Fig. 3.

SeV infection induces tetherin degradation. (a) HeLa cells were transfected, infected (m.o.i. = 20) and analysed as in Fig. 1(a). Moreover, total RNA extracts were isolated and tetherin mRNA was determined by quantitative PCR (see Methods). (b) MDCK Flag-tetherin cells were mock-infected or infected with SeV (m.o.i. = 20). At 20 h p.i., cells were pulse radiolabelled for 15 min and chased for the indicated periods of time. Radiolabelled Flag-tetherin was immuno-precipitated, analysed by PAGE and revealed by autoradiography. Lower part: quantified values of tetherin were plotted. Bars represent mean sd from at least two independent experiments. (c) Mock-treated or SeV-infected (m.o.i. = 20) HeLa cells were incubated for 24 h in the presence of chloroquine (0, 25 and 50 µM) or the proteasome inhibitor MG132 (0, 0.4 and 0.8 µM). Tetherin levels were analysed by Western blot. Viral proteins and cellular actin were also monitored as controls. (d) HeLa cell samples were transfected with scrambled (Sc), anti-TIP47 (T), anti-tetherin (B) or anti-TIP47+anti-tetherin (TB) siRNAs 40 h prior to infection (m.o.i. = 20). Twenty-four hours p.i., cells and cell supernatants were collected. Cell extracts were analysed by Western blot for tetherin, actin and viral protein (CE) and infected cell supernatants for virus particles (VP). (e) Infectious titres in supernatants of infected cells in (d). *, Partially saturated signals (see Methods).