The recently discovered Canis familiaris papillomavirus type 2 (CfPV2) provides a unique opportunity to study papillomavirus (PV) gene functions in vitro and in vivo. Unlike the previously characterized canine oral PV (COPV), CfPV2 contains an E5 ORF and is associated with progression to squamous cell carcinoma. In the current study, we have expressed and characterized the CfPV2-encoded E5 protein: a small, hydrophobic, 41 amino acid polypeptide. Similar to the E5 protein from high-risk human PV type 16 (HPV-16), we demonstrate that the CfPV2 E5 protein is localized in the endoplasmic reticulum. Differential-detergent permeabilization studies indicate that its N- and C-termini are exposed at the cytoplasmic ER surface, suggesting that this hydrophobic polypeptide most likely forms a short intramembrane loop. CfPV2 E5 seems to be unique from other PV E5 proteins, as it is neither capable of in vitro cellular transformation, nor is it located in detergent-resistant membranes. Although, our attempts to develop a method for analyzing in vivo transformation were unsuccessful, they did lead to the development of a novel whole-genome pseudovirus preparation that can be utilized to elucidate many aspects of the PV life cycle, including the role of E5. Similar to the HPV-16 E5 protein, expression of the canine E5 protein slows keratinocyte proliferation; with a concomitant increase in G1 phase and decrease in the S phase of the cell cycle. We have employed a new real-time PCR method, based upon the splicing of XBP1 mRNA, to demonstrate that CfPV2 E5 expression induces ER stress, providing a plausible explanation for cell growth inhibition. Interestingly, the growth inhibition and ER stress induced by CfPV2 E5 are tempered significantly by co-expression of canine PV E6 and E7 genes, suggesting that differential expression of E6/E7 genes during keratinocyte differentiation might spatially modulate E5 activity. In addition, E5-induced ER stress may play an important role in the viral life cycle as well as in the progression of viral lesions toward malignancy.
|Advisor:||Schlegel, Richard, Cole, Michael|
|Commitee:||Casey, John, Fonzi, William, McBride, Alison|
|Department:||Microbiology & Immunology|
|School Location:||United States -- District of Columbia|
|Source:||DAI-B 70/05, Dissertation Abstracts International|
|Keywords:||E5, Papillomavirus, Unfolded protein resonse, Xbp1|
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