Antibodies

antibody species application (dilution) source

anti-actin rabbit WB (1 : 10 000) Sigma-Aldrich

anti-GAPDH mouse WB (1 : 1 000) Abcam

anti-GFP rabbit WB (1 : 10 000) Invitrogen

anti-RABV G

(HCA05/1) rabbit WB (1 : 10 000) custom-made, Metabion

anti-RABV N (S86) rabbit WB (1 : 10 000) custom-made, J. Cox (BFAV, Tübingen) anti-RABV N

(FITC-labeled) CentocorTM

mouse IF (1 : 200) FDI Fujirebio Diagnostics

anti-RABV P

(FCA05/1) rabbit WB (1 : 10 000) custom-made, Metabion

anti-RABV P (160-5) rabbit WB (1 : 50 000) S. Finke (FLI, Riems) anti-RABV RNP (S50) rabbit WB (1 : 25 000) custom-made, J. Cox

(BFAV, Tübingen)

anti-rabbit HRP goat WB (1 : 10 000) Jackson ImmunoResearch

Laboratories

2.1.6 Oligos

DNA oligos used in this work were ordered from Metabion. Sequences are listed in the appendix (6.1).

2.1.7 Plasmids

Plasmids commercially available (alphabetical order)

pCR3 Eucaryotic expression vector Invitrogen

pEGFP-C3 CMV promoter-dependent eGFP expression Clontech pRL-CMV CMV promoter-dependent Renilla luciferase NEB pSUPER H1 Pol-III-driven shRNA vector

(Brummelkamp et al., 2002) Oligoengine

pTRE2hyg Tet-inducible expression vector (TET-On) Clontech

Plasmids kindly provided (alphabetical order)

pCAGGS Eukaryotic expression vector with chicken-β-

actin promoter (Niwa et al., 1991)

pCAGGS-P RABV P in pCAGGS vector K. Brzozka

pCMV-

RL-FF-N/P Expression plasmid for a bicistronic mRNA Renilla-N/P-firefly under control of a CMV promoter; RABV N/P gene border is inserted between the coding sequences for Renilla and firefly luciferase.

A. Marschalek

pCMV-FF-PV-RL Like pCMV-RL-FF-N/P; N/P gene border

replaced by poliovirus IRES sequence A. Marschalek

pCR3-P RABV P in pCR3 vector K. Brzozka

pEGFP-miR23-2 Expression plasmid for eGFP with MCMV

miR-23-2 sequence in the 3’-UTR. L. Dölken pSAD G_DsRed DsRed cloned into extra transcription unit in

pSAD L16 (N/P gene border) (Klingen et al., 2008) pSAD L16 Full-length cDNA of RABV SAD L16 cloned

into pBluescript (Schnell et al., 1994)

pSAD PVP(bi) (or pSAD PVP) N/P gene border in pSAD L16

replaced by poliovirus IRES (Marschalek et al., 2009) pSC6 T7-neo CMV promoter-dependent expression of T7-

pol (Radecke et al., 1995)

pSDI CNPL RABV minigenome (negative sense); CAT and

firefly luciferase reporter genes (Finke et al., 2000) pSDI(+) (or pSDI-1plus) RABV minigenome positive

sense (Schnell et al., 1994)

pSDI-1 RABV minigenome (negative sense) (Conzelmann and Schnell, 1994)

psiCHECK-23-2 Dual-luciferase Reporter construct to

measure miRNA-dependent downregulation L. Dölken (Dölken et al., 2010) pTIT-L T7-pol and EMCV IRES-dependent expression

of RABV L (Finke and Conzelmann, 1999)

pTIT-N T7-pol and EMCV IRES-dependent expression

of RABV N (Finke and Conzelmann, 1999))

pTIT-P T7-pol and EMCV IRES-dependent expression

of RABV P (Finke and Conzelmann, 1999)

pX8∂T Plasmid comprising HDV and T7 terminator

sequence (Schnell et al., 1994)

The plasmids, cloned for this work, together with cloning strategies are listed in the appendix (6.2)

2.1.8 Viruses

Viruses kindly provided (alphabetical order)

SAD L16 Recombinant RABV, cDNA derived from strain SAD B19 (Schnell et al., 1994)

SAD PVP(bi) (or SAD PVP) N/P gene border replaced by poliovirus IRES element (Marschalek et al., 2009)

Viruses rescued in this work (alphabetical order)

SAD EL EMCV IRES element in 5’-UTR of L gene (downstream G/L gene border)

SAD EP(mono) EMCV IRES element in 5’-UTR of P gene (downstream N/P gene border)

SAD G_dHH-HSmm-

SC shRNA against GFP (guide strand and passenger strand are replaced in their positions) flanked by a defect 5’-HHRz and a 3’-SC1 ribozyme in extra transcription unit. Ribozymes in direct proximity to shGFP. SAD G_dHH-SH-SC shRNA against GFP flanked by a defect 5’-HHRz and a 3’-SC1 ribozyme

in extra transcription unit. Ribozymes in direct proximity to shGFP. SAD G_eGFP eGFP in extra transcription unit between G gene and L gene, with N/P

gene border SAD G_eGFP-miR23-

2 eGFP with MCMV miR23-2 in 3’-UTR in extra transcription unit SAD G_HH-HSmm-

SC shRNA against GFP (guide strand and sense strand are replaced in their positions) flanked by a 5’-HHRz and a 3’-SC1 ribozyme in extra transcription unit. Ribozymes in direct proximity to shGFP.

SAD G_HH-shGFP-

SC1 shRNA against GFP flanked by a 5’-HHRz and a 3’-SC1 ribozyme in extra transcription unit. Spacer nts between ribozymes and shGFP. SAD G_HH-SH-SC shRNA against GFP flanked by a 5’-HHRz and a 3’-SC1 ribozyme in

extra transcription unit. Ribozymes in direct proximity to shGFP. SAD G_shGFP shRNA against GFP in extra transcription unit

SAD L16 Recombinant RABV, cDNA derived from strain SAD B19 (Schnell et al., 1994).

SAD N100(mut)EN

G_eGFP As SAD N100EN G_eGFP, but AUG at leader-N gene junction mutated to UUG. SAD N100EN

G_eGFP First 100 nts of N gene and EMCV IRES inserted between leader-N gene junction and N gene (leader-N gene junction restored to wild- type). EGFP in extra transcription unit between G and L.

SAD N200(mut)EN

G_eGFP As SAD N200EN G_eGFP, but AUG at leader-N gene junction mutated to UUG. SAD N200EN

G_eGFP First 200 nts of N gene and EMCV IRES inserted between leader-N gene junction and N gene (leader-N gene junction restored to wild- type). EGFP in extra transcription unit between G and L.

SAD N200EN(Fse) First 200 nts of N gene and EMCV IRES inserted between leader-N gene junction and N gene (FseI-site at leader-N gene junction). SAD N2AP N/P gene border replaced by 2A-like sequence

SAD NEN Second N gene and EMCV IRES inserted between leader-N gene junction and N gene (FseI-site at leader-N gene junction).

SAD NEN G_eGFP Second N gene and EMCV IRES inserted between leader-N gene junction and N gene (leader-N gene junction restored to wild-type). EGFP in extra transcription unit between G and L.

SAD NEN_EL Bicistronic N-EMCV IRES-N gene (with wild type-like leader-N gene junction) and monocistronic EMCV IRES-L gene.

SAD NEN2AP Tricistronic N-EMCV IRES-N-2A-like-P gene (with wild type-like leader- N gene junction)

SAD NEN2APEL Tricistronic N-EMCV IRES-N-2A-like-P gene and monocistronic EMCV IRES-L gene.

SAD NENEP Bicistronic N-EMCV IRES-N gene (with wild type-like leader-N gene junction) and monocistronic EMCV IRES-P gene.

SAD NENEPEL Bicistronic N-EMCV IRES-N gene, monocistronic EMCV IRES-P gene and monocistronic EMCV IRES-L gene.

SAD NEP(bi)EL Derived from rescue of pCAGGS-T7_NEP(bi)_EL_SC: Bicistronic N- EMCV IRES-P gene and monocistronic EMCV IRES-L gene.

SAD NEP(bi)EL 1 Derived from rescue of pSAD T7-HH_NENEPEL_SC, rearranged variant: Bicistronic N-EMCV IRES-P gene and monocistronic EMCV IRES-L gene. SAD NEP(mono)EL Monocistronic EMCV IRES-P gene and monocistronic EMCV IRES-L

gene.

SAD NPVN Second N gene and poliovirus IRES inserted between leader-N gene junction and N gene (FseI-site at leader-N gene junction).

SAD PVP(bi) (or SAD PVP) N/P gene border replaced by poliovirus IRES element (Marschalek et al., 2009).

SAD PVP(mono) Poliovirus IRES element in 5’-UTR of P gene (downstream N/P gene border)