Shoshana Levy, PhD: Research Areas
Abstract
Our research is aimed at understanding the mechanism of action of tetraspanins. This is a multi-gene family, which has shown remarkable conservation over evolution and whose members are expressed in mammals, insects and nematodes. Tetraspanins are also widely expressed in most cell types, forming molecular associations with different proteins in the different cell types.
The tetraspanin CD81 was originally identified in our laboratory as a receptor that controls cell growth. To better define the role of CD81 we created CD81-deficient mice. These mice have impairments in their immune, nervous and reproductive systems.
CD81 has been implicated in the pathogenesis of two major human diseases: hepatitis C virus (HCV) and malaria. CD81 is the putative receptor for HCV, CD81 is also required for infection by malaria. Plasmodium sporozoites mature in the liver to merozoites, the stage that infects red blood cells, this maturation step is CD81-dependent.
Recent Studies
CD81 is a widely expressed and evolutionary conserved molecule that associates with different proteins in different cell types (ref/abstract). We hypothesize that CD81 affects a multitude of cell functions because it is essential for the spatial orientation of its associated/partner proteins in membranes. To determine its role we generated CD81 knockout (cd81-/-) mice.
Lack of CD81 affects cell-cell interactions in multiple systems
- CD81 is required for normal immune responses, lack of this molecule
results in weaker early antibody responses to protein antigens, specifically
Th2 responses (ref/abstract).
The impairment in Th2 responses is physiologically important, as cd81-/-
mice do not develop allergen-induced airway hyper-reactivity (ref/abstract).
CD81 enhances cognate T-B cell interactions and greatly augments intracellular
activation pathways leading to Th2 polarization (ref/abstract).
CD81 is required for the normal expression of the CD19 signaling molecule
in B cells (ref/abstract).
Moreover, it is necessary for partitioning of coligated CD19/CD21-B
cell receptor complexes into signaling lipid rafts (ref/abstract).
- CD81 is a required for normal development of the brain. Lack of CD81
results in an increase in brain size and glial cell number (ref/abstract).
It is a critical component of astrocyte responses to neuronal differentiation
signals as it regulates neuron-induced astrocytic differentiation (ref/abstract).
- CD81 is required for membrane reorganization during fertilization,
cd81-/- eggs do not fuse with sperm, resulting in female infertility
(ref/abstract).
- CD81 is required for infection of hepatocyte by malaria. cd81-/- mice are resistant to infection by Plasmodium yoelii sporozoites. CD81 is also critical for infection of human hepatocytes by Plasmodium falciparum sporozoites, highlighting the importance of this molecule in the most widespread infectious disease known to mankind (ref/abstract).
CD81 is a putative receptor for hepatitis C virus (HCV)
Hepatitis C virus (HCV) infection affects an estimated 160 million people worldwide. It is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma and of mixed cryoglobulinemia, a B lymphocyte proliferative disorder. The life cycle of HCV is currently not known. A major difficulty in the study of HCV is that the virus infects only humans and chimpanzees and cannot be propagated in small animal models. Moreover, HCV cannot be readily grown in tissue culture. Cloning of the virus enabled the production of the recombinant envelope glycoprotein E2, which in turn, was shown to bind to human CD81. My laboratory has been engaged in HCV related research since the identification of CD81 as the putative receptor of this virus.
- We determined the structural basis for the interactions of the viral
envelope proteins with human CD81. We mapped the critical amino acid
of CD81 involved in binding the viral envelope protein. The F186L mutation
resulted in loss of binding. (ref/abstract).
- We demonstrated the inhibitory effect of HCV infection on the maturation
and function of dendritic cells in patients (ref/abstract).
- We analyzed the individual B cell immune response of HCV-infected
patients to the viral envelope proteins. Single B cells immortalized
as hybridomas and selected for binding E2 (ref/abstract)
were analyzed for their V gene usage. Remarkably, these anti-E2 hybridomas
preferentially expressed the immunoglobulin VH1-69 gene (ref/abstract).
This VH gene was previously shown to be highly expressed in HCV-associated
lymphoproliferative disorders. These data strengthen the hypothesis
that the HCV-associated lymphomas are derived from clonally expanded
B cells stimulated by HCV.
- We cloned the B cell receptors from HCV-associated lymphoma cases
and expressed them as soluble immunoglobulins (Igs). The rescued Igs
were then tested for their ability to bind the HCV-E2 envelope glycoprotein.
One of two lymphoma Ig test cases bound the E2 protein in a manner identical
to a bona fide human anti-E2 antibody. Moreover, it bound E2 from multiple
viral genotypes, suggesting reactivity with a conserved E2 epitope.
These findings support the hypothesis that some HCV-associated lymphomas
may originate from B cells that were initially activated by the HCV-E2
protein, and might explain the association between HCV infection and
some B cell lymphoproliferative disorders (ref/abstract).
- We characterized the human mAb that recognize conformation-dependent
epitopes within E2, which in turn, provided insight into the conformational
state of the HCV envelope glycoproteins (ref/abstract).
Specifically, we identified one specific human mAb that recognizes the
heterodimeric HCV envelope proteins, E1E2, but not misfolded E1E2 aggregates.
This implies that the patient’s immune response recognizes specifically
the native E1E2 heterodimeric form.
- We developed a method to isolate E1E2 complexes that are properly folded and demonstrated the biological activity of the purified E1E2 heterodimers (ref/abstract).