FIG 4.

SHFV nsp1β stimulates −2/−1 PRF in pDluc reporter systems. (A) Schematic diagram of dual luciferase (pDluc) constructs. A 79-nt sequence (WT) containing the SHFV putative slippery sequence and downstream C-rich motif was inserted between Renilla luciferase (Rluc) and firefly luciferase (Fluc; in the −2 frame relative to Rluc) ORFs. The in-frame control (IFC) construct was generated by inserting two Us after the slippery sequence. In addition to the stop product translated without frameshifting, −2FS and −1FS products containing Renilla luciferase could be translated via −2 PRF and −1 PRF, respectively. (B) Identification of critical residues in SHFV nsp1β involved in −2/−1 PRF. HEK-293T cells were cotransfected with plasmid pDluc-SHFV/WT containing wild-type SHFV −2/−1 PRF signal and a plasmid expressing SHFV nsp1β or mutants thereof; empty vector (EV) was used as the control. Nonframeshift, −1 PRF, and −2 PRF products were detected by Western blotting using anti-Renilla luciferase (Rluc) mAb and are indicated as stop, −1FS, and −2FS, respectively. FLAG-tagged nsp1β was detected with mAb M2, and GAPDH was detected as a loading control. (C) SHFV/WT mRNA transcribed from plasmid pDluc-SHFV/WT was translated in rabbit reticulocyte lysate (RRL) in the presence of different concentrations of GST-tagged nsp1β (μM) or with dilution buffer (DB) as a control. (D) Efficiencies of −2 PRF and −1 PRF were calculated based on the quantified bands using ImageQuant TL software (GE Healthcare). Bars and error bars show mean values and standard errors of the means (SEM). (E) In vitro translation of SHFV/WT mRNA was performed with 1 μM GST-nsp1β or its mutant. (C and E) 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.