Specific interaction between hnRNP H and HPV16 L1 proteins: Implications for late gene auto-regulation enabling rapid viral capsid protein production

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Abstract

Heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP H, are RNA-binding proteins that function as splicing factors and are involved in downstream gene regulation. hnRNP H, which binds to G triplet regions in RNA, has been shown to play an important role in regulating the staged expression of late proteins in viral systems. Here, we report that the specific association between hnRNP H and a late viral capsid protein, human papillomavirus (HPV) L1 protein, leads to the suppressed function of hnRNP H in the presence of the L1 protein. The direct interaction between the L1 protein and hnRNP H was demonstrated by complex formation in solution and intracellularly using a variety of biochemical and immunochemical methods, including peptide mapping, specific co-immunoprecipitation and confocal fluorescence microscopy. These results support a working hypothesis that a late viral protein HPV16 L1, which is down regulated by hnRNP H early in the viral life cycle may provide an auto-regulatory positive feedback loop that allows the rapid production of HPV capsid proteins through suppression of the function of hnRNP H at the late stage of the viral life cycle. In this positive feedback loop, the late viral gene products that were down regulated earlier themselves disable their suppressors, and this feedback mechanism could facilitate the rapid production of capsid proteins, allowing staged and efficient viral capsid assembly.

Highlights

► The RNA-binding hnRNP H regulates late viral gene expression. ► hnRNP H activity was inhibited by a late viral protein. ► Specific interaction between HPV L1 and hnRNP H was demonstrated. ► Co-localization of HPV L1 and hnRNP H inside cells was observed. ► Viral capsid protein production, enabling rapid capsid assembly, was implicated.

Introduction

hnRNP H is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family. hnRNP H has been shown to form heterodimers during the regulation of mRNA splicing [1], [2]. During this process, hnRNP H binds to intronic G triplets or G3: for example, the six “GGG” repeats in RNA as shown in Fig. 1A [3], [4]. hnRNP H enhances the binding of cleavage/polyadenylation specificity factor (CPSF) to RNA polyadenylation sites [5]. This binding event leads to further enhancement of pre-mRNA cleavage and polyadenylation [6], [7], [8]. As previously reported [3], [9], in mRNA, the polyadenylation site sequence procedes (i.e., is 5′ to) the GU-rich sequence, which includes several intronic G triplets. The binding of CPSF to the polyadenylation site failed to stimulate cleavage and polyadenylation [10], demonstrating that the binding of hnRNP H is required to initiate cleavage and polyadenylation [3], [9]. In several viruses, hnRNP H has been shown to play critical roles in the splicing regulation of several viral mRNAs, including the splicing of simian vacuolating virus at 40 pAL [11], human papillomavirus (HPV) at pAE [3], Rous sarcoma virus pre-mRNA [12] and HIV type 1 tat-specific exon 2 and tev-specific exon 6D [13], [14].

Over 100 serotypes of HPV exist, with more than 50 serotypes being human pathogens [15]. The most studied serotype, HPV16, is a high-risk serotype and the most common serotype that causes cervical cancer in women. In all HPV serotypes, there are two late viral proteins (L1 and L2) that form the viral capsid, with L1 being the predominant protein. The HPV16 L2 sequence encodes a polyadenylation element, which encompasses multiple GGG (or G3) motifs in the RNA. In addition, hnRNP H has been shown to interact with the G3-containing motifs, and it is speculated that hnRNP H regulates polyadenylation at the HPV16 early polyadenylation signal, which suppresses the expression of the late proteins during the early stage of the viral life cycle [3]. Whether this regulatory protein interacts with late gene products (once expressed) at the protein level, providing a feedback loop, remains unclear.

In this study, we assessed the possible association between the HPV L1 capsid protein and hnRNP H, the suppressor of the late gene expression. To ensure that the reporter module pPG was suitable for this purpose, the tuning of hnRNP H activity using RNAi and other means was demonstrated with the varying level of the resultant green fluorescent protein (GFP) gene or protein. Once validated, the impact of HPV16 L1 co-expression with pPG was studied, and this co-expression demonstrated effective suppression of hnRNP H activity in the presence of the L1 protein. Moreover, the direct association between HPV16 L1 and hnRNP H was demonstrated on a molecular level by various techniques, including peptide mapping after specific pull-down experiments, co-immunoprecipitation and intracellular co-localization.

Section snippets

Cell lines, proteins, and antibodies

HeLa cells (ATCC) were cultured with RPMI-1640 (GIBCO) containing 10% fetal calf serum at 37 °C with 5% CO2. Recombinant HPV16 L1 self assembles into virus-like particles upon over-expression in various host cells, including E. coli [16], bacuolovirus [17] and yeast [18], [19]. E. coli-expressed HPV16 L1 [20] was used for specific pull-down analysis. The HPV16 L1-specific mouse monoclonal antibodies (mAbs), denoted PB9 and PB11, were produced in-house using hybridoma technology. The purified IgG

Plasmid construction and validation

A plasmid with multiple G3 elements was constructed to evaluate the impact of various factors on the functioning of hnRNP H. The reporter plasmid, pPG (Fig. 1A), linking the reporter gene expression to hnRNP H function, was constructed to elucidate the mechanism of late gene expression. The synergistic effect of hnRNP H and cleavage/polyadenylation specificity factor (CPSF) enhanced RNA splicing and adenylation, leading to the premature termination of GFP reporter transcription, as shown in

Conclusion

Direct evidence of HPV16 L1: hnRNP H binding was obtained through a combination of biochemical, biophysical and immunochemical methods. The toolbox of methods that we used to probe interactions at different levels while studying a gene-regulating protein could be applied to other systems. Using HPV as a model system, the protein product of a gene that was regulated by a suppressor, hnRNP H, was shown to play a role in binding that suppressor and diminishing its function. This action, in turn,

Acknowledgments

We thank Dr. John T. Schiller (NCI/NIH) for providing HPV16 L1 eukaryotic expression plasmids (p16L1 h). We appreciate the helpful comments from Drs. Zhiqiang An (University of Texas Health Science Center at Houston) and Tong-ming Fu (Merck). This work was supported by the Chinese National Science Fund (81273327), Chinese Major New Drug R&D project (2012ZX09101-316), and International Cooperation Projects (2011DFG33050).

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    These authors contributed equally to this work.

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