The HPV-16 E5 protein resides in membranes of the endoplasmic reticulum (ER) and modulates cell growth and viral replication. In order to help define its biological activities, we analyzed E5-induced changes in human keratinocyte gene expression. Our studies identified the downregulation of spliced XBP-1 transcripts, a key player in the ER stress response, as a biochemical marker of E5 expression. IRE1 alpha, the endoribonuclease responsible for XBP-1 RNA splicing, was also downregulated. Furthermore, cDNA microarray analysis revealed the repression of COX-2, another member of the ER stress pathway. In contrast, these genes were not altered either by the low-risk HPV-6b E5, or a C-terminal HPV-16 E5 mutant, in which the histidine and alanine residues (conserved in high-risk HPVs) were replaced with tyrosine and isoleucine (conserved in low-risk HPVs). HPV-16 E5 was also able to lower COX-2 mRNA levels in cells co-expressing E6/E7, suggesting that it might exert similar activity during viral replication. Interestingly, the E6/E7 genes were independently able to lower COX-2 transcripts compared to vector cells, indicating that multiple pathways of COX-2 repression exist. COX-2 downregulation by E5 could be overcome by thapsigargin or tunicamycin treatments, which initiate ER stress via calcium fluxes and abnormal protein glycosylation respectively, making it unlikely that E5 specifically tempers these pathways. Overall, our data indicate that E5 represses the cellular ER stress response and suggest a potential role for E5 during productive HPV infection. (C) 2010 Elsevier Inc. All rights reserved.