Best, Steven; (1994) The positional cloning of Fv-1, a mouse retroviral restriction gene. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The mouse Fv-1 gene restricts the replication of murine leukaemia viruses (MuLVs) and has been mapped to the distal region of chromosome four. This gene somehow prevents integration of viral DNA into the host genome. Several genes, notably four endogenous proviral elements (Xmvs) and Pnd were linked to Fv-1 and so offered a route to clone the gene by a positional approach. A backcross was analysed to position Fv-1 more accurately relative to the linked genes. This data revealed that Pnd and Xmv-9 were within -1,200 kilobases of Fv-1. Yeast artificial chromosome (YAC) clones carrying linked genes were isolated. Attempts were made to generate a contig of the area, this but was complicated by the presence of mouse B1, LINE and zinc finger repeats, and an area of instability in the clones. YACs judged to lie closest to Fv-1 were introduced, by fusion, into mouse cells. It was anticipated that the presence of Fv-1 on a YAC would be detected by the ability to restrict an infectious virus. Of the cell lines tested, L cells gave the best fusion results, with a high frequency of complete YAC integration. The L cells produced their own reverse transcriptase (RT) from an endogenous virus. The virus infectivity assay, which monitored virus production by RT activity, was unusable. An alternative assay was developed which used colony formation as an measure of infection. This led to the identification of a 190 kilobase YAC which conferred Fv-1 activity. Identification of the Fv-1 gene is currently underway. Cloning Fv-1 will allow the biochemical mechanisms involved in virus restriction to be investigated, and will offer a wider insight into the mechanisms of retroviral integration. The implications for the prevention of human retroviral disease are potentially important.

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