Figure 4

The catalytic factor requirements for RNase activity mediated by PrV vhs. To increase the sensitivity, PrV vhs was synthesized using the TNT^® T7 Quick coupled Translation system (RRL) and simultaneously labeled with S³⁵ (A). (B) Rabbit reticulocyte (RRL) translated PrV vhs protein displays ribonuclease activity in vitro. RNA internally labeled with α-[P³²]-ATP was incubated with only assay buffer (mock control), RRL (as a vhs negative control), or in vitro translated PrV vhs at 37 °C for the indicated times (0, 15, and 30 min). The RNA reaction products were resolved by agarose gel electrophoresis followed by autoradiography. (C) Contribution of positive ions (e.g. Mg²⁺, K⁺) and ATP to PrV vhs mediated RNase activity. To deplete the residual ions in RRL, desalted plain RRL or RRL translated vhs, as indicated by asterisks, were prepared using Sephadex G-25 spin columns. The RNase activities of the desalted lysates were analyzed in assay buffer D or buffer F containing all, or none of the three factors (Mg²⁺, K⁺, ATP), respectively. To identify the requirements in terms of individual co-factor for vhs-dependent RNase activity, the RNA degradation activity of desalted PrV vhs was further analyzed in two systems: either using buffers missing one of the three co-factors, namely buffers B, E, and C without Mg²⁺, K⁺, or ATP, respectively (D), or in buffers containing only one co-factors (buffer F supplemented with Mg²⁺, K⁺, or ATP) (E). To confirm the contribution of Mg²⁺, the divalent chelator EDTA was added to the reaction containing Mg²⁺. Nuclease activity was measured using a Kodak image analyzer system and the amounts of RNA remaining (%) of three independent assays were plotted (F).