Cell Host & Microbe, Volume 6 Supplemental Data Tetherin-Driven Adaptation of Vpu and Nef Function and the Evolution of Pandemic and Nonpandemic HIV-1 Strains

To construct infectious HIV-1 molecular clones expressing heterologous vpu genes without overlapping env sequences, we first eliminated this overlap (NL4-3 UEOel; vpU Env Overlap eliminated) and subsequently inserted an IRES element downstream of vpu (NL4-3 UIE; vpU-IRES- Env) to restore Env expression (Figure S4A). Western blot analysis of 293T cells transfected with these proviral constructs confirmed that the IRES element facilitated efficient Env expression, although the p55 and p24 gag levels were moderately reduced compared to the parental and UEOel NL4-3 constructs (Figure S4B). Despite this, the infectivity of the NL4-3 UIE derived virus was only 2.5-fold reduced compared to the parental virus (Figure S4C). An intact vpu gene did not significantly enhance the release or infectivity of progeny virus from transfected 293T cells in both the presence and absence of CD4 expression (Fig. S4C, and data not shown) but enhanced the release of progeny virions (as measured by p24 antigen production) from HeLa-derived TZM-bl cells (which express endogenous tetherin) by 9-fold (Figure S4D). On average, production of the NL4-3 UIE virus from transfected TZM-bl cells was 6.6-fold reduced compared to that of wildtype NL4-3. Importantly, virus release was further reduced by about 10-fold when the vpu gene was inactivated (Figure S4D). Next, we examined the functional activity of the NL4-3 UIE constructs at different levels of tetherin expression. After transfection with a high dose (1000 ng) of the tetheri