Figure 5. Analysis of TNTH (lane 2) and RIPA (lane 3) products for JV/V5. The N-term/V5 product is approximately 42 kDa in size, larger than the predicted size of 35.3 kDa. Molecular weight marker is represented in lane 1. doi:10.1371/journal.pone.0002169.g005 [29], for which a commercially available monoclonal antibody is used for detection. Following in vitro transcription and translation of JV V5 a new product, approximately 42 kDa in size, was visible (Figure 5, lane 2). This product was not observed in any prior analyses of JV. To confirm that this protein was V5/N-term associated the TNTH reaction was subjected to RIPA using the anti-V5 antibody (Figure 5, lane 3). This confirmed expression of the N-term protein in vitro. To confirm expression of the V5/N-term protein in cell culture capped RNA was synthesised from the JV V5 T7 cDNA construct, which was used to transfect CRFK cells. As there is currently no host cell line in which to propagate JV the CRFK cell line was used as it has been shown to support the replication of feline Analysis of the 59 GS of JV Figure 7. Western analysis from lysates of CRFK cells trans- fected with no RNA (lane 2), JV RNA (lane 3) and JV/V5 RNA (lane 4) using the anti-V5 monoclonal antibody. Bradford analysis was performed on the lysates to ensure equal loading. The appearance of the 42 kDa V5 product confirms ORF1 translation and activity of the 3C protease in vivo. Molecular weight marker is represented in lane 1. doi:10.1371/journal.pone.0002169.g007 calicivirus [30]. Confocal immunofluorescence of transfected cells using the anti-V5 antibody demonstrated expression of the V5/N- term protein in cultured cells (Figure 6). Expression of the V5/N- term protein was diffuse and did not co-localise with the Golgi/ ER/plasma membrane marker wheat germ agglutinin (WGA) and therefore displays a different pattern of cellular expression compared to Norwalk virus [15]. Cells transfected with the wild type full length JV RNA were negative for fluorescence (data not shown). Lysates of cells transfected with wild type JV and JV/V5 RNA were subjected to western blot using the anti-V5 antibody (Figure 7). No product was present for cells transfected with wild type JV RNA, but a protein of approximately 42 kDa in size was visible in cells that had been transfected with JV/V5 RNA, confirming N-term expression and size as seen in the in vitro system. In addition, this important observation also confirms for the first time that the JV 3C protease was active in cells transfected with capped RNA as the size of the V5/N-term indicated successful cleavage of the protein from the ORF1 polyprotein. To address the issue of potential rapid degradation of the JV N- term protein CRFK cells were transfected with JV V5 RNA and were harvested at designated time points following the addition of the protein synthesis inhibitor cycloheximide. Cell lysates were analysed by Western blot using the anti-V5 antibody (Figure 8). The consistent appearance of the N-term/V5 protein suggested that it is stable and insensitive to degradation by viral and host cell proteases. The predicted molecular weight of the JV N-term is 35.3 kDa, based on the site of initiation of translation and location of conserved cleavage sites. The appearance, therefore, of a Figure 6. Confocal immunofluorescence of CRFK cells transfected with JV/V5 RNA. A = cells stained with wheat germ agglutinin (plasma and Golgi/ER membrane marker, red) and DAPI (blue). B = cells stained with anti-V5 (green). C = merged. doi:10.1371/journal.pone.0002169.g006 PLoS ONE | www.plosone.org 5 May 2008 | Volume 3 | Issue 5 | e2169