Modulating Phosphorylation of Caveolin 1 and Its Effect on the Intracellular Trafficking of HPV16




Elinor Y. Mossop

Introduction

Papillomaviruses are a group of nonenveloped, double-stranded DNA viruses known to cause warts and, in some cases, cancer (Bosch et al., 2002). Approximately 99% of all cervical cancers are attributed to HPV infections (American Cancer Society, 2013). HPV types 16 and 18 alone are reported to be responsible for 70% of cervical cancer, 85% of anal cancer, and more than 50% of oropharyngeal cancer (Muñoz et al., 2004; Schiffman et al., 2007; Watson et al., 2008). In developing countries cervical cancer is the 2nd leading cause of death for women, causing 245,000 women to die in 2010 (World Health Organization, 2010).

HPV16 infection occurs in actively dividing cells in the skin (Schiller et al., 2010). Many of the initial steps of the virus binding to cells, entering cells, and trafficking to the nucleus remain vague. Caveolin 1 is a small (~25 KDa) scaffolding protein that can be involved in endocytosis, intracellular trafficking, can act as a platform for signaling molecules, and has been shown to stabilize focal adhesions when phosphorylated on tyrosine 14 (Parton et al., 2013; Boscher et al., 2012; Bailey et al., 2008; Goetz et al., 2008; del Pozo et al., 2005; Lisanti et al., 2004). Research has shown that HPV16 does not seem to be endocytosed in a caveolin 1-dependent manner (Schelhaas et al., 2012; Laniosz et al., 2009). However, when caveolin 1 protein level is reduced in human basal keratinocytes via shRNA, there is a significant decrease in infection (Laniosz et al., 2009). Recent work has shown an increase in the phosphorylation of caveolin 1 on tyrosine 14 at early timepoints of 10 to 30 minutes after virus binding (Mossop, Meneses, unpublished data).

This study aims to shed light on whether the phosphorylation of caveolin 1 on tyrosine 14 is a necessary event for HPV16 infection of human basal keratinocytes. Four cDNAs encoding caveolin 1 were generated: the wild type (WT), a cDNA with a single base change that does not result in an amino acid change (Y14Y), and two mutant cDNAs that result in a change of the 14th amino acid from a tyrosine to a phenylalanine (Y14F1 and Y14F2). The mutants containing a phenylalanine at the 14th position instead of a tyrosine cannot be phosphorylated at this residue. The expression of the Y14F mutant caveolin 1 has been shown to act in a dominant-negative manner in human lung cancer cells by inhibiting cell invasion and migration (Shatz et al., 2010). In this study, preliminary experiments with the described generated caveolin 1 mutants rely on the possibility that the mutant caveolin 1 may also act in a dominant-negative manner in HaCaT cells (human basal keratinocytes) as HaCaT cells already express caveolin 1.


Materials and Methods

Homo sapiens caveolin 1 cDNA sequence was taken from the NCBI database (NCBI Reference Sequence: NM_001753.4). Four forward primers were designed starting at the ATG start codon at the beginning of the cDNA. The first is for the wild type cDNA, the other three have a one or two bases change for the fourteenth amino acid (normally a tyrosine). The second forward primer changes the fourteenth codon from TAC to TAT, which still encodes for a tyrosine. The third primer changes the fourteenth codon from TAC to TTC and the fourth primer changes the codon from TAC to TTT, both of which encode a phenylalanine instead of a tyrosine. The four forward primers also have the Kozak consensus sequence (GCCGCCACC) before the start codon for the cDNA at the 5’ end. One reverse primer was designed, starting with the stop codon for the cDNA. See Figure 1 for the full primer sequences.

RT-PCR was conducted with the described primers and PCR product was ligated into the pTARGET™ vector (a mammalian expression vector) and transformed in JM109 cells. Full mini-preps were performed on colonies with inserts and the purified plasmids were sequenced in one direction using the T7 primer by GENEWIZ, Inc. (South Plainfield, NJ). Sequences were aligned with the wild type caveolin 1 cDNA with Basic Local Alignment Search Tool (BLAST, NCBI) (Figure 3).

HaCaT cells (human basal keratinocytes) and 293TTs (human embryonic kidney cells) were provided by Dr. Meneses’ lab, Department of Biological Sciences, Fordham University. Cells were plated in 12-well plates (100,000 per well) and grown at 37°C for 48 hours. The cells were transfected (lipofectamine-based) with 1µg of the wild type or mutant cDNAs carried out at 37°C for 24 hours or 48 hours before infection with the HPV16 pseudovirions, in the case of the HaCaT cells, or harvesting for western blots, in the case of the 293TTs (Figure 4).

Results

Each of the primers successfully amplified the caveolin 1 mRNA (Figure 1). PCR product from each reaction was ligated into the pTARGET vector and transformed into JM109 cells. Based on the sequencing results a clone from each of the wild type or mutant cDNAs was chosen that did not have any mutations other than the base change at the 14th amino acid position (Figure 2).

To check for expression of the cDNAs the chosen clones were transfected into 293TT cells, which do not normally express caveolin 1. As is seen in the western blot in Figure 3A, all four constructs express caveolin 1 at similar amounts at 48 hours (lanes 2 – 5) as compared with the untransfected 293TT cells in lane 1. Only the mutants with the change from tyrosine to phenylalanine at position 14 (lanes 2 and 3 in figure 3B) do not show a band when probed an antibody specific against tyrosine 14 phosphorylated caveolin 1.

Figure 4 shows flowcytometry HPV16 infection data of a 48 hour transfection with 1 μg of DNA of each of the constructs and a control with a transfection of 1μg of the empty vector. There is a statistically significant increase in HPV16 infection in the cells transfected with each of the four caveolin 1 contructs compared to the empty vector control. There is no observed decrease in infection with the expression of the mutant caveolin 1 constructs that cannot be phosphorylated on tyrosine 14.


Discussion

As briefly described in the introduction of this paper, caveolin 1 currently has an undefined role in the HPV16 infection process. It is clear that caveolin 1 is important for infection, but it is not clear exactly how or when during the intracellular trafficking the protein plays its part. Caveolin 1 phosporylated on tyrosine 14 has been shown to localize to focal adhesions and is hypothesized to stabilize focal adhesions, aiding in cell migration, invasion, and adhesion (Shatz et al., 2010). The observed increase in phosphorylation of caveolin 1 on tyrosine 14 upon binding of HPV16 in HaCaT cells led us to believe this phosphorylation event may be necessary for HPV16 infection.

The aim of this study was to assess the effect of expressing a mutant caveolin 1 that cannot be phosphorylated on tyrosine 14 on HPV16 infection. To reach this goal the necessary tools first had to be produced. The correct mutant caveolin 1 cDNAs were successfully generated. The Y14Y and the Y14F1 mutant were transfected into 293TT cells and expression of the protein was successfully detected by western blotting. The absence of tyrosine 14 phosphorylation on the mutant Y14F1 was confirmed via western blotting and probing with a tyrosine 14 specific caveolin 1 antibody. While transfecting 293TT cells and confirming the expression of the proteins from the constructs was a necessary step, an infection assay using 293TT cells is irrelevant to HPV16 biology because 293TTs readily infect with the virus despite not expressing caveolin 1. The infection of 293TTs may occur through an alternative pathway than in HaCaT cells.

It was important to test the effect of the mutant caveolin 1 on infection in HaCaT cells because these cells more closely resemble the cells that HPV16 would be infecting in vivo. In the preliminary infection experiments, the expression of the caveolin 1 constructs mutated at tyrosine 14 to phenylalanine do not seem to impact infection with HPV16 whereas the overexpression of caveolin 1 in general (wild type or mutant) does seem to cause an increase in HPV16 infection (Figure 4). These results need to be repeated. There is a possibility that the expression of the mutant caveolin 1 is not behaving in a dominant negative manner in the HaCaT cells as was reported in human lung cancer cells (Shatz et al., 2010). If the expression of the mutant caveolin 1 is not causing the loss of function of the endogenous caveolin 1 in the cells, then it is possible that infection can still occur because there is still enough functional endogenous caveolin 1. Possible future studies will involve siRNA knockdown of the endogenous caveolin in HaCaT cells, with siRNA sequences targeting the wild type codon (TAC) for caveolin 1, along with expressing the wild type and mutant caveolin 1 cDNAs.

Consensus has not yet been made about the necessary steps for HPVs to bind, enter, and traffic in cells in order to cause an infection (Raff et al., 2013). This study aimed to clarify the involvement of one of the candidate proteins in the HPV16 infection process, caveolin 1. The data generated in this study suggest that while caveolin 1 is involved in HPV16 infection, the phosphorylation of the protein on tyrosine 14 is not necessary for infection to occur.


References

American Cancer Society. Cancer Facts and Figures 2013. Atlanta, Ga: American Cancer Society; 2013.

Bailey, K. M. and J. Liu. 2008. "Caveolin-1 Up-Regulation during Epithelial to Mesenchymal Transition is Mediated by Focal Adhesion Kinase." The Journal of Biological Chemistry 283 (20): 13714-13724.

Bosch, F. X., A. Lorincz, N. Muñoz, C. J. L. M. Meijer, and K. V. Shah. 2002. "The Causal Relation between Human Papillomavirus and Cervical Cancer." Journal of Clinical Pathology 55 (4): 244-265.

Boscher, C. and I. R. Nabi. 2012. "Caveolin-1: Role in Cell Signaling." Advances in Experimental Medicine and Biology 729: 29-50.

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Laniosz, Valerie, Sarah A. Dabydeen, Mallory A. Havens, and Patricio I. Meneses. 2009. "Human Papillomavirus Type 16 Infection of Human Keratinocytes Requires Clathrin and Caveolin-1 and is Brefeldin A Sensitive." Journal of Virology 83 (16): 8221-8232.

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Shatz, M., G. Lustig, R. Reich, and M. Liscovitch. 2010. "Caveolin-1 Mutants P132L and Y14F are Dominant Negative Regulators of Invasion, Migration and Aggregation in H1299 Lung Cancer Cells." Experimental Cell Research 316 (10): 1748-1762.

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Figures


Figure 1-A. Primer sequences. Each forward primer is preceded by a Kozak consensus sequence for optimal translation (in red). The base changes in each forward primer are in red. B. RT-PCR with caveolin 1 primers. GAPDH as control. Each reaction was run in triplicate with 10 ng of DNA and a control with 0ng of DNA (lanes 1, 3, 7, 11, 15). Lanes 1 and 2 are with GAPDH primers. Lanes 3 – 6 are with primers for the wild type caveolin 1 sequence. Lanes 7 – 10 are with the forward primer with one base change that does not cause an amino acid. Lanes 11 – 14 are with the primer that causes one base change and an amino acid change. Lanes 15 – 18 are with the primer that causes 2 bases changes and one amino acid change. Lanes 3 – 18 have the same reverse primer for caveolin 1.


Figure 2-Cloned wild type caveolin 1 and mutant caveolin 1 cDNAs aligned with the wild type caveolin 1 cDNA sequence from NCBI. The codon for the 14th amino acid is in red. These selected clones have no mutations other than the expected mutation due to the primers. Part A has the cloned wild type caveolin 1 (TAC at codon 14). Part B of the figure aligns the sequence with a one base mutation but still encodes for a tyrosine (TAT). Part C shows the one base change from the wild type tyrosine (TAC) to TTC, encoding a phenylalanine. Part D shows the two base change from the wild type tyrosine (TAC) to TTT, also encoding phenylalanine.


Figure 3-Western blot with lysates from 293TT cells untransfected, transfected with the caveolin 1 WT construct, transfected with the Y14Y construct with a base change that does not change the amino acid sequence (Y→Y), transfected with the caveolin 1 mutant Y14F1 with one base change that results in a change from a tyrosine to a phenylalanine at position 14 (Y→F1), or transfected with the caveolin 1 mutant Y14F2 with two base changes that result in a change from a tyrosine to a phenylalanine at position 14 (Y→F2). The western blot in part A was incubated with a polyclonal antibody against total caveolin 1 and the same blot in part B was incubated with a monoclonal antibody against caveolin 1 phosphorylated on tyrosine 14. The membrane was also incubated with a monoclonal anti-actin antibody.


Figure 4-HaCaT cells were transfected with the empty DNA vector, caveolin 1 WT, caveolin 1 Y14Y, caveolin Y14F1, or caveolin 1 Y14F2. The cells were incubated for 48 hours before infection with HPV16 pseudovirions. After 48 hours with the PsVs, the cells were harvested and GFP expression measured on a flowcytometer. * denotes p < 0.05, ** denotes p < 0.005.


Abstract

Human papillomavirus type 16 (HPV16) infection of basal keratinocytes causes lesions that can progress into cancer, most commonly of the cervix. The exact mechanisms of entry and intracellular trafficking of HPV16 in human basal keratinocytes continue to be elusive. Caveolin 1 has been shown in previous studies to be a possible player in the intracellular trafficking of HPV16 through colocalization and protein knockdown experiments. Additionally, the level of caveolin 1 that is phosphorylated on tyrosine 14 has been shown to increase upon binding of HPV16 to human basal keratinocytes. In this study, a mutant caveolin 1 was generated that cannot be phosphorylated on tyrosine 14 and the effect of this mutant on the infectivity of HPV16 was explored.

Full Paper

Acknowledgments

I would like to thank Dr. Rubin, Kate Reid, Catharina Grubaugh, and Dr. Anderson for all of their help, guidance, and time spent with this project. Thank you to Dr. Meneses for his guidance and for the use of the cells, pseudovirions, and other materials necessary for the project completion. This work was supported by the Department of Biological Sciences at Fordham University.


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