In Vitro Studies of SIV nef Function

Akhtar Karim, Shamim (2003). In Vitro Studies of SIV nef Function. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f561

Abstract

There is strong evidence that a functional Nef protein of human and simian immunodeficiency viruses is a critical factor in contributing to viral pathogenesis. A variety of properties have been ascribed to Nef that may contribute towards these effects. However, the exact mechanisms by which Nef functions remain unclear.
A number of clones of SIV nef have been described which were generated after spontaneous evolution both in vivo and in vitro based upon changes relative to the wild type SIVmacJ5 virus (Rud et ah, 1994, Arnold et al, 1999, N. Almond, personal communication). Furthermore, additional clones exist which were based upon the repair mutants reported by Whatmore et al (1995) after infection of macaques with the attenuated SlVmacCS virus (Rud et al, 1994).
The aims of this study were two-fold. First, to utilise a panel of SIV Nef specific monoclonal antibodies (Arnold et al, 1999) to elucidate structural differences between wild type and attenuated SIV Nef clones and further information on the epitope recognised by the conformational dependant monoclonal antibody, KX70 (Arnold et al, 1999). Second, by utilising yeast-2-hybrid technology to investigate interactions of the SIV Nef clones with selected cellular components and relate these findings to the manner in which the virus behaves in vivo.
The results indicate that the alpha helical region around which the attenuation in SrVmacCS lies is a key component of the epitope recognised by KK70 as well as being a key region for interaction of cellular components with SIV Nef. The repair mutants reported by Whatmore et al (1995) did not regain the epitope recognised by KK70 as the amino acids evolved to a near wild type sequence. This indicates that it may be an overall structural effect that these mutations are having which is affecting antigenicity. Interestingly, one of the repair mutants (J5(EIYL)) did regain the ability to interact with the cellular components. This was lost when this repair was presented in conjunction with the R191E mutation (J5(EIYL+R>E)). Interestingly, the R191E mutation alone (J5(R>E)) caused non-specific trans-activation of the GAL4-AD. Furthermore, GX2, which harbours a 66 nucleotide deletion at the 5’ end of Nef (disrupting the binding of KK75, a monoclonal antibody that recognises a linear epitope) also caused non-specific trans-activation of the GAL4-AD. These results suggest that the mutations in GX2 and J5(R>E) may have a significant effect on the overall structure of SIV Nef.
This study describes the use of a novel technique for the fine mapping of novel antigenic epitopes using monoclonal antibodies. The structural and yeast-2-hybrid data together highlight important observations of SIV Nef which can be related to structure and function in vivo, as well as being used as a basis for future in vivo studies.