Fig 1.
Functional domains in L-proteins and related complexes of segmented negative-sense RNA viruses.
The RNA polymerase complex of influenza contains three subunits with the following functions: cap-snatching endonuclease (PA; 726 residues, 83 kDa), RdRp (PB1; 752 residues, 84 kDa) and cap-binding (PB2; 770 residues, 88 kDa). Functional equivalent domains are evident in the monomeric L-protein (2263 residues, 263 kDa) of the La Crosse Virus although the cap binding function remains elusive. The L-protein (3945 residues, 448 kDa) of the Crimean-Congo Haemorrhagic Fever Virus contains an additional OTU domain at its N-terminus. The structural information for the CCHFV L protein is limited and is indicated by the dotted boxes and lines. The sizes of the functional domains are not shown in scale.
Fig 2.
Purification and characterization of the RdRp activity of recombinant CCHFV L wild type and D2517N mutant proteins.
(A) A partial sequence alignment of RdRp of segmented viruses shows the conserved SDD sequence within motif C. Residue D2517 within CCHFV L protein is highlighted in purple. (B) SDS PAGE migration pattern of the purified enzyme preparation stained with Coomassie Brilliant Blue G-250 dye. The band migrating above the 250 kDa molecular weight marker (lane 1) contains CCHFV L wild type or D2517N mutant full-length protein (as indicated) confirmed by mass spectrometry (see S1 and S2 Figs for details). Lane 2 contains 20x less protein than lane 1. The protein band is still visible, suggesting that there is ~20 times more of the CCHFV L protein than impurities. This corresponds to ~95% pure protein preparation. (C) RNA primer/template used in the RNA synthesis assays is shown above the gel. Template and primer were both mono-phosphorylated (p) at their 5’-ends. C indicates incorporation of the radiolabeled nucleotide opposite template position 5. RNA synthesis was monitored with purified wild type CCHFV L protein in the presence of [α-32P]CTP, RNA primer/templates and NTP combinations designed to generate either intermediate or full template length products. Note that 10x less of wild type CCHFV L protein was used in reactions started with MnCl2. Lane m illustrates the migration pattern of the radiolabeled 4 nucleotide-long primer. (D) RNA primer/template used in the RNA synthesis assays is shown above the gel. Template and primer were both phosphorylated (p) at their 5’-ends. G indicates incorporation of the radiolabeled nucleotide opposite template position 5. RNA synthesis was monitored with purified CCHFV L wt and D2517N mutant proteins in the presence of [α-32P]GTP, RNA primer/template and NTP combinations designed to generate full template length products. Lane m illustrates the migration pattern of the radiolabeled 4 nucleotide-long primer. Lane “[α-32P]GTP-only’ illustrates the background signal associated with the [α-32P]GTP preparation in the absence of enzyme.
Fig 3.
Patterns of inhibition of RNA synthesis with Ribavirin- and Favipiravir-TP.
(A) RNA primer/templates used in the RNA synthesis assays to test incorporation of Ribavirin-MP and Favipiravir-MP as A-, G-, and UMP-analogues are shown above the respective gels. C indicates incorporation of the radiolabeled nucleotide opposite template position 5. Position i allows incorporation of A-, G-, and UMP or nucleotide analogue inhibitors. Ribavirin-MP and Favipiravir-MP incorporation as A-, G-, and UMP-analogues was monitored with purified CCHFV L protein in the presence of [α-32P]GTP, RNA primer/template, 5 mM MgCl2 and various combinations of 100 μM NTP and 100 μM NTP substrate analogues. The presence of three natural NTPs allows full-length product formation up to position 14. The presence of two natural NTPs provides a control for mis-incorporations. Lane m illustrates the migration pattern of the radiolabeled 4 nucleotide-long primer. Asterisks indicate limited ongoing primer extensions.
Fig 4.
Selective incorporation of ribavirin- and favipiravir-MP opposite template U and C. (A, C) RNA primer/template used in the RNA synthesis assays to test incorporation of ribavirin-MP and favipiravir-MP as AMP or GMP-analogues are shown above the respective gels. C indicates incorporation of the radiolabeled nucleotide opposite template position 5. Position i allows incorporation of AMP or GMP or nucleotide analogue inhibitors. NTP incorporation was monitored with purified CCHFV L protein in the presence of [α-32P]CTP, RNA primer/template, 5 mM MgCl2 and increasing concentrations of NTP and NTP substrate analogues. Lane m illustrates the migration pattern of the radiolabeled 4 nucleotide-long primer. (B) Graphic representation of the data for incorporation of AMP and ribavirin- or favipiravir-MP opposite template U. (C) Graphic representation of the data for incorporation of GMP and ribavirin- or favipiravir-MP opposite template C. Error bars represent standard deviation of data from three independent experiments.
Table 1.
CCHFV L protein selectivity values for Ribavirin-TP and Favipiravir-TP.
Fig 5.
Patterns of inhibition of RNA synthesis with 2’deoxy-2’-fluoro-CTP and 2’deoxy-2’-amino-CTP.
(A) RNA primer/template used in the RNA synthesis assays to test incorporation of with 2’deoxy-2’-fluoro- CTP and 2’deoxy-2’-amino- CTP as a CMP-analogue across templating G at position 8 is shown above the gel. G indicates incorporation of the radiolabeled nucleotide opposite template position 5. Position i allows incorporation of CMP or nucleotide analogue inhibitors. 2’deoxy-2’-fluoro- CTP and 2’deoxy-2’-amino- CTP incorporation was monitored with purified CCHFV L protein in the presence of [α-32P]GTP, RNA primer/template, 5 mM MgCl2 and various combinations of 100 μM NTP and 100 μM NTP substrate analogues. The presence of three natural NTPs allows full-length product formation up to position 14. The presence of two natural NTPs provides a control for mis-incorporations across templating G at position 8. Lane m illustrates the migration pattern of the radiolabeled 4 nucleotide-long primer. (B) Graphic representation of the data for selective incorporation of 2’deoxy-2’-fluoro-CMP and 2’deoxy-2’-amino-CMP. Error bars represent standard deviation of data from three independent experiments.
Fig 6.
Competition between CTP and 2’deoxy-2’fluoro-CTP.
(A) RNA primer/template used in the RNA synthesis assays to test competition of CTP and 2’deoxy-2’fluoro-CTP is shown above the gel. G indicates incorporation of the radiolabeled nucleotide opposite template position 5. Template G allows incorporation of CTP or 2’deoxy-2’fluoro-CTP and their competition for incorporation when both nucleotides are present in the reaction mixture. RNA synthesis was monitored with purified CCHFV L protein in the presence of [α-32P]GTP, RNA primer/template, 5 mM MgCl2 and 3.7, 11, 33 and 100 μM ATP, CTP, UTP and increasing concentrations of 2’deoxy-2’fluoro-CTP. The presence of three natural NTPs in the absence of 2’deoxy-2’fluoro-CTP allows full-length product formation up to position 14. Lane m illustrates the migration pattern of the radiolabeled 4 nucleotide-long primer. (B) Full-length-template product was quantified as a fraction of total signal in the lane, normalized to the full-template-length product fraction in the absence of 2’deoxy-2’fluoro-CTP and plotted versus log concentrations of the 2’deoxy-2’fluoro-CTP. Data were fitted to a dose response function in GraphPad (Prism 6.0) to determine the concentration of 2’deoxy-2’fluoro-CTP at which the amount of full-length-template product decreased by 50% (IC50).
Table 2.
CCHFV L protein selectivity values for 2'amino-CTP and 2'fluoro-CTP.
Fig 7.
Qualitative analysis of polyUb chain hydrolysis by CCHFV L protein and the isolated CCHFV OTU domain.
Purified CCHFV L protein (RdRp) was incubated with either (A) K48polyUb or (B) K63polyUb chains in the presence or absence of OTU-specific inhibitor CC.4. Ub chain hydrolysis was also assessed for the purified OTU domain using (C) K48polyUb chains or (D) K63polyUb chains in the presence or absence of CC.4. Reactions were incubated at 37°C and samples taken at 0, 15 and 30 minutes as indicated. Ub chain lengths following digestion are indicated with arrows. Samples were resolved on a 10% tris-tricine gel and visualized by silver stain.
Fig 8.
De-ubiquitinating (DUB) activity of purified CCHFV full-length L and the isolated OTU domain.
(A) Schematic representation of the assay. A C-terminus-derived ubiquitin peptide conjugated with 4-amino-4-methylcoumarin (AMC) is used as a fluorogenic substrate for CCHFV L DUB-activity. Once AMC is released from the conjugated substrate it can emit fluorescence at 445 nn upon excitation at 355 nm. (B) Time dependent formation of the fluorescence signal in reactions containing ~5 nM CCHFV L protein or the isolated OTU domain and 500 nM ubiquitin-AMC substrate. Slopes (red dotted lines) of linear portions of signal formation were used to determine the velocity of substrate cleavage. DUB activity of both full length protein and OTU domain is inhibited by ubiquitin variant CC.4. (C) Ubiquitin variant CC.4 inhibits DUB activity of both full length protein and OTU domain to a similar extent as illustrated by the 50% inhibitory concentrations (IC50).
Fig 9.
Polymerase and de-ubiquitinating (DUB) activities of purified CCHFV full-length L and the isolated OTU domain in the presence of respective inhibitors.
(A) Ubiquitin variant CC.4 does not inhibit polymerase activity of CCHFV L protein. (B) Graphic representation of data shown in (A). (C) The presence of NTP substrates and nucleotide analogue inhibitors do not affect DUB activity. (D) DUB activity of CCHFV L protein wt and D2517N mutant.