Projects

Below is a list of the current and future studies that will be developed in the laboratory:

1.  Understanding the initial events leading to viral endocytosis and trafficking. We have been working on defining the process by which the viral genome reaches the nucleus and our studies have defined a series of events that were novel to previous interpretations.  The main difference was regarding what happens after the viral particles are endocytosed into the cytoplasm. There is strong evidence that the initial endocytosis event results in the viral particles residing in an early endosome [1-3]. Laboratories previously suggested that the viral particle exited the endosomes into the cytoplasm and moved towards the nucleus with the aid of motor proteins.  Our data supported the loss of colocalization of endosome marker and viral particles, but supported a scenario in which the viral particles were moved to the endoplasmic reticulum via a caveolin-1 vesicle intermediate [4, 5].  In fact our data showed that lack of movement of viral particles into the caveolin-1 positive vesicle resulted in loss of infection.  Because the different interpretations of acquired data lead to different mechanisms, it was of importance to meticulously dissect the different stages of viral trafficking.  Our original findings were supported by our subsequent data showing that indeed the viral particles remain within a vesicle as they traverse the cytoplasm. The mechanism of trafficking within the cytoplasm now focuses on an endosome to Golgi movement, and final residence in an ERwith virus reaching the nucleus after ce Our long-term goal is to understand the various events leading to PV infection by focusing on HPV16 the most common human oncogenic genotype, and BPV1 the prototypic animal genotype.  In this project we will focus on the events occurring upon initial virus binding leading to viral internalization and trafficking in the cytoplasm. Our rationale is that understanding the precise events leading to infection can translate into powerful antiviral approaches directed at reducing the burden of PV associated diseases including cancers. We have also made great strides in identifying the cell surface receptors [6].

2.Determining the role of the viral capsid proteins and identify auxiliary cellular molecules. Our goal is to understand the basic processes that are necessary to establish HPV infection.  The objective here is to understand how the capsid proteins and auxiliary cellular proteins regulate viral entry and intracellular trafficking. Our central hypothesisis that the minor capsid protein L2 plays a role in initiating viral entry into cells and the intracellular trafficking of the infectious viral particles.  Our hypothesis is based on our published, and exciting preliminary data from ongoing studies showing that altering of L2 sequences results in improper entry, sorting and trafficking of viral particles.  The rationale for the proposed research is that upon identifying the mechanism by which the highly conserved papillomavirus capsid protein L2 and auxiliary cellular molecules direct the entry, sorting and trafficking, we will be able to develop targeted and innovative approaches to prevent infection by many or most papillomavirus genotypes[7] .

3.  L2 as a regulator of cellular differentiation and division.  The viral life cycle of PV is linked to the differentiation events of the squamous epithelium, the natural host cells of PV infection.  Primary PV infection occurs in the basal layer of the epithelia, the only cells capable of dividing, allowing the virus to persist as an extrachromosomal DNA element.  As the cells rise to the surface of the epithelium, the virus enters its vegetative phase in which the viral DNA is amplified, late genes are expressed, and mature virions are generated.  We hypothesize that L2 is involved in regulating the cellular environment necessary for viral production and/or cellular differentiation by regulating the transcriptome. The inhibition of differentiation in PV-infected keratinocytes by L2 at initial infection or at the vegetative state, may provide an environment for viral DNA replication, and/or may be beneficial for the transformation mechanism regulated by PV E6 and E7 proteins that interfere with the function of the Rb and p53 proteins respectively.  We explore the role of L2 in human keratinocyte differentiation and cell division by focusing on L2s effect on cellular mRNA expression.  We have published a transcriptome study showing the changes in genes involved in regulating cell fate including epithelial to mesenchyme transition (observed in many cancers)[8].

4.  Antiviral strategies targeting viral entry.  The L1 capsid protein is primarily, if not exclusively, responsible for the initial binding of PVs to the cell membrane.  Neutralization of infection with antibodies directed against L1 prevents the virus interaction with the plasma membrane. In contrast, neutralizing antibodies directed against L2 do not prevent viral binding to the cell surface.  We recently showed that pseudovirions generated with L2 mutants that are unable to interact in vivo with the ER resident protein syntaxin 18 are unable to infect cells [9].  These L2 mutant pseudovirions bind to the surface of the cell, enter the cell into some unidentified vesicle, but do not reach the perinuclear region as assessed by confocal microscopy using capsid antibodies and cellular markers as guides.  We hypothesize that the inability to interact with syntaxin 18 is responsible for the loss of ER trafficking of the virus and loss of infection. In support of this hypothesis, we have shown that a synthetic peptide, PM1, encompassing the ER trafficking domain of L2 retards infection by PVs and an antibody to this region is also able to block infection [10].  This region of the L2 protein is highly conserved among all PV genotypes, and is also found in viral proteins believed to be involved in viral entry of picornaviruses, HIV, and bluetongue virus.  PV viral particles reach the perinuclear region by 7 hours after infection in the presence of a non-specific peptide, whereas virions remain at the periphery of the cells in the presence of PM1, or the antibody to this region.  We will pursue these studies by identifying the organelle where infection with the defective virions and neutralizing antibody is blocked, and by exploring the possibility that infection can be blocked permanently by targeting this L2 region.  We have also been able to target the c-terminus of the L1 protein for neturalization of infection.

Studies in early states, i.e., future directions.

1. Host immunity and infection: Infection by HPV is thought to occur through a microtear/trauma in the mucosa.  Through these microtears the virus reaches the target cells in the basement membrane: the undifferentiated keratinocytes.  In addition to reaching the undifferentiated keratinocytes, the virus encounters the mucosal immunity including antigen presenting cells (APCs, e.g., dendritic cells, B lymphocytes and macrophages), CD4+ helper T cells and CD8+ cytotoxic lymphocytes, and CD4+/CD25+ regulatory T-cells.  The events that occur after the virus infects the cells it encounters are the focus of this project. We propose to determine how HPV manipulates the immune response by studying the changes infection has on keratinocytes’ gene transcription, protein expression, and cell cycle, on the effects of infection of HPV on immune cells, and to determine changes in the interaction of keratinocytes with immune cells.  This approach will allow us to determine changes caused by HPV in keratinocytes, peripheral blood mononuclear cells (PBMCs), and the interplay between these cells.

2. Individual susceptibility: Although most HPV infections are readily cleared in infected individuals, in certain individuals HPV infections persist for years and decades leading to the development of carcinoma.  Worldwide there is an increase in the level of oral carcinoma, cervical cancer, and anal carcinoma in the HIV infected population as compared to the HIV negative population. This leads to the hypothesis that the HIV population is at risk of acquiring a new HPV infection or that existing HPV infections are re-activating in the immuno-suppressed HIV infected individual.  Studies have suggested that both scenarios are occurring supporting the notion that alterations of the immune system are beneficial for HPV infection. We would like to profile variances in infected/uninfected individuals immune system, and those of patients suffering from carcinoma. Is there a difference in cells profile, e.g., T-Regs that has shifted the system from a quiescent infection towards carcinoma?

3. Clinical expression of HPV genes: We are establishing a collaboration with Dr. Tergas, a clinician Gynecologist at Columbia University. We have preliminary data suggesting that post- HPV vaccine there is a worrysome drift towards other HPV genotypes than those in vaccines.  We also want to compare our in-vitro viral protein expression with the expression of genes in patient samples including pre-cancerous to cancerous.

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