**, < 0.01; ***, < 0.001. The participation of Cdc42, Rab5, and Rab7 in BRV UK infectivity was confirmed by overexpressing constitutively active (CA) and dominant negative (DN) mutants of these SIRT-IN-1 proteins (49). (LEs) to infect the SIRT-IN-1 cell. The requirement for Rab7 was also shared by other RV strains of human and porcine origin. Of interest, most RV strains that reach LEs were also found to depend on the activities of Rab9, the cation-dependent mannose-6-phosphate receptor (CD-M6PR), and cathepsins B, L, and S, suggesting that cellular factors from the test with GraphPad Prism (version 5.0) software (GraphPad Software, Inc.). values of less than 0.05 were considered significant. RESULTS Rotavirus strains UK and RRV reach different endocytic compartments during cell infection. RRV and BRV UK enter cells using different endocytic mechanisms (36, 38). It is not known, however, if both viruses follow the same vesicular traffic upon cell entry. RRV has been shown to exit the endocytic compartment after reaching a mature form of EEs (40), while BRV UK was shown to colocalize with Rab5a, a marker of EEs, suggesting that this virus might also reach the SIRT-IN-1 EE compartment (39). To define the vesicular traffic of BRV UK, we tested the effect of siRNAs directed to various Rab GTPases involved in different SIRT-IN-1 steps of the vesicular traffic. For this, MA104 cells were treated with the indicated siRNAs and then were either infected with BRV UK-TLPs or lipofected with BRV UK DLPs, and the resulting virus infectivity was measured by either immunoperoxidase or immunofluorescence focus-forming assays, as indicated in Materials BACH1 and Methods and the appropriate figure legends. The level of gene silencing by the siRNAs used was evaluated either by Western blotting or by qRT-PCR; all siRNAs used had a silencing efficiency of 80% or more (data not shown). All siRNAs against the three isoforms of Rab5, the early endosomal antigen (EEA1), and the GTPase Cdc42 decreased BRV UK infectivity (Fig. 1A). In contrast, in cells transfected with transcriptionally active BRV UK DLPs, used to bypass the virus entry step, the same siRNAs did not have a significant effect on virus replication. These results suggest that all three isoforms of Rab5, EEA1, and the GTPase Cdc42 that regulates different types of endocytosis are required for BRV UK cell entry. Thus, as recently reported for RRV (39, 40), BRV UK also seems to reach EEs after entering the cell via clathrin-dependent endocytosis (36). However, unlike RRV, whose entry pathway was described to be restricted either to EEs (39) or to an ME compartment (40), the infectivity of BRV UK determined by immunoperoxidase focus-forming assays was found to decrease when the expression of Rab7a and Rab9a was silenced, suggesting that BRV UK needs to reach LEs to infect the cell (Fig. 1B). The infectivity of transfected BRV UK DLPs was not affected by these siRNAs, indicating again that their effect is limited to virus entry. In these assays, we also evaluated the participation of CD-M6PR in the infectivity of both RRV and UK RV strains. CD-M6PR is involved in the transport of proteins from the TGN to LEs and contributes to the maturation of this compartment (26). Interestingly, while the infectivity of RRV was not affected by the siRNA against CD-M6PR, that of BRV UK was decreased by this treatment (Fig. 1B). Open in a separate window FIG 1 Evaluation of vesicular traffic components on the infectivity of BRV UK. (A) MA104 cells were transfected with the indicated siRNA and then infected with BRV UK at an MOI of 3 or lipofected with BRV UK DLPs. At 6 hpi, cells were fixed and virus infectivity was determined by an immunofluorescence assay, as indicated in Materials and Methods. (B) MA104 cells transfected with the indicated siRNA were infected with RV strains RRV and UK at an MOI of 0.02 or lipofected with BRV UK DLPs. At 14 hpi, the cells were fixed and the virus was detected by an immunoperoxidase assay as described in Materials and Methods. In panels A and B, data are expressed as the percent.