Herpesvirus Entry Mediator HVEM Mediates Cell-Cell Spread in BHK(TK−) Cell Clones

95-19 and U_S11cl19.3 are BHK(TK⁻)-derived cell lines that are highly resistant to postattachment entry of herpes simplex virus type 1 (HSV-1) and HSV-2 but not to later steps in single-step replication. The resistance properties of these two cell types are not identical. U_S11cl19.3 cells are fully susceptible to pseudorabies virus (PRV), as shown by single-step growth experiments, whereas 95-19 cells are resistant to entry of free PRV but not to entry by cell-cell spread. We have tested the ability of HVEM to overcome the block to infection in both cell lines following transient and stable transfection. HVEM was able to mediate entry of free HSV-1 into both cell lines, as shown by an increase in the number of β-galactosidase-expressing cells in cultures transiently transfected with an HVEM expression plasmid and infected with lacZ-expressing HSV-1. In stably transfected 95-19 cells, HVEM enhanced infection by free HSV-1, as shown by an increase in the number of infectious centers obtained following infection. In both cell types, HVEM strongly enhanced entry of HSV-1 and HSV-2 by cell-cell spread, suggesting that HVEM can function as an entry mediator both in entry of free virus and in entry by cell-cell spread.     

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

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.