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HPV-18 E6 mutants reveal p53 modulation of viral DNA amplification in organotypic cultures

Eun-Young Kho1, Hsu-Kun Wang, N Sanjib Banerjee

  • 1Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.

Proceedings of the National Academy of Sciences of the United States of America
|April 11, 2013
PubMed

Insights

Human papillomavirus (HPV) E6 protein impacts viral DNA amplification by destabilizing p53. This study identifies critical E6 targets and reveals p53

Area of Science:

  • Virology
  • Molecular Biology
  • Oncology

Background:

  • Human papillomaviruses (HPVs) are oncogenic viruses that replicate in differentiated epithelial cells.
  • The HPV E7 protein promotes viral DNA amplification by inactivating tumor suppressors like p130.
  • The high-risk HPV E6 protein targets the p53 tumor suppressor, but its precise role in viral DNA amplification remains unclear.

Purpose of the Study:

  • To investigate the role of HPV-18 E6 protein in viral DNA amplification.
  • To identify critical host protein targets of E6 involved in viral replication.
  • To characterize the function of HPV-18 E6 mutants in keratinocyte cultures.

Main Methods:

  • Utilized Cre-loxP recombination to generate HPV-18 genomic plasmids in primary human keratinocytes.
  • Established organotypic epithelial cultures to model productive HPV-18 infection.
  • Generated and analyzed four distinct HPV-18 E6 mutations, including an E6 null mutant and three missense mutants.
  • Assessed p53 levels and viral DNA amplification efficiency in cells expressing wild-type (WT) E6 and E6 mutants.
  • Employed p53 siRNA to modulate p53 levels and rescue viral amplification.

Main Results:

  • An E6 null mutant exhibited high p53 levels and significantly impaired viral DNA amplification.
  • Partial restoration of amplification was observed with p53 siRNA or expression of WT E6.
  • Three missense E6 mutants, which poorly destabilized p53, showed limited complementation of the E6 null mutant.
  • Unexpectedly, two missense E6 mutants demonstrated some viral amplification in cis, but only in p53-undetectable cells.
  • High levels of viral DNA amplification were essential for late protein expression.

Conclusions:

  • p53 and other host proteins play crucial roles in regulating HPV DNA amplification.
  • E6's interaction with p53 is critical for efficient viral DNA amplification.
  • Overexpression of E6 may have an inhibitory effect on viral amplification.
  • Identified novel viral RNAs, including bicistronic transcripts, suggesting complex viral gene expression patterns.
Keywords:
HPV transcriptshuman papillomavirus DNA amplificationtrans complementation

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