FIG. 6.

Reduced levels of VP1 or VP3 decrease the formation of DLPs and TLPs, whereas the amounts of low-density particles remain similar. MA104 cells were transfected with the indicated siRNAs, and at 72 hpi the cells were infected with RRV at an MOI of 3. At 6 hpi cells were radiolabeled for 6 h with 25 μCi/ml of Easy-tag Express-³⁵S and then harvested. Viral particles from each condition were separated by CsCl gradients (see Materials and Methods). Twelve 400-μl fractions were collected from each gradient and analyzed by PAGE and autoradiography (A). The amount of VP6 present in each fraction was calculated by densitometric analysis. The numbers below each lane represent the relative amounts of VP6 with respect to the amounts of VP6 present in the TLP fraction of the control gradient, which was taken as 100%. (B) Total RNA from the TLPs and low-density particle fractions was Trizol extracted and quantified by qRT-PCR as described in Materials and Methods using primer pairs specific for the positive (Gene 6 RNA+) and negative (Gene 6 RNA−) strands of rotavirus gene segment 6. The qRT-PCR results are expressed as the increase relative to the amount of positive or negative strand found in the TLP fraction produced in cells transfected with the control siRNA, which was taken as 100%. Data shown represent the arithmetic means ± standard deviation of three independent experiments. (C). Fractions 10 and 11 (low-density fractions) from gradients of the indicated conditions were resolved by SDS-7% PAGE and detected by autoradiography. An asterisk indicates the position of the silenced proteins. For siRNAs, siVP1 represents the siRNA directed against VP1; other designations follow the same form. Irr, irrelevant control siRNA.