Fig 1.
L-RdRp gene codon optimization and recovery of CCHFV from DNA.
(A) Reporter minigenome luciferase activity was measured 48 h after transfecting BSR-T7/5 cells seeded in 10 cm2 wells. Cells were transfected with 250 ng of pC-L or pC-L opti, together with 500 ng of pC-N, 50 ng of pT7-M-Renilla [19], and 30 ng of internal control pGL3 per well. Data are represented as fold increase in Renilla luciferase expression over control transfections in which pC-L was omitted. Error bars indicate means ± standard deviation (n = 3) (B) V5-tagged L-RdRp and N protein levels in cell lysates from panel A as described before [9]. (C) BSR-T7/5 cells were transfected as presented in S1C Fig (pC-L support, upper panel), and also with pC-L opti in place of WT pC-L (lower panel). Four days post transfection, BSR-T7/5 cell supernatants were passaged onto SW13 cells, and viral antigens were detected with a GP38 domain-specific mAb (BSR-T7/5 IFA). Three days after passaging, viral cytopathic effect and CCHFV were visualized by bright field (SW13) or immunofluorescence (SW13 IFA) microscopy with anti-CCHFV hyperimmune mouse ascetic fluid (HMAF).
Fig 2.
Growth kinetics of CCHFV derived from cDNA.
(A) BSR-T7/5 and (B) A549 cells were infected with 0.001 of 50% tissue culture infective dose (TCID50)/cell of cDNA-derived CCHFV (circles) or parental virus isolate from Nigeria (squares). Viral titers were measured daily. Dashed line indicates the limit of detection.
Fig 3.
Optimization of support plasmid ratios for CCHFV rescue in BSR-T7/5.
(A) BSR-T7/5 cells were transfected with 1 μg pT7-S, 2.5 μg pT7-M, 1 μg pT7-L, 0.66 μg pC-N, and 0.33 μg pC-L opti. Cell supernatants were collected and viral titers measured by determining TCID50 at the indicated times post transfection. (B) In the experiments using 2:1 ratio of pC-N to pC-L opti, cells were transfected as in panel A except that 1 μg of pC-T7 was added to the transfection mix. In the experiment using a 19:1 pC-N:pC-L opti ratio, the same plasmid mix was used as for the 2:1 ratio, but with 0.95 μg of pC-N and 0.05 μg of pC-L opti. Error bars indicate means ± standard deviation. Statistical significance was evaluated using Student’s unpaired t test. Asterisk (*) indicates P < 0.05 at 3 days post transfection (2:1 versus 19:1). Dashed line indicates the limit of detection.
Fig 4.
CCHFV rescue efficiency using varying ratios of plasmids producing complementary genome segments in BSR-T7/5.
BSR-T7/5 cells were transfected with a total of 4.5 μg of pT7-S, pT7-M, and pT7-L at the indicated S:M:L ratios, together with 0.66 μg pC-N, 0.33 μg pC-L opti, and 1 μg pC-T7. Supernatants were collected 4 days after transfection, and viral titers were measured by TCID50 determination. Dashed line indicates the limit of detection.
Fig 5.
CCHFV M segment polyprotein processing.
(A) Signal peptide (SP) and predicted transmembrane domains are indicated in yellow. Arrows indicate locations of cleavage motifs recognized by mammalian convertases. The RSKR motif (boxed) was mutated to ASKA to block processing. (B) Signal peptidase (SPase) cleavages result in production of Gn and Gc precursors (PreGn and PreGc). Binding regions of the anti-glycoprotein antibodies used in this study (7F5, Gn tail, 11E7, and 6C11) are also represented. (C) Mature glycoprotein products (GP160/85, GP38, Gn, and Gc) resulting from mammalian convertase cleavage. (D) S1P cleavage is required for the production of GP160/85 and Gn, while GP38 requires cleavage by both S1P and furin-like PCs. An unidentified mammalian convertase is required for PreGc maturation to Gc.
Fig 6.
Furin enhances CCHFV propagation.
CHO-derived cell lines used were parental clone 6 (Par6), furin-deficient (FD11), and FD11 stably expressing furin (FD11-Fur). Each cell line was infected with CCHFV at multiplicity of infection (MOI) = 1 or with Rift Valley fever virus expressing EGFP in place of NSs (RVFV-EGFP; MOI = 0.1). Percentage of infected cells was determined by immunostaining for CCHFV, or by EGFP detection for RVFV at 24 h (A) and 48 h (B) post infection. Black bars represent CCHFV-infected cells; white bars represent RVFV-infected cells. Error bars indicate means ± standard deviation (n = 3). Statistical significance was evaluated using Student’s unpaired t test. Asterisk (*) indicates P < 0.01.
Fig 7.
Effects of blocking furin cleavage on CCHFV glycoprotein maturation.
(A) SW13 cells were infected with WT CCHFV or CCHFV-ASKA at MOI = 0.1, and immunoblots of structural proteins were performed on lysates collected 24 h post infection. Ratios of Gn:Pre and Gc:PreGc were obtained by densitometry of the bands (AlphaView; Alpha Innotech). (B) Immunoprecipitation of secreted non-structural proteins containing the GP38 domain with 6C11 mAb.
Fig 8.
Furin effect on CCHFV-WT and-ASKA growth.
FD11 and FD11-Fur cells were infected with CCHFV-WT or CCHFV-ASKA (MOI = 0.1). Cell supernatants were collected daily, and RNA S-segment copy numbers and infectious virus titers were measured by qRT-PCR and TCID50 determination, respectively. Means ± standard deviation (n = 3) are plotted. Statistical significance was evaluated using Student’s unpaired t test. Asterisk (*) indicates P < 0.05.