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The Fv-1 murine gene controls predominantly the replication of leukemia viruses of murine cells. Forced passage by B-tropic Friend leukemia virus in the restrictive host cells (NIH, Fv-1n/n) results in viral progeny capable of replicating efficiently in murine cells of any Fv-1 type, which are denoted as NB-tropic virus. We have studied the RNase T1-resistant oligonucleotide pattern of a series of NB-tropic Friend virus isolates and have been able to show changes from the parental B-tropic virus which occur at the 5' end of the genome. Cloned NB-tropic virus falls into three classes, demonstrating either four, one, or no apparent changes in the genome. These results suggest the possibility that conversion to NB tropism occurs by a recombination mechanism but, since change to NB tropism can occur without any observable oligonucleotide alteration, they do not confirm that any single oligonucleotide is diagnostic of NB tropism.

The regulation of expression of the Friend virus complex (FV) was studied in clones of H-2 congenic erythroleukemia cell lines. Molecular probes specific for each of the two FV components were generated and hybridized to RNA extracted from clones differing with respect to virus production or viral envelope antigen expression. Sequences representing the defective component of FV continued to be transcribed in cells regardless of virus production. The presence of helper virus-specific sequences, however, was shown to be directly related to virus expression. Molecular hybridization with cytoplasmic and nuclear RNA extracted from the H-2 congenic clones representing various stages of viral expression demonstrated a direct correlation between the level of transcription of FMuLV-specific sequences and the degree of virus expression. These data suggest that regulation of FV expression is primarily related to the cessation of helper virus transcription in the cell lines studied. In this paper we provide further evidence that the defective viral components is responsible for the rapid malignancy associated with Friend disease.

The fate of input Friend leukemia virus RNA was studied using labeled input virus. The appearance of nuclear RNA-DNA hybrid molecules and the apparent integration of input virion RNA with host cell DNA was studied using a series of inhibitors of DNA or protein synthesis, cell growth conditions, and an intercalating agent. Under all these conditions of infection, little to no viral-specific RNA-DNA hybrid molecules were formed. These data demonstrate that the formation of such RNA-DNA hybrid structures requires conditions of infection that allow provirus synthesis and integration. Furthermore, they suggest that at least a fraction of input virion RNA may transiently become integrated with host cell DNA.

Fv-1 gene-mediated host restriction of Friend leukemia virus replication was investigated in terms of coat protein synthesis. By using the assay of pseudotype formation with vesicular stomatitis virus. it was shown that under restricting growth conditions the availablity of leukemia virus coat protein for pseudotype formation was decreased. These studies appear to eliminate a pure assembly defect as the mechanism of Fv-1 host restriction.

The murine gene Fv-1 exerts a major control over the replication of Friend murine leukemia virus (F-MuLV). An effect of the gene product has been determined to be at the level of accumulation and integration of viral DNA. Aphidicolin, an inhibitor of eucaryotic DNA polymerase alpha, was studied in murine cells infected either permissively or nonpermissively with regard to the Fv-1 genotype. Results indicated that inhibition of DNA polymerase alpha did not affect the accumulation of form III viral DNA in either permissive or nonpermissive cells. However, the normal accumulation of circular form I DNA in permissive cells was inhibited. The block in the accumulation of form I DNA resembled that occurring in some F-MuLV Fv-1-nonpermissive infections. Additionally, aphidicolin treatment resulted in the accumulation of novel low-molecular-weight viral DNA species, normally detectable in a nonpermissive infection of NIH cells with B-tropic F-MuLV. These data suggest that the Fv-1 gene product may interact with host DNA polymerase alpha to prevent viral replication.

The murine gene Fv-1 has been shown to exert a major influence over the replication of ecotropic murine leukemia viruses. Studies of the replication of Friend murine leukemia virus have shown that the restriction of viral replication occurs intracellularly after the initiation of viral DNA synthesis. The precise mechanism of the block imposed by the Fv-1 gene product is not completely understood. Our studies of Fv-1 restrictive infection have shown a variable decrease in the accumulation of intracellular unintegrated form I viral DNA. Analysis by microinjection of the viral DNA formed in nonpermissively infected BALB/c cells indicates that this DNA is infectious. These studies indicate that the form I DNA accumulated in nonpermissively infected BALB/c cells contains the complete viral sequences necessary for the production of viral progeny, and therefore, they suggest that the Fv-1 host restrictive mechanism recognizes viral factors other than form I DNA alone. These results support the possibility that Fv-1 host restriction occurs after formation of infectious viral DNA, perhaps at the integration step itself.

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.

The host response to murine ecotropic leukemia viruses is mainly controlled by the mouse Fv-1 gene. This locus controls virus replication at an intracellular stage and prevents provirus integration. Biological studies suggest that the Fv-1 effector molecule recognizes at least one virion structural protein. We have produced host range variants of B-tropic Friend murine leukemia virus in order to study the primary structure of potential viral target proteins. Our results show that conversion of B-tropism to NB-tropism is associated with changes in the primary structure of three gag proteins--p15, p12, and p30. These results suggest that host range conversion is due to a recombinational event, presumably between the parental virus and an endogenous murine virus. They also open the possibility that p12 and p30 may be involved in host range restriction.

Host restriction of exogenous infection by murine leukemia viruses is controlled in vitro predominantly by the murine Fv-1 locus. The mechanism of this host restriction was investigated by comparing the early events in the replication of N-tropic versus B-tropic Friend leukemia virus in NIH 3T3 cells. These cells, which are Fv-1nn in type, are permissive for the N-tropic strain, but nonpermissive for the B-tropic strain, which replicates permissively in Balb/c cells. We have studied the synthesis, intracellular location, and molecular form of virus-specific DNA early in replication by means of molecular hybridization with a virus-specific DNA probe. Our results suggest that in the permissive infection viral DNA rapidly becomes integrated with cellular DNA. However, in the nonpermissive infection, although almost equal amounts of both positive and negative strand viral DNA are synthesized, integration of the provirus does not occur.

Concanavalin A (Con A) has been used to rapidly and selectively agglutinate murine and avian oncornavirions from culture medium or plasma. The agglutinated virus was concentrated rapidly and gently by low-speed centrifugation and solubilization with alpha-methyl mannoside. Infectious virus was purified 2.3 times with respect to nucleic-acid content, and more than 60% of its infectivity was recovered. Infectious particles of densities 1.18 and 1.16 g/cm(3) were found in mouse cells infected with Friend virus. Con A reacted only with particles of density 1.16 g/cm(3), indicating heterogeneity with respect to carbohydrate content or structure as well as buoyant density. Electron microscopy of virus agglutinated with Con A showed a zone of Con A-glycoprotein complexes averaging 12-15 nm in thickness.

Host restriction of oncogenesis of RNA tumor viruses in vivo is associated with several gene loci. One of these genes, the Fv-1 locus in mice, is expressed in vitro and may be studied in mouse-embryo cultures that are restrictive or permissive for replication of Friend leukemia virus. Two strains of Friend leukemia virus, N-or B-tropic, show reciprocal ability to replicate successfully in either NIH Swiss (N-type) or BALB/c (B-type) cells that differ at the Fv-1 locus. These two strains of virus and two cell lines form a system to measure host restriction in vitro. Measurement of adsorption of Friend leukemia virus to permissive or restrictive cells reveals no difference in rate or total amount of virus bound. Furthermore, studies with virions of vesicular stomatitis virus phenotypically mixed within an envelope containing Friend leukemia virus protein show no differences in penetration or replication of vesicular stomatitis virus. These results strongly suggest that host restriction of Friend leukemia virus is due to an intracellular event in the viral replication cycle.