarcoma virus, they were relatively sensitive to helper component murine leukemia virus. To amphotropic murine leukemia virus and to focus formation by amphotropic murine sarcoma virus, G mouse cells were fully permissive. When the cell lines were established starting from the individual embryos, most cell lines were not resistant to the murine leukemia viruses. Only one resistant line was established. Cloning of this cell line indicated that the resistant cells constantly segregated sensitive cells during the culture; i.e., the G mouse cell cultures were probably always mixtures of sensitive and resistant cells. Among the sensitive cell clones, some were devoid of Fv-1 restriction. Such dually permissive cells, and also feral mouse-derived SC-1 cells, retained glucose-6-phosphate dehydrogenase-1 and apparently normal number 4 chromosomes. The loss of Fv-1 restriction in these mouse cells was not brought about by any gross structural changes in the vicinity of Fv-1 on number 4 chromosomes.     

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.     

Fv-1 host restriction of Friend leukemia virus: analysis of unintegrated proviral DNA.

The murine gene Fv-1 predominantly controls the outcome of infection by murine ecotropic retroviruses. The inhibition of virus replication by the Fv-1 gene product has been determined to be at an early stage in virus replication. Mechanistically, its effect appears to be on the accumulation of unintegrated proviral DNA or its integration or both. We investigated the synthesis of unintegrated proviral DNA, using several clones of B-, N-, or NB-tropic Friend murine leukemia virus. Our results indicate that the accumulation of B-tropic proviral DNA in NIH cells may be inhibited at either the level of linear (form III) or covalently closed circular DNA (form I), depending upon the degree of restriction of the clone of virus used. We confirmed that there is an effect of the Fv-1 gene on the accumulation of form I DNA of either B- or N-tropic Friend murine leukemia virus. However, the decrease in infectious centers effected by the Fv-1 gene did not correlate quantitatively with the effect on form I proviral DNA produced by N-tropic Friend murine leukemia virus in nonpermissive cells. Lastly, we demonstrated in nonpermissively infected NIH cells that a rapidly migrating doublet of viral DNA is formed.     

Susceptibility of inbred strains of mice to murine AIDS (MAIDS) correlates with target cell expansion and high expression of defective MAIDS virus.

Murine AIDS (MAIDS) is readily induced by the Duplan strain of defective murine leukemia virus in susceptible C57BL/6 mice. To identify mouse strains resistant to MAIDS, and to understand the genetic factors controlling susceptibility to the disease, we screened more than 20 inbred strains of mice for their susceptibility to MAIDS. For this study, mice of the Fv-1n/n, Fv-1b/b, or Fv-1n/b genotype were inoculated with stocks of defective MAIDS virus pseudotyped with N-tropic, B-tropic, or NB-tropic helper murine leukemia virus, respectively. Strains could be classified as susceptible, resistant, or moderately resistant. None of the individual H-2 haplotypes examined appears to explain resistance to MAIDS by itself. However, a very good correlation between the susceptibility or resistance phenotype and the presence or absence of defective proviral DNA and RNA in the spleen of these animals was found. Since the presence of defective proviral DNA and RNA reflects the oligoclonal proliferation of the cells infected by the defective MAIDS virus, our results strongly suggest that this target cell expansion is genetically controlled and is necessary and perhaps even sufficient for the development of the disease.     

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.     

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.     

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.     

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.     

Analysis of wild-derived mice for Fv-1 and Fv-2 murine leukemia virus restriction loci: a novel wild mouse Fv-1 allele responsible for lack of host range restriction.

Wild-derived mice originally obtained from Asia, Africa, North America, and Europe were typed for in vitro sensitivity to ecotropic murine leukemia viruses and for susceptibility to Friend virus-induced disease. Cell cultures established from some wild mouse populations were generally less sensitive to exogenous virus than were cell cultures from laboratory mice. Wild mice also differed from inbred strains in their in vitro sensitivity to the host range subgroups defined by restriction at the Fv-1 locus. None of the wild mice showed the Fv-1n or Fv-1b restriction patterns characteristic of most inbred strains, several mice resembled the few inbred strains carrying Fv-1nr, and most differed from laboratory mice in that they did not restrict either N- or B-tropic murine leukemia viruses. Analysis of genetic crosses of Mus spretus and Mus musculus praetextus demonstrated that the nonrestrictive phenotype is controlled by a novel allele at the Fv-1 locus, designated Fv-10. The wild mice were also tested for sensitivity to Friend virus complex-induced erythroblastosis to type for Fv-2. Only M. spretus was resistant to virus-induced splenomegaly and did not restrict replication of Friend virus helper murine leukemia virus. Genetic studies confirmed that this mouse carries the resistance allele at Fv-2.     

Biochemical analysis of the p30''s of N-, B-, and B leads to NB-tropic murine leukemia viruses of BALB/c origin.

Previous analysis of the virion proteins of an N- and a B-tropic type C virus of BALB/c mice, of 16 N-tropic recombinants (XLPN viruses) between these viruses, and of eight NB-tropic viruses derived from the B-tropic virus suggested that among these closely related viruses N-, B-, or NB-tropism was associated with the electrophoretic mobility of p30 on sodium dodecyl sulfate-polyacrylamide gels, and thus that p30 might determine this phenotype. To obtain further evidence for the association of structural markers of p30 with N-, B-, or NB-tropism, we have analyzed the p30's of these same viruses by using two-dimensional tryptic peptide mapping and slab gel isoelectric focusing. The results of these analyses suggest that (i) a single peptide unique to the N-tropic virus p30- is present in the p30 of all N-tropic recombinants; (ii) a single peptide unique to the B virus p30 is not present in p30's of the N-tropic recombinants, and this peptide is also absent in p30's of NB-tropic viruses derived from the B-tropic virus; and (iii) p30's of NB-tropic viruses possess a new tryptic peptide not found in the p30 of their B-tropic virus progenitors, and this new peptide is not found in the p30 of the N-tropic virus of BALB/c or the XLPN viruses. These results are consistent with the possibility that p30 may determine the N-, B-, or NB-tropism of murine leukemia viruses. In addition, these studies indicate that some of the N-tropic recombinants have experienced recombination within the p30 gene.     

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

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

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

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

Molecular characterization of the Akvr-1 restriction gene: a defective endogenous retrovirus-borne gene identical to Fv-4r.

A dominant restriction allele, Akvr-1r, from California wild mice (Mus musculus domesticus) confers resistance to exogenous ecotropic murine leukemia virus (MuLV) infection. The presence of an ecotropic MuLV envelope-related glycoprotein in uninfected virus-resistant cells suggests that viral interference is a possible mechanism for this resistance. We molecularly cloned the ecotropic MuLV envelope-related sequence from the genomic DNA of a wild mouse homozygous for the Akvr-1r locus. The cloned provirus was defective and contained a C-terminal end of the pol gene, a complete envelope gene, and a 3' long terminal repeat. The presence of this provirus was directly correlated with Akvr-1r-mediated virus resistance in cell cultures and hybrid mice. The Akvr-1r provirus restriction map and partial DNA sequence were identical to those of the Fv-4r allele, an ecotropic MuLV resistance locus from Japanese feral mice (M. musculus molossinus), which was previously shown to be allelic with the Akvr-1r gene. The 3' host flanking sequences of Fv-4r and Akvr-1r also had identical restriction maps. These findings indicate that Akvr-1r and Fv-4r are the same gene. It was probably acquired by interbreeding of these feral species in recent times. Conservation of this locus might be favored by the useful function that it performs in protection against ecotropic MuLV infection endemic in both populations of wild mice.     

Genetic evidence for a product of the Fv-1 locus that transfers resistance to mouse leukemia viruses.

Extracts of mouse cells have been shown to transfer to N- or B-trophic host range types of mouse leukemia viruses. The genetic specificity of the inhibition was tested in two ways: (i) by correlating the Fv-1 genotype of a number of mouse strains with the restriction-transferring activity of extracts of the respective embryo cell cultures, and (ii) by correlating the Fv-1 genotype of BLC3F2 (C57BL/6 female [Fv-1bb] by C3H male [Fv-1nn] parental strains) mouse embryos, which segregate the Fv-1 alleles in a 12:1 ratio, with the inhibitor activity of extracts of the cells from each embryo. Five independent matings, totaling 45 individual embryos, were tested. Each embryo was cultured, and the Fv-1 genotype was determined independently by titration of N- and B-tropic viruses; the extracts of replicate secondary cultures were tested for their effect on infection of permissive cells by N- and B-tropic viruses. The specific-restriction-transferring activity of the embryos was found to segregate with the appropriate Fv-1 genotype. These res-lts confirm the suggestion that the inhibitor of the leukemia virus host range types in the cellular extracts is a product of the Fv-1 locus.     

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.     

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.