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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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Infection of Cos-7 and 293 TT cells by HPV 31 and HPV 16 pseudovirions. (A). HPV 31 pseudovirions were used to infect Cos-7 cells (left panel) or 293 TT cells (right panel). (B). HPV 16 pseudovirions were used to infect Cos-7 cells or 293 TT cells (right panel). After 24 hours, the media was changed. The infection was allowed to proceed for a total of 72 hours where maximal GFP expression occurred. The cells were fixed and stained with DAPI. Shown is a representative field of infected cells detected by GFP fluorescence.
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Figure 3: Infection of Cos-7 and 293 TT cells by HPV 31 and HPV 16 pseudovirions. (A). HPV 31 pseudovirions were used to infect Cos-7 cells (left panel) or 293 TT cells (right panel). (B). HPV 16 pseudovirions were used to infect Cos-7 cells or 293 TT cells (right panel). After 24 hours, the media was changed. The infection was allowed to proceed for a total of 72 hours where maximal GFP expression occurred. The cells were fixed and stained with DAPI. Shown is a representative field of infected cells detected by GFP fluorescence.

Mentions: We next used our HPV 31 pseudovirions to examine the parameters that influence entry. HPV 31 pseudovirion preparations were added to sub-confluent, monolayer cultures of Cos-7 or 293TT cells and incubated for various periods of time. The cells were then fixed and analyzed by fluorescence microscopy. We observed initial fluorescence at approximately 36 hours post infection that peaked at 72 hours. Figure 3 shows representative GFP expression in Cos-7 and 293 TT cells that are positive for HPV 31 pseudovirion infection at 72 hours post-infection. GFP expression occurs only if the genome has been transported to the nucleus and the reporter plasmid has been transcribed [25]. Production of HPV 31 pseudovirions and infectivity was found to be significantly higher in 293 TT cells than Cos-7 cells.


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

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

Infection of Cos-7 and 293 TT cells by HPV 31 and HPV 16 pseudovirions. (A). HPV 31 pseudovirions were used to infect Cos-7 cells (left panel) or 293 TT cells (right panel). (B). HPV 16 pseudovirions were used to infect Cos-7 cells or 293 TT cells (right panel). After 24 hours, the media was changed. The infection was allowed to proceed for a total of 72 hours where maximal GFP expression occurred. The cells were fixed and stained with DAPI. Shown is a representative field of infected cells detected by GFP fluorescence.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Infection of Cos-7 and 293 TT cells by HPV 31 and HPV 16 pseudovirions. (A). HPV 31 pseudovirions were used to infect Cos-7 cells (left panel) or 293 TT cells (right panel). (B). HPV 16 pseudovirions were used to infect Cos-7 cells or 293 TT cells (right panel). After 24 hours, the media was changed. The infection was allowed to proceed for a total of 72 hours where maximal GFP expression occurred. The cells were fixed and stained with DAPI. Shown is a representative field of infected cells detected by GFP fluorescence.
Mentions: We next used our HPV 31 pseudovirions to examine the parameters that influence entry. HPV 31 pseudovirion preparations were added to sub-confluent, monolayer cultures of Cos-7 or 293TT cells and incubated for various periods of time. The cells were then fixed and analyzed by fluorescence microscopy. We observed initial fluorescence at approximately 36 hours post infection that peaked at 72 hours. Figure 3 shows representative GFP expression in Cos-7 and 293 TT cells that are positive for HPV 31 pseudovirion infection at 72 hours post-infection. GFP expression occurs only if the genome has been transported to the nucleus and the reporter plasmid has been transcribed [25]. Production of HPV 31 pseudovirions and infectivity was found to be significantly higher in 293 TT cells than Cos-7 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