Oncogenic potential diverge among human papillomavirus type 16 natural variants

We compared E6/E7 protein properties of three different HPV-16 variants: AA, E-P and E-350G. Primary human foreskin keratinocytes (PHFK) were transduced with HPV-16 E6 and E7 and evaluated for proliferation and ability to grow in soft agar. E-P infected keratinocytes presented the lowest efficiency in colony formation. AA and E-350G keratinocytes attained higher capacity for in vitro transformation. We observed similar degradation of TP53 among HPV-16 variants. Furthermore, we accessed the expression profile in early (p5) and late passage (p30) transduced cells of 84 genes commonly involved in carcinogenesis. Most differences could be attributed to HPV-16 E6/E7 expression. In particular, we detected different expression of ITGA2 and CHEK2 in keratinocytes infected with AA and AA/E-350G late passage cells, respectively, and higher expression of MAP2K1 in E-350G transduced keratinocytes. Our results indicate differences among HPV-16 variants that could explain, at least in part, differences in oncogenic potential attributed to these variants.     

Down-regulation of MHC class I is a property common to papillomavirus E5 proteins

The E5 protein family of papillomaviruses comprises small hydrophobic proteins which are associated with the cell endomembrane compartments. The functions of the E5 proteins, particularly those of HPV, are still far from clear. We have reported that the E5 proteins of BPV-1, BPV-4, HPV-16 and HPV-6 down-regulate MHC class I, potentially helping the virus evade the host immune response. Others have described MHC class I down-regulation by HPV-2 E5. We report here that another E5 protein, HPV-83 E5, likewise down-regulates MHC class I and propose that interference with expression, assembly and/or transport of MHC class I is a common property of all E5 proteins evolved by the virus to circumvent host immunosurveillance and thus establish productive infection.     

Are transforming properties of the bovine papillomavirus E5 protein shared by E5 from high-risk human papillomavirus type 16?

The E5 proteins of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 16 (HPV-16) are small (44-83 amino acids), hydrophobic polypeptides that localize to membranes of the Golgi apparatus and endoplasmic reticulum, respectively. While the oncogenic properties of BPV-1 E5 have been characterized in detail, less is known about HPV-16 E5 due to its low expression in mammalian cells. Using codon-optimized HPV-16 E5 DNA, we have generated stable fibroblast cell lines that express equivalent levels of epitope-tagged BPV-1 and HPV-16 E5 proteins. In contrast to BPV-1 E5, HPV-16 E5 does not activate growth factor receptors, phosphoinositide 3-kinase or c-Src, and fails to induce focus formation, although it does promote anchorage-independent growth in soft agar. These variant activities are apparently unrelated to differences in intracellular localization of the E5 proteins since retargeting HPV-16 E5 to the Golgi apparatus does not induce focus formation.     

Adenovirus E4orf4 induces HPV-16 late L1 mRNA production

The adenovirus E4orf4 protein regulates the switch from early to late gene expression during the adenoviral replication cycle. Here we report that overexpression of adenovirus E4orf4 induces human papillomavirus type 16 (HPV-16) late gene expression from subgenomic expression plasmids. E4orf4 specifically overcomes the negative effects of two splicing silencers at the two late HPV-16 splice sites SD3632 and SA5639. This results in the production of HPV-16 spliced L1 mRNAs. We show that the interaction of E4orf4 with protein phosphatase 2A (PP2A) is necessary for induction of HPV-16 late gene expression. Also an E4orf4 mutant that fails to bind the cellular splicing factor ASF/SF2 fails to induce L1 mRNA production. Collectively, these results suggest that dephosphorylation of SR proteins by E4orf4 activates HPV-16 late gene expression. Indeed, a mutant ASF/SF2 protein in which the RS-domain had been deleted could itself induce HPV-16 late gene expression, whereas wild type ASF/SF2 could not.     

Sequence variants and functional analysis of human papillomavirus type 16 E5 gene in clinical specimens

Summary.  Previously, we found that the E5 protein can be expressed in HPV-16 infected precancerous lesions and cervical cancer [4]. In this study, we investigated the presence of sequence variants of E5 in HPV-16 infected tissues. Toward this end, we amplified the E5 gene by polymerase chain reaction from 29 HPV-16 infected tissues including eight normal tissues, seven high grade neoplastic tissues (high grade squamous intraepithelial lesions (HSIL) and 14 cervical cancer tissues. Sequence analysis demonstrated that there were three mutational hot spots at positions 3979, 4042, and 4077 of the HPV-16 DNA; these and other mutations resulted in six variants in the E5 sequence. This resulted in four E5 protein mutants, named WTE5 [wild type E5 protein], 14E5, 21E5 and 56E5. Functional analysis of these four mutant proteins revealed that the transforming activities of 14E5, 21E5 and 56E5 were 0.95, 0.59, and 0.89 fold of WTE5, respectively. Although E5 was expressed in all of the HSIL and cervical cancer tissues, but in only one of the eight normal tissues tested, only WT E5 protein was found in HSIL while in cervical cancer tissues both WT and mutant E5 proteins were detected. Since these E5 proteins exhibited the same in vitro transforming activity, these data suggest that expression of E5 is important in development and progression toward malignancy but mutation of E5 does not affect the transformation process. Received January 24, 2000/Accepted May 30, 2000     

特异siRNA抑制宫颈癌细胞中人乳头状瘤病毒16型E6表达的研究

目的 优化HPV-16 E6癌基因特异的U6质粒表达的siRNA，抑制HPV癌基因表达及其对子宫颈癌细胞生长繁殖的影响。方法 选择4个分别针对HPV-16 E6 mRNA外显子和内含子序列为靶序列，合成DNA链，构建表达HPV-16 E6短发卡样dsRNA的重组pSilencer1．0-U6载体，导入HPV-16DNA阳性的宫颈癌细胞株CaSki中，观察该细胞中HPV-16 E6、E7基因表达水平及其蛋白含量的变化，并观察细胞生长被抑制的情况。结果 4种HPV-16 E6 siRNA均能降低宫颈癌细胞CaSki的生长速率。通过细胞生长曲线观察到HPV-16 E6 shRNA表达质粒导入细胞0-96h内，可降低细胞生长速度。荧光定量RT-PCR检测HPV-16 E6 siRNA可使宫颈癌细胞株CaSki中HPV-16 E6、E7基因转录的mRNA水平降低，其中针对E6 mRNA内含子的重组shRNA只抑制E6基因的表达水平。Western blot分析表明，4个HPV-16 E6 siRNA作用72h后，未能检测到宫颈癌细胞中HPV-16 E6蛋白。结论 HPV-16 E6 siRNA能使宫颈癌细胞CaSki生长缓慢；选择针对E6内含子的siRNA作用位点，特异性抑制E6表达；而针对E6外显子的siRNA作用位点，可抑制E6和E7基因的表达，是用于治疗HPV阳性宫颈癌细胞的理想靶位。     

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

To facilitate the investigations of HPV-16 late gene expression HPV-16 reporter plasmids were generated using previously described sub-genomic HPV-16 plasmids, named pBEL and pBELM, that, similar to the full viral genome, produce primarily HPV-16 early mRNAs and very little, if any, late mRNAs in cervical cancer cells. The HPV-16 late L1 gene was replaced by the chloramphenicol acetyltransferase (CAT) reporter gene, or green fluorescent protein (GFP), preceded by the poliovirus internal ribosome entry site (IRES). Results show that the reporter genes mimic the expression of L1 from these plasmids. For example, overexpression of adenovirus E4orf4 protein (E4orf4), polypyrimidine tract binding protein (PTB), arginine/serine-rich SRp30c protein (SRp30c) or alternative splicing factor/splicing factor 2 (ASF/SF2) induced an increased expression of CAT or GFP. Stable cell lines with reporter plasmids pBELCAT and pBELMCAT were also generated. An induction of CAT was observed in HPV-16 reporter cell lines in the presence of the small molecule phorbol 12-myristate 13-acetate (TPA). Further experiments identified the TPA-inducible, hnRNP A2/B1 protein as a regulator of HPV-16 late gene expression. In conclusion, the HPV-16 reporter plasmids and reporter cell lines described herein can be used to identify small molecules and cellular factors that regulate HPV-16 gene expression.     

Effects of E5a and E7 genes of human papillomavirus type 11 on immortalized human epidermal keratinocytes and NIH 3T3 cells

Summary The E5a gene of HPV-11 expressed in NIH 3T3 cells led to tumorigenesis in nude mice; whereas when expressed in keratinocytes, E5a induced anchorage independent growth, but was nontumorigenic in nude mice. The E7 gene of HPV-11 expressed NIH 3T3 cells led to tumors in nude mice and morphological transformation, but not anchorage independent growth. Keratinocytes expressing the E7 gene induced colony formation in soft agarose, but not tumorigenesis in nude mice. Comparison of transforming activities of HPV-11 E5a and E7 genes of HPV-11 showed that the efficiency of cell transformation by E7 was weaker than that of E5a.     

The human papillomavirus type 58 E7 oncoprotein modulates cell cycle regulatory proteins and abrogates cell cycle checkpoints

HPV type 58 (HPV-58) is the third most common HPV type in cervical cancer from Eastern Asia, yet little is known about how it promotes carcinogenesis. In this study, we demonstrate that HPV-58 E7 significantly promoted the proliferation and extended the lifespan of primary human keratinocytes (PHKs). HPV-58 E7 abrogated the G1 and the postmitotic checkpoints, although less efficiently than HPV-16 E7. Consistent with these observations, HPV-58 E7 down-regulated the cellular tumor suppressor pRb to a lesser extent than HPV-16 E7. Similar to HPV-16 E7 expressing PHKs, Cdk2 remained active in HPV-58 E7 expressing PHKs despite the presence of elevated levels of p53 and p21. Interestingly, HPV-58 E7 down-regulated p130 more efficiently than HPV-16 E7. Our study demonstrates a correlation between the ability of down-regulating pRb/p130 and abrogating cell cycle checkpoints by HPV-58 E7, which also correlates with the biological risks of cervical cancer progression associated with HPV-58 infection.     

IFN-tau exhibits potent suppression of human papillomavirus E6/E7 oncoprotein expression.

The effects of interferon-tau (IFN-tau) on tumor suppressor factors and virus oncoprotein expression were compared with two other type I IFN in human papillomavirus (HPV-16)-transformed cells. Nontumorigenic human keratinocytes, HuKc/HPV-16d-2C (d-2C), treated with recombinant human IFN-alpha2a (Roferon), a human recombinant alpha IFN hybrid, alpha B/D (IFN-alphaB/D), or ovine IFN-tau were evaluated for their effects on the levels of E6 and E7 expression. IFN-tau was comparable to IFN-alpha2a in decreasing intracellular levels of E6 and E7, and IFN-alphaB/D was more effective than IFN-a2a in suppressing E7 levels. All three IFN were capable of increasing the cellular concentration of wild-type p53 tumor suppressor with the magnitude IFN-tau > IFN-alpha2a > IFN-alphaB/D. Increases in p53 concentrations correlated with the observed decreases in E6 mRNA and protein levels. The antiviral effects observed in this study reveal that IFN-tau has potent antipapillomavirus activity. Sequences/structures unique to IFN-tau could allow for alternative IFN/receptor interactions and may explain the differences in biologic function.     

The biochemical and biological functions of human papillomavirus type 16 E5 protein

Summary. Human papillomavirus type 16 (HPV-16) E5 protein, along with the more publicized E6 and E7 proteins of this virus, has been found to be oncogenic. E5 is a highly hydrophobic membrane-bound protein of 83 amino acids associated with the Golgi apparatus, endoplasmic reticulum, and nuclear membrane in infected cells. E5 can activate epidermal growth factor receptor (EGFR) through binding to the 16kD subunit of protein pump ATPase leading to a reduced downregulation of EGFR receptors. The activation of EGFR can initiate biochemical cascades that lead to overexpression of a variety of protooncogenes and stimulate rapid cell growth. Moreover, E5 can inhibit the expression of tumor suppressor gene p21^{(WafI/SdiI/CipI)} and impair the control of cell cycle checkpoint. E5 protein has been identified as a potential tumor vaccine target antigen.Received November 6, 2002; accepted March 10, 2003 Published online June 2, 2003     

The E5 Protein of HPV-6, but Not HPV-16, Associates Efficiently with Cellular Growth Factor Receptors

The transforming activity of the prototype E5 protein of bovine papillomavirus type 1 (BPV-1) is associated with its binding to, and activation of, both the platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. The E5 proteins of human papillomavirus types 6 and 16 (HPV-6, HPV-16) also transform rodent cells in the presence of the EGF receptor. In this study we examined whether epitope-tagged HPV E5 proteins could associate with three different tyrosine kinase-containing growth factor receptors: the EGF receptor, the erbB2 receptor, and the PDGF receptor. The HPV-6 E5 protein was found to associate efficiently with all three of these growth factor receptors, while the HPV-16 E5 protein did not. These findings suggest either that the in vitro transforming activities of HPV-6 and HPV-16 E5 proteins involve a similar mechanism unrelated to receptor binding (e.g., binding to the 16-kDa membrane pore protein) or that they proceed along distinct pathways, with receptor binding being important for HPV-6. Regardless of the ultimate mechanisms, the differences between the HPV-6 and HPV-16 E5 proteins in binding to growth factor receptors may potentially contribute to the distinctive morphologies of their respective neoplastic lesions.     

The human papillomavirus type 16 E5 oncoprotein synergizes with EGF-receptor signaling to enhance cell cycle progression and the down-regulation of p27

E5 oncoprotein activity from high risk human papillomaviruses (HPVs) is associated with growth factor receptor signaling, but the function of this protein is not well understood. In this study, we investigated the role of HPV-16 E5 on the cell cycle progression during EGF-stimulation. Wild-type and NIH 3T3 cells over-expressing human EGF-receptor were transfected with HPV-16 E5 gene and the cell cycle progression was characterized. This analysis showed that the E5-expressing cells increased DNA synthesis (S-phase) by around 40%. Cell cycle protein analysis of E5-expressing cells showed a reduction in the half-life of p27^Kip1 protein as compared to control cells (18.4 vs. 12.7 h), an effect that was enhanced in EGF-stimulated cells (12.8 vs. 3.6 h). Blockage of EGF-receptor activity abrogated E5 signals as well as p27^Kip1 down-regulation. These results suggest that E5 and the EGF-receptor cooperate to enhance cell cycle entry and progression through regulating p27^Kip1 expression at protein level.