Virus Research 149 (2010) 217–223 Contents lists available at ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres The early noncoding region of human papillomavirus type 16 is regulated by cytoplasmic polyadenylation factors Jacob A. Glahder ∗ , Karen Kristiansen 1 , Marjorie Durand 2 , Jeppe Vinther 3 , Bodil Norrild Department of Cellular and Molecular Medicine, Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark article info Article history: Received 12 November 2009 Received in revised form 30 January 2010 Accepted 1 February 2010 Available online 8 February 2010 Keywords: Human papillomavirus Cytoplasmic polyadenylation Post-transcriptional abstract All human papillomavirus type 16 (HPV-16) early mRNAs are polyadenylated at the poly(A) signal within the early 3 ′ untranslated region (3 ′ UTR). The 3 ′ end of the early E5 open reading frame and the 3 ′ UTR of HPV-16 is very AU-rich, with five regions similar to cytoplasmic polyadenylation elements (CPEs). We show here that a fragment of the early 3 ′ end comprising four of the five CPE-like regions when inserted downstream of a reporter gene confers regulation of the gene expression. A key protein involved in cytoplasmic polyadenylation is CPEB. We show that the human CPEB1 can repress the activity of the reporter construct containing the HPV-16 early sequences. This repression can be counteracted by a human cytoplasmic poly(A) polymerase, hGLD-2 fused to CPEB1. The hGLD-2/CPEB1 fusion protein facilitates furthermore poly(A) elongation of early HPV transcripts. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Human papillomaviruses (HPV) are all strictly epitheliotropic, infecting only cutaneous or mucosal epithelia (zur Hausen, 1996; McGlennen, 2000). The HPV types infecting mucosal epithelium can be subdivided into low- and high-risk types depending on their malignant potential, where the high-risk HPVs, of which HPV type 16 (HPV-16) is the most common in the western world, are associated with invasive carcinomas of the genital region, espe- cially the cervix uteri (de Villiers, 1994; zur Hausen, 1999). Their genomes consist of eight open reading frames (ORFs), divided into an early and a late region. A small noncoding region (SNR) con- taining the early poly(A) signal (pAE) separates the early and late regions (Maki et al., 1996). The expression of early proteins is very tightly regulated, both transcriptionally (Norrild et al., 2007) and post-transcriptionally, which is the focus of the present study. Viral cis-acting elements that affect HPV gene expression have pre- viously been found within both coding and noncoding regions. Within the HPV-16 L1 and L2 coding regions, there has been iden- tified negative regulatory elements (Sokolowski et al., 1998; Tan et al., 1995a). A region in the 5 ′ end of the L2 ORF influenced the sta- ∗ Corresponding author. E-mail address: jglahder@hotmail.com (J.A. Glahder). 1 Current affiliation: BioCentrum-DTU, Technical University of Denmark, Building 208, DK-2800 Lyngby, Denmark. 2 Current affiliation: Magistère de Biotechnologies, Université de Paris-Sud, Paris, France. 3 Current affiliation: Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark. bility of L2 containing mRNAs increasing degradation (Sokolowski et al., 1998). This region was furthermore shown to enhance the utilization of the pAE (Öberg et al., 2005). A negative regulatory ele- ment (NRE) was furthermore found in the 3 ′ end of HPV-16 L1 gene extending into the late 3 ′ UTR. This element was shown to decrease stability of mRNAs, thereby downregulating late protein expres- sion in undifferentiated cell lines (Kennedy et al., 1990; Kennedy et al., 1991). The NRE consists of four 5 ′ splice sites bound by U1 snRNP proteins, possibly reducing 3 ′ end processing (Cumming et al., 2003). Another negative acting element was found to be situated in the 5 ′ end of the L1 gene, downregulating late gene expression independently of the inhibitory NRE sequence previously identified (Collier et al., 2002). In addition, a splicing enhancer was found in the E4 ORF important for splicing at nt. 3358 as well as polyadeny- lation of early mRNAs (Rush et al., 2005). In the cutaneous HPV-1 an AU-rich inhibitory RNA-element (termed h1ARE) containing AUUUA and UUUUU motifs was identified in the late 3 ′ UTR (Tan and Schwartz, 1995b). This element was bound by both nuclear and cytoplasmic cellular proteins, downregulating mRNA levels as well as inhibiting translation (Sokolowski et al., 1997, 1999; Wiklund et al., 2002; Zhao et al., 1996). The early 3 ′ end including the early 3 ′ UTR (3 ′ eUTR) of HPV-16 from nt. 4005-4213 were previously shown to have a de-stabilizing effect on the human -globin mRNA (Jeon and Lambert, 1995), although this is questioned by Vinther et al. (2005) not seeing a decrease in the half-life of a GUS reporter with the HPV-16 sequence from nt. 4063-4214 inserted downstream. The HPV-16 3 ′ eUTR also contains an enhancing upstream sequence element (USE) of 57 nucleotides from nt. 4155-4212 (just upstream of the pAE) (Zhao et al., 2005). The sequence interacted with hFip1 (a component of 0168-1702/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2010.02.001