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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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Western analysis of HPV 31 L2. Cos-7 cells were transfected with GFP tagged L2 in pcDNA 3.1(-) expression vectors. After 72 hours, lysates were harvested and analyzed by Western blot with an antibody to GFP. (Panel A). Lane A: wild type HPV 31 L2. Lane B: wild type HPV 31 L2 N-terminus. Lane C: wild type HPV 31 L2 C-terminus. Lane D: codon optimized HPV 31 L2. (Panel B). Western analysis of transfections of GFP in frame fusions to 198 base pair fragments of the C-terminal domain of wild type L2. Each fragment overlapped with the adjacent fragment by 99 base pairs. Cell extracts were examined by Western analysis using a GFP antibody. Lane A: fragment A, GFP fusion to nucleotides 4771–4969. Lane B: fragment Bnucleotides 4870–5068. Lane C: fragment C, nucleotides4969–5167. Lane D: fragment D, nucleotides5068–5266. Lane E: fragment E, nucleotides5167–5365. Lane F: fragment F nucleotides 5266–5464. Lane G: fragment G, nucleotides5365–5568. (Panel C). RT-PCR analysis of RNAs isolated from cells transfected with plasmids shown in panel B. Primers to common GFP sequences were used in this analysis. (Panel D) Lane A: mock transfected cells. Lane B: complete wild type HPV 31 L2 fused to GFP nucleotides 4171–5568. Lane C: N-terminal domain wild type L2 (nucleotides 4171–4870) fusion to GFP. Lane D: C-terminal domain wild type L2 (nucleotides 4870–5568) fused to GFP. Lane E: N-terminal domain wild type L2 (nucleotides 4171–4969) fused to GFP. Lane F: N-terminal domain wild type L2 (nucleotides 4171–5068) fused to GFP. Lane G: N-terminal domain wild type L2 (nucleotides 4171–5166) fused to GFP. Lane H: N-terminal domain wild type L2 (nucleotides 4171–5265). Lane I: N-terminal domain wild type L2 (nucleotides 4171–5465). Cartoon identifies fragments of L2 fused to GFP examined in lanesE – I. Nucleotide numbers are those in HPV 31 genome.
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Figure 2: Western analysis of HPV 31 L2. Cos-7 cells were transfected with GFP tagged L2 in pcDNA 3.1(-) expression vectors. After 72 hours, lysates were harvested and analyzed by Western blot with an antibody to GFP. (Panel A). Lane A: wild type HPV 31 L2. Lane B: wild type HPV 31 L2 N-terminus. Lane C: wild type HPV 31 L2 C-terminus. Lane D: codon optimized HPV 31 L2. (Panel B). Western analysis of transfections of GFP in frame fusions to 198 base pair fragments of the C-terminal domain of wild type L2. Each fragment overlapped with the adjacent fragment by 99 base pairs. Cell extracts were examined by Western analysis using a GFP antibody. Lane A: fragment A, GFP fusion to nucleotides 4771–4969. Lane B: fragment Bnucleotides 4870–5068. Lane C: fragment C, nucleotides4969–5167. Lane D: fragment D, nucleotides5068–5266. Lane E: fragment E, nucleotides5167–5365. Lane F: fragment F nucleotides 5266–5464. Lane G: fragment G, nucleotides5365–5568. (Panel C). RT-PCR analysis of RNAs isolated from cells transfected with plasmids shown in panel B. Primers to common GFP sequences were used in this analysis. (Panel D) Lane A: mock transfected cells. Lane B: complete wild type HPV 31 L2 fused to GFP nucleotides 4171–5568. Lane C: N-terminal domain wild type L2 (nucleotides 4171–4870) fusion to GFP. Lane D: C-terminal domain wild type L2 (nucleotides 4870–5568) fused to GFP. Lane E: N-terminal domain wild type L2 (nucleotides 4171–4969) fused to GFP. Lane F: N-terminal domain wild type L2 (nucleotides 4171–5068) fused to GFP. Lane G: N-terminal domain wild type L2 (nucleotides 4171–5166) fused to GFP. Lane H: N-terminal domain wild type L2 (nucleotides 4171–5265). Lane I: N-terminal domain wild type L2 (nucleotides 4171–5465). Cartoon identifies fragments of L2 fused to GFP examined in lanesE – I. Nucleotide numbers are those in HPV 31 genome.

Mentions: We next examined if these changes in codon usage resulted in increased HPV 31 L1 and L2 protein synthesis. Codon optimized HPV 31 L1 and L2 were cloned into SV40 based expression vectors and transfected into Cos-7 cells along with wild type expression vectors. As shown in Figure 1, alteration of the coding sequence resulted in high level of expression of codon optimized L1. Since our L2 antibody had a high background, we tagged wild type and codon optimized L2 with a GFP tag and screened L2-GFP protein levels using an antibody to GFP. Similar to our observations with HPV 31 L1, Cos-7 cells transfected with codon optimized L2 tagged to GFP expressed high levels of protein in comparison to tagged wild type L2 which were not detectable (Figure 2).


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

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

Western analysis of HPV 31 L2. Cos-7 cells were transfected with GFP tagged L2 in pcDNA 3.1(-) expression vectors. After 72 hours, lysates were harvested and analyzed by Western blot with an antibody to GFP. (Panel A). Lane A: wild type HPV 31 L2. Lane B: wild type HPV 31 L2 N-terminus. Lane C: wild type HPV 31 L2 C-terminus. Lane D: codon optimized HPV 31 L2. (Panel B). Western analysis of transfections of GFP in frame fusions to 198 base pair fragments of the C-terminal domain of wild type L2. Each fragment overlapped with the adjacent fragment by 99 base pairs. Cell extracts were examined by Western analysis using a GFP antibody. Lane A: fragment A, GFP fusion to nucleotides 4771–4969. Lane B: fragment Bnucleotides 4870–5068. Lane C: fragment C, nucleotides4969–5167. Lane D: fragment D, nucleotides5068–5266. Lane E: fragment E, nucleotides5167–5365. Lane F: fragment F nucleotides 5266–5464. Lane G: fragment G, nucleotides5365–5568. (Panel C). RT-PCR analysis of RNAs isolated from cells transfected with plasmids shown in panel B. Primers to common GFP sequences were used in this analysis. (Panel D) Lane A: mock transfected cells. Lane B: complete wild type HPV 31 L2 fused to GFP nucleotides 4171–5568. Lane C: N-terminal domain wild type L2 (nucleotides 4171–4870) fusion to GFP. Lane D: C-terminal domain wild type L2 (nucleotides 4870–5568) fused to GFP. Lane E: N-terminal domain wild type L2 (nucleotides 4171–4969) fused to GFP. Lane F: N-terminal domain wild type L2 (nucleotides 4171–5068) fused to GFP. Lane G: N-terminal domain wild type L2 (nucleotides 4171–5166) fused to GFP. Lane H: N-terminal domain wild type L2 (nucleotides 4171–5265). Lane I: N-terminal domain wild type L2 (nucleotides 4171–5465). Cartoon identifies fragments of L2 fused to GFP examined in lanesE – I. Nucleotide numbers are those in HPV 31 genome.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Western analysis of HPV 31 L2. Cos-7 cells were transfected with GFP tagged L2 in pcDNA 3.1(-) expression vectors. After 72 hours, lysates were harvested and analyzed by Western blot with an antibody to GFP. (Panel A). Lane A: wild type HPV 31 L2. Lane B: wild type HPV 31 L2 N-terminus. Lane C: wild type HPV 31 L2 C-terminus. Lane D: codon optimized HPV 31 L2. (Panel B). Western analysis of transfections of GFP in frame fusions to 198 base pair fragments of the C-terminal domain of wild type L2. Each fragment overlapped with the adjacent fragment by 99 base pairs. Cell extracts were examined by Western analysis using a GFP antibody. Lane A: fragment A, GFP fusion to nucleotides 4771–4969. Lane B: fragment Bnucleotides 4870–5068. Lane C: fragment C, nucleotides4969–5167. Lane D: fragment D, nucleotides5068–5266. Lane E: fragment E, nucleotides5167–5365. Lane F: fragment F nucleotides 5266–5464. Lane G: fragment G, nucleotides5365–5568. (Panel C). RT-PCR analysis of RNAs isolated from cells transfected with plasmids shown in panel B. Primers to common GFP sequences were used in this analysis. (Panel D) Lane A: mock transfected cells. Lane B: complete wild type HPV 31 L2 fused to GFP nucleotides 4171–5568. Lane C: N-terminal domain wild type L2 (nucleotides 4171–4870) fusion to GFP. Lane D: C-terminal domain wild type L2 (nucleotides 4870–5568) fused to GFP. Lane E: N-terminal domain wild type L2 (nucleotides 4171–4969) fused to GFP. Lane F: N-terminal domain wild type L2 (nucleotides 4171–5068) fused to GFP. Lane G: N-terminal domain wild type L2 (nucleotides 4171–5166) fused to GFP. Lane H: N-terminal domain wild type L2 (nucleotides 4171–5265). Lane I: N-terminal domain wild type L2 (nucleotides 4171–5465). Cartoon identifies fragments of L2 fused to GFP examined in lanesE – I. Nucleotide numbers are those in HPV 31 genome.
Mentions: We next examined if these changes in codon usage resulted in increased HPV 31 L1 and L2 protein synthesis. Codon optimized HPV 31 L1 and L2 were cloned into SV40 based expression vectors and transfected into Cos-7 cells along with wild type expression vectors. As shown in Figure 1, alteration of the coding sequence resulted in high level of expression of codon optimized L1. Since our L2 antibody had a high background, we tagged wild type and codon optimized L2 with a GFP tag and screened L2-GFP protein levels using an antibody to GFP. Similar to our observations with HPV 31 L1, Cos-7 cells transfected with codon optimized L2 tagged to GFP expressed high levels of protein in comparison to tagged wild type L2 which were not detectable (Figure 2).

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