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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 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.

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 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.

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 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.