The Regulatory Element in the 3′-Untranslated Region of Human Papillomavirus 16 Inhibits Expression by Binding CUG-binding Protein 1

Abstract

The 3-untranslated regions (UTRs) of human papillomavirus 16 (HPV16) and bovine papillomavirus 1 (BPV1) contain a negative regulatory element (NRE) that inhibits viral late gene expression. The BPV1 NRE consists of a single 9-nucleotide (nt) U1 small nuclear ribonucleoprotein (snRNP) base pairing site (herein called a U1 binding site) that via U1 snRNP binding leads to inhibition of the late poly(A) site. The 79-nt HPV16 NRE is far more complicated, consisting of 4 overlapping very weak U1 binding sites followed by a poorly understood GU-rich element (GRE). We undertook a molecular dissection of the HPV16 GRE and identify via UV cross-linking, RNA affinity chromatography, and mass spectrometry that is bound by the CUG-binding protein 1 (CUGBP1). Reporter assays coupled with knocking down CUGBP1 levels by small interfering RNA and Dox-regulated shRNA, demonstrate CUGBP1 is inhibitory in vivo. CUGBP1 is the first GRE-binding protein to have RNA interfering knockdown evidence in support of its role in vivo. Several fine-scale GRE mutations that inactivate GRE activity in vivo and GRE binding to CUGBP1 in vitro are identified. The CUGBP1⅐GRE complex has no activity on its own but specifically synergizes with weak U1 binding sites to inhibit expression in vivo. No synergy is seen if the U1 binding sites are made weaker by a 1-nt down-mutation or made stronger by a 1-nt up-mutation, underscoring that the GRE operates only on weak sites. Interestingly, inhibition occurs at multiple levels, in particular at the level of poly(A) site activity, nuclear-cytoplasmic export, and translation of the mRNA. Implications for understanding the HPV16 life cycle are discussed.The human epidermis consists of layers of keratinocytes that broadly speaking represent separate stages of differentiation (1). The basal layer of cells are the rapidly dividing progeny of epithelial stem cells that, as they transit upwards, slow down in cell growth and division, and flatten out to eventually become terminally differentiated keratinocytes at the stratum corneum. The life cycle of papillomaviruses, small ϳ8000-bp DNA viruses, is intimately connected to the differentiation program of keratinocytes (2) such that they can readily infect almost any type of mammalian cultured cell but are unable to complete the viral life cycle. Human papillomaviruses (HPVs) 2 infect squamous epithelia in the basal layer giving rise to papillomas that can lead to benign or malignant lesions (3). Of the more than 100 types of HPVs that are sequenced, only a handful account for nearly all cervical carcinomas and intraepithelial neoplasias, with HPV type 16 (HPV16) causing about 60% of the cases, making it the most clinically significant of the HPVs and one of the most studied (4, 5). In addition to HPV16, HPV1 that causes non-malignant lesions and bovine papillomavirus 1 (BPV1) have also been intensively studied and serve as prototypes for understanding HPVs (6).The genomic structure of papillomaviruses shares many common features. For example, all of the viral open re...

Human Papillomavirus Type 16 E2 Protein Transcriptionally Activates the Promoter of a Key Cellular Splicing Factor, SF2/ASF

“…cis-Acting RNA regulatory motifs have been identified in the HPV16 genome that may regulate capsid protein expression at one or more posttranscriptional levels (10,11,12,29,45,50,55,65). The elements are proposed to act via interactions with cellular RNA splicing factors, including U1 snRNP, hnRNP A1, SF2/ASF, PTB hnRNP C1/C2, and CUG-BP1 (9,10,12,19,30,41,56,64). Such RNA-protein interactions are proposed to be essential in regulating virus late gene expression, for example by regulating alternative splicing and polyadenylation of virus late mRNAs (20).…”

Serine/arginine-rich protein 30c activates human papillomavirus type 16 L1 mRNA expression via a bimodal mechanism

“…The identification of host cellular factors binding to these regulatory sequences would help to resolve the mechanism of HPV-16 late gene suppression in undifferentiated cells, including cancer cells. The following factors have been shown to bind to the HPV-16 and/or HPV-31 late 39UTR: HuR, U2 small nuclear ribonucleoprotein auxiliary factor 65 kDa subunit (U2AF 65 ), cleavage stimulation factor-64 (CstF-64), alternative splicing factor/splicing factor 2 (ASF/SF2) and CUGbinding protein (Cumming et al, 2002;Goraczniak & Gunderson, 2008;Koffa et al, 2000;McPhillips et al, 2004), whilst heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2, HuR and poly(A)-binding protein (PABP) bind to the late 39UTR of HPV-1 (Sokolowski & Schwartz, 2001;Sokolowski et al, 1997Sokolowski et al, , 1999. hnRNP C1/ C2, polypyrimidine tract binding-protein (PTB), CstF-64 and human Fip-1 have been shown to bind to a U-rich region of the early 39UTR of HPV-16, a sequence that enhances recognition of the early poly(A) signal pAE .…”

CELF1, a Multifunctional Regulator of Posttranscriptional Networks

“…Subsequent searches for the RNA-binding specificities of CELF1 and CELF2 used systemic evolution of ligands exponential enrichment (SELEX), revealing that CELF1 and CELF2 both bound preferentially to GU-rich RNA sequences [60]. Binding by CELF1 to GU-rich sequences in vitro and in vivo was abrogated by mutation of G nucleotides to C [30], [61]. Takanashi et.…”