Fig 2: Effect of MDV1 infection on paip2 expression, PAPB1 level and localization.(A) Chicken embryo fibroblasts (CEF) were transfected with oncogenic BAC clone pRB1B5 of MDV1 or mock-transfected for 72 h. Total proteins were harvested and analysed by immunoblotting with the indicated antibodies. Quantification of the immunoblots from panel A using ImageQuant software is shown to the right. The results are from two independent experiments each in duplicate. (B) Total proteins were extracted from control samples or from samples taken from chicken infected with the oncogenic BAC clone pRB1B5 derived from archive samples. Proteins were analysed by immunoblotting as in panel A. Quantification of the immunoblots from panel B using ImageQuant software is shown to the right. The results are repeats from two different archive samples derived from the same chicken challenge experiment. (C) Indirect immunofluorescence of pRB1B5-infected CEF 72 h posttransfection. A series of optical sections were taken sequentially for each channel along the z-axis using a step size of 0.290 µm. The resulting 3D confocal image was reconstructed using IMARIS software. DAPI-staining shows the nucleus in blue, PABP1 in red and pp14 in green, the scale bar: 10 µm.

Fig 3: Reverse genetic mutation analysis shows that MDV1 miRNAs from Lat-cluster are responsible for paip2 repression.(A) Schematic representation of the bicistronic transcripts that we and others have cloned as cDNA and that encode for pp14a and pp14b isoforms, modified from Tahiri-Alaoui et al, J. Virol. Dec. 2009, Vol.83, No. 24, p12769-12778. (B) & (C) Chicken embryo fibroblasts (CEF) were transfected with BAC clone pRB1B5 Lat-miR-Revertant or pRB1B5 Lat-miR- deletion, respectively. RNA and proteins were simultaneously extracted using Trizol at the indicated time points. Viral and host proteins were detected by immunoblotting with the indicated antibodies. (D) Quantitative RT-PCR of host (paip2) and of viral transcripts (pp14a and pp14b isoforms) at the indicated time points. GAPDH is used as the endogenous control and time zero is used as the calibrator. All experiments were repeated three times and the error bars indicate the SEM.

Fig 4: Model depicting the closed loop topology for the bicistronic immediate-early transcript (IE) that encodes RLORF9 and the pp14 from MDV1.In this model, only the pp14b isoform is shown, which is under the translation control of the 5L IRES. The internal poly(A) of the 5L IRES recruits PABP1 to the 5' end of the mRNA, which may be concomitant with the recruitment of PABP1 to the poly(A) tail of the message, leading to circularization of the bicistronic dual IRES IE-mRNA. A subset of viral miRNAs down-regulate the expression level of paip2 which is a well-known inhibitor of PABP1. This down regulation of paip2 indirectly contributes to an increased level of the available pool of active PABP1 which interacts with the internal poly(A) sequence of the 5L IRES hence leading to increased IRES activity.

Fig 5: Depletion of paip2 by RNA interference in cells infected with Lat-miR BAC mutants.Chicken Embryo Fibroblasts were co-transfected with BAC clone of pRB1B5 Lat-miR-Revertant, pRB1B5 Lat-miR- deletion, si-paip2 RNA or siRNA control as indicated. The cells were lysed 72 hours post-transfection and the extracts were analysed by immunoblotting with the indicated antibodies against viral as well as host proteins.