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Fv-lb restriction in BALB/3T3 cells is temporarily abrogated following infection with N-tropic murine leukemia virus. The mechanism of this phenomenon was investigated by comparing the inactivation rates for viral infectivity and for the ability of the same virus to abrogate Fv-1 restriction. Inactivation of the abrogating ability of N-tropic murine leukemia virus following graduated doses of gamma radiation proceeded at half the rate of that for viral infectivity. This result indicates that viral RNA must function in abrogating Fv-lb restriction but that only a portion of the viral genome is required. The inactivation kinetics of Ntropic murine leukemia virus were also determined following incubation of virus at 43°C. Abrogating ability of N-tropic murine leukemia virus was found to be about six times as stable under these conditions as was viral infectivity. Interestingly, virion-associated reverse transcriptase activity was inactivated at the same rate as was viral infectivity, indicating that this enzyme may not need to function during abrogation. Virus heated at 43°C was used to study the kinetics of the abrogation phenomenon itself. Abrogation was shown to be transient, requiring 6 to 9 h after virus infection to become maximally effective and beginning to disappear after about 18 h. The data reported here confirm the idea that abrogation of Fv-1 restriction can be separated experimentally from virus replication, and they raise the possibility that a separate biochemical pathway exists for incoming viral RNA in Fv-1 restrictive cells. Certain murine leukemia viruses (MuLV's) have reported that Fv-i restriction is not assoare inhibited in their ability to replicate in ciated with a two-hit requirement for productive certain mouse cells by a naturally occurring re-infection of nonpermissive celLs (18, 31). striction system dependent upon the presence of To understand and to characterize Fv-1 rethe Fv-i mouse gene (16, 24). Studies of Fv-i striction more fully, we have initiated a series of restriction thus far have shown that MuLV's are experiments designed to examine the nature of probably inactivated after they adsorb to and the two-hit requirement for MuLV replication penetrate nonpermissive cells (17,21), but before in nonpermissive cells. This work has been DNA transcribed from incoming viral RNA is greatly facilitated by the use of defective murine integrated into the host cell genome (19, 32). sarcoma virus (MSV) pseudotypes which have The exact mechanism of Fv-i restriction, howbeen rendered phenotypically sensitive to Fv-1 ever, has yet to be elucidated. restriction following mixed infection with Nor Fv-1 restriction characteristically alters the B-tropic MuLV during the "rescue" process (1, titration patterns of N-and B-tropic MuLV's. 7). Although these virus stocks infect corresponding In a previous report we used an N-tropic pseupermissive cells with one-hit kinetics, they give dotype of Moloney MSV, rescued from transtwo-hit or multi-hit dose-response curves in Fv-formed S+L-cells by N-tropic MuL...

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The mechanism of Fv-1 restriction in DBA/Z ( F v -l n ) mouse cells was investigated by quantitative infectious center assays employing a newly isolated continuous cell line. Titration of B-tropic murine leukemia virus (MuLV) i n DBAjZ cells showed twohit kinetics and, at sufficient multiplicities of infection (MOls), the entire cell population could be productively infected with the restricted virus.

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The molecular basis of abrogation of Fv-1 restriction in mouse cells by murine leukemia virus was investigated. Two different lines of experimentation indicated that high-molecular-weight viral RNA is required for abrogation. First, the decay of abrogating ability of virus stocks heated at 43 degrees C was quantitatively correlated with a loss of intact virion 35S RNA. Second, Act D virions, which lack such RNA although they contain normal structural proteins, failed to abrogate. These findings imply that abrogation does not result from the mere entry of virion structural proteins into a cell. Additional data indicate that the role of viral RNA in abrogation is not that of a template for DNA synthesis. Virus particles lacking reverse transcriptase activity as a result of either mutation or heat inactivation exhibit abrogating activity even though they do not synthesize detectable viral DNA. In addition, abrogation was shown to take place in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. Thus, abrogation does not depend on viral or cellular DNA synthesis, and the role of viral RNA in this process must involve some other function. The nature of this viral function and its occurrence in Fv-1 permissive cells are discussed.

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