A 57-nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hFip1, CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein

We have investigated the role of the human papillomavirus type 16 (HPV-16) early untranslated region (3' UTR) in HPV-16 gene expression. We found that deletion of the early 3' UTR reduced the utilization of the early polyadenylation signal and, as a consequence, resulted in read-through into the late region and production of late L1 and L2 mRNAs. Deletion of the U-rich 3' half of the early 3' UTR had a similar effect, demonstrating that the 57-nucleotide U-rich region acted as an enhancing upstream element on the early polyadenylation signal. In accordance with this, the newly identified hFip1 protein, which has been shown to enhance polyadenylation through U-rich upstream elements, interacted specifically with the HPV-16 upstream element. This upstream element also interacted specifically with CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 early polyadenylation signal. Mutational inactivation of the early polyadenylation signal also resulted in increased late mRNA production. However, the effect was reduced by the activation of upstream cryptic polyadenylation signals, demonstrating the presence of additional strong RNA elements downstream of the early polyadenylation signal that direct cleavage and polyadenylation to this region of the HPV-16 genome. In addition, we identified a 3' splice site at genomic position 742 in the early region with the potential to produce E1 and E4 mRNAs on which the E1 and E4 open reading frames are preceded only by the suboptimal E6 AUG. These mRNAs would therefore be more efficiently translated into E1 and E4 than previously described HPV-16 E1 and E4 mRNAs on which E1 and E4 are preceded by both E6 and E7 AUGs.     

The l2 minor capsid protein of human papillomavirus type 16 interacts with a network of nuclear import receptors

The L2 minor capsid proteins enter the nucleus twice during viral infection: in the initial phase after virion disassembly and in the productive phase when, together with the L1 major capsid proteins, they assemble the replicated viral DNA into virions. In this study we investigated the interactions between the L2 protein of high-risk human papillomavirus type 16 (HPV16) and nuclear import receptors. We discovered that HPV16 L2 interacts directly with both Kapbeta(2) and Kapbeta(3). Moreover, binding of Ran-GTP to either Kapbeta(2) or Kapbeta(3) inhibits its interaction with L2, suggesting that the Kapbeta/L2 complex is import competent. In addition, we found that L2 forms a complex with the Kapalpha(2)beta(1) heterodimer via interaction with the Kapalpha(2) adapter. In agreement with the binding data, nuclear import of L2 in digitonin-permeabilized cells could be mediated by either Kapalpha(2)beta(1) heterodimers, Kapbeta(2), or Kapbeta(3). Mapping studies revealed that HPV16 L2 contains two nuclear localization signals (NLSs), in the N terminus (nNLS) and C terminus (cNLS), that could mediate its nuclear import. Together the data suggest that HPV16 L2 interacts via its NLSs with a network of karyopherins and can enter the nucleus via several import pathways mediated by Kapalpha(2)beta(1) heterodimers, Kapbeta(2), and Kapbeta(3).     

Intranasal vaccination with recombinant adeno-associated virus type 5 against human papillomavirus type 16 L1

Adeno-associated viruses (AAV) have been developed and evaluated as recombinant vectors for gene therapy in many preclinical studies, as well as in clinical trials. However, only a few approaches have used recombinant AAV (rAAV) to deliver vaccine antigens. We generated an rAAV encoding the major capsid protein L1 (L1h) from the human papillomavirus type 16 (HPV16), aiming to develop a prophylactic vaccine against HPV16 infections, which are the major cause of cervical cancer in women worldwide. A single dose of rAAV5 L1h administered intranasally was sufficient to induce high titers of L1-specific serum antibodies, as well as mucosal antibodies in vaginal washes. Seroconversion was maintained for at least 1 year. In addition, a cellular immune response was still detectable 60 weeks after immunization. Furthermore, lyophilized rAAV5 L1h successfully evoked a systemic and mucosal immune response in mice. These data clearly show the efficacy of a single-dose intranasal immunization against HPV16 based on the recombinant rAAV5L1h vector without the need of an adjuvant.     

Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles.

The L1 genes of two human papillomavirus type 16 (HPV16) isolates derived from condylomata acuminata were used to express the L1 major capsid protein in insect cells via recombinant baculoviruses. Both L1 major capsid proteins self-assembled into virus-like particles (VLP) with high efficiency and could be purified in preparative amounts on density gradients. The yield of VLP was 3 orders of magnitude higher than what has been obtained previously, using L1 derived from the prototype HPV16. DNA sequence comparison identified a single nonconserved amino acid change to be responsible for the inefficient self-assembly of the prototype L1. VLP were also obtained by expressing L1 of HPV6, HPV11, and cottontail rabbit papillomavirus, indicating that L1 from a variety of papillomaviruses has the intrinsic capacity to self-assemble into VLP. Coexpression of HPV16 L1 plus L2 by using a baculovirus double-expression vector also resulted in efficient self-assembly of VLP, and the average particle yield increased about fourfold in comparison to when L1 only was expressed. Coimmunoprecipitation of L1 and L2 and cosedimentation of the two proteins in a sucrose gradient demonstrated that L2 was incorporated into the particles. The ability to generate preparative amounts of HPV16 L1 and L1-L2 VLP may have implications for the development of a serological assay to detect anti-HPV16 virion immune responses to conformational epitopes and for immunoprophylaxis against HPV16 infection.     

Immunogenic assessment of plant‐produced human papillomavirus type 16 L1/L2 chimaeras

Cervical cancer is caused by infection with human papillomaviruses (HPV) and is a global concern, particularly in developing countries, which have ~80% of the burden. HPV L1 virus‐like particle (VLP) type–restricted vaccines prevent new infections and associated disease. However, their high cost has limited their application, and cytological screening programmes are still required to detect malignant lesions associated with the nonvaccine types. Thus, there is an urgent need for cheap second‐generation HPV vaccines that protect against multiple types. The objective of this study was to express novel HPV‐16 L1‐based chimaeras, containing cross‐protective epitopes from the L2 minor capsid protein, in tobacco plants. These L1/L2 chimaeras contained epitope sequences derived from HPV‐16 L2 amino acid 108–120, 56–81 or 17–36 substituted into the C‐terminal helix 4 (h4) region of L1 from amino acid 414. All chimaeras were expressed in Nicotiana benthamiana via an Agrobacterium‐mediated transient system and targeted to chloroplasts. The chimaeras were highly expressed with yields of ~1.2 g/kg plant tissue; however, they assembled differently, indicating that the length and nature of the L2 epitope affect VLP assembly. The chimaera containing L2 amino acids 108–120 was the most successful candidate vaccine. It assembled into small VLPs and elicited anti‐L1 and anti‐L2 responses in mice, and antisera neutralized homologous HPV‐16 and heterologous HPV‐52 pseudovirions. The other chimaeras predominantly assembled into capsomeres and other aggregates and elicited weaker humoral immune responses, demonstrating the importance of VLP assembly for the immunogenicity of candidate vaccines.     

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

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.     

An intron element modulating 5' splice site selection in the hnRNP A1 pre-mRNA interacts with hnRNP A1.

The hnRNP A1 pre-mRNA is alternatively spliced to yield the A1 and A1b mRNAs, which encode proteins differing in their ability to modulate 5' splice site selection. Sequencing a genomic portion of the murine A1 gene revealed that the intron separating exon 7 and the alternative exon 7B is highly conserved between mouse and human. In vitro splicing assays indicate that a conserved element (CE1) from the central portion of the intron shifts selection toward the distal donor site when positioned in between the 5' splice sites of exon 7 and 7B. In vivo, the CE1 element promotes exon 7B skipping. A 17-nucleotide sequence within CE1 (CE1a) is sufficient to activate the distal 5' splice site. RNase T1 protection/immunoprecipitation assays indicate that hnRNP A1 binds to CE1a, which contains the sequence UAGAGU, a close match to the reported optimal A1 binding site, UAGGGU. Replacing CE1a by different oligonucleotides carrying the sequence UAGAGU or UAGGGU maintains the preference for the distal 5' splice site. In contrast, mutations in the AUGAGU sequence activate the proximal 5' splice site. In support of a direct role of the A1-CE1 interaction in 5'-splice-site selection, we observed that the amplitude of the shift correlates with the efficiency of A1 binding. Whereas addition of SR proteins abrogates the effect of CE1, the presence of CE1 does not modify U1 snRNP binding to competing 5' splice sites, as judged by oligonucleotide-targeted RNase H protection assays. Our results suggest that hnRNP A1 modulates splice site selection on its own pre-mRNA without changing the binding of U1 snRNP to competing 5' splice sites.     

Expression of human papillomavirus type 16 L1 protein in transgenic tobacco plants

To develop a plant expression system for the production of the human papillomavirus type 16 (HPV16) vaccine, we investigated whether the HPV16 L1 protein can be expressed in tobacco plants and whether it can be used as the cheapest form of edible vaccine. The HPV16 L1 coding sequence was amplified by PCR using specific primers from the plasmid pGEM-T-HPV16 containing the template sequence, and subcloned into the intermediate vector pUCmT and binary vector pBI121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector contained the constitutive Cauliflower mosaic virus (CaMV) 35S promoter and the neomycin phosphotransferase npt Ⅱ gene, which allowed the selection of transformed plants using kanamycin. The tobacco plants were transformed by cocultivating them, using the leaf disc method, with Agrobacterium tumefaciens LBA4404, which harbored the plant expression plasmid. The regenerated transgenic tobacco plants were selected using kanamycin, and confirmed by PCR. The results of the Southern blot assay also showed that the HPV16 L1 gene was integrated stably into the genome of the transformed tobacco plants. The Western blot analysis showed that the transformed tobacco leaves could express the HPV 16 L1 protein. Furthermore, it was demonstrated by ELISA assay that the expressed protein accounted for 0.034%-0.076% of the total soluble leaf protein, was able to form 55nm virus-like particles compatible with HPV virus-like particle (VLP), and induced mouse erythrocyte hemagglutination in vitro. The present results indicate that the HPV 16 L1 protein can be expressed in transgenic tobacco plants and the expressed protein possesses the natural features of the HPV16 L1 protein, implying that the HPV16 L1 transgenic plants can be potentially used as an edible vaccine.     

人乳头瘤病毒16型L1蛋白的克隆及表达

采用PCR技术从宫颈癌组织中扩增人乳头瘤病毒16型(Human papillomavirus type16,HPV16)L1全长基因片段,目的片段克隆到pMD18T载体后经酶切鉴定及测序确认。构建重组原核表达质粒pGEX4T1-L1,转化大肠杆菌E.coliBL21,IPTG诱导表达出以非可溶性蛋白形式存在的表达蛋白,该重组蛋白的表达量占菌体总蛋白的17%,免疫印迹检测表明,表达蛋白与宫颈癌病人血清出现特异性反应。成功构建了重组原核表达质粒pGEX4T1-L1,并且在原核细胞中得到表达,为进一步研究L1蛋白的免疫学活性及疫苗的研发奠定了基础。     

Stable high-level expression of truncated human papillomavirus type 16 L1 protein in Drosophila Schneider-2 cells

To improve the existing human papillomavirus type 16 (HPV16) virus-like particle (VLP) preparation, the Drosophila inducible/secreted expression system, a highly efficient, economical method, was used to produce HPV16 VLPs. Drosophila Schneider-2 cells were cotransfected with pMT/BiP/V5-His expression vector containing the target gene encoding HPV16L1 protein without nucleus localization sequence and the selection vector pCoHygro plasmids at the ratio of 4:1. The stabled hygromycin-resistant cell line was obtained 1 month later, and the protein expression was induced by copper sulfate. The molecular mass of expressed HPV16L1 protein was 66 kDa, as revealed by SDS-PAGE, and confirmed by Western blot analysis. The yield of HPV16L1 protein was 0.554 mg per 1×10⁷ cells. The characteristics of HPV16L1 protein were further analyzed by mouse erythrocyte hemagglutination assay, hemagglutination inhibition assay, and transmission electron microscopy. Results showed that the truncated protein was as biologically active as natural HPVL1 protein, inducing murine erythrocyte agglutination and VLP formation. These findings indicate that the Drosophila inducible/secreted expression system is promising as a convenient and economical method for the preparation of HPV16VLP vaccine.     

Effects of downstream processing on structural integrity and immunogenicity in the manufacture of papillomavirus type 16 L1 virus-like particles

There is increasing demand for virus-like particles (VLPs) as a platform for prophylactic vaccine production. However, little attention has been paid to how downstream processing affects the structure and immunogenicity of the VLPs. In this study, we compared three methods of purifying human papillomavirus type 16 (HPV16) VLPs, each including the same cation-exchange chromatography (CEC) step. Method T-1 uses both ammonium sulfate precipitation (ASP) and a step to remove precipitated contaminating proteins (SRPC) prior to CEC, while T-2 uses only the SRPC step prior to CEC and T-3 includes neither step. We compared the structural integrity and immunogenicity of the HPV16 VLPs resulting from these three methods. All three preparations were highly pure. However, the final yields of the VLPs obtained with T-2 were 1.5 and 2 fold higher than with T-1 and T-3, respectively. With respect to structural integrity, T-1 and T-2 HPV16 VLPs had smaller hydrodynamic diameters and higher reactivity towards monoclonal anti-HPV16 neutralizing antibodies than T-3 VLPs, indicating higher potentials of T-1 and T-2 VLPs for eliciting anti-HPV16 neutralizing antibodies. Moreover, it was confirmed that the T-1 and T-2 HPV16 VLPs elicit anti-HPV16 neutralizing antibodies more efficiently than T-3 HPV16 VLPs do in mice immunizations: the abilities for eliciting neutralizing antibodies were in the order T-2 VLP > T-1 VLP > T-3 VLP. We conclude that the process design for purifying HPV VLPs is a critical determinant of the quality of the final product.     

Interactions of noncanonical motifs with hnRNP A2 promote activity-dependent RNA transport in neurons

A key determinant of neuronal functionality and plasticity is the targeted delivery of select ribonucleic acids (RNAs) to synaptodendritic sites of protein synthesis. In this paper, we ask how dendritic RNA transport can be regulated in a manner that is informed by the cell’s activity status. We describe a molecular mechanism in which inducible interactions of noncanonical RNA motif structures with targeting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 form the basis for activity-dependent dendritic RNA targeting. High-affinity interactions between hnRNP A2 and conditional GA-type RNA targeting motifs are critically dependent on elevated Ca²⁺ levels in a narrow concentration range. Dendritic transport of messenger RNAs that carry such GA motifs is inducible by influx of Ca²⁺ through voltage-dependent calcium channels upon β-adrenergic receptor activation. The combined data establish a functional correspondence between Ca²⁺-dependent RNA–protein interactions and activity-inducible RNA transport in dendrites. They also indicate a role of genomic retroposition in the phylogenetic development of RNA targeting competence.     

The L1 major capsid protein of human papillomavirus type 11 recombinant virus-like particles interacts with heparin and cell-surface glycosaminoglycans on human keratinocytes

The L1 major capsid protein of human papillomavirus (HPV) type 11, a 55-kDa polypeptide, forms particulate structures resembling native virus with an average particle diameter of 50-60 nm when expressed in the yeast Saccharomyces cerevisiae. We show in this report that these virus-like particles (VLPs) interact with heparin and with cell-surface glycosaminoglycans (GAGs) resembling heparin on keratinocytes and Chinese hamster ovary cells. The binding of VLPs to heparin is shown to exhibit an affinity comparable to that of other identified heparin-binding proteins. Immobilized heparin chromatography and surface plasmon resonance were used to show that this interaction can be specifically inhibited by free heparin and dextran sulfate and that the effectiveness of the inhibitor is related to its molecular weight and charge density. Sequence comparison of nine human L1 types revealed a conserved region of the carboxyl terminus containing clustered basic amino acids that bear resemblance to proposed heparin-binding motifs in unrelated proteins. Specific enzymatic cleavage of this region eliminated binding to both immobilized heparin and human keratinocyte (HaCaT) cells. Removal of heparan sulfate GAGs on keratinocytes by treatment with heparinase or heparitinase resulted in an 80-90% reduction of VLP binding, whereas treatment of cells with laminin, a substrate for alpha6 integrin receptors, provided minimal inhibition. Cells treated with chlorate or substituted beta-D-xylosides, resulting in undersulfation or secretion of GAG chains, also showed a reduced affinity for VLPs. Similarly, binding of VLPs to a Chinese hamster ovary cell mutant deficient in GAG synthesis was shown to be only 10% that observed for wild type cells. This report establishes for the first time that the carboxyl-terminal portion of HPV L1 interacts with heparin, and that this region appears to be crucial for interaction with the cell surface.