Francesca Cocchi

The novel receptors that mediate the entry of herpes simplex viruses and animal alphaherpesviruses into cells.

An extended array of cell surface molecules serve as receptors for HSV entry into cells. In addition to the heparan sulphate glycosaminoglycans, which mediate the attachment of virion to cells, HSV requires an entry receptor. The repertoire of entry receptors into human cells includes molecules from three structurally unrelated molecular families. They are (i) HveA (herpesvirus entry mediator A), (ii) members of the nectin family, (iii) 3‐O‐sulphated heparan sulphate. The molecules have different attributes and play potentially different roles in HSV infection and spread to human tissues. All the human entry receptors interact physically with the virion envelope glycoprotein D (gD). (i) HveA is a member of the TNF‐receptor family. It mediates entry of a restricted range of HSV strains. Its expression is restricted to few lineages (e.g. T‐lymphocytes). (ii) The human nectin1α (HIgR), nectin1δ (PRR1‐HveC), and the nectin2α (PRR2α‐HveB) and nectin2δ (PRR2δ) belong to the immunoglobulin superfamily. They are homologues of the poliovirus receptor (CD155), with which they share the overall structure of the ectodomain. The human nectin1α‐δ are broadly expressed in cell lines of different lineages, are expressed in human tissue targets of HSV infection, serve as receptors for all HSV‐1 and HSV‐2 strains tested and mediate entry not only of free virions, but also cell‐to‐cell spread of virus. (iii) The 3‐O‐sulphated heparan sulphate is expressed in some selected human cell lines (e.g. endothelial and mast cells) and human tissues, and mediates entry of HSV‐1, but not HSV‐2. The human nectin2α and nectin2δ serve as receptors for a narrow range of viruses. A characteristic of the human nectin1α‐δ is the promiscuous species non‐specific receptor activity towards the animal alphaherpesviruses, pseudorabies virus (PrV) and bovine herpesvirus 1 (BHV‐1). By contrast with the human nectin1δ, its murine homologue (mNectin1δ) does not bind gD at detectable level, yet it mediates entry of HSV, as well as of PrV and BHV‐1. This provides the first example of a mediator of HSV entry independent of a detectable interaction with gD. Copyright

Nectin2α (PRR2α or HveB) and Nectin2δ Are Low-Efficiency Mediators for Entry of Herpes Simplex Virus Mutants Carrying the Leu25Pro Substitution in Glycoprotein D

ABSTRACT The receptors for entry of herpes simplex viruses 1 and 2 (HSV-1 and -2), widely expressed in human cell lines, are members of a subset of the immunoglobulin superfamily exemplified by herpesvirus entry mediator C (HveC) and the herpesvirus immunoglobulin-like receptor (HIgR). This report focuses on two members of this subset, herpesvirus entry mediator B (HveB), recently designated nectin2/PRR2α, and its splice variant isoform, nectin2/PRR2δ. Nectin2α and -δ share the ectodomain but differ in the transmembrane and cytoplasmic regions. HveB was reported to enable entry of HSV-1 carrying mutations in glycoprotein D (gD) and of HSV-2, but not of wild-type (wt) HSV-1. We report that (i) both nectin2α and -δ served as receptors for the entry of HSV-1 mutant viruses HSV-1(U10) and -(U21) and AP7r that carry the Leu25Pro substitution in gD but not for HSV-1 mutants U30 and R5000 that carry the Ser140 or Ala185 substitution in gD. All of these mutants were able to overcome the block to entry mediated by expression of wt gD. (ii) Infection of cells expressing nectin2α or -δ required exposure to multiplicities of infection about 100-fold higher than those required to infect cells expressing HveC or HIgR. (iii) gD from HSV-1(U21) bound in vitro soluble forms of nectin2. The association was weaker than that to the soluble form of HveC/HIgR. Binding of wt HSV-1 gD to soluble nectin2 was not detectable. (iv) A major region of nectin2 functional in virus entry mapped to the V domain, located at the N terminus.

Cell-to-Cell Spread of Wild-Type Herpes Simplex Virus Type 1, but Not of Syncytial Strains, Is Mediated by the Immunoglobulin-Like Receptors That Mediate Virion Entry, Nectin1 (PRR1/HveC/HIgR) and Nectin2 (PRR2/HveB)

ABSTRACT The immunoglobulin-like receptors that mediate entry of herpes simplex virus type 1 (HSV-1) into human cells were found to mediate the direct cell-to-cell spread of wild-type virus. The receptors here designated Nectin1α and -δ and Nectin2α were originally designated HIgR, PRR1/HveC, and PRR2α/HveB, respectively. We report the following. (i) Wild-type HSV-1 spreads from cell to cell in J cells expressing nectin1α or nectin1δ but not in parental J cells that are devoid of entry receptors. A monoclonal antibody to nectin1, which blocks entry, also blocked cell-to-cell spread in nectin1-expressing J cells. Moreover, wild-type virus did not spread from a receptor-positive to a receptor-negative cell. (ii) The antibody to nectin1 blocked transmission of wild-type virus in a number of human cell lines, with varying efficiencies, suggesting that nectin1 is the principal mediator of wild-type virus spread in a variety of human cell lines. (iii) Nectin1 did not mediate cell fusion induced by the syncytial strains HSV-1(MP) and HFEM-syn. (iv) Nectin2α could serve as a receptor for spread of a mutant virus carrying the L25P substitution in glycoprotein D, but not of wild-type virus, in agreement with its ability to mediate entry of the mutant but not of wild-type virus.

Novel, Soluble Isoform of the Herpes Simplex Virus (HSV) Receptor Nectin1 (or PRR1-HIgR-HveC) Modulates Positively and Negatively Susceptibility to HSV Infection

ABSTRACT A novel member of the nectin family, nectin1γ, was molecularly cloned. The cDNA has the same ectodomain as nectin1α and nectin1β, the two known transmembrane isoforms that serve as receptors for herpes simplex virus (HSV) entry into human cell lines (nectin1α and nectin1β, also called PRR1-HveC and HIgR, respectively). The 1.4-kb transcript, which originated by alternative splicing, is expressed in human cell lines, and appears to have a narrow distribution in human tissues. The sequence does not have a hydrophobic anchoring region, and the protein is secreted in the culture medium of cells transfected with the cDNA. Nectin1γ, purified from culture medium, can compete with membrane-bound nectin1β and reduce HSV infectivity. The expression of nectin1γ cDNA in cells resistant to HSV infection and lacking HSV receptors enables HSV to enter the cell, which implies that it is present at the cell surface. Thus, nectin1γ has the potential both to mediate and to reduce HSV entry into cells.

The Herpes Simplex Virus JMP Mutant Enters Receptor-Negative J Cells through a Novel Pathway Independent of the Known Receptors nectin1, HveA, and nectin2

ABSTRACT The herpes simplex virus type 1(JMP) [HSV-1(JMP)] mutant was selected for its ability to grow and form plaques in receptor-negative J cells. It enters J cells through a novel gD-dependent pathway, independent of all known HSV receptors, nectin1, nectin2, and HveA. Evidence that the pathway is dependent on a nectin3 binding site on HSV-1(JMP) and requires three mutations in gD rests on the following. We derived monoclonal antibodies to nectin3 and show that J cells express nectin3. HSV-1(JMP) entry and cell-to-cell spread were inhibited by soluble nectin3-Fc, demonstrating that virions carry a binding site for nectin3. The site is either directly involved in HSV-1(JMP) entry, or nectin3 binding to its site affects the gD domains involved in entry (entry site). HSV-1(JMP) entry and cell-to-cell spread in J cells were also inhibited by soluble nectin1-Fc, showing that the nectin1 binding site on gDJMP overlaps with the entry site or that nectin1 binding to gD affects the entry site. gDJMP carries three mutations, S140N, R340H, and Q344R. The latter two lie in the C tail and are present in the parental HSV-1(MP). HSV-1 strain R5000 carrying the S140N substitution was not infectious in J cells, indicating that this substitution was not sufficient. We constructed two recombinants, one carrying the three substitutions and the other carrying the two C-tail substitutions. Only the first recombinant infected J cells with an efficiency similar to that of HSV-1(JMP), indicating that the three mutations are required for the novel entry pathway. The results highlight plasticity in gD which accounts for changes in receptor usage.