Human papillomavirus (HPV) is a non-enveloped, double-stranded DNA virus. HPVs carry out their life cycle in either mucosal or cutaneous epithelia. They have icosahedral capsids composed of 72 capsomeres, surrounding a circular DNA genome of ~7900bp (Miller et al., 2012) (Figure 1). To date 180 HPV genotypes have been cloned from clinical lesions and grouped as, high-risk and low-risk HPVs. The high-risk HPVs are known to cause cancer and the low-risk HPVs are known to cause skin warts (Stanley MA, 2012). High-risk HPV infection accounts for approximately 5% of all cancers worldwide and all cervical cancers are associated with high-risk HPV infection. HPV16 and HPV18 are the most cancer causing HPV types.
Integrin family of proteins function as αβ heterodimers and they act as adhesion receptors that bind to extracellular matrix ligands, cell-surface ligands, and soluble ligands. They mediate proliferation, migration and differentiation as well as cell-cell interactions and attachment to extracellular matrix (Aoudjit and Vuori, 2012). At least 18α and 8β subunits have been found in humans and 24 heterodimers been identified (Figure 2).
The expression and distribution of integrins at the cell surface is controlled by endocytosis and recycling. Recycling of integrins is especially important for migrating cells. Integrins are first endocytosed, then moved to endosomes and finally recycled back to the plasma membrane (Riggs et al., 2012).
Integrins can act as receptors for non-enveloped and enveloped viruses for their attachment and/or entry to host cells. Specifically, α6 integrin has been shown to act as a receptor for HPV16 and mediate infection of keratinocytes (Evander et al., 1996).
Toll-like receptors (TLRs) are the key players in innate immune response. To date 10 TLRs have been identified in humans. TLRs can recognize a wide range of pathogens and activate antigen-presenting cells to produce pro-inﬂammatory cytokines. This pathway eventually leads to establishment of adaptive immunity (Kaisho and Akira, 2005). Eight TLRs have been demonstrated to be involved in viral recognition and of those, TLR9 has been shown to be down-regulated upon HPV infection (Hasan et al., 2007).
In this study six integrin subunit genes (α2, α3, αv, β1, β4 and β6) and TLR9 expression profiles upon HPV infection were investigated in immortalized human keratinocyte cells (HaCaT) by reverse transcriptase PCR (RT-PCR).
Materials and Methods
HaCaT cell line was kindly provided by Dr. Meneses Lab, Department of Biological Sciences, Fordham University. Cells were cultured in Dulbecco's Modified Eagle's media (DMEM) either with or without 10% Fetal Bovine Serum (FBS, DMEM-10).
HPV16 Pseudovirus Infection
Pseudovirus (PsV) is also kindly provided by Dr. Meneses Lab, Department of Biological Sciences, Fordham University and produced as described by Buck et al (Buck et al., 2004).
200,000 HaCaT cells were plated on 6-well plates and incubated at 37°C in DMEM-10 for 2 days to reach ~80% confluency. Then DMEM-10 was aspirated and cells were starved in DMEM for 1 hour on ice. After 1 hour, PsV at a multiplicity of infection (MOI) 0.15 is added to each well except the uninfected controls. Cells were further incubated for an additional 1 hour on ice. After incubation each well was washed 3 times with 1X PBS and cells were incubated at 37°C in DMEM-10 for either 1 hour or 4 hours.
The total RNA was extracted at corresponding time points using RNeasy® Plus Mini Kit (QIAGEN), according to the manufacturer’s instructions.
RT-PCR was performed using QIAGEN® One-Step RT-PCR Kit following the instructions. GAPDH was used as the loading control.
RT-PCR products were confirmed by sequencing and all experiments were replicated at least three times. The transcript level of integrins (α2, α3, αv, β1, β4 and β6) remained unchanged upon 4 hours of PsV infection; whereas up-regulation in the level of TLR9 transcript was observed (Figure 3 and 4).
Integrins have been shown to act as viral receptors and α6 integrin has been shown to act as a receptor for HPV16. Upon binding to their ligand, integrins are internalized and recycle back to the cell membrane. Their internalization is thought to mediate the HPV16 infection. The cells are expected to regulate the integrin distribution on the cell surface in the presence of HPV16. For this, the cell may increase the transcript levels of HPV16 associated integrin subunits, stabilize their mRNAs and/or also can change the recycling rate.
The results show that all of the selected integrin subunit genes are expressed in HaCaT cells, but their expression levels remain unchanged after 4 hours following HPV infection, which indicates that there may be other mechanisms involved in regulation of integrin distribution, such as mRNA stabilization or increasing the recycling rate. Alternatively these results can also indicate that the selected integrin subunits are not involved in HPV16 binding and internalization.
It has been shown by others that TLR9 expression is down-regulated upon HPV16 infection. In this study, however, the results indicate an up-regulation for TLR9 upon HPV16 infection. The discrepancy might have stemmed from two reasons: First, the longest time-point in this study is 4h while it is 24h/48h in the previous studies; second, for this study PsV is used for infection, so there is no HPV16 genome surrounded by the capsid, whereas the previous studies have utilized the HPV16 genome. The down-regulation of TLR9, as shown by others, is believed to be caused by viral proteins (E6, E7) that are produced in the cell following the infection, but since the pseudovirus used in this study lacks these genes and the time-points are not long enough for the virus to be localized in the nucleus. These results might show that the observed TLR9 up-regulation is due to a different mechanism and most likely the result of a quick immune-response of the host cell.
Aoudjit F, Vuori K. (2012). Integrin signaling in cancer cell survival and
chemoresistance. Chemother Res Pract. 2012:283181.
Buck CB, Pastrana DV, Lowy DR, Schiller JT. (2004). Efficient intracellular assembly of papillomaviral vectors. J Virol. 78(2):751–7.
Evander M, Frazer IH, Payne E, Qi YM, Hengst K, McMillan NA. (1997). Identification of the alpha6 integrin as a candidate receptor for papillomaviruses. J Virol. 71(3):2449-56.
Kaisho, T. and Akira, S. (2005). Toll-like Receptors. eLS.
Miller DL, Puricelli MD, Stack MS. (2012). Virology and molecular pathogenesis of HPV (human papillomavirus)-associated oropharyngeal squamous cell carcinoma. Biochem J.,443(2):339-53.
Riggs KA, Hasan N, Humphrey D, Raleigh C, Nevitt C, Corbin D, Hu C. (2012). Regulation of Integrin Endocytic Recycling and Chemotactic Cell Migration by Syntaxin 6 and VAMP3 Interaction. J Cell Sci.
Stanley MA. (2012). Epithelial cell responses to infection with human papillomavirus. Clin Microbiol Rev. 25(2):215-22.
Takada Y, Ye X, Simon S. (2007). The integrins. Genome Biol. 8(5):215.
Uzma A. Hasan, Elizabeth Bates, Fumihiko Takeshita, Alexandra Biliato, Rosita Accardi, Veronique Bouvard, Mariam Mansour, Isabelle Vincent, Lutz Gissmann, Thomas Iftner, Mario Sideri, Frank Stubenrauch and Massimo Tommasino. (2007). TLR9 Expression and Function Is Abolished by the Cervical Cancer-Associated Human Papillomavirus Type 16. J Immunol. 178;3186-3197.