Foto del docente

Maria Gabriella Campadelli

Professor emeritus

Alma Mater Studiorum - Università di Bologna

Research

Molecular basis of herpes simplex virus (HSV) entry into the cell and exit out of the cell

Multivesicular bodies as a cellular compartment for Herpes simplex virion assembly and egress out of the cell.

Genetic engineering and activity of oncolytic  HSVs retargeted to tumor-specific  receptors

Molecular basis of herpes simplex virus (HSV) entry into the cell and exit out of the cell

Since the late 1980s, the work of G Campadelli-Fiume centered on the molecular basis of herpes simplex virus entry into the cells, exit out of the cell, and modification of compartments like the exocytic pathway, cytoskeleton, etc. Recent work aims to apply the accrued knowledge to the engineering of oncolytic herpes simplex viruses specifically retargeted to receptors expressed on cancer cell, and (the structure-based design of fusion inhibitors specifically active towards herpes simplex virus.

In the late 1980s G campadelli-Fiume  laboratory provided the first evidence that, out of the numerous glycoproteins that decorate the HSV envelope, gD is the one that interacts with entry receptor(s). In 1998, we and others discovered nectin1, an immunoglobulin-like intercellular adhesion molecule, as a receptor for HSV gD, and characterized its murine hortholog. gD may also employ an alternative receptor, HVEM (Herpesvirus entry mediator). Recent studies highlighted that gD plays another function, e.i it signals receptor-recognition to gB, and thus triggers fusion. To perform the two tasks, gD ectodomain is organized in two topologically and functionally distinct regions. The N-terminus (aa 1-260 of mature gD) carries the receptor binding sites. The C-terminus (aa 260-310) carries the pro-fusion domain (PFD) required for the triggering of fusion but not for receptor binding. The unexpected finding that brought the latter activity to light was that soluble forms of gD can rescue the infectivity of a gD-null virus, provided that they carry not only the receptor binding region, but also the downstream region, at least up to aa 285. Biochemical and structural data clearly indicate that the most prominent properties of gD C-terminus are the ability to bind the gD N-terminus in the unlinganded gD, and the ability to be displaced from its binding site when gD interacts with one of its receptors. The model that emerged envisions that, in the native unliganded gD, the C-terminus folds back on itself and wraps the N-terminus; this enables gD to adopt an autoinhibited closed conformation. Upon gD binding to either receptor, the interaction between the N- and C-termini is lost, the C-terminus is displaced from its binding site on the N-terminus and released from restrains; gD adopts an opened conformation. Downstream of gD, HSV entry requires three additional glycoproteins, gB, gH and gL. Key to decipher how gD signals receptor recognition to the downstream glycoproteins has been the identification of a supramolecular complex that forms at receptor-gD binding among the glycoprotein quartet. The glycoproteins are recruited to the complex in a specific order. The receptor-bound gD recruits gB; in turrn, the receptor-gD-gB recruits gH.gL. Complex assembly absolutely requires one of the gD receptor, nectin1 or HVEM.

Fusion execution: gH and gB

A bioinformatic search by means of a neural network based predictor was applied to the glycoprotein quartet. gH emerged as the only HSV glycoprotein carrying in the ectodomain a highly hydrophobic membrane α-helix (named α -H1), and two heptad repeats (HR-1 and HR-2). A thorough characterization of these structural elements highlighted alpha-helix1 as the candidate fusion peptide in gH. The two heptad repeats interact with each other and form a coiled coil. This property appears to be critical in guiding the massive glycoprotein refolding that takes place at fusion. Recently, the crystal structure of gB was solved. It reveals a trimer with a coiled coil core, and resembles closely that of vesicular stomatitis virus envelope glycoprotein (G). This came as a surprise, since, a priori, there was no reason to anticipate that gB carries a structure typical of viral fusion glycoproteins. At present, the most likely scenario is that gH and gB form a complex, and that the two together function as fusion executors.

Rational design of fusion inhibitors

The identification of gH as a candidate fusion executor in HSV, and the finding that synthetic peptides mimicking the heptad repeats block infection has been the basis for a European Community-approved project (effective January 2007), named TargetHerpes. One of the aim of the project is the identification of molecular targets for the rational design of antiherpes chemotherapeutics (Campadelli-Fiume is the European Coordinator).

Multivesicular bodies as a cellular compartment for Herpes simplex virion assembly and egress out of the cell.

Multivesicular bodies (MVBs) biogenesis is topologically equivalent to virion budding. Hence, a number of viruses exploit the MVBs pathway to build their envelope and exit from the cell. By expression of dominant negative forms of two components of the multivesicular bodies pathway,  infectious herpes simplex virus (HSV) assembly/egress was impaired. Furthermore, HSV-1 infection resulted in morphological changes to MVB. gB, one of the most highly conserved glycoprotein across the herpesviridae family, was sorted to MVB membranes. gB appeared to be  ubiquitinated in both infected and transfected cells. Ubiquitination was in part dependent on ubiquitin lysine63, a signal for cargo sorting to MVBs.  This line of research supports the view that sorting of gB to MVB membranes may represent a critical step in HSV envelopment and egress, and that modified MVBs membranes constitute a platform for HSV cytoplasmic envelopment, or, alternatlvely, that MVBs components are recruited to the site(s) of envelopment.

 

Retargeting of HSV tropism to heterologous receptors

The structural bases of HSV entry have paved the way for a successful modification of HSV tropism to heterologous receptors. HSVs are being employed as oncolytic viruses, mainly against glioblastoma. The safety profile has been achieved by debilitating the viral replication, such that the virus can only replicate in dividing cells of the tumor, and not in the non dividing cells of the brain. These viruses have been tested in phase 1 and 2 clinical studies, with promising results, even though their effects are marred by the low replication. The goal now is the construction of HSV specifically retargeted to receptors that are expressed in the tumor cells, but not in normal cells, such that less debilitated viruses can be employed. The successful strategy for modification of HSV tropism devised by Roizman and Zhou has been the insertion of a novel ligand in a position of gD that leaves the glycoprotein capable of responding to receptor recognition, and to trigger fusion. In the US , two ligands were introduced in gD, IL13 and urokinase plasminogen activator. In the past few years, our laboratory has engineered a HSV specifically retargeted to the HER2/neu receptor, a member of the EGFR family overexpressed in mammary and ovary tumors. The rational is that about 25-30 % of mammary tumors (those with the worse prognosis and highest metastaticity) express this receptor. Because HER2/neu lacks a natural ligand, the engineered ligand that we selected was a single chain antibody (scFv) to HER2/neu. Overall, the insertion almost doubled the size of the gD ectodomain.

Recently, HSV fully retargeted to HER2 and detargeted from the natural HSV receptors, nectin1 and HVEM, were successfully engineered. These recombinants exhibit oncolytic activity in vivo, in mice xenografted with HER-2-positive human tumors.