Deletion of the vaccinia virus growth factor gene reduces virus virulence

Abstract

the construction of a vaccinia virus mutant which lacks a functional VGF gene. The VGFmutant replicated less efficiently than did the wild-type (WT) virus in resting Swiss 3T3 cells and exhibited an attenuated phenotype following intracranial (i.c.) and intradermal inoculations into mice and rabbits, respectively. MATERIALS AND METHODS Cells and viruses. The vaccinia virus WR strain, originally obtained from the American Type Culture Collection Rockville, Md., was propagated in HeLa cells and purified as reported previously (22). CV-1 and Swiss 3T3 cells were grown in Dulbecco modified Eagle medium (DMEM; Quality Biologic Inc.) supplemented with 10% fetal bovine serum (FBS). A431 human epitheloid carcinoma cells, which have a high density of EGF receptors on the cell surface, were grown in DMEM supplemented with 5% FBS or 10% bovine serum (14). BS-C-1 cells were grown in minimum essential medium (MEM; GIBCO Laboratories) containing 10% FBS. Enzymes and chemicals. Restriction enzymes were supplied by Bethesda Research Laboratories, Inc., Gaithersburg, Md., New England BioLabs, Inc., Beverly, Mass., or Boehringer Mannheim Biochemicals, Indianapolis, Ind., and used as specified by the manufacturers. The Klenow fragment of DNA polymerase I, T4 DNA ligase, 5-bromo-4chloro-3-indolyl- ,-D-galactopyranoside (X-Gal), and isopropyl-j3-D-thiogalactopyranoside (IPTG) were obtained from Boehringer Mannheim. The BglII linker was purchased from New England BioLabs, and EGF was obtained from Collaborative Research, Inc., Waltham, Mass. Animals. Male BALB/cByJ and A/J mice between 6 and 10 weeks of age were obtained from Jackson Laboratory, Bar Harbor, Maine. Outbred black rabbits of both sexes were obtained from Spring Valley Laboratories. Guidelines in the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" were followed for animal husbandry. Virus infectivity assays. Virus infectivity was estimated as 866

The Vaccinia Virus O1 Protein Is Required for Sustained Activation of Extracellular Signal-Regulated Kinase 1/2 and Promotes Viral Virulence

“…The major phenotypic changes resulting from O1L deletion in CVA were very similar to the reported phenotypes of a vaccinia virus deletion mutant lacking the EGF-like viral growth factor gene in the VACV-WR strain background (4,43). These VACV-⌬VGF mutants also showed reduced plaque size but almost wildtype-like viral yields and lower lethality after intracranial infection of mice, as well as smaller skin lesions in rabbits.…”

“…Poxviruses generally appear to benefit from activation of this pathway in their natural hosts, since poxviruses from most genera have been shown to express a secreted growth factor similar to mammalian EGF, resulting in upregulation of ERK1/2 activation (40,57). The vaccinia virus growth factor (VGF) (4,5) represents the best-studied example. Using a VGF deletion mutant of VACV-WR, it has been demonstrated that VGF is required for most of the ERK1/2 activation observed in VACV-infected cells (1,43).…”

“…Using a VGF deletion mutant of VACV-WR, it has been demonstrated that VGF is required for most of the ERK1/2 activation observed in VACV-infected cells (1,43). In addition, VGF expression caused cell proliferation in VACV-infected tissue (5) and enhanced viral virulence (4). The advantage for poxviruses in directing the expression of such a factor was long seen as providing a metabolically active cellular environment supporting viral replication.…”

Vaccinia Virus Entry, Exit, and Interaction with Differentiated Human Airway Epithelia

“…Vaccinia virus contains two copies of the vaccinia growth factor (VGF) gene, one at each end of its genome, flanked by inverted terminal repeats (5). Previous studies showed that VGF, an epithelial growth factor-like peptide, binds to and activates erbB1 (also called the EGF receptor) (48), induces cellular proliferation of infected cells (5,6), and was required for viral replication (5,26). Variola virus produces the closely related smallpox growth factor that also binds erbB1 (24).…”

“…Thus, we hypothesized that the increase in cell number following infection was due to VGF. We infected well-differentiated epithelia with virus that retains the coding regions for VGF (vp37-eGFP) or a mutant virus with the two VGF genes disrupted (VSC20) (5). By 96 h after infection with the VSC20 virus, epithelia appeared irregular, and proliferative changes were not apparent (Fig.…”