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Abstract

Vaccinia virus (VACV) intracellular enveloped virus (IEV) particles are transported to the cell periphery on microtubules where they fuse with the plasma membrane to form cell-associated enveloped virus (CEV). Two IEV-specific proteins, F12L and A36R, are implicated in mediating transport of IEV. Without F12L, virus morphogenesis halts after formation of IEV, and CEV is not formed, whereas without A36R, IEV was reported not to be transported, yet CEV was formed, To address the roles of A36R and F12L in IEV transport, viruses with deletions of either (vΔF12L) or (vΔA36R) were labelled with enhanced green fluorescent protein (EGFP) fused to the core protein A5L, and used to follow CEV production with time. Without F12L, CEV production was inhibited by >99 %, whereas without A36R, CEV were produced at ∼60 % of wild-type levels at 24 h post-infection. Depolymerization of microtubules, but not actin, inhibited CEV formation in vΔA36R-infected cells. Moreover, vΔA36R IEV labelled with EGFP fused to the B5R protein co-localized with microtubules, showing that the A36R protein is not required for the interaction of IEV with microtubules. Time-lapse confocal microscopy confirmed that both wild-type and vΔA36R IEV moved in a stop–start manner at speeds consistent with microtubular movement, although the mean length of vΔA36R IEV movement was shorter. These data demonstrate that VACV IEV is transported to the cell surface using microtubules in the absence of A36R, and therefore IEV must attach to microtubule motors using at least one protein other than A36R.

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2005-11-01
2020-01-21
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Genome analysis of recombinant viruses. (a) Virus genomes were analysed by PCR using oligonucleotides that flanked the gene, amplified the gene or amplified the chimeric gene, producing DNA fragments of 2.3, 0.7 and 2.0 kb for vDA36R-EGFPA5L, vDF12L-EGFPA5L and the vEGFPA5L positive control, respectively. The sizes of these DNA fragments confirmed that the chimeric gene had inserted into the gene locus of vDA36R and vDF12L (see Methods for oligonucleotide sequences and a key to primer numbers). (b) Virus genomes were analysed by PCR using oligonucleotides that flanked the gene, amplified the chimeric gene or amplified the SCR regions 2–4. The reactions produced DNA fragments of 1.7 and 1.6 kb for vDA36R-EGFPB5R and the vEGFPB5R positive control, respectively. A 0.5 kb SCR2–4 band was only found for the vEGFPA5L positive control that has a wild-type gene. The sizes of these DNA fragments confirmed that the chimeric gene had inserted into the gene locus of vDA36R (see Methods for oligonucleotide sequences and a key to primer numbers).[ PDF file] (679 KB)

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Time-lapse confocal movies corresponding to the cells shown in Fig. 5. Ptk cells infected with vB5REGFP (WR) [ Windows Media Audio/Video file] (4.232 MB). Ptk cells infected with vΔA36R-B5REGFP (vΔA36R) [ Windows Media Audio/Video file] (4.482 MB).

Time-lapse confocal movies corresponding to the cells shown in Fig. 5. Ptk cells infected with vB5REGFP (WR) [ Windows Media Audio/Video file] (4.232 MB). Ptk cells infected with vΔA36R-B5REGFP (vΔA36R) [ Windows Media Audio/Video file] (4.482 MB).

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