I am a highly active individual with a vast array of experiences and am currently pursuing my PhD in biomedical science at the University of Southern California Keck School of Medicine. I specialize in cancer immunotherapy of tumor associated antigen cancers, immunology, and virology of the human papillomavirus (HPV), human immunodeficiency virus (HIV), and the herpes simplex virus (HSV).
Skills and Expertise
PCRCell CultureWestern Blot AnalysisGene ExpressionCancer BiologyFlow CytometryImmunohistochemistryImmunofluorescenceMolecular Cell BiologyRNA IsolationTransfectionMolecular CloningPrimary Cell CultureTissue CultureImmunoprecipitationCancer Cell BiologyImmunofluorescence StainingHuman Cell CultureGraphPad PrismMammalian Cell TransfectionCo-ImmunoprecipitationAssay DevelopmentCervical Cancer PreventionSmall Animal HandlingCell IsolationLentiviral TransductionCervical CancerRetroviral Transduction
Awards & Achievements (6)
Award · Jun 2017
American Association of Immunologists Young Investigator Award - Immunology LA 2017
Award · Mar 2017
Best Poster in Basic Science Research - 31st International HPV Conference
Award · Feb 2016
USC Graduate Research Symposium - Presentation Award
Award · Jan 2016
Graduate Student Teaching Assistantship
Award · Aug 2015
BeHEARD 2015 Rare Disease Science Challenge - Ultimate Technology Prize
Currently investigating the negative signaling factors involved in the suppression of Langerhans cells by the Annexin A2/S100A10 heterotetramer.
Research Item (23)
Herpes Simplex Virus (HSV) was originally implicated in the etiology of cervical cancer, and although high-risk human papillomavirus (HPV) is now the accepted causative agent, the epidemiologic link between HSV and HPV-associated cancers persists. The Annexin A2 heterotetramer (A2t) has been shown to mediate infectious HPV type 16 (HPV16) uptake by human keratinocytes, and secretory leukocyte protease inhibitor (SLPI), an endogenous A2t ligand, inhibits HPV16 uptake and infection. Interestingly, HSV infection induces a sustained downregulation of SLPI in epithelial cells, which we hypothesized promotes HPV16 infection through A2t. Here we show that in vitro infection of human keratinocytes with HSV type 1 or 2, but not with an HSV-1 ICP4 deletion mutant that does not downregulate SLPI, leads to a >70% reduction of SLPI mRNA, and a >60% decrease in secreted SLPI protein. Consequently, we observed a significant increase in the uptake of HPV16 virus-like particles and gene transduction by HPV16 pseudovirions (2 and 2.5 fold, respectively) in HSV-1 and HSV-2 infected human keratinocyte cell cultures compared to uninfected cells, whereas exogenously added SLPI reversed this effect. Using a Single Molecule Pulldown (SiMPull) assay, we demonstrate that endogenously secreted SLPI interacts with A2t on epithelial cells in an autocrine/paracrine manner. These results suggest that on-going HSV infection and resultant downregulation of local levels of SLPI may impart a greater susceptibility for keratinocytes to HPV16 infection through the host cell receptor A2t, providing a mechanism that may, in part, provide an explanation for the etiological link between HSV and HPV-associated cancers.
- Feb 2016
Carcinomas of the anogenital tract, in particular cervical cancer, remains one of the most common cancers in women, and represent the most frequent gynecological malignancies and the fourth leading cause of cancer death in women worldwide. Human papillomavirus (HPV)-induced lesions are immunologically distinct in that they express viral antigens, which are necessary to maintain the cancerous phenotype. The causal relationship between HPV infection and anogenital cancer has prompted substantial interest in the development of therapeutic vaccines against high-risk HPV types targeting the viral oncoproteins E6 and E7. This review will focus on the most recent clinical trials for immunotherapies for mucosal HPV-induced lesions as well as emerging therapeutic strategies that have been tested in pre-clinical models for HPV-induced diseases. Progress in peptide- and protein-based vaccines, DNA-based vaccines, viral/bacterial vector-based vaccines, immune checkpoint inhibition, immune response modifiers, and adoptive cell therapy for HPV will be discussed.
In the last three decades, extensive research on human immunodeficiency virus (HIV) has highlighted its capability to exploit a variety of strategies to enter and infect immune cells. Although CD4(+) T cells are well known as the major HIV target, with infection occurring through the canonical combination of the cluster of differentiation 4 (CD4) receptor and either the C-C chemokine receptor type 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4) coreceptors, HIV has also been found to enter other important immune cell types such as macrophages, dendritic cells, Langerhans cells, B cells, and granulocytes. Interestingly, the expression of distinct cellular cofactors partially regulates the rate in which HIV infects each distinct cell type. Furthermore, HIV can benefit from the acquisition of new proteins incorporated into its envelope during budding events. While several publications have investigated details of how HIV manipulates particular cell types or subtypes, an up-to-date comprehensive review on HIV tropism for different immune cells is lacking. Therefore, this review is meant to focus on the different receptors, coreceptors, and cofactors that HIV exploits to enter particular immune cells. Additionally, prophylactic approaches that have targeted particular molecules associated with HIV entry and infection of different immune cells will be discussed. Unveiling the underlying cellular receptors and cofactors that lead to HIV preference for specific immune cell populations is crucial in identifying novel preventative/therapeutic targets for comprehensive strategies to eliminate viral infection.
Human papillomaviruses (HPV) establish persistent infections because of evolved immune evasion mechanisms , particularly HPV-mediated suppression of the immune functions of Langerhans cells (LC), the antigen presenting cells of the epithelium. Polyinosinic-polycytidilic acid (Poly-I:C) is broadly immunostimulatory with the ability to enhance APC expression of costimulatory molecules and inflammatory cytokines resulting in T cell activation. Here we investigated the activation of primary human LC derived from peripheral blood monocytes after exposure to HPV16 virus like particles followed by treatment with stabilized Poly-I:C compounds (s-Poly-I:C), and their subsequent induction of HPV16-specific T cells. Our results indicate that HPV16 particles alone were incapable of inducing LC activation as demonstrated by the lack of costimulatory molecules, inflammatory cytokines, chemokine-directed migration, and HPV16-specific CD8 þ T cells in vitro. Conversely, s-Poly-I:C caused significant upregulation of costimulatory molecules and induction of chemokine-directed migration of LC that were pre-exposed to HPV16. In HLA-A*0201-positive donors, s-Poly-I:C treatment was able to induce CD8 þ T cell immune responses against HPV16-derived peptides. Thus, s-Poly-I:C compounds are attractive for translation into therapeutics in which they could potentially mediate clearance of persistent HPV infection. & 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Mucosotropic, high-risk human papillomaviruses (HPV) are sexually transmitted viruses that are causally associated with the development of cervical cancer. The most common high-risk genotype, HPV16, is an obligatory intracellular virus that must gain entry into host epithelial cells and deliver its double stranded DNA to the nucleus. HPV capsid proteins play a vital role in these steps. Despite the critical nature of these capsid protein-host cell interactions, the precise cellular components necessary for HPV16 infection of epithelial cells remains unknown. Several neutralizing epitopes have been identified for the HPV16 L2 minor capsid protein that can inhibit infection after initial attachment of the virus to the cell surface, which suggests an L2-specific secondary receptor or cofactor is required for infection, but so far no specific L2-receptor has been identified. Here, we demonstrate that the annexin A2 heterotetramer (A2t) contributes to HPV16 infection and co-immunoprecipitates with HPV16 particles on the surface of epithelial cells in an L2-dependent manner. Inhibiting A2t with an endogenous annexin A2 ligand, secretory leukocyte protease inhibitor (SLPI), or with an annexin A2 antibody significantly reduces HPV16 infection. With electron paramagnetic resonance, we demonstrate that a previously identified neutralizing epitope of L2 (aa 108-120) specifically interacts with the S100A10 subunit of A2t. Additionally, mutation of this L2 region significantly reduces binding to A2t and HPV16 pseudovirus infection. Furthermore, downregulation of A2t with shRNA significantly decreases capsid internalization and infection by HPV16. Taken together, these findings indicate that A2t contributes to HPV16 internalization and infection of epithelial cells and this interaction is dependent on the presence of the L2 minor capsid protein.
- May 2018
High-risk human papillomavirus-associated cancers express viral oncoproteins (e.g., E6 and E7) that induce and maintain the malignant phenotype. The viral origin of these proteins makes them attractive targets for development of a therapeutic vaccine. Camelid-derived single-domain antibody fragments (nanobodies or VHHs) that recognize cell surface proteins on antigen-presenting cells (APCs) can serve as targeted delivery vehicles for antigens attached to them. Such VHHs were shown to induce CD4+ and CD8+ T-cell responses against model antigens conjugated to them via sortase, but antitumor responses had not yet been investigated. Here, we tested the ability of an anti-CD11b VHH (VHHCD11b) to target APCs and serve as the basis for a therapeutic vaccine to induce CD8+ T cell responses against HPV+ tumors. Mice immunized with VHHCD11b conjugated to an H-2Db-restricted immunodominant E7 epitope (E749-57) had more E7-specific CD8+ T cells compared to those immunized with E749-57 peptide alone. These CD8+ T cells acted prophylactically and conferred protection against a subsequent challenge with HPV E7-expressing tumor cells. In a therapeutic setting, VHHCD11b-E749-57 vaccination resulted in greater numbers of CD8+ tumor-infiltrating lymphocytes compared to mice receiving E749-57 peptide alone in HPV+ tumor-bearing mice, as measured by in vivo noninvasive VHH-based immune-positron emission tomography (immunoPET), which correlated with tumor regression and survival outcome. Together, these results demonstrate that VHHs can serve as a therapeutic cancer vaccine platform for HPV-induced cancers.
Nano-Pulse Stimulation (NPS) is a non-thermal pulsed electric field modality that has been shown to have cancer therapeutic effects. Here we applied NPS treatment to the human papillomavirus type 16 (HPV 16)-transformed C3.43 mouse tumor cell model and showed that it is effective at eliminating primary tumors through the induction of immunogenic cell death while subsequently increasing the number of tumor-infiltrating lymphocytes within the tumor microenvironment. In vitro NPS treatment of C3.43 cells resulted in a doubling of activated caspase 3/7 along with the translocation of phosphatidylserine (PS) to the outer leaflet of the plasma membrane, indicating programmed cell death activity. Tumor-bearing mice receiving standard NPS treatment showed an initial decrease in tumor volume followed by clearing of tumors in most mice, and a significant increase in overall survival. Intra-tumor analysis of mice that were unable to clear tumors showed an inverse correlation between the number of tumor infiltrating lymphocytes and the size of the tumor. Approximately half of the mice that cleared established tumors were protected against tumor re-challenge on the opposite flank. Selective depletion of CD8⁺ T cells eliminated this protection, suggesting that NPS treatment induces an adaptive immune response generating CD8⁺ T cells that recognize tumor antigen(s) associated with the C3.43 tumor model. This method may be utilized in the future to not only ablate primary tumors, but also to induce an anti-tumor response driven by effector CD8⁺ T cells capable of protecting individuals from disease recurrence.
Project - Viral modifications to epithelial cells that result in enhanced HPV infection
During sexual transmission of human immunodeficiency virus (HIV), macrophages are initial targets for HIV infection. Secretory leukocyte protease inhibitor (SLPI) has been shown to protect against HIV infection of macrophages through interactions with annexin A2 (A2), which is found on the macrophage cell surface as a heterotetramer (A2t) consisting of A2 and S100A10. Therefore, we investigated potential protein-protein interactions between A2 and HIV-1 gp120 through a series of co-immunoprecipitation assays and a single molecule pulldown (SiMPull) technique. Additionally, inhibitors of A2t (A2ti) that target the interaction between A2 and S100A10 were tested for their ability to impair productive HIV-1 infection of macrophages. Our data suggest that interactions between HIV-1 gp120 and A2 exist, though this interaction may be indirect. Furthermore, an anti-A2 antibody impaired HIV-1 particle production in macrophages in vitro, whereas A2ti did not indicating that annexin A2 may promote HIV-1 infection of macrophages in its monomeric rather than tetrameric form.
Human papillomavirus type 16 (HPV16) infections are intra-epithelial, and thus, HPV16 is known to interact with Langerhans cells (LCs), the resident epithelial antigen-presenting cells (APCs). The current paradigm for APC-mediated induction of T cell anergy is through delivery of T cell receptor signals via peptides on MHC molecules (signal 1), but without costimulation (signal 2). We previously demonstrated that LCs exposed to HPV16 in vitro present HPV antigens to T cells without costimulation, but it remained uncertain if such T cells would remain ignorant, become anergic, or in the case of CD4+ T cells, differentiate into Tregs. Here we demonstrate that Tregs were not induced by LCs presenting only signal 1, and through a series of in vitro immunizations show that CD8+ T cells receiving signal 1+2 from LCs weeks after consistently receiving signal 1 are capable of robust effector functions. Importantly, this indicates that T cells are not tolerized but instead remain ignorant to HPV, and are activated given the proper signals.
- Sep 2015
Human papillomavirus (HPV)-mediated suppression of Langerhans cell (LC) function can lead to persistent infection and development of cervical intraepithelial neoplasia (CIN). Women with HPV-induced high-grade CIN2/3 have not mounted an effective immune response against HPV, yet it is unknown if LC-mediated T cell activation from such women is functionally impaired against HPV. We investigated the functional activation of in vitro generated LC and their ability to induce HPV16-specific T cells from CIN2/3 patients after exposure to HPV16 followed by treatment with stabilized Poly-I:C (s-Poly-I:C). LC from patients exposed to HPV16 demonstrated a lack of costimulatory molecule expression, inflammatory cytokine secretion, and chemokine-directed migration. Conversely, s-Poly-I:C caused significant phenotypic and functional activation of HPV16-exposed LC, which resulted in de novo generation of HPV16-specific CD8(+) T cells. Our results highlight that LC of women with a history of persistent HPV infection can present HPV antigens and are capable of inducing an adaptive T cell immune response when given the proper stimulus, suggesting s-Poly-I:C compounds may be attractive immunomodulators for LC-mediated clearance of persistent HPV infection.
- Feb 2015
Objectives: High-risk human papillomavirus (HPV) infection leads to the development of several human cancers that cause significant morbidity and mortality worldwide. HPV type 16 (HPV16) is the most common of the cancer-causing genotypes and gains entry to the basal cells of the epithelium through a non-canonical endocytic pathway that involves the annexin A2/S100A10 heterotetramer (A2t). A2t is composed of two annexin A2 mono- mers bound to an S100A10 dimer and this interaction is a potential target to block HPV16 infection. Here, recently identified small molecule inhibitors of A2t (A2ti) were investigated for their ability to prevent HPV16 infection in vitro. Methods: A2ti were added to HeLa cells in increasing concentrations prior to the addition of HPV16. Cytotoxicity was evaluated via trypan blue exclusion. HPV16 pseudovirion infection and fluorescently labelled HPV16 capsid internalization was measured with flow cytometry. Results: A2ti blocked HPV16 infection by 100% without substantial cellular toxicity or reduction in cell growth. Furthermore, A2ti blocked HPV16 entry into epithelial cells by 65%, indicating that the observed inhibition of HPV16 infection is in part due to a block in entry and that non-infectious entry may occur in the absence of A2t binding. Conclusions: These results demonstrate that targeting A2t may be an effective strategy to prevent HPV16 infection.
- Nov 2014
LIGHT, a ligand for lymphotoxin-β receptor (LTβR) and herpes virus entry mediator, is predominantly expressed on activated immune cells and LTβR signaling leads to the recruitment of lymphocytes. The interaction between LIGHT and LTβR has been previously shown to activate immune cells and result in tumor regression in a virally-induced tumor model, but the role of LIGHT in tumor immunosuppression or in a prostate cancer setting, where self antigens exist, has not been explored. We hypothesized that forced expression of LIGHT in prostate tumors would shift the pattern of immune cell infiltration toward an anti-tumoral milieu, would inhibit T regulatory cells (Tregs) and would induce prostate cancer tumor associated antigen (TAA) specific T cells that would eradicate tumors.
The systemic treatment of soft tissue sarcomas other than Gastrointestinal Stromal Tumors (GIST) has not changed for several decades. The recently demonstrated effectiveness of immune checkpoint inhibitors in melanoma has led to its application to other solid tumors with many ongoing studies that await completion. Soft tissue sarcomas possess several classes of immunogenic antigens that could provide a basis for future immunotherapy trials. The presence of Cancer Testis Antigens (CTA) and other immunogenic antigens unique to soft tissue sarcomas will be reviewed here along with a review of past studies that may shed light on the design and conduct of future immunotherapy trials in sarcoma.
Historically, Herpes Simplex Virus (HSV) was the first etiologic agent implicated in the development of cervical cancer, and although Human Papillomavirus (HPV) is now the globally accepted causative agent, ongoing epidemiologic data still suggests that HSV is linked to cervical cancer development. An uptake receptor for HPV, the Annexin A2/S100A10 heterotetramer (A2t), has recently been shown as an important component in infectious HPV uptake by epithelial cells. A2t has also been documented as having an endogenous ligand, secretory leukocyte protease inhibitor (SLPI). Here we show that infection of epithelial cells with HSV leads to a reduction in levels of SLPI mRNA expression by greater than 90% and total secreted protein levels by over 60% within human epithelial cells cultured in vitro , as well as an increase in infection by HPV16 pseudovirus by 230%. Additionally we provide direct evidence that SLPI binding to A2t is facilitated through interaction with the Annexin A2 subunit of the heterotetramer, and that restoration of SLPI levels to the cell culture medium leads to an ablation of the increase in HPV16 infection seen in HSV-1 infected cell cultures. These results provide mechanistic evidence that suggests that in humans, an on-going HSV-1 infection may cause a greater susceptibility of keratinocytes for HPV16 infection through down-modulation of the HPV A2t receptor ligand SLPI which can explain the observed synergism between these two highly prevalent sexually transmitted viruses in the etiology of cervical cancer.
Question - How can I isolate WBC for RNA extraction?
Given that PBMCs are some of the most transcriptionally active cells in whole blood I would start with them.
Everything that Pamela said above is spot on, but if you want to look at PBMCs add the following at the beginning (due to the amount of RBCs present):
1. A ficoll separation.
2. Several washes with RPMI1640 or similar (utilize patient plasma as a replacement for FBS if you have enough)
3. Treatment with 1x RBCL buffer for 3-5 minutes followed by 2 additional washes (50 mL).
4. Move on to RNA isolation.
Not sure what profile you're looking for with the RNA, but I don't think ficoll will effect them too much. I attached pdf with a good overview of RNA isolation from blood.
High-risk human papillomaviruses (HPVs) are sexually transmitted viruses causally associated with several cancers. During its natural life cycle, HPV16, the most common high-risk genotype, infects the epithelial basal cells in a process facilitated through a recently identified receptor, the annexin A2 heterotetramer (A2t). During infection, HPV16 also interacts with Langerhans cells (LC), the APC of the epithelium, inducing immune suppression, which is mediated by the HPV16 L2 minor capsid protein. Despite the importance of these virus-immune cell interactions, the specific mechanisms of HPV16 entry into LC and HPV16-induced immune suppression remain undefined. An N-terminal peptide of HPV16 L2 (aa 108-126) has been shown to specifically interact with A2t. In this study, we show that incubation of human LC with this peptide blocks binding of HPV16. Inhibiting this interaction with an A2t ligand or by small interfering RNA downregulation of A2t significantly decreases HPV16 internalization into LC in an L2-dependent manner. A2t is associated with suppression of LC maturation as demonstrated through attenuated secretion of Th1-associated cytokines and decreased surface expression of MHC class II on LC exposed to A2t. Conversely, small molecule inhibition of A2t prevents HPV16-induced suppression of LC immune function as indicated by significantly increased secretion of inflammatory cytokines and surface expression of CD86 in HPV16 treated LC pre-exposed to A2t inhibitors. These results demonstrate that HPV16 suppresses LC maturation through an interaction with A2t, revealing a novel role for this protein.
Question - What are the possibilities of eradicating cervical cancer in the next two decades if we can plan on implementing HPV vaccination at a larger scale?
I was not trying to imply that the immune response is compromised if an individual is already infected with HPV at the time of immunization, but I do not believe it is a standalone method that can be used in an immunotherapy setting. You can stimulate T-cells all you want, but you need infiltration as well in those who develop tumors.
Ultimately I was just trying to build on the idea that vaccination is not enough to eliminate HPV-related cancers due to the fact that you need to address those who are already infected, and those individuals can have their HPV status monitored through DNA screening etc.
Additionally, thank you for the correction!
Question - What are the possibilities of eradicating cervical cancer in the next two decades if we can plan on implementing HPV vaccination at a larger scale?
The answers so far have just been based on vaccination uptake and the assumption that a multivalent vaccine will be sufficient to eradicate HPV. A most accurate answer is a bit more complicated.
While it is correct that gardasil only protects against the most prevalent hrHPV types (16, 18) as well as 6 and 11, it does not work as an effective immunotherapy for those who already have an HPV infection (a process which takes an estimated 10 years of recurrent infection). In the best case scenario we get >80 percent vaccination uptake in the younger population and generate a herd immunity for that generation and those that will come after, however we still have the older population which did not have that protection and will still go on to develop an HPV-related cancer.
Realistically, if we can succeed at developing an immunotherapy-based vaccine strategy/treatment that takes care of those who have infections as well as generate a greater uptake in number of children vaccinated against the most-prevalent HPV types we could eliminate HPV-related cancers, but it is going to take more than 20 years given the current mis-information and confusing stigma that has been placed on vaccines.
I know I did not address the idea that "another HPV-type will just take 16 or 18's place," however given that more that 60% of cervical cancers are caused by 16 alone this is a great starting point.
A retrospective case series of three patients with chronic low back pain who received baseline MRI scans revealing multifidus muscle atrophy with fatty replacement is provided. Each patient received spinal manipulative therapy, and two were compliant with low back exercises targeting the multifidus. A follow-up scan performed >1 year later was compared to the baseline scan revealing a decrease in atrophy with fatty replacement in the two patients who performed multifidus-focused low back exercises (15% and 39% on the left and 7% and 32% on the right respectively), and an increase in the patient who underwent spinal manipulation alone (41% and 53%). Interestingly, the decrease in atrophy in the two patients that performed the exercises correlated to functional improvements. Though limited, these results highlight the utility of MRI in quantifying positive and negative long-term changes in multifidus atrophy, which may be an indicator of recovery in chronic low back pain patients.
Question - Can anyone help: my western blot result of a snail protein is higher than 30 KD?
My first couple of questions are:
1. What steps did you take to denature prior to loading?
2. What ladder did you use?
3. What was your running buffer, the buffer in which you run (MOPS, MES, etc.) will result in different ladder-running profiles.
4. How much of a difference in size did you see?
Question - How can I get an even spread of cells in a 24 well plate?
One final idea here that builds on the 'figure 8'; after letting cells rest in the hood do your figure 8 distribution directly on the incubator shelf, just leave them when your done. One of the reasons you'll get any residual clustering in the center is due to the motion generated when transporting the cells.
- Mar 2014
Human papillomavirus (HPV) has evolved mechanisms that allow it to evade the human immune system. Studies have shown HPV-mediated suppression of activation of Langerhans cells (LC) is a key mechanism through which HPV16 evades initial immune surveillance. However, it has not been established whether high- and low-risk mucosal and cutaneous HPV genotypes share a common mechanism of immune suppression. Here, we demonstrate that LC exposed to capsids of HPV types 18, 31, 45, 11, (alpha-papillomaviruses) and HPV5 (beta-papillomavirus) similarly suppress LC activation, including lack of costimulatory molecule expression, lack of cytokine and chemokine secretion, lack of migration, and deregulated cellular signaling. In contrast, HPV1 (mu-papillomavirus) induced costimulatory molecule and cytokine upregulation, but LC migration and cellular signaling was suppressed. These results suggest that alpha and beta HPV genotypes, and partially a mu genotype, share a conserved mechanism of immune escape that enables these viruses to remain undetected in the absence of other inflammatory events.
Human papillomaviruses (HPVs) infect epithelia and can lead to the development of lesions, some of which have malignant potential. HPV type 16 is the most oncogenic genotype and causes various types of cancer, including cervical, anal, and head and neck cancers. However, despite significant research, our understanding of the mechanism by which HPV16 binds to and enters host cells remains fragmented. Over several decades, many HPV receptors and entry pathways have been described. This review puts those studies in context and offers a model of HPV16 binding and entry as a framework for future research. Our model suggests that HPV16 binds to heparin-sulfate proteoglycans (HSPGs) either on the epithelial cell surface or basement membrane through interactions with the L1 major capsid protein. Growth factor receptors may also become activated through HSPGs/growth factor/HPV16 complexes that initiate signaling cascades during early virion-host cell interactions. After binding to HSPGs, the virion undergoes conformational changes leading to isomerization by cyclophilin B and proprotein convertase-mediated L2 minor capsid protein cleavage that increases L2 N-terminus exposure. Along with binding to HSPGs, HPV16 binds to α6 integrins, which initiate further intracellular signaling events. Following these primary binding events, HPV16 binds to a newly identified L2-specific receptor, the annexin A2 heterotetramer. Subsequently, clathrin-, caveolin-, lipid raft-, flotillin-, cholesterol-, dynamin-independent endocytosis of HPV16 occurs.
HPV16 L1 and L2 immunoblot analysis of HPV16 WT PsV and HPV16 L1–L2(GGDD) mutant PsV. An equal amount of infectious particles of wildtype and mutant pseudovirions were separated by SDS-PAGE and transferred to PVDF membranes. Blots were separately probed with a mouse anti-L1 antibody followed by IR800 (green)-labeled goat anti-mouse IgG secondary antibody, or a rabbit anti-HPV16 L2 antibody followed by AlexaFluor 680 (red)-labeled goat anti-rabbit IgG secondary antibody. Blots were scanned on the Licor Odyssey infrared imaging system. The L2 immunoblot shows that incorporation of the L2 protein into pseudovirions is not affected by the L2108–111 LVEE → GGDD mutation. (TIFF)