title: Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion.
abstract: Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion.
authors.affiliation: Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521, USA. jtr1@cdc.gov
authors.forename: John T
authors.initials: JT
authors.lastname: Roehrig
authors.forename: Siritorn
authors.initials: S
authors.lastname: Butrapet
authors.forename: Nathan M
authors.initials: NM
authors.lastname: Liss
authors.forename: Susan L
authors.initials: SL
authors.lastname: Bennett
authors.forename: Betty E
authors.initials: BE
authors.lastname: Luy
authors.forename: Thomas
authors.initials: T
authors.lastname: Childers
authors.forename: Karen L
authors.initials: KL
authors.lastname: Boroughs
authors.forename: Janae L
authors.initials: JL
authors.lastname: Stovall
authors.forename: Amanda E
authors.initials: AE
authors.lastname: Calvert
authors.forename: Carol D
authors.initials: CD
authors.lastname: Blair
authors.forename: Claire Y-H
authors.initials: CY
authors.lastname: Huang
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D009033:Culicidae; D004252:DNA Mutational Analysis; D003716:Dengue Virus / Q000502:physiology*; D006497:Heparan Sulfate / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D053586:Virus Internalization
chemical_list: D006497:Heparan Sulfate; D050505:Mutant Proteins; D014759:Viral Envelope Proteins