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. 2012 May;86(9):5371–5375. doi: 10.1128/JVI.06933-11

Fig 3.

Fig 3

M064 is an early/late viral factor that is packaged into the progeny virions. (A) Construction of the MYXV with V5-tagged M064. A V5 tag was inserted before the stop codon of M064R, and an EGFP expression cassette driven by a vaccinia virus p11 late promoter was inserted after the V5-tagged M064R. The purity of the recombinant virus was confirmed by PCR, and this recombinant virus remains the wild-type phenotype of MYXV in vitro. (B) M064 is expressed early during viral infection, and the expressed protein stably accumulates throughout the course of infection. RK-13 cells were pretreated with AraC or not pretreated, followed by mock infection or infection with vMyxM064RV5 at an MOI of 5 in the presence or absence of AraC. At given time points (1, 2, 4, 8, 12, and 25 h p.i.), cell lysates were harvested for Western blotting. V5-tagged M064 was detected by probing with the anti-V5 antibody. Serp-1 expression was probed to show the effective AraC treatment that abolishes late gene expression. (C) M064 appears to be packaged into MYXV virions. Gradient-purified MYXV virions (0.15 optical density [OD] units for each virus) were used to coarsely separate the membrane and core components in the presence of detergent and DTT. The resulting fractions were separated on 12% SDS-polyacrylamide gels for Western blotting. V5-tagged protein was probed by anti-V5 antibody, and a known membrane component of MYXV virion, M071, was also probed as the control for a successful separation of virion core and membrane. Abbreviations: C, core component; M, membrane fraction. (D) M064 does not appear to be located in the membrane component of the purified MYXV virion. Gradient-purified MYXV virions (0.15 OD units for each virus) were used to separate only membrane and core component along with the intervened network structure which is insoluble in the absence of DTT, designated the H fraction. H+C, core fraction and H fraction that are insoluble in the absence of DTT; M, outer membrane component that is soluble in the presence of detergent while without DTT.