Activity of the Human Papillomavirus Type 16 Late Negative Regulatory Element Is Partly due to Four Weak Consensus 5′ Splice Sites That Bind a U1 snRNP-Like Complex

Abstract

The human papillomavirus (HPV) life cycle is tightly linked to differentiation of the squamous epithelia that it infects. Capsid proteins, and hence mature virions, are produced in the outermost layer of differentiated cells. As late gene transcripts are produced in the lower layers, posttranscriptional mechanisms likely prevent capsid protein production in less differentiated cells. For HPV type 16 (HPV-16), a 79-nucleotide (nt) negative regulatory element (NRE) inhibits gene expression in basal epithelial cells. To identify key NRE sequences, we carried out transient transfection in basal epithelial cells with reporter constructs containing the HPV-16 late 3 untranslated region with deletions and mutations of the NRE. Reporter gene expression was increased over 40-fold by deletion of the entire element, 10-fold by deletion of the 5 portion of the NRE that contains four weak consensus 5 splice sites, and only 3-fold by deletion of the 3 GU-rich region. Both portions of the element appear to be necessary for full repression. Inactivating mutations in the 5 splice sites in the 5 NRE partially alleviated repression in the context of the 79-nt NRE but caused full derepression when assayed in a construct with the 3 NRE deleted. All four contribute to the inhibitory effect, though the second splice site is most inhibitory. Sm proteins, U1A and U1 snRNA, but not U1 70K, could be affinity purified with the wild-type NRE but not with the NRE containing mutations in the 5 splice sites, indicating that a U1 snRNP-like complex forms upon the element.Human papillomaviruses (HPVs) are small DNA viruses that specifically infect squamous epithelial cells, giving rise to warts or papillomas (16). They may be divided into mucosal or cutaneous types and also into high-or low-risk types, depending on the probability of the lesions that they cause becoming malignant. High-risk mucosal types, which include HPV type 16 (HPV-16), HPV-18, HPV-31, HPV-33, and HPV-45, may give rise to cervical intraepithelial neoplasias and cervical carcinomas. HPV-16 is the most clinically significant of these, being found in over 50% of such cervical lesions (36).The early genes of the 8-kb HPV-16 genome encode proteins involved in the regulation of DNA replication, episomal maintenance of the genome, and control of host cell division. The late region encodes the major and minor capsid proteins, L1 and L2. The noncoding region contains the late gene 3Ј untranslated region (late 3Ј UTR) at its 5Ј end and regulatory sequences controlling activity of the viral promoter toward its 3Ј end (Fig. 1A). The major promoter in HPV-16, P 97 , is constitutively active from early in the viral life cycle (25). A second promoter, P 670 , also becomes active in differentiated cells (12). The genome consists of a single transcription unit, and alternative splicing generates individual mRNAs (7,17,23). Transcripts may be polyadenylated at the early polyadenylation [poly(A)] site; alternatively one of the tandem late poly(A) sites downstream of the L1 coding region may be...

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

“…1B. The mutated HPV16 U1 binding sites are identical to a previously published mutation (21,27) that inactivates this element because it inactivates base pairing to U1 snRNP. In contrast there is no published fine-scale substitution mutant of the GRE.…”

“…In reporter plasmids where the 3Ј-UTR and sequences past the poly(A) site are derived from the corresponding HPV16 sequences, expression of the reporter is low. Deletion of the NRE but not other parts of the 3Ј-UTR resulted in a large increase in expression ranging from 40-to 100-fold depending on the transfection conditions and cell types used (18,21,26,27). As diagrammed in Fig.…”

“…U1 binding site) led to extensive mapping efforts to identify NREs in HPVs, including the 79-nt HPV16 NRE that spans from the last few nucleotides of the L1 coding region into the viral late 3Ј-UTR (Fig. 1B) (21,26,27). In reporter plasmids where the 3Ј-UTR and sequences past the poly(A) site are derived from the corresponding HPV16 sequences, expression of the reporter is low.…”

A bipartite U1 site represses U1A expression by synergizing with PIE to inhibit nuclear polyadenylation

“…This is the first example of an endogenous cellular gene having a functional U1 site. Up to now, only two types of U1 sites had been reported: U1 sites in papillomaviruses (Furth et al 1994;Cumming et al 2003) and artificial U1 sites used to silence expression of reporter and specific endogenous genes (Beckley et al 2001, Fortes et al 2003. Prior work from multiple labs showed that these U1 sites use the same mechanism, namely inhibition of pre-mRNA maturation by inhibition of nuclear polyadenylation resulting in lower mRNA levels.…”

“…To date, naturally existing U1 sites have only been reported in papillomaviruses (Furth et al 1994;Cumming et al 2003) with no functional ones being reported in cellular genes. Here, we use bioinformatics and a reporter gene and in vitro polyadenylation assays to identify and characterize a U1 site in the terminal exon of the human U1A gene.…”