1887

Abstract

Infection with (VV) produces several distinct virions called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). In this report, we have investigated how incoming virus cores derived from IMV are transported within the cell. To do this, recombinant VVs (vA5L-EGFP-N and vA5L-EGFP-C) were generated in which the A5L virus core protein was fused with the enhanced green fluorescent protein (EGFP) at the N or C terminus. These viruses were viable, induced formation of actin tails and had a plaque size similar to wild-type. Immunoblotting showed the A5L-EGFP fusion protein was present in IMV particles and immunoelectron microscopy showed that the fusion protein was incorporated into VV cores. IMV made by vA5L-EGFP-N were used to follow the location and movement of cores after infection of PtK cells. Confocal microscopy showed that virus cores were stained with anti-core antibody only after they had entered the cell and, once intracellular, were negative for the IMV surface protein D8L. These cores co-localized with microtubules and moved in a stop–start manner with an average speed of 51·8 (±3·9) μm min, consistent with microtubular movement. Treatment of cells with nocodazole or colchicine inhibited core movement, but addition of cytochalasin D did not. These data show that VV cores derived from IMV use microtubules for intracellular transport after entry.

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2003-09-01
2019-10-17
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Carter (2003). , , part 9, pp. 2443–2458.

Virus genomes were analysed by PCR using oligonucleotides that flank the A5L or EGFP gene. PCR with primers flanking the A5L gene produced DNA fragments of 1.6, 2.3, 2.3, 1.6 and 1.6 kb for WT, vA5L-EGFP-C, vA5L-EGFP-N, vA5L-EGFP-C-rev and vA5L-EGFP-N-rev, respectively, whereas EGFP-specific primers produced a DNA fragment of 0.7 kb for vA5L-EGFP-C and vA5L-EGFP-N but not for WR or revertant viruses. The sizes of these DNA fragments confirmed that the chimaeric vA5L-EGFP-C and vA5L-EGFP-N gene was inserted into the A5L gene locus.



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