Host restriction factors such as Fv1 in mice and Trim5α in primates block retroviral infection through their interaction with capsids. One major form of Fv1, encoded by the Fv1b allele, restricts only N-tropic MLV (N-MLV) when expressed at the lower endogenous level, but also restricts B-tropic MLV (B-MLV) and NB-tropic MLV (NB-MLV) at the higher levels seen in cells transduced with Fv1 b-expressing retroviral vector. However, a previous pull- down study detected almost equal binding of Fv1b to all three MLV capsids. In this study, the relationships between Fv1 binding, restriction and expression level were studied in detail. To study the restriction specificity of Fv1 b at different expression levels, new Tet-On vectors were developed to allow doxycycline-inducible expression of Fv1. These vectors allowed restriction studies from a very low Fv1 b level where no restriction could be observed, to a high level where inhibitions of B-MLV and NB-MLV are also observed. Similar phenomenon is also observed in other Fv1 mutants. By contrast, Fv1 n, even when over-expressed, restricted only B-MLV but not NB-MLV or N-MLV. The binding of Fv1b to different capsids at different Fv1 concentration were compared using a new pull-down assay performed on microtitre plates. Fv1b appears to bind to all MLV capsids at the lowest concentration of Fv1 where binding could be detected. The study was extended to include other Fv1 mutants, and as expected binding could be detected for all restrictive Fv1-MLV pairs. Interestingly, a few outliers including Fv1b vs B-MLV demonstrated strong binding but either weak or low restriction, suggesting there may be other factors causing the lack of restriction. Together, these data suggest that although Fv1b have very similar apparent binding affinities to different MLV capsids, different amount of Fv1b are required for the restriction of N-, B- and NB-MLV.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:647323 |
| Date | January 2015 |
| Creators | Li, H. L. W. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1468721/ |
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