Ocular Virology Laboratory
Dr. Vaibhav Tiwari wins first place at COM Research Day 2008
Study Shows How Herpes Infects Cornea, Evades Immune Cells
New research on herpes simplex virus has implications for treating ocular herpes
Research conducted in my laboratory focuses on understanding the molecular mechanism of virus entry into cells. Our goal is to understand how alphaherpesviruses, such as human herpes simplex viruses (HSV-1 and HSV-2) enter into susceptible cells to cause infections. Herpesviruses encode about 100 proteins from a DNA genome, which is packaged within an icosahedral capsid and a lipid-protein envelope. These human viruses cause various forms of disease, from lesions on the lips, eyes, or genitalia, to encephalitis or disseminated disease. Ocular herpes can produce a painful sore on the eyelid or surface of the eye and cause inflammation of the cornea. The less severe forms of ocular herpes include blepharitis, conjunctivitis, and epithelial keratitis. The more severe form of ocular herpes is stromal keratitis, which causes scarring of the cornea, which can lead to loss of vision and possibly blindness. The entry of HSV into cells begins with an initial binding of viral glycoproteins gB or gC to heparan sulfate proteoglycans (HSPG) on cell surface (figure below). After this initial binding, a third glycoprotein gD binds to one of its receptors. The gD-receptor binding triggers fusion of viral envelope with the cell membrane. Once fused, viral capsid and some tegument proteins are released into the cytoplasm of the cell. The cellular receptors for gD are very diverse in nature. They come from three different classes of cell surface receptors, (I) HVEM, the first known gD receptor is a member of the tumor necrosis factor receptor (TNFR) family, (II) two others (Nectin-1 and -2) are members of the immunoglobulin (Ig) superfamily related to the poliovirus receptor; and (III) some unique sites in heparan sulfate (HS) resulting from the action of specific 3-O-sulfotransferases also produce HSV-1 specific receptor. Interaction of gD with any one of those receptors triggers the membrane fusion reaction, which by an unknown mechanism also requires a concerted action of three other envelope glycoproteins namely, gB and gH-gL. We use genetics, molecular biology, biochemistry, and cell biology to identify viral and cellular mediators of entry and to decipher their precise roles in the process of viral entry, both in human and mouse cells. A thorough understanding of the intimate details of virus entry and the presence of a well-characterized animal model will help design novel anti-viral therapies to prevent entry and the spread of HSV.
Laboratory Personnel
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Susmita Bommireddy |
Education
MS Virology
Sri Venkateswara University, India
Area of Research
Our goal is to define the molecular events during the entry of Herpes Simplex Viruses. It is an enveloped DNA virus that causes mucosal lesions affecting ocular, oral and genital areas. Very rarely it causes life-threatening disease that includes encephalitis.I work in the laboratory of Dr. Deepak Shukla. My assignment deals with variety of cell culture techniques and to produce stable cell lines expressing target protein molecules. In molecular approaches I focus on PCR-based cloning, gel-electrophoresis, blotting techniques, immunological techniques and ELISA. I am also involved with preparation and titration of virus stocks for entry assays.
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Christian Clement |
Education
Bachelor of Science (Hons): Biochemistry with Chemistry, University of Ghana Legon
Master of Philosophy: Biochemistry University of Ghana.
Experience
As a member of faculty at the University of Ghana I actively researched into isolation and characterization of bacteriophages, screened secondary plant metabolites of medicinal value especially for medical conditions such as asthma and diabetes as well as antibacterial agents. Ghana is a major gold producing country, therefore an aspect of my study has given me practical knowledge in bioremediation of industrial/mining effluents. I have vast experience in the preparation of environmental assessments and pollution monitoring/emergency measures. I have worked on a novel approach in the re-vegetation of surface mine sites and leach pads in association with researchers at the Council for Scientific and Industrial Research-Ghana.
Currently
Ph.D. Student, Microbiology and Immunology Department, University of Illinois at Chicago
Research
Heparan sulfate an ubiquitous sugar molecule expressed on surfaces of a variety of cell lines and implicated in initial attachment of various viruses, has been shown to act as a receptor for entry of Herpes Simplex Virus (HSV-1), when modified by an isoform of D- glucosaminyl 3-O-sulfotransferases. In the search for drugs for the treatment of HSV related illness, targeting entry is significant in preventing infection. The study is focused at investigating the effect on viral entry by co-expression of some of the isoforms of these transferases in stably transfected cell lines as a mode of elucidating the entry mechanism.The HSV viral glycoprotein gD is critical for entry, novel peptides that will interact with gD in a specific way are being selected using the Phage Display (PhD) technology to study the structural-functional properties of this glycoprotein.
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Perry M. Scanlan |
Education
Bachelor of Science
Medical Microbiology and Immunology: University of Wisconsin-Madison
Clinical Laboratory Science: University of Wisconsin-Madison
Certifications
Clinical Laboratory Scientist MT (ASCP)
PhD. Student
Microbiology and Immunology: University of Illinois- Chicago
Research
I am studying Herpes Simplex Virus (HSV) entry and pathogenesis. I am working with the viral glycoproteins of gD, gH, gL, gB, and many others, studying virus entry in human corneal cells. HSV and HSV related illness is one of the leading causes of blindness in developed countries. It is important to study entry of these viruses so that prophylactic treatment can be used to prevent blindness, particularly in newborn infants. In addition to these important considerations, these viruses could be used as gene delivery vectors. Some current work being done with these viruses is involved in brain cancer treatment. The mutant virus has the ability to infect the dividing cancer cells leaving the other cells intact. This technique may someday be a possible way to treat brain cancer. Discovering ways to prevent infection would be a major breakthrough in this field. As a PhD. Student I look forward to adding to this knowledge and hopefully someday discover or contribute to the treatment or cure of this widespread disease.
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Vaibhav Tiwari |
Education
Ph.D. (Biotechnology)
School of Biotechnology, Banaras Hindu University, India
Research
We are trying to understand the molecular mechanism of Herpes Simplex Virus (HSV) entry. This enveloped virus is member of neurotropic subgroup (alpha herpesvirus) of herpes family having short replication cycle and broad host range and hence HSV is one of the most opportunistic agents involved in the pathogenesis that uses multiple cell receptors on many different cell types. Herpes Simplex Virus infections are endemic and receive a great deal of attention due to spread and ubiquitous prevalence (infecting 60-90% of world population) in human population. The virus uses mucosal epithelium membranes as a port of entry and replicates in epidermis and dermis and establishes life long latency in the neurons of dorsal ganglia. Two major types of HSV can be distinguished by known tissue type-specific antisera or by biologic effects on tissue cultures. Type 1 (HSV-1) is responsible for most infections above the waist, including ocular, oral, labial, upper respiratory tract, and central nervous system involvement. Type 2 (HSV-2) is responsible for most genital or below waist infections and neonatal infections. However 12-50% of genital herpes is from HSV-1, and HSV-2 can cause any nongenital herpetic infection.
The large genome (152 Kb) of HSV encodes variety of structural proteins including 11 glycoproteins (gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, gM) out of which five enveloped glycoproteins (gB, gC, gD and heterodimer gH /gL) are implicated in viral entry. The entry of virus in a cell is a chain of dynamic and sequential events in which first viral glycoprotein probably gC or gB binds to heparan sulfate (a glycosaminoglycan; GAGs) moieties on cell surface proteoglycans which are ubiquitously expressed on mammalian cells and tissues. After initial attachment second glycoprotien gD interacts with one of the cellular receptors. Interestingly gD receptors identified belongs to three different families; the herpesvirus entry mediator A (Hve A or HVEM) is tumor necrosis factor (TNF a) receptor family and is principal receptor for entry of HSV-1 and HSV-2 in to human lymphoid cells, while Nectin-1 (Hve B) and Nectin-2 (Hve C) are poliovirus receptor-related proteins and belong to Immunoglobulins (Ig) superfamily. Nectin-1 allows entry of all a-herpes viruses; in contrast HSV-2 and mutant strains (Rid 1) and Psuedorabies virus (PRV) use Nectin-2. Recently a novel sugar receptor 3-0-sulfated Heparan Sulfate (HS) was identified which generates gD binding sites after being modified by an enzyme D-glucosaminyl-3-0-sulfotransferase isform 3 (3-0ST-3). This specific type of HS can substitute Hve A or Hve C and binds to gD to allow entry of HSV-1. The third interaction involves at least 4 glycoproteins (gB, gD and gH/gL) for pH independent fusion of the virion envelop with plasma membrane. However membrane fusion by herpesvirus is not well understood because it is possible that it requires the activity of several herpesvirus proteins or capable of several modes of fusion. A considerable effort has been made to understand the fusion mechanism by transfecting cells to express viral glycoproteins. The development of such system for HSV would provide a model system for studying either virus -cell interaction or cell-to-cell spread.
Our goal is to figure out the viral-cellular requirements for entry of HSV in ocular tissue and further links to tropism and pathogenesis. HSV is most common infectious cause of corneal blindness in developed countries. Approximately 50,000 reported cases of new or recurrent ocular HSV (Herpes simplex virus type 1 and 2) disease per year, predominantly with stromal scarring of the cornea leading to significant vision loss in about 6,000 patient per year. HSV keratitis is the leading cause for corneal transplantation in US and other developed countries.
Recent publications
Previous publicationsXia G, Chen J, Tiwari V,Ju W, Li J-P, Malmstrom A, Shukla D and Liu J (2002)
Heparan Sulfate 3-0-Sulfotransferase Isoform 5 Generates Both an
Antithrombin-binding Site and an Entry Receptor for Herpes Simplex Virus,Type
1. Journal of Biological Chemistry,Vol 277 No.40 pp. 37912-37919
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Tiwari V, Deo Singh B, Nath Tiwari K (1998) Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monniera (L.) Wettst.] Plant Cell Reports Spriger-Verlag 17:538-543
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Tiwari KN, Sharma NC, Tiwari V, Singh BD (2000) Micropropagation of Centella asiatica (L.), a valuable medicinal herb Plant Cell Tissue Organ and Organ Culture Kluwer Academic Publisher 16: 179-185
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Tiwari V, Tiwari KN, Singh BD (2001) Comparative studies of cytokinins on in vitro propagation of Bacopa monniera Plant Cell Tissue Organ and Organ Culture Kluwer Academic Publisher 66: 9-1
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Tiwari V, Tiwari KN, Singh BD (2001) Suitability of liquid cultures for in vitro multplication of Bacopa monniera (L.) Wettst. Phytomorphology 50: 337-342
