Figure 2. Analysis of CD4 downregulation and tetherin antagonism of 304 natural Vpu alleles.

Representatives of all amino acid sequences obtained through SGA were cloned and tested in standardised assays for the two major functions of Vpu: (A) cell surface CD4 downregulation and (B) tetherin counteraction. The time point of each sample, in years post-seroconversion, is indicated beneath the graph with the patient identification and progression group. The function of each Vpu is represented relative to the prototypical Vpu from NL4.3, as indicated by a dashed line at 100%, and the functions of all other Vpus are represented as a percentage thereof. The NL4.3 S52,56A Vpu mutant, defective for both CD4 downregulation and tetherin counteraction activity, is indicated by a dashed line (at 0% on the CD4 downregulation graph and 13% on the tetherin counteraction graph). Each symbol represents the average of a minimum of three independent experiments, weighted to represent the number of sequences obtained per sample with that particular amino acid sequence (allele frequency), to give an overall proportional representation of function per time point. Means for overall Vpu function for each time point are shown as short horizontal lines. Significant differences between time points from each individual are indicated by asterisks. Briefly, the assays were performed as follows: (A) HeLa-TZMbl cells were co-transfected with 100 ng of pCRVI-Vpu or empty vector (EV) plasmid and 100 ng of pCR3.1-eGFP. 24 hours later cell surface CD4 levels were analysed by flow cytometry. CD4 downregulation was determined by comparing median fluorescent intensities of CD4 in the presence and absence of Vpu, with the downregulation achieved by NL4.3 Vpu set at 100%. Note that the absolute value of CD4 reduction achieved by NL4.3 was 80%±6. (B) 293T cells were transfected with a fixed dose (50 ng) of pCR3.1-hu-tetherin in combination with 500 ng of NL4.3-del Vpu proviral plasmid and 25 ng of pCRVI-Vpu. 48 hours later the supernatants are removed from the cells and assayed on Hela-TZMbl cells for the quantity of infectious virus. The 100% line represents the amount of infectious virus released in the presence of NL4.3, to allow direct comparisons between the CD4 downregulation and tetherin counteraction assays. 25 ng of pCRVI-Vpu was used as this quantity produced the same amount of Vpu protein as that of the full-length NL4.3 molecular clone, as determined by Western blot analysis.