Replacing the SCR domains of vaccinia virus protein B5R with EGFP causes a reduction in plaque size and actin tail formation but enveloped virions are still transported to the cell surface.
A vaccinia virus (VV) recombinant is described in which the outer envelope of extracellular enveloped virus (EEV), cell-associated enveloped virus (CEV) and intracellular enveloped virus (IEV) is labelled with the enhanced green fluorescent protein (EGFP) derived from Aequorea victoria. To construct this virus, EGFP was fused to the VV B5R protein from which the four short consensus repeats (SCRs) of the extracellular domain had been deleted. Cells infected with the recombinant virus expressed a B5R-EGFP fusion protein of 40 kDa that was present on IEV, CEV and EEV, but was absent from IMV. The recombinant virus produced 2- and 3-fold reduced levels of IMV and EEV, respectively. Analysis of infected cells by confocal microscopy showed that actin tail formation by the mutant virus was reduced by 86% compared to wild-type (WT). The virus formed a small plaque compared to WT, consistent with a role for actin tails in promoting cell-to-cell spread of virus. However, the enveloped virions were still transported to the cell surface, confirming that this process is independent of actin tail formation. Lastly, we compared the mutant virus with a recombinant VV in which the B5R SCR domains were deleted and show that, contrary to a previous report, the plaque size of the latter virus was reduced compared to WT. This observation reconciles an inconsistency in the field and confirms that viruses deficient in formation of actin tails form small plaques.
Rodger
Gaener
G
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK1.
Smith
Geoffrey L
GL
Department of Infectious Diseases, Division of Investigative Science, Faculty of Medicine, Imperial College, St Mary's Campus, Norfolk Place, London W2 1PG, UK2.|Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK1.
D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018613:Microscopy, Confocal; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D014615:Vaccinia / Q000821:virology; D014616:Vaccinia virus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010948:Viral Plaque Assay; D014771:Virion / Q000378:metabolism
D000199:Actins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C074512:42kDa protein, Vaccinia virus; D049452:Green Fluorescent Proteins