Identification and characterization of an extracellular envelope glycoprotein affecting vaccinia virus egress.
Sequence analysis of the vaccinia virus strain Western Reserve genome revealed the presence of an open reading frame (ORF), SalL4R, which has the potential to encode a transmembrane glycoprotein with homology to C-type animal lectins (G. L. Smith, Y. S. Chan, and S. T. Howard, J. Gen. Virol. 72:1349-1376, 1991). Here we show that the SalL4R gene is transcribed late during infection from a TAAATG motif at the beginning of the ORF. Antisera raised against a TrpE-SalL4R fusion protein identified three glycoprotein species of Mr 22,000 to 24,000 in infected cells. Immunogold electron microscopy demonstrated that SalL4R protein is present in purified extracellular enveloped virus particles but not in intracellular naked virus (INV). A mutant virus was constructed by placing a copy of the SalL4R ORF downstream of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible vaccinia virus promoter within the thymidine kinase locus and subsequently deleting the endogenous SalL4R gene. The growth kinetics of this virus demonstrated that SalL4R was nonessential for the production of infectious INV but was required for virus dissemination. Consistent with this finding, the formation of wild-type-size plaques by this mutant was dependent on the presence of IPTG. Electron microscopy showed that without SalL4R expression, the inability of the virus to spread is due to a lack of envelopment of INV virions by Golgi-derived membrane, a morphogenic event required for virus egress.
Duncan
S A
SA
Sir William Dunn School of Pathology, University of Oxford, United Kingdom.
Smith
G L
GL
D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D004279:DNA, Viral / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D006056:Golgi Apparatus / Q000378:metabolism; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D037102:Lectins / Q000235:genetics; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000502:physiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000235:genetics / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000502:physiology*; D015678:Viral Structural Proteins / Q000235:genetics; D014779:Virus Replication
D004279:DNA, Viral; D037102:Lectins; D008562:Membrane Glycoproteins; D009838:Oligodeoxyribonucleotides; D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins