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. 2012 Sep;86(18):10173–10185. doi: 10.1128/JVI.05560-11

Fig 1.

Fig 1

Hantavirus cap-snatching assay. (A) A diagrammatic representation of the cap-snatching assay. Step 1, Vero E6 cells were transfected with cap donor plasmid (pCDNAGFPns or pCDNAGFP) followed by SNV infection 4 h posttransfection (step 2). Step 3, cells were lysed 48 h postinfection, and total RNA was purified as described in Materials and Methods. Twenty-five nanograms of the purified RNA was reverse transcribed using a primer specific to the S-segment-derived mRNA. The cDNA was PCR amplified using a forward primer specific to the 5′ terminus of GFP mRNA (purple) and a reverse primer specific to the N gene (black). See Materials and Methods for details. (B) As expected, the PCR product was generated only from the cells which were transfected with either pCDNAGFPns or pCDNAGFP plasmid, followed by viral infection. (C) The Vero E6 cells were transfected with cap donor plasmid pCDNAGFPns or pCDNAGFP, followed by SNV infection 4 h posttransfection, as described for panel A. Cells were lysed 48 h postinfection, and total RNA was purified. Intrinsic mRNA levels expressed from cap donor plasmids pCDNAGFPns and pCDNAGFP were quantified by real-time PCR analysis using a primer set specific to the GFP open reading frame, as described in Materials and Methods. (D) Similarly, the cap-donating potential of the transcripts expressed from cap donor plasmids pCDNAGFPns and pCDNAGFP was determined by real-time PCR using a primer set shown in panel A. (E) The PCR product from panel B was cloned in a TA cloning vector (Invitrogen), and plasmid DNA from 20 random clones was sequenced to examine the cap-UTR junction. As shown at the bottom, the capped primers were terminated at the 3′ G residue. (F) The GFP mRNA and nonsense GFP mRNA were synthesized by in vitro T7 transcription, as described in Materials and Methods. To distinguish their 5′ UTRs, two nucleotides in the 5′ UTR of nonsense GFP mRNA were mutated (bold and underlined). Vero E6 cells were cotransfected with GFP mRNA and nonsense GFP mRNA, followed by virus infection. The cap-donating potential of these two transcripts was examined by 5′RACE, as described in Materials and Methods.