Figure 5. CD4-downregulation, MHC class I-downregulation and infectivity enhancement mediated by SIV Nef mutants with impaired anti-tetherin activity.

The surface expression of CD4 (A) and MHC class I (B) was compared on cells expressing each of the SIV Nef mutants. Jurkat cells were electroporated with a bicistronic vector (pCGCG) expressing either wild-type Nef or the indicated Nef mutants together with GFP. Cells were stained with a PerCP-conjugated monoclonal antibody to CD4 and an APC-conjugated monoclonal antibody to HLA class I molecules. Cells were gated on the GFP⁺ cell population and the percentage of CD4 and MHC I on the cell surface was determined relative to cells transfected with empty vector. Error bars indicate the standard deviation of duplicate transfections, and the black and red dotted lines correspond, respectively, to 3 and 5 standard deviations over the activity of wild-type Nef. (C) The Nef mutants with impaired anti-tetherin activity were also tested for infectivity enhancement. Viruses were generated by transient transfection of 293T cells with SIV_mac239 Δnef proviral DNA and constructs expressing each of the Nef mutants, wild-type Nef or an empty vector. The infectivity of these viruses was then determined on GHOST X4/R5 cells, which express GFP upon SIV infection, 48 hours post-inoculation by flow cytometry. The relative infectivity of SIV Δnef trans-complemented with the Nef mutants was calculated relative to SIV Δnef trans-complemented with wild-type Nef. Virus infectivity in the absence of Nef is indicated by the white bars, and the activities mediated by wild-type Nef and the G₂A myristoylaton site mutant are indicated by black and red bars, respectively. Error bars indicate the standard deviation of duplicate infections, and the black and red dotted lines correspond, respectively, to 3 and 5 standard deviations over the infectivity of SIV Δnef without trans-complementation. Substitutions in the N-terminal domain are indicated by green bars, substitutions in the globular core domain are indicated by blue bars, and substitutions in the flexible loop region are indicated by orange bars.