Down-modulation of the costimulatory molecule, CD28, is a conserved activity of multiple SIV Nefs and is dependent on histidine 196 of Nef.

In this study Nef proteins derived from simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) were compared to assess their abilities to down-modulate the cell surface levels of the T-cell costimulatory molecule CD28. We demonstrate that in addition to Nef derived from the prototypic SIVmac239, Nef proteins encoded by the pathogenic SIVsmmPBj molecular clone and the SIVsmmB670 isolate also down-modulate cell surface CD28. In contrast, Nef proteins derived from HIV failed to down-modulate CD28. We have also identified H196 as a critical residue which influences the capacity of SIVmac Nef to down-modulate CD28. Nef derived from SIVmacJ5 failed to down-modulate cell surface CD28, whereas a Q196H substitution mutant of SIVmacJ5 Nef was able to down-modulate cell surface CD28. Conversely, substitution of H196 to Q196 in SIVmac239 Nef resulted in a mutant that had minimal effect on cell surface CD28 expression, despite retaining the capacity to down-modulate cell surface CD3epsilon. H196 lies immediately adjacent to a documented di-leucine endocytic motif and mutation of this motif also abrogated the ability of SIVmac239 Nef to down-modulate CD28. These findings demonstrate that down-modulation of the costimulatory molecule, CD28, and clonotypic TCR/CD3 complex are conserved attributes of SIV Nef.