Fig 2.
Knocking out M064R from the MYXV genome leads to a delayed expression of viral gene products during infection of rabbit cells. (A) M064R knockout virus (vMyxM064-KO) infection in rabbit cells leads to a reduced RNA transcription for viral early/late genes. RK-13 cells were infected with either wild-type MYXV (vMyxGFP) or vMyxM064-KO at an MOI of 5. At given time points (1, 2, 4, 8, and 12 h p.i.), cell lysates were harvested for RNA extraction and DNase treatment, followed by reverse transcription and then Sybr green real-time PCR to detect the transcription of an early/late gene, M-T7. The comparative CT method was used to calculate and compare the relative level of RNA. The results of the two independent experiments combined are shown. (B) The delay of vMyxM064-KO infection is at the early stage of viral gene expression. RK-13 cells were treated with AraC before and during the infection with either vMyxGFP or vMyxM064-KO. At given time points (1, 2, 4, 8, and 12 h p.i.), cell lysates were harvested and processed as described above for panel A. The results shown are representative of the results of two independent experiments. (C) The delay of viral gene expression by infection of vMyxM064-KO can be detected at the protein level. RK-13 cells were infected by either vMyxGFP (wt) or vMyxM064-KO at an MOI of 10. At given time points (1, 4, 8, 12, and 25 h p.i.), cell lysates were harvested for Western blotting. Early/late gene expression (M-T7, M156, and M135) and late gene expression (SERP-1 and M130) were compared between wt and vMyxM064-KO. The results shown are representative of the results of two independent experiments. The positions of molecular size markers in daltons are shown to the right of the gels (e.g., 50K, 50,000).