PMID:7684466

Citation	Dubuisson, J and Rice, CM (1993) Sindbis virus attachment: isolation and characterization of mutants with impaired binding to vertebrate cells. J. Virol. 67:3363-74
Abstract	Sindbis virus can infect a broad range of insect and vertebrate cell types. The ability to restrict tissue tropism and target virus infection to specific cell types would expand the usefulness of engineered alphaviruses as gene expression vectors. In this study, virus pools derived from libraries of full-length Sindbis virus cDNA clones containing random insertion mutations in the PE2 or E1 virion glycoprotein gene were screened for mutants defective for binding to vertebrate cells. Binding-competent mutants were depleted by serial adsorption to chicken embryo fibroblast (CEF) monolayers at 4 degrees C, and the remaining population was amplified by immune-enhanced infection of P388D1 cells. From the PE2 libraries, 12 candidate mutants showing reduced cytopathic effects on CEF monolayers were isolated and three representative mutants, NB1, NB2, and NB12, were characterized in detail. Insertion mutations for NB1 and NB12 were found near the PE2 cleavage site, whereas the insertion in NB2 occurred between residues 69 and 74 of E2. Although virion assembly and release occurred normally for all three mutants, PE2 cleavage was completely (NB1) or partially (NB12) blocked for the mutants with insertions near the PE2 cleavage site. Both NB1 and NB2 were defective for binding to CEF and BHK-21 cells. Mild trypsin digestion of isolated NB1 virions resulted in PE2 cleavage and partially restored binding to CEF. Besides defective binding, NB1 also exhibited slower CEF penetration kinetics. Consistent with previous work, these results implicate PE2 cleavage and domains in the N-terminal portion of E2 as important determinants of alphavirus binding and penetration. Binding-defective mutants such as NB2, which exhibit normal particle assembly, release, and penetration, may be useful for future efforts to target Sindbis virus infection.
Links	PubMed PMC237680
Keywords	Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibodies, Viral; Base Sequence; Cells, Cultured; Chick Embryo; Chromosome Mapping; Epitopes; Glycoproteins/genetics; Glycoproteins/metabolism; Molecular Sequence Data; Mutagenesis, Insertional; Receptors, Virus/metabolism; Selection, Genetic; Sindbis Virus/chemistry; Sindbis Virus/genetics; Sindbis Virus/growth & development; Viral Envelope Proteins/genetics; Viral Envelope Proteins/immunology; Viral Structural Proteins/analysis; Virion/chemistry; Virus Replication
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
SINDV:POLS		GO:0019062: virion attachment to host cell	ECO:0000315:		P		Mutant strains of sindbis virus with insertion sequences in the PE2 glycoprotein gene have significantly reduced binding capabilities. PE2 cleavage is necessary for E2/E3 specific spike protein with E2 specific for receptor binding. See Fig. 3; Fig. 6; Fig. 7; Fig. 8	complete CACAO 9625
SINDV:POLS	involved_in	GO:0019062: virion attachment to host cell	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete

References

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