Fig 1.
Organization and signal expression of the Crimean-Congo hemorrhagic fever virus (CCHFV) minigenome and support plasmids in transfected cells, and in cells treated with transcription- and entry-competent virus-like particles (tecVLP).
(A) Basic organization of CCHFV helper plasmids and minigenomes used in these experiments. Helper plasmids encoding the nucleocapsid protein (NP), the viral RNA-dependent RNA-polymerase (L), and glycoprotein precursor (GPC) are under the control of the chicken β-actin polymerase II (pol II) promoter. Replication machinery genes (NP and L) are from the CCHFV strain IbAr10200, and the surface GPC are from IbAr10200, Oman, Turkey, or Afg09 CCHFV strains, as indicated. The minigenome plasmids encode the NanoLuc (Luc) reporter gene in reverse, or non-coding, sense; NanoLuc is flanked by non-coding regions (NCR) from one of the 3 CCHFV genomic segments, S, M, or L. Minigenomes of all 3 segment NCRs from strain IbAr10200, and only the L segment NCR from strains Afg09 and Oman, were used in these experiments. (B) Absolute NanoLuc signal levels, in relative light units (RLU), following transfection of BSR-T7 cells with minigenome and replication plasmids (NP + L) or minigenome and tecVLP assembly plasmids (L + NP + GPC). Data are reported as standard error of the mean. ** p < 0.01, ***p < 0.001 (average of 2 experiments). (C) Titration experiments from 2 tecVLP production experiments passaged into SW-13 cells. S, M, and L NCR minigenomes are shown. (D) NanoLuc signal (in RLU) in SW-13 cells treated with supernatants from BSR-T7 cells transfected with tecVLP assembly plasmids (average of 2 experiments). NanoLuc signal data in tecVLP-treated cells are presented as absolute RLU values which are calculated as signal in SW-13 cells treated with entry-competent VLPs (i.e., containing NP, L, and GPC) minus signal in SW-13 cells treated with VLPs containing only NP and L. All data are reported as standard error of the mean.
Fig 2.
CCHFV tecVLP morphology and glycoprotein-mediated entry.
(A) Representative electron microscopy images of tecVLP. (B) Neutralization of tecVLP entry into SW-13 cells by previously reported monoclonal antibodies (mAbs). 11E7 and 12A9 are CCHFV neutralizing antibodies targeting the glycoprotein Gc; 13G8 is a non-neutralizing mAb targeting the immature glycoprotein PreGn; 9D5 targets the NP protein. Data are displayed as standard error of the mean (average of 2 experiments).
Fig 3.
CCHFV strain IbAr10200 replication machinery does not significantly discriminate against minigenomes from other CCHFV strains.
(A) Absolute NanoLuc signal in relative light units (RLU) in BSR-T7 cells transfected with IbAr10200 NP, L, and GPC plasmids, and with minigenomes encoding L NCR of other CCHFV strains, as indicated. Data are reported as standard error of the mean (n = 8 from 2 experiments). (B) TCID50 determination in SW-13 cells treated with supernatants from cells in (A). Results from 2 experiments are shown. (C) NanoLuc signal in SW-13 cells treated as in (B) (n = 8 from 2 experiments). NanoLuc signal data in VLP-treated cells are presented as absolute RLU values which are calculated as signal in SW-13 cells treated with entry-competent VLPs (i.e., containing NP, L, and GPC) minus signal in SW-13 cells treated with VLPs containing only NP, L and the corresponding minigenome. All data are reported as standard error of the mean.
Fig 4.
Effects of GPCs from various CCHFV strains on minigenome activity and transcription and tecVLP titers.
(A) Absolute NanoLuc signal (in RLU) in BSR-T7 cells transfected with L NCR minigenome and plasmids encoding GPC from Turkey, Oman, Afg09, or IbAr10200 strains. Data are reported as standard error of the mean (average of 2 experiments). (B) tecVLP titers determined by TCID50 in SW-13 cells treated with supernatants from BSR-T7 cells in (A). Shown are titers from 2 tecVLP generation experiments. (C) Western blot of CCHFV proteins in concentrated tecVLP supernatants produced by BSR-T7 cells from (A) (ΔGPC obtained from cells transfected with NP and L helper plasmids only). (D) Western blot of Gn precursor and mature Gn produced in cell lysates of transfected BSR-T7 cells. CCHFV proteins were detected by antibodies against IbAr10200 Gn, Gc, or NP, as indicated.
Fig 5.
Exchanging surface glycoproteins has a greater impact on tecVLP titer and NanoLuc signal than exchanging minigenomes.
(A) TCID50 titers/mL and NanoLuc signals in relative light units (RLU) measured in SW-13 cells treated with tecVLPs with Afg09 GPC and with L segment NCR minigenomes from indicated CCHFV strains. NanoLuc data are presented as standard error of the mean (average of 3 experiments), and results of 3 TCID50 experiments are shown. (B) TCID50 titers/mL and NanoLuc signals measured in SW-13 cells treated with tecVLPs with Oman GPC and L segment NCR minigenomes from indicated CCHFV strains. NanoLuc signal data in tecVLP-treated cells are presented as absolute RLU values which are calculated as signal in SW-13 cells treated with entry-competent VLPs (i.e., containing NP, L, and GPC) minus signal in SW-13 cells treated with VLPs containing only NP, L and the corresponding minigenome. NanoLuc data are reported as standard error of the mean (average of 3 experiments), and results of 3 TCID50 experiments are shown.
Fig 6.
tecVLPs are capable of entering into several immortalized cell lines and primary cells.
(A) NanoLuc signal in relative light units (RLU) in indicated cell lines treated with CCHFV tecVLPs containing GPC from several CCHFV strains (average of 3 experiments for each cell line). (B) NanoLuc signal in indicated primary cells treated with tecVLPs containing GPC from various CCHFV strains (average of 4 experiments for each cell line). NanoLuc signal data in VLP-treated cells are presented as absolute RLU values which are calculated as signal in SW-13 cells treated with entry-competent VLPs (i.e., containing NP, L, and GPC) minus signal in SW-13 cells treated with VLPs containing only NP and L. NanoLuc data are reported as standard error of the mean. * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 7.
Inhibitors of CCHFV entry and transcription inhibit tecVLP signal production.
(A) Neutralization assays of tecVLPs treated with mAbs. Percent inhibition was calculated by comparing the NanoLuc signal strength in SW-13 cells treated with tecVLP and mAbs to signal in cells treated with tecVLPs and a control mAb. (B) Percent inhibition (compared to untreated tecVLP) in SW-13 cells treated with tecVLPs and indicated inhibitors (top panel), and 50% effective concentration (EC50), 50% cytotoxicity concentration (CC50), and selectivity index (SI) for ribavirin and chloroquine in these cells (bottom panel, average of 3 experiments).