Crimean-Congo hemorrhagic fever virus-encoded ovarian tumor protease activity is dispensable for virus RNA polymerase function

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus (genus Nairovirus, family Bunyaviridae) associated with high case fatality disease outbreaks in regions of Africa, Europe, and Asia. The CCHFV genome consists of three negative-strand RNA segments, S, M, and L. The unusually large virus L polymerase protein and the need for biosafety level 4 (BSL-4) containment conditions for work with infectious virus have hampered the study of CCHFV replication. The L protein has an ovarian tumor (OTU) protease domain located in the N terminus, which has led to speculation that the protein may be autoproteolytically cleaved to generate the active virus L polymerase and additional functions. We report the successful development of efficient CCHFV helper virus-independent S, M, and L segment minigenome systems for analysis of virus RNA and protein features involved in replication. The virus RNA segment S, M, and L untranslated regions were found to be similar in support of replication of the respective minigenomes. In addition, the OTU domain located in the N terminus of the expressed virus L protein was shown to be a functional protease. However, no evidence of L protein autoproteolytic processing was found, and the OTU protease activity was dispensable for virus RNA replication. Finally, physiologically relevant doses of ribavirin inhibited CCHFV minigenome replication. These results demonstrated the utility of the minigenome system for use in BSL-2 laboratory settings to analyze CCHFV biology and in antiviral drug discovery programs for this important public health and bioterrorism threat.

FIG. 1.

CCHFV minigenome expression systems. (A) T7-cRNA minigenome expression vector diagram. The vector V0.0/B was used to produce cRNA minigenome under the control of a T7 RNA polymerase promoter (T7-p) which is expected to synthesize minigenomes containing two nonviral Gs at the 5′ terminus. Hepatitis D virus ribozyme (HDV-Rz) sequence was added to obtain minigenome RNAs with the 3′ termini identical to CCHFV cRNA 3′ termini. T7 terminator (T7-Ter) was also added to stop T7 RNA polymerase transcription. The segment ORF was replaced by either the secreted Gaussia luciferase (Luc) or EGFP (GFP). (B) vRNA minigenome expression vector diagram. This minigenome was cloned in negative orientation relative to the T7 promoter (shown as upside-down Luc), and one nonviral G is incorporated at the 5′ end; ribozyme cleavage would yield the correct 3′ end. (C) PolI-vRNA minigenome expression diagram. vRNA minigenome was cloned into PolI expression vector (pRF207); murine PolI promoter (PolI-p) and human PolI terminator (Pol-ter) are present to ensure the fidelity of the termini of minigenome RNAs. (D) In vitro vRNA minigenome diagram. This vRNA minigenome was generated from the DNA construct shown in panel B.

FIG. 2.

Replication and packaging of CCHFV minigenomes. BSR-T7/5 cells were transfected with cRNA minigenomes (cS, cM, and cL) and subsequently infected (black bars) or not (white bars) with CCHFV. Supernatants were passaged to Huh-7 monolayers. Following CCHFV passage, luciferase activity was measured (n = 3) (A) and an EGFP picture was taken (B) at 36 h after the passage.

FIG. 3.

WT and mutant L protein colocalize with the nucleoprotein (N). BSR-T7/5 cells were cotransfected with either pC-V5-L (L), pC-V5-L-ΔDD (L-ΔDD), or pC-V5-L-C40A (L-C40A) together with empty plasmid or N-expressing vector, pC-N (N). The V5 fusion L proteins were detected with V5-FITC MAb (green), anti-N MAb (red), and Golgi compartment marker (white).

FIG. 4.

WT and mutant L proteins display similar expression levels. BSR-T7/5 cells were cotransfected (as described in the legend of Fig. 3) 24 h posttransfection, and cell lysate proteins were separated by NuPAGE electrophoresis under reducing conditions. Western blot analyses were performed with anti-V5 (upper panel) and anti-CCHFV N (lower panel) MAbs. Apparent molecular masses are indicated in kilodaltons (kDa).

FIG. 5.

Recombinant L-RdRp activity. (A) BSR-T7/5 cells were cotransfected with either the V0.0/B-vS-Gluc (T7), pRF207-vS-Gluc (PolI), or empty plasmid (RNA) together with empty plasmid (−), RdRp-inactive L protein (L-ΔDD) or WT L protein (L) and N, and secreted AP was used as an internal control. At 24 h after transfection, cells that had been transfected with empty plasmid were transfected with vS-Gluc minigenome in vitro transcripts (RNA). Conditioned medium was collected 24 h after DNA (T7 and PolI) or RNA (RNA) transfections for luciferase assays. Average RLU and standard deviation (n = 4) values are shown. (B) Cells were transfected as described in panel A (RNA) except that vM-Gluc and vL-Gluc in vitro transcript minigenomes were tested in parallel with the vS-Gluc minigenome. Average RLU of Gluc (open bars) and AP (black bars) activity with standard deviation were determined (n = 6).

FIG. 6.

Inhibition of the L-RdRp activity by anti-CCHFV drug. BSR-T7/5 cells were transfected as described in the legend of Fig. 5, except that complete medium with increasing doses of ribavirin was added 4 h after transfection of the vS-Gluc minigenome in vitro transcripts. Conditioned medium was collected, and average activities (RLU) of Gluc and AP were determined (n = 4).

FIG. 7.

Active L-OTU domain is not required for L-RdRp function. (A and B) BSR-T7/5 cells were cotransfected with either WT L or L-C40A mutant together with N. L and L-C40A were immunoprecipitated with V5 MAb. Equal aliquots of immunoprecipitated material were incubated with either Ub-AMC or ISG15-AMC substrates. AMC release was measured every 30 s for 600 s and plotted as relative fluorescence units (RFU). (C) Cells were transfected as described for panels A and B and subsequently transfected with vS-Gluc minigenome in vitro transcripts. Conditioned medium was collected, and average activities (RLU) of Gluc and AP were determined (n = 4).