Heterogeneous nuclear ribonucleoprotein C proteins interact with the human papillomavirus type 16 (HPV16) early 3'-untranslated region and alleviate suppression of HPV16 late L1 mRNA splicing.

In order to identify cellular factors that regulate human papillomavirus type 16 (HPV16) gene expression, cervical cancer cells permissive for HPV16 late gene expression were identified and characterized. These cells either contained a novel spliced variant of the L1 mRNAs that bypassed the suppressed HPV16 late, 5'-splice site SD3632, produced elevated levels of RNA-binding proteins SRSF1 (ASF/SF2), SRSF9 (SRp30c) and HuR that are known to regulate HPV16 late gene expression, or were shown by a gene expression array analysis to overexpress the RALYL RNA-binding protein of the heterogeneous nuclear ribonucleoprotein C (hnRNP C)-family. Overexpression of RALYL or hnRNP C1 induced HPV16 late gene expression from HPV16 subgenomic plasmids and from episomal forms of the full-length HPV16 genome. This induction was dependent on the HPV16 early untranslated region. Binding of hnRNP C1 to the HPV16 early, untranslated region activated HPV16 late 5'-splice site SD3632 and resulted in production of HPV16 L1 mRNAs. Our results suggested that hnRNP C1 controls HPV16 late gene expression

Eight nucleotide substitutions inhibit splicing to HPV-16 3'-splice site SA3358 and reduce the efficiency by which HPV-16 increases the life span of primary human keratinocytes.

The most commonly used 3'-splice site on the human papillomavirus type 16 (HPV-16) genome named SA3358 is used to produce HPV-16 early mRNAs encoding E4, E5, E6 and E7, and late mRNAs encoding L1 and L2. We have previously shown that SA3358 is suboptimal and is totally dependent on a downstream splicing enhancer containingmultiple potential ASF/SF2 binding sites. Here weshow that only one of the predicted ASF/SF2 sites accounts for the majority of the enhancer activity. We demonstrate that single nucleotide substitutions in this predicted ASF/SF2 site impair enhancer function and that this correlates with less efficient binding to ASF/SF2 in vitro. We provide evidence that HPV-16 mRNAs that arespliced to SA3358 interact with ASF/SF2 in living cells. In addition,mutational inactivation of the ASF/SF2 site weakened the enhancer at SA3358 in episomal forms of the HPV-16 genome, indicating that the enhancer is active in the context of the full HPV-16 genome.This resulted in induction of HPV-16 late gene expression as a result of competition from late splice site SA5639. Furthermore, inactivation of the ASF/SF2 site of the SA3358 splicing enhancer reduced the ability of E6- and E7-encoding HPV-16 plasmids to increase the life span of primary keratinocytes in vitro, demonstrating arequirement for an intact splicing enhancer of SA3358 forefficient production of the E6 and E7 mRNAs. These results link the strength of the HPV-16 SA3358 splicing enhancer to expression of E6 and E7 and to the pathogenic properties of HPV-16

One of the predicted ASF/SF2 binding sites downstream of HPV-16 SA3358 accounts for the majority of the enhancer activity.

<p>(<b>A</b>). CAT protein levels produced in HeLa cells transfected with the indicated mutant pBELMCAT-derived plasmids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072776#pone.0072776-Orru1" target="_blank">[38]</a>. CAT was monitored as described in Materials and Methods. Mean values and standard deviations are shown. Note the logarithmic scale. (<b>B</b>) RT-PCR with primers 757s and L1A on cDNA of cytoplasmic RNA extracted from HeLa cells transfected with the indicated plasmids. L1 and L1i mRNAs are indicated. M, molecular weight marker; GAPDH, cDNA amplified as internal control. (<b>C, D</b>) CAT protein levels produced in HeLa cells transfected with the indicated mutant pBELMCAT-derived plasmids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072776#pone.0072776-Orru1" target="_blank">[38]</a>. CAT was monitored as described in Materials and Methods. Mean values and standard deviations are shown.</p

of the HPV 3′splice site that corresponds to HPV-16 SA3358.

*<p>pyrimidines upstream of the invariable AG of the 3′-splice site are underlined.</p

Nucleotide substitutions in ASF/SF2 binding site III in the splicing enhancer at SA3358 induced late gene expression from the full-length,episomalform of the HPV-16 genome.

<p>(<b>A</b>). Schematic representation of the HPV-16 genomic plasmids pHPV16ANSL and pHPV16MANSL. The early and late viral promoters p97 and p670 are indicated. Numbers indicate nucleotide positions of 5′- (filled arrow heads) and 3′-splice sites (open arrow heads) or the early and late poly (A) sites pAE and pAL, respectively. L1M represents a previously described mutant HPV-16 L1 sequence in which a number of nucleotide substitutions inactivate splicing silencer elements downstream of late 3′-splice site SA5639. IRES, the poliovirus internal ribosome entry site sequence; sLuc, secreted luciferase gene; LCR, long control region. Sequences below plasmid maps represent wild type HPV-16 ASF/SF2 site III (capitals, black) and nucleotide substitutions (lower case, red) in the various plasmids indicated to the left. (<b>B, C</b>). Cell culture medium ofhuman primary keratinocytes collected at day 5 posttransfection with the various indicated plasmids was subjected to secreted-luciferase assay as described in Materials and Methods. Transfections were performed in the presence of pCAGGS-nlscre. Mean values and standard deviations of sLuc activity in the cell culture med of triplicate transfections are shown.</p

Predicted ASF/SF2 sites that are mutated in the various HPV-16 reporter plasmids.

<p>Predicted ASF/SF2 sites that are mutated in the various HPV-16 reporter plasmids.</p