Characterising RNA-Early stage Antiviral Factor (REAF) as an inhibitor of Human Immunodeficiency Virus (HIV) replication

PhD thesisREAF is a potent inhibitor of HIV-1, HIV-2 and SIV infection, however the mechanism by which REAF mediates restriction is unknown. Defining the mechanisms by which previously identified restriction factors mediate restriction has shown that many restriction factors target and disrupt specific steps in the viral life cycle. It is known that REAF mediates restriction at or before reverse transcription, that REAF is capable of binding nucleic acids, and that capsid stability is a determinant of susceptibility to REAF-mediated restriction. Counter mechanisms, employed by viruses to evade or overcome the inhibition mediated by specific restriction factors, have also been identified and characterised. For many archetypal restriction factors - SAMHD1, Tetherin and APOBEC3G - viral factors which directly counteract their restriction have been defined. This thesis characterises REAF as a potent inhibitor of HIV-1 infection, and proposes that HIV-1, and potentially other immunodeficiency viruses, downmodulate REAF during the early stages of infection, increasing cell suspectibility to a second viral infection in the process. Furthermore, evidence is presented that the viral mechanism mediating downmodulation of REAF involves Vpr. Analogies are also drawn between Vpx-mediated degradation of SAMHD1 and a potential mechanism for Vpr-mediated degradation of REAF. Like SAMHD1, Vpx and Vpr, REAF is here shown to localise to the nucleus, for example. Additionally, REAF carries a short motif (‘KRPR’) which functions as a nuclear localisation signal and regulatory element found on SAMHD1. Many previously identified restriction factors have been defined as inducible factors functioning as effector proteins of the immune response. Here REAF is demonstrated to be an inducible antiviral factor operating outside of the dominant IFN-mediated antiviral response, but nonetheless induced by various immune stimulants, some of which are also capable of inducing the IFN-mediated response. REAF is shown to be exceptionally sensitive to certain immune stimulants, in terms of levels of induction and rapidity of response. Furthermore, large and significant variations in REAF expression levels are observed across donors in primary immune cells. Variation in inducibility in these cells is also clear and significant. It should therefore be considered that REAF is a potentially physiologically important aspect of the induced immune response, and that variation in REAF levels across individuals could potentially be clinically important.Life Sciences InstituteRosetrees trus