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.
