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. 2019 Jul 30;93(16):e00370-19. doi: 10.1128/JVI.00370-19

FIG 6.

FIG 6

The slippery sequence and downstream C-rich RNA motif are required for SHFV −2/−1 PRF. (A) Schematic diagram of dual luciferase (pDluc) constructs. A 79-nt sequence (WT) containing the SHFV putative slippery sequence and downstream C-rich motif assumed to be necessary for −2/−1 PRF was inserted between Renilla luciferase (Rluc) and firefly luciferase (Fluc; in the −2 frame relative to Rluc) ORFs. Constructs SS1 and SS2 contain substitutions (red) at the slippery sequence to mimic natural PRF signal variations identified in simarteriviruses other than SHFV. The CC1 construct contains substitutions (red) to disrupt the C-rich motif, while the in-frame control (IFC) construct was generated by inserting two Us after the slippery sequence. (B) Effect of mutations in the slippery sequence (SS1 and SS2) or C-rich motif (CC1) on SHFV −2/−1 PRF in the pDluc reporter system. HEK-293T cells were cotransfected with pDluc-SHFV/WT or mutants thereof and a plasmid expressing SHFV nsp1β; empty vector (EV) was used as a control. Nonframeshift, −1 PRF, and −2 PRF products (indicated as stop, −1FS, and −2FS, respectively) were detected by Western blotting using anti-Renilla luciferase (Rluc) mAb. FLAG-tagged nsp1β was detected with anti-FLAG mAb, and GAPDH was detected as a loading control. (C) In vitro translation of mRNA derived from the pDluc-SHFV/WT construct or its mutant in the presence of GST-nsp1β. In vitro translation products were resolved in 12% SDS-PAGE and visualized by autoradiography. Products generated without frameshifting or from −1 or −2 PRF are indicated as stop, −1FS, and −2FS, respectively. (D) The expression of nsp2TF in KRCV-1 stimulated by nsp1β. HEK-293T cells were cotransfected with a plasmid expressing HA-tagged KRCV-1 nsp2 and a plasmid expressing FLAG-tagged wild-type nsp1β; empty vector (EV) was used as a control. The expression of nsp2 and nsp2TF was detected by Western blot analysis using a mAb against the HA tag, and FLAG-tagged nsp1β was detected with anti-FLAG M2 antibody. GAPDH was monitored as a loading control. (E) MARC-145 cells were infected with SHFV or mutants (vSS1, vSS2, and vCC1) at an MOI of 0.01, and cell lysates were harvested at 36 hpi. The nsp2-related products were detected by anti-PLP2 mAb 134-260. SHFV nsp2TF was also detected by rabbit pAb against the nsp2TF C-terminal peptide (nsp2TFC). GAPDH was monitored as a loading control.