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. 2018 Oct 30;9(5):e01761-18. doi: 10.1128/mBio.01761-18

FIG 5.

FIG 5

Viral replicase activity inhibits subsequent infection. (A) Subconfluent 293T cell monolayers were transfected with: plasmids encoding the complete set of viral replicase proteins (PB2, PB1, PA, and NP) along with a plasmid that encodes a reporter vRNA (eGFP ORF plus 5′ and 3′ viral UTR sequences; pHH21::eGFPvRNA) under the control of the polymerase I (PolI) promoter (RNP + eGFPvRNA), plasmids encoding the complete set of viral replicase proteins along with a plasmid (pHH21::eGFPORF) that encodes the eGFP ORF without the viral UTRs (RNP + eGFPORF), plasmids encoding an incomplete set of three viral replicase proteins (PB2, PB1, and NP only; no PA) along with the viral reporter RNA plasmid pHH21::eGFPvRNA (RNPPA- + eGFPvRNA), or plasmids encoding the complete set of viral replicase proteins along with an empty vector control (RNP + pHH21). At 24 h posttransfection, cells were infected with rH3N2 at MOI = 0.2 TCID50/cell. At 8 hpi, cells were harvested and assessed for rH3N2 infection via flow cytometry. For each group, rH3N2 infectivities are presented as the percentages of the mean rH3N2 infectivity in the RNP + pHH21 vector control group. (B) Experiment performed as described for panel A with cotransfection of plasmids encoding the complete set of viral replicase proteins plus either pHH21::eGFPvRNA or pHH21::HAvRNA. Data representing rH3N2 infectivity in cotransfected cells (eGFP+ or HA+) are normalized to uncotransfected cells (eGFP− or HA−) in the same samples. (C) Experiment performed as described for panel A with transfection of pHH21 vector, pHH21::eGFPvRNA, pHH21::HAvRNA, pHH21::HAcRNA, or pHH21::HAvRNA together with pHH21::HAcRNA. rH3N2 infectivities are presented as the percentages of the mean rH3N2 infectivity in the vector control group. (D) Experiment performed as described for panel A with transfection of pCI vector, pCI::eGFPORF, or pCI::HAORF. Data representing rH3N2 infectivity in transfected cells (eGFP+ or HA+) are normalized to untransfected cells (eGFP− or HA−) in the same samples. Data are presented as mean values (n = 2 cell culture wells) ± standard deviations.