Identification of an hnRNP A1-Dependent Splicing Silencer in the Human Papillomavirus Type 16 L1 Coding Region That Prevents Premature Expression of the Late L1 Gene

Abstract

We have previously identified cis-acting RNA sequences in the human papillomavirus type 16 (HPV-16) L1 coding region which inhibit expression of L1 from eukaryotic expression plasmids. Here we have determined the function of one of these RNA elements, and we provide evidence that this RNA element is a splicing silencer which suppresses the use of the 3 splice site located immediately upstream of the L1 AUG. We also show that this splice site is inefficiently utilized as a result of a suboptimal polypyrimidine tract. Introduction of point mutations in the L1 coding region that altered the RNA sequence without affecting the L1 protein sequence resulted in the inactivation of the splicing silencer and induced splicing to the L1 3 splice site. These mutations also prevented the interaction of the RNA silencer with a 35-kDa cellular protein identified here as hnRNP A1. The splicing silencer in L1 inhibits splicing in vitro, and splicing can be restored by the addition of RNAs containing an hnRNP A1 binding site to the reaction, demonstrating that hnRNP A1 inhibits splicing of the late HPV-16 mRNAs through the splicing silencer sequence. While we show that one role of the splicing silencer is to determine the ratio between partially spliced L2/L1 mRNAs and spliced L1 mRNAs, we also demonstrate that it inhibits splicing from the major 5 splice site in the early region to the L1 3 splice site, thereby playing an essential role in preventing late gene expression at an early stage of the viral life cycle. We speculate that the activity of the splicing silencer and possibly the concentration of hnRNP A1 in the HPV-16-infected cell determines the ability of the virus to establish a persistent infection which remains undetected by the host immune surveillance.Human papillomaviruses (HPVs) are a group of nonenveloped, double-stranded DNA tumor viruses with tropism for epithelial cells (21, 62). HPV type 16 (HPV-16) is one of the most common sexually transmitted HPV types and is also the HPV type most frequently detected in cervical cancers (43). While many of the early HPV gene products are present in all layers of the squamous epithelium, expression of the late mRNAs encoding the L1 and L2 capsid proteins is restricted to the terminally differentiated cells in the upper layers of the epithelium (21). For this reason, it has been difficult to grow HPVs in vitro, and only organotypic raft cultures in which infected or transfected keratinocytes are grown in the airliquid interface to induce terminal cell differentiation produce HPV virions (27). Expression of the late genes is regulated at the levels of transcription and RNA processing (1,38,39,42,55). Members of our group and others have previously shown that the late HPV-16 mRNAs contain cis-acting regulatory RNA elements (23, 24, 51), both in the 3Ј untranslated region (UTR) and in the L1 and L2 coding regions (9,33,46,51). It is likely that these elements are involved in the regulation of HPV late gene expression. Inhibitory RNA elements in the late 3Ј UTR have been identif...

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

“…pBEL encoded viral early and late genes after a human cytomegalovirus (CMV) immediate-early promoter (Fig. 1a), but primarily produced the early E4 mRNA upon transfection of HeLa cells, as described previously Somberg & Schwartz, 2010;Somberg et al, 2008Somberg et al, , 2009Zhao et al, 2004Zhao et al, , 2005. HPV-16 late L1 and L2 mRNAs were undetectable (Fig.…”

Development and validation of a novel reporter assay for human papillomavirus type 16 late gene expression

“…pBEL, pBELM, have been described previously (Zhao et al, 2004). In order to construct the reporter plasmids pBELCAT and pBELMCAT, PCR was first performed with oligonucleotides IRESs and IRESa (Table 1) (Fig.…”

“…Total RNA was then reverse transcribed at 42°C by using Superscript II and gene specific primers ( to PCR amplify cDNAs with 757s and E4a primers (Zhao et al, 2004), whereas an annealing temperature of 57°C was used with primers 757s and L1Ma primers . cDNA synthesized with oligonucleotide…”

“…In order to generate a simple and reliable bioassay to study HPV-16 late gene expression, two previously described plasmids named pBEL and pBELM (Zhao et al, 2004) were used (Fig. 1B).…”

“…HPV-16 late gene expression is also indirectly inhibited by RNA elements that stimulate early mRNA splicing and polyadenylation Terhune et al, 1999;Zhao et al, 2005). It was speculated that premature induction of HPV-16 late gene expression in the persistently In previous studies two subgenomic HPV-16 expression plasmids were described carrying the viral early and late genes, except E6 and E7, driven by the strong human cytomegalovirus (CMV) immediate early promoter, called pBEL and pBELM (Zhao et al, 2004) (Fig. 1B).…”