The effects of targeting the vaccinia virus B5R protein to the endoplasmicreticulum on virus morphogenesis and dissemination

The consequence of redirecting the vaccinia virus (VV) B5R protein to the e ndoplasmic reticulum (ER) has been investigated by the addition of an ER re trieval signal KKSL (K2X2) to the B5R C-terminus. This mutant B5R gene and a version of the gene with the inactive ER retrieval sequence KKSLAL (K2X4) were inserted into the thymidine kinase locus of a VV mutant lacking the B 5R gene, v Delta B5R. Similar levels of B5R protein were made by each virus , but the B5R-K2X2 protein remained sensitive to endoglycosidase H and colo calised with protein disulphide isomerase in the ER. In contrast, the B5R-K 2X4 protein colocalised with 1,4-galactosyltransferase in the trans-Golgi n etwork. Electron microscopy revealed that even when the B5R protein was red irected to the ER, intracellular mature virus particles were wrapped by cel lular membranes to form intracellular enveloped virus particles, although m ore incompletely wrapped particles were evident compared with wild type. Th ese intracellular enveloped virus particles were, however, unable to effici ently induce the polymerisation of actin and the plaque size formed by vBBR -K2X2 was small. Nevertheless, the amount and specific infectivity of EEV p roduced by VB5R-K2X2 were similar to those of wild type, despite the dramat ic reduction in the amount of B5R protein present in VB5R-K2X2 EEV. (C) 199 9 Academic Press.