title: A herpes simplex virus 2 glycoprotein D mutant generated by bacterial artificial chromosome mutagenesis is severely impaired for infecting neuronal cells and infects only Vero cells expressing exogenous HVEM.
abstract: We constructed a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) clone, bHSV2-BAC38, which contains full-length HSV-2 inserted into a BAC vector. Unlike previously reported HSV-2 BAC clones, the virus genome inserted into this BAC clone has no known gene disruptions. Virus derived from the BAC clone had a wild-type phenotype for growth in vitro and for acute infection, latency, and reactivation in mice. HVEM, expressed on epithelial cells and lymphocytes, and nectin-1, expressed on neurons and epithelial cells, are the two principal receptors used by HSV to enter cells. We used the HSV-2 BAC clone to construct an HSV-2 glycoprotein D mutant (HSV2-gD27) with point mutations in amino acids 215, 222, and 223, which are critical for the interaction of gD with nectin-1. HSV2-gD27 infected cells expressing HVEM, including a human epithelial cell line. However, the virus lost the ability to infect cells expressing only nectin-1, including neuronal cell lines, and did not infect ganglia in mice. Surprisingly, we found that HSV2-gD27 could not infect Vero cells unless we transduced the cells with a retrovirus expressing HVEM. High-level expression of HVEM in Vero cells also resulted in increased syncytia and enhanced cell-to-cell spread in cells infected with wild-type HSV-2. The inability of the HSV2-gD27 mutant to infect neuronal cells in vitro or sensory ganglia in mice after intramuscular inoculation suggests that this HSV-2 mutant might be an attractive candidate for a live attenuated HSV-2 vaccine.
authors.affiliation: Medical Virology Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA. kwang@niaid.nih.gov
authors.forename: Kening
authors.initials: K
authors.lastname: Wang
authors.forename: Justin D
authors.initials: JD
authors.lastname: Kappel
authors.forename: Caleb
authors.initials: C
authors.lastname: Canders
authors.forename: Wilmer F
authors.initials: WF
authors.lastname: Davila
authors.forename: Dean
authors.initials: D
authors.lastname: Sayre
authors.forename: Mayra
authors.initials: M
authors.lastname: Chavez
authors.forename: Lesley
authors.initials: L
authors.lastname: Pesnicak
authors.forename: Jeffrey I
authors.initials: JI
authors.lastname: Cohen
mesh_terms: D000818:Animals; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D018258:Herpesvirus 2, Human / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D016296:Mutagenesis; D000075983:Nectins; D009474:Neurons / Q000821:virology*; D009841:Oligonucleotides / Q000235:genetics; D017354:Point Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D053240:Receptors, Tumor Necrosis Factor, Member 14 / Q000378:metabolism; D014709:Vero Cells / Q000378:metabolism / Q000821:virology*; D014759:Viral Envelope Proteins / Q000235:genetics*; D053586:Virus Internalization
chemical_list: D015815:Cell Adhesion Molecules; C000619010:NECTIN1 protein, human; C000619015:Nectin1 protein, mouse; D000075983:Nectins; D009841:Oligonucleotides; D053240:Receptors, Tumor Necrosis Factor, Member 14; D014759:Viral Envelope Proteins