FIG 7.

NoV RdRp cosediments with intracellular membranes, associates with the intracellular membrane fraction, and behaves as an integral membrane protein. Intracellular membranes were harvested from BSR-T7/5 cells transfected with either pT7-N1-HA or pT7-N1Δ12–64-HA by differential centrifugation. Proteins were separated on SDS-PAGE gels and detected by immunoblotting with antibodies specific for the HA tag or for the mitochondrial outer membrane protein MAO, as indicated in the figure. The relative positions of the protein standards are indicated at the right. (A) Proteins were examined in PNLs, membrane fractions P20 and P100 (pellets from the 20,000 and 100,000 × g spins, respectively), and the cytosolic fraction S100 (supernatant from 100,000 × g spins). The blot for pT7-N1Δ12–64-HA (right) was an overexposure necessitated by the production of lower levels of the mutant protein than the WT. (B) Intracellular membranes were fractionated on an equilibrium density gradient, as described in Materials and Methods, and proteins present in the LD and HD fractions were examined. (C) Membrane fractions were treated with high salt, high pH, or high salt with TX-100 prior to fractionation by equilibrium density gradient.