Molecular basis oh herpes simplex virus entry into the cell and retargeting of the viral tropism for the design of oncolytic herpesviruses

Herpes simplex virus 1 (HSV-1) infects oral epitelial cells, then spreads to the nerve endings and estabilishes latency in sensory ganglia, from where it may, or may not reactivate. Diseases caused by virus reactivation include mild diseases such as muco-cutaneous lesions, and more severe, and even life-threatening encephalitis, or systemic infections affecting diverse organs. Herpes simplex virus represents the most comprehensive example of virus receptor interaction in Herpesviridae family, and the prototype virus encoding multipartite entry genes. In fact, it encodes 11-12 glycoproteins and a number of additional membrane proteins: five of these proteins play key roles in virus entry into subsceptible cells. Thus, glycoprotein B (gB) and glycoprotein C (gC) interact with heparan sulfate proteoglycan to enable initial attachment to cell surfaces. In the next step, in the entry cascade, gD binds a specific surface receptor such as nectin1 or HVEM. The interaction of glycoprotein D with the receptor alters the conformation of gD to enable the activation of gB, glycoprotein H, and glycoprotein L, a trio of glycoproteins that execute the fusion of the viral envelope with the plasma membrane. In this thesis, I described two distinct projects: I. The retargeting of viral tropism for the design of oncolytic Herpesviruses: • capable of infecting cells through the human epitelial growth factor receptor 2 (HER2), overexpressed in highly malignant mammary and ovarian tumors and correlates with a poor prognosis; • detargeted from its natural receptors, HVEM and nectin1. To this end, we inserted a ligand to HER2 in gD. Because HER2 has no natural ligand, the selected ligand was a single chain antibody (scFv) derived from MAb4D5 (monoclonal antibody to HER2), herein designated scHER2. All recombinant viruses were targeted to HER2 receptor, but only two viruses (R-LM113 and R-LM249) were completely detargeted from HVEM and nectin1. To engineer R-LM113, we removed a large portion at the N-terminus of gD (from aa 6 to aa 38) and inserted scHER2 sequence plus 9-aa serine-glycine flexible linker at position 39. On the other hand, to engineer R-LM249, we replaced the Ig-folded core of gD (from aa 61 to aa 218) with scHER2 flanked by Ser-Gly linkers. In summary, these results provide evidence that: i. gD can tolerate an insert almost as big as gD itself; ii. the Ig-like domain of gD can be removed; iii. the large portion at the N-terminus of gD (from aa 6 to aa 38) can be removed without loss of key function; iv. R-LM113 and R-LM249 recombinants are ready to be assayed in animal models of mammary and ovary tumour. This finding and the avaibility of a large number of scFv greatly increase the collection of potential receptors to which HSV can be redirected. II. The production and purification of recombinant truncated form of the heterodimer gHgL. We cloned a stable insect cell line expressing a soluble form of gH in complex with gL under the control of a metalloprotein inducible promoter and purified the heterodimer by means of ONE-STrEP-tag system by IBA. With respect to biological function, the purified heterodimer is capable: • of reacting to antibodies that recognize conformation dependent epitopes and neutralize virion infectivity; • of binding a variety cells at cell surface. No doubt, the availability of biological active purified gHgL heterodimer, in sufficient quantities, will speed up the efforts to solve its crystal structure and makes it feasible to identify more clearly whether gHgL has a cellular partner, and what is the role of this interaction on virus entry

A Herpes Simplex Virus Recombinant That Exhibits a Single-Chain Antibody to HER2/neu Enters Cells through the Mammary Tumor Receptor, Independently of the gD Receptors

The human epidermal growth factor receptor 2/neuregulin (HER2/neu) receptor is overexpressed in highly malignant mammary and ovarian tumors and correlates with a poor prognosis. It is a target for therapy; humanized monoclonal antibodies to HER2 have led to increased survival of patients with HER2/neu-positive breast cancer. As a first step in the design of an oncolytic herpes simplex virus able to selectively infect HER2/neu-positive cells, we constructed two recombinants, R-LM11 and R-LM11L, that carry a single-chain antibody (scFv) against HER2 inserted at residue 24 of gD. The inserts were 247 or 256 amino acids long, and the size of the gD ectodomain was almost doubled by the insertion. We report the following. R-LM11 and R-LM11L infected derivatives of receptor-negative J or CHO cells that expressed HER2/neu as the sole receptor. Entry was dependent on HER2/neu, since it was inhibited in a dose-dependent manner by monoclonal antibodies to HER2/neu and by a soluble form of the receptor. The scFv insertion in gD disrupted the ability of the virus to enter cells through HVEM but maintained the ability to enter through nectin1. This report provides proof of principle that gD can tolerate fusion to a heterologous protein almost as large as the gD ectodomain itself without loss of profusion activity. Because the number of scFv's to a variety of receptors is continually increasing, this report makes possible the specific targeting of herpes simplex virus to a large collection of cell surface molecules for both oncolytic activity and visualization of tumor cells

Construction of a Fully Retargeted Herpes Simplex Virus 1 Recombinant Capable of Entering Cells Solely via Human Epidermal Growth Factor Receptor 2 ▿ †

A novel frontier in the treatment of tumors that are difficult to treat is oncolytic virotherapy, in which a replication-competent virus selectively infects and destroys tumor cells. Herpes simplex virus (HSV) represents a particularly attractive system. Effective retargeting to tumor-specific receptors has been achieved by insertion in gD of heterologous ligands. Previously, our laboratory generated an HSV retargeted to human epidermal growth factor receptor 2 (HER2), a receptor overexpressed in about one-third of mammary tumors and in some ovarian tumors. HER2 overexpression correlates with increased metastaticity and poor prognosis. Because HER2 has no natural ligand, the inserted ligand was a single-chain antibody to HER2. The objective of this work was to genetically engineer an HSV that selectively targets the HER2-expressing tumor cells and that has lost the ability to enter cells through the natural gD receptors, HVEM and nectin1. Detargeting from nectin1 was attempted by two different strategies, point mutations and insertion of the single-chain antibody at a site in gD different from previously described sites of insertion. We report that point mutations at gD amino acids 34, 215, 222, and 223 failed to generate a nectin1-detargeted HSV. An HSV simultaneously detargeted from nectin1 and HVEM and retargeted to HER2 was successfully engineered by moving the site of single-chain antibody insertion at residue 39, i.e., in front of the nectin1-interacting surface and not lateral to it, and by deleting amino acid residues 6 to 38. The resulting recombinant, R-LM113, entered cells and spread from cell to cell solely via HER2

The multipartite system that mediates entry of herpes simplex virus into the cell

The multipartite entry-fusion system of herpes simplex virus is made of a quartet of glycoproteins-gD, gB, gH.gL-and three alternative gD receptors, herpesvirus entry mediator (HVEM), nectin1 and modified sites on heparan sulphate. This multipartite system recapitulates the basic steps of virus-cell fusion, i.e. receptor recognition, triggering of fusion and fusion execution. Specifically, in addition to serving as the receptor-binding glycoprotein, gD triggers fusion through a specialised domain, named pro-fusion domain (PFD), located C-terminally in the ectodomain. In the unliganded gD the C-terminal region folds around the N-terminal region, such that gD adopts a closed autoinhibited conformation. In HVEM- and nectin1-bound gD the C-terminal region is displaced (opened conformation). gD is the tool for modification of HSV tropism, through insertion of ligands to heterologous tumour-specific receptors. It is discussed whether gD responds to the interaction with the natural and the heterologous receptors by adopting similar conformations, and whether the closed-to-open switch in conformation is a generalised mechanism of activation. A peculiar recombinant highlighted that the central Ig-folded core of gD may not encode executable functions for entry and that the 219-314 aa segment may be sufficient to trigger fusion. With respect to fusion execution, gB appears to be a prospective fusogen based on its coiled-coil trimeric structure, similar to that of another fusion glycoprotein. On the other hand, gH exhibits molecular elements typical of class 1 fusion glycoproteins, in particular heptad repeats and strong tendency to interact with lipids. Whether fusion execution is carried out by gB or gH.gL, or both glycoproteins in complex or sequentially remains to be determined

Herpes Simplex Virus Glycoproteins H/L Bind to Cells Independently of αVβ3 Integrin and Inhibit Virus Entry, and Their Constitutive Expression Restricts Infection ▿

Herpes simplex virus (HSV) fusion with cells requires the gD, gB, and gH/gL glycoprotein quartet. gD serves as a receptor binding glycoprotein. gB and gH/gL execute fusion in an as-yet-unclear manner. To better understand the role of gH/gL in HSV entry, we produced a soluble version of gH/gL carrying a One-STrEP tag (gHt.st/gL). Previous findings implicated integrins as possible ligands to gH/gL (C. Parry et al., J. Gen. Virol. 86:7-10, 2005). We report that (i) gHt.st/gL bound a number of cells in a dose-dependent manner at concentrations similar to those required for the binding of soluble gB or gD. (ii) gHt.st/gL inhibited HSV entry at the same concentrations required for binding. It also inhibited cell-cell fusion in transfected cells. (iii) The absence of β3 integrin did not prevent the binding of gHt.st/gL to CHO cells and infection inhibition. Conversely, integrin-negative K562 cells did not acquire the ability to bind gHt.st/gL when hyperexpressing αVβ3 integrin. (iv) Constitutive expression of wild-type gH/gL (wt-gH/gL) restricted infection in all of the cell lines tested, a behavior typical of glycoproteins which bind cellular receptors. The extent of restriction broadly paralleled the efficiency of gH/gL transfection. RGD motif mutant gH/gL could not be differentiated from wt-gH with respect to restriction of infection. Cumulatively, the present results provide several lines of evidence that HSV gH/gL interacts with a cell surface cognate protein(s), that this protein is not necessarily an αVβ3 integrin, and that this interaction is required for the process of virus entry/fusion