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Mechanisms regulating expression of the HPV 31 L1 and L2 capsid proteins and pseudovirion entry.

Hindmarsh PL, Laimins LA - Virol. J. (2007)

Bottom Line: Similar to studies in HPV 16, expression of wild type HPV 31 L1 and L2 from heterologous promoters resulted in very low levels of synthesis.In contrast, modification of the codons in the capsid genes to ones more commonly used in cellular genes resulted in high-level synthesis.This suggests that high-risk HPV types may enter cells through common mechanisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. phindm@lsuhsc.edu

ABSTRACT
Human papillomaviruses (HPV) infect stratified epithelia and restrict expression of late capsid genes to highly differentiated cells. In order to begin to understand the processes regulating HPV 31 infection we examined the synthesis of the HPV 31 capsid proteins, L1 and L2, using heterologous expression systems. Similar to studies in HPV 16, expression of wild type HPV 31 L1 and L2 from heterologous promoters resulted in very low levels of synthesis. In contrast, modification of the codons in the capsid genes to ones more commonly used in cellular genes resulted in high-level synthesis. Through the use of chimeric proteins that fused fragments of wild type L1 to Green Fluorescent Protein (GFP) coding sequences, a short region was identified that was sufficient to inhibit high level synthesis and similar elements were detected in L2. One element was localized to the 3' end of the L1 gene while a series of elements were localized at the 3' end of the L2 coding sequences. These observations are most consistent with negative RNA regulatory elements controlling the levels of L1 and L2 synthesis that are distinct from those identified in HPV 16. Expression vectors for the codon modified HPV 31 capsid proteins were then transfected together with GFP reporter plasmids to generate HPV 31 pseudoviruses. Infection of cells with HPV 31 pseudoviruses in the presence of the inhibitors, chlorpromazine, nystatin or methyl-beta-cyclodextrin, demonstrated that HPV 31, like HPV 16, enters human and monkey cells through a clathrin-mediated pathway rather than through caveolae as previously reported. This suggests that high-risk HPV types may enter cells through common mechanisms.

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Inhibition of HPV 31 pseudovirion infection. (A) Cos-7 cells or (B) 293 TT cells were treated with inhibitors chlorpromazine, nystatin or methyl-β-cyclodextran at the concentrations shown for 30 min prior to infection. The cells were infected over night with HPV 31 pseudovirions. Media was changed at 24 hours, fresh media and drug was added. The infection was allowed to continue for an additional 48 hours for a total of 72 hours. Percentage GFP positive cells relative to control is shown. Each panel is an average of 3 experiments.
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Figure 5: Inhibition of HPV 31 pseudovirion infection. (A) Cos-7 cells or (B) 293 TT cells were treated with inhibitors chlorpromazine, nystatin or methyl-β-cyclodextran at the concentrations shown for 30 min prior to infection. The cells were infected over night with HPV 31 pseudovirions. Media was changed at 24 hours, fresh media and drug was added. The infection was allowed to continue for an additional 48 hours for a total of 72 hours. Percentage GFP positive cells relative to control is shown. Each panel is an average of 3 experiments.

Mentions: We next wanted to confirm, using our experimental protocols, that HPV 16 pseudoviruses enter cells by the clathrin mediated pathway as has been reported [10-12]. In these experiments we added the inhibitors (either nystatin, methyl-β-cyclodextrin or chlorpromazine) 30 minutes prior to infection and maintained for 72 h. In Figure 4, averaged results from three experiments are shown in which Cos-7 and 293 TT cells were infected with HPV 16 pseudovirions in the presence or absence of inhibitors. Similar results were seen in multiple experiments. As the concentration of chlorpromazine was increased, we observed a significant decrease in HPV 16 pseudovirion entry (Figure 4). In contrast, the presence of nystatin and methyl-β-cyclodextrin exhibited no effect on HPV 16 pseudovirion infectivity. These concentrations have been reported to inhibit the infection by BPV-1, HPV 33 and EBV [10,11,21,26]. We next performed identical experiments with HPV 31 pseudoviruses and the results are shown in Figure 5. As we observed with HPV 16 pseudoviruses, increasing concentrations of chlorpromazine decreased HPV 31 pseudovirion entry in a dose-dependent manner into either Cos-7 or 293 TT cells. The concentrations of inhibitors used in Figure 5 had no effect on cell viability but higher concentrations of chlorpromazine were observed to be toxic to the Cos-7 and 293 TT cells (data not shown). As shown in Figure 5, no decrease on HPV 31 pseudovirion infectivity was observed as nystatin or methyl-β-cyclodextrin levels were increased. Interestingly, at intermediate concentrations of nystatin or methyl-β-cyclodextrin, we observed a reproducible increase in infectivity over untreated cells. In addition we observed that treatment with the drugs was reversible, as infections in the presence of chlorpromazine for 2–6 hours followed by a wash with fresh media and incubation for a total of 72 hours had negligible effect on infectivity (data not shown). Our results suggest a role for clathrin-mediated entry of HPV 16 and 31 pseudovirions in the entry of human and monkey cells.


Mechanisms regulating expression of the HPV 31 L1 and L2 capsid proteins and pseudovirion entry.

Hindmarsh PL, Laimins LA - Virol. J. (2007)

Inhibition of HPV 31 pseudovirion infection. (A) Cos-7 cells or (B) 293 TT cells were treated with inhibitors chlorpromazine, nystatin or methyl-β-cyclodextran at the concentrations shown for 30 min prior to infection. The cells were infected over night with HPV 31 pseudovirions. Media was changed at 24 hours, fresh media and drug was added. The infection was allowed to continue for an additional 48 hours for a total of 72 hours. Percentage GFP positive cells relative to control is shown. Each panel is an average of 3 experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1808446&req=5

Figure 5: Inhibition of HPV 31 pseudovirion infection. (A) Cos-7 cells or (B) 293 TT cells were treated with inhibitors chlorpromazine, nystatin or methyl-β-cyclodextran at the concentrations shown for 30 min prior to infection. The cells were infected over night with HPV 31 pseudovirions. Media was changed at 24 hours, fresh media and drug was added. The infection was allowed to continue for an additional 48 hours for a total of 72 hours. Percentage GFP positive cells relative to control is shown. Each panel is an average of 3 experiments.
Mentions: We next wanted to confirm, using our experimental protocols, that HPV 16 pseudoviruses enter cells by the clathrin mediated pathway as has been reported [10-12]. In these experiments we added the inhibitors (either nystatin, methyl-β-cyclodextrin or chlorpromazine) 30 minutes prior to infection and maintained for 72 h. In Figure 4, averaged results from three experiments are shown in which Cos-7 and 293 TT cells were infected with HPV 16 pseudovirions in the presence or absence of inhibitors. Similar results were seen in multiple experiments. As the concentration of chlorpromazine was increased, we observed a significant decrease in HPV 16 pseudovirion entry (Figure 4). In contrast, the presence of nystatin and methyl-β-cyclodextrin exhibited no effect on HPV 16 pseudovirion infectivity. These concentrations have been reported to inhibit the infection by BPV-1, HPV 33 and EBV [10,11,21,26]. We next performed identical experiments with HPV 31 pseudoviruses and the results are shown in Figure 5. As we observed with HPV 16 pseudoviruses, increasing concentrations of chlorpromazine decreased HPV 31 pseudovirion entry in a dose-dependent manner into either Cos-7 or 293 TT cells. The concentrations of inhibitors used in Figure 5 had no effect on cell viability but higher concentrations of chlorpromazine were observed to be toxic to the Cos-7 and 293 TT cells (data not shown). As shown in Figure 5, no decrease on HPV 31 pseudovirion infectivity was observed as nystatin or methyl-β-cyclodextrin levels were increased. Interestingly, at intermediate concentrations of nystatin or methyl-β-cyclodextrin, we observed a reproducible increase in infectivity over untreated cells. In addition we observed that treatment with the drugs was reversible, as infections in the presence of chlorpromazine for 2–6 hours followed by a wash with fresh media and incubation for a total of 72 hours had negligible effect on infectivity (data not shown). Our results suggest a role for clathrin-mediated entry of HPV 16 and 31 pseudovirions in the entry of human and monkey cells.

Bottom Line: Similar to studies in HPV 16, expression of wild type HPV 31 L1 and L2 from heterologous promoters resulted in very low levels of synthesis.In contrast, modification of the codons in the capsid genes to ones more commonly used in cellular genes resulted in high-level synthesis.This suggests that high-risk HPV types may enter cells through common mechanisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. phindm@lsuhsc.edu

ABSTRACT
Human papillomaviruses (HPV) infect stratified epithelia and restrict expression of late capsid genes to highly differentiated cells. In order to begin to understand the processes regulating HPV 31 infection we examined the synthesis of the HPV 31 capsid proteins, L1 and L2, using heterologous expression systems. Similar to studies in HPV 16, expression of wild type HPV 31 L1 and L2 from heterologous promoters resulted in very low levels of synthesis. In contrast, modification of the codons in the capsid genes to ones more commonly used in cellular genes resulted in high-level synthesis. Through the use of chimeric proteins that fused fragments of wild type L1 to Green Fluorescent Protein (GFP) coding sequences, a short region was identified that was sufficient to inhibit high level synthesis and similar elements were detected in L2. One element was localized to the 3' end of the L1 gene while a series of elements were localized at the 3' end of the L2 coding sequences. These observations are most consistent with negative RNA regulatory elements controlling the levels of L1 and L2 synthesis that are distinct from those identified in HPV 16. Expression vectors for the codon modified HPV 31 capsid proteins were then transfected together with GFP reporter plasmids to generate HPV 31 pseudoviruses. Infection of cells with HPV 31 pseudoviruses in the presence of the inhibitors, chlorpromazine, nystatin or methyl-beta-cyclodextrin, demonstrated that HPV 31, like HPV 16, enters human and monkey cells through a clathrin-mediated pathway rather than through caveolae as previously reported. This suggests that high-risk HPV types may enter cells through common mechanisms.

Show MeSH
Related in: MedlinePlus