人乳头瘤病毒16型早期基因E6、E7的克隆及表达

高危人乳头瘤病毒16型的感染与宫颈癌的发病密切相关。HPV16E6和E7蛋白在大多数HPV16相关宫颈癌及其癌前病变中持续表达,因此E6和E7蛋白可作为制备HPV16相关肿瘤及其癌前病变治疗性疫苗的靶抗原〔2〕。采用套式PCR方法,从宫颈癌患者组织中扩增出E6、E7基因并成功构建了包含E6、E7的表达质粒PET-E6、PET-E7。SDS聚丙烯酰胺凝胶电泳和Western-blotting分析结果表明,外源基因E6,E7在T7启动子控制下可获得稳定表达。     

Vesicular stomatitis virus-based therapeutic vaccination targeted to the E1, E2, E6, and E7 proteins of cottontail rabbit papillomavirus

Persistent human papillomavirus (HPV)-associated benign and malignant lesions are a major cause of morbidity and mortality worldwide. Vaccination against HPV early proteins could provide an effective means of treating individuals with established infections. Recombinant vesicular stomatitis virus (VSV) vectors have been used previously to elicit strong humoral and cellular immune responses and develop prophylactic vaccines. We have shown that VSV vectors also can be used to elicit therapeutic immunity in the cottontail rabbit papillomavirus (CRPV)-rabbit model of high-risk HPV infection. In the present study, three new VSV vectors expressing the CRPV E1, E2, or E7 protein were produced and compared to the previously generated VSV-E6 vector for therapeutic efficacy. To determine whether vaccine efficacy could be augmented by simultaneous vaccination against two CRPV proteins, the four vaccines were delivered individually and in all possible pairings to rabbits 1 week after CRPV infection. Control rabbits received the recombinant wild-type VSV vector or medium only. Cumulative papilloma volumes were computed for analysis of the data. The analyses showed that VSV-based vaccination against the E1, E2, E6, or E7 protein significantly reduced papilloma volumes relative to those of the controls. Furthermore, VSV-based CRPV vaccination cured all of the papillomas in 5 of 30 rabbits. Of the individual vaccines, VSV-E7 was the most effective. The VSV-E7 vaccine alone was the most effective, as it reduced cumulative papilloma volumes by 96.9% overall, relative to those of the controls, and ultimately eliminated all of the disease in all of the vaccinees. Vaccine pairing was not, however, found to be beneficial, suggesting antigenic competition between the coexpressed CRPV proteins. These preclinical results, obtained in a physiologically relevant animal model of HPV infection, demonstrate that VSV vectors deserve serious consideration for further development as therapeutic antitumor vaccines.     

Expressions of E2 and E7-HPV16 proteins in pre-malignant and malignant lesions of the uterine cervix

Continuous production of the E7 protein from different types of high risk human papilloma virus (HPV) is required for progression of malignancy. We developed antibodies against HPV type 16 E7 and E2 proteins to evaluate their utility as markers for diagnosis during early stages of cervical cancer. Forty biopsies from uterine cervices were diagnosed as low grade intraepithelial lesion (LSIL), high grade intraepithelial lesion (HSIL), squamous carcinoma (SC), in situ adenocarcinoma (ISA) and invasive adenocarcinoma (AC), all of which were infected with HPV 16. Immunohistochemistry was used to investigate the expressions of E7 and E2 (both from HPV 16) and p16. P16 was expressed in eight of 12 LSILs, in all HSILs, in 16 of 18 SC and in all ACs. E2 was expressed in six of 12 LSILs. E7 was positive in eight of 12 LSILs and in all HSIL and carcinomas. The expressions of E2 and E7 of HPV16 related to p16 expression confirmed the value of the viral oncogenic proteins as complementary to histology and support the carcinogenic model of the uterine cervix, because HPVDNA integration into cellular DNA implies the destruction of the gene encoding E2, which suppresses the expression of the E6 and E7 oncoproteins. E2 from HPV16 could be marker for LSILs, while E7 could be a marker for progression of LSILs to HSILs in patients infected by HPV16, because viral typing has little positive predictive value.     

Bap31 is a novel target of the human papillomavirus E5 protein

The E5 proteins of human papillomaviruses (HPVs) are small hydrophobic proteins that are expressed in the early and late stages of the viral life cycle; however, their role in HPV pathogenesis is not clearly understood. In this study, a split-ubiquitin yeast (Saccharomyces cerevisiae) two-hybrid system was used to identify B-cell-associated protein 31 (Bap31) as a binding partner of HPV E5 proteins. The association of these proteins was confirmed by coimmunoprecipitation of complexes of Bap31 with either HPV type 16 (HPV16) or HPV31 E5. In addition, Bap31 and E5 were found to colocalize in perinuclear patterns consistent with localization to the endoplasmic reticulum. Mutational analysis of E5 identified amino acids in the extreme C terminus as important for stabilizing the interaction with Bap31. Deletion of these C-terminal amino acids of E5 in the context of complete HPV31 genomes resulted in impaired proliferative capacity of HPV-positive keratinocytes following differentiation. When small interfering RNAs were used to reduce the levels of Bap31, the proliferative ability of HPV-positive keratinocytes upon differentiation was also reduced, implicating Bap31 as a regulator of this process. These studies identify a novel binding partner of the high-risk HPV E5 proteins and provide insight into how the E5 proteins may modulate the life cycle in differentiating cells.     

Oligomerization properties of the viral oncoproteins adenovirus E1A and human papillomavirus E7 and their complexes with the retinoblastoma protein

Human papillomavirus 16 E7 (HPV16 E7) and adenovirus 5 E1A (Ad5 E1A) are encoded by highly divergent viruses yet are functionally similar in their ability to bind the retinoblastoma (pRB) tumor suppressor protein, causing the aberrant displacement of E2F trancription factors. The amino acid residues of HPV16 E7 that are necessary for stability, for inhibition of pRB function, and for cell transformation are also necessary for E7 oligomerization. However, neither the specific oligomerization state of HPV16 E7 nor of Ad5 E1A as a function of pRB-binding has been characterized. To gain insight into HPV16 E7 and Ad5 E1A oligomerization properties, sedimentation equilibrium experiments were performed with recombinant HPV16 E7 and Ad5 E1A proteins. These studies reveal that, despite the overall functional similarities between these proteins, monomers, dimers, and tetramers of HPV16 E7 were detected while only reversible monomer-dimer association was identified for Ad5 E1A. The apparent K(d(monomer)-(dimer)) of HPV16 E7 is approximately 100-fold lower than that of a comparable region of Ad5 E1A, and it is concluded that under physiological protein concentrations HPV16 E7 exists primarily as a dimer. Sedimentation equilibrium experiments of pRB/Ad5 E1A and of pRB/HPV16 E7 complexes demonstrate that the tight association of pRB with the viral oncoproteins does not disturb their inherent oligomerization properties. Taken together, this study demonstrates significant differences between the Ad5 E1A and HPV16 E7 oligomerization states that are potentially related to their distinct structures and specific mechanisms of pRB-inactivation.     

Mutational analysis of human papillomavirus E4 proteins: identification of structural features important in the formation of cytoplasmic E4/cytokeratin networks in epithelial cells.

We have previously demonstrated that human papillomavirus type 1 (HPV 1) and 16 (HPV 16) E4 proteins form cytoplasmic filamentous networks which specifically colocalize with cytokeratin intermediate-filament (IF) networks when expressed in simian virus 40-transformed keratinocytes. The HPV 16 (but not the HPV 1) E4 protein induced the collapse of the cytokeratin networks. (S. Roberts, I. Ashmole, G. D. Johnson, J. W. Kreider, and P. H. Gallimore, Virology 197:176-187, 1993). The mode of interaction of E4 with the cytokeratin IFs is unknown. To identify E4 sequences important in mediating this interaction, we have constructed a large panel of mutant HPV (primarily HPV 1) E4 proteins and expressed them by using the same simian virus 40-epithelial expression system. Mutation of HPV 1 E4 residues 10 to 14 (LLGLL) abrogated the formation of cytoplasmic filamentous networks. This sequence corresponds to a conserved motif, LLXLL, found at the N terminus of other E4 proteins, and similar results were obtained on deletion of the HPV 16 motif, LLKLL (residues 12 to 16). Our findings indicate that this conserved motif is likely to play a central role in the association between E4 and the cytokeratins. An HPV 1 E4 mutant protein containing a deletion of residues 110 to 115 induced the collapse of the cytokeratin IFs in a manner analogous to the HPV 16 E4 protein. The sequence deleted, DLDDFC, is highly conserved between cutaneous E4 proteins. HPV 1 E4 residues 42 to 80, which are rich in charged amino acids, appeared to be important in the cytoplasmic localization of E4. In addition, we have mapped the N-terminal residues of HPV 1 E4 16-kDa and 10/11-kDa polypeptides expressed by using the baculovirus system and shown that they begin at tyrosine 16 and alanine 59, respectively. Similar-sized E4 proteins are also found in vivo. N-terminal deletion proteins, which closely resemble the 16-kDa and 10/11-kDa species, expressed in keratinocytes were both cytoplasmic and nuclear but did not form cytoplasmic filamentous networks. These findings support the postulate that N-terminal proteolytic processing of the E1-- E4 protein may modulate its function in vivo.     

Overexpression,purification, and structural analysis of the hydrophobic E5 protein from human papillomavirus type 16

The E5 proteins of human papillomavirus (HPV) are highly hydrophobic transmembrane proteins that display weak transforming activity. The HPV E5 proteins are localized largely to intracellular membranes, such as the Golgi apparatus and endoplasmic reticulum, but also appear in the plasma membrane. Infection with HPV16 is the cause of over 90% of human cervical cancers. HPV E5 is known to interact with growth factor receptors and gap junction proteins and is believed to play a role during the initiation of neoplasia. The structure of HPV E5 and the mechanism of its interactions with growth factor receptors remain largely unknown. In the present studies, the E5 protein of HPV16 was cloned into the pBAD/TOPO vector fused to an N-terminal thioredoxin leader and a C-terminal His-tag, and expressed in Escherichia coli. The identity of the protein was confirmed by immunoblotting using antibodies against a V5-epitope tag engineered into the protein. Due to formation of high molecular mass superaggregates of the protein, two chromatography steps were employed for its purification: (1) gel filtration chromatography to separate the superaggregated protein from other soluble proteins and (2) Ni-chelate affinity chromatography in the presence of detergent. The superaggregates of the E5-fusion protein were broken down to monomers and various oligomers by sonication in the presence of 0.2% SDS. The purified E5-fusion protein was then reconstituted into lipid vesicles and initial structural analysis of the protein was performed using circular dichroism spectroscopy.     

HnRNP D activates production of HPV16 E1 and E6 mRNAs by promoting intron retention

Human papillomavirus type 16 (HPV16) E1 and E6 proteins are produced from mRNAs with retained introns, but it has been unclear how these mRNAs are generated. Here, we report that hnRNP D act as a splicing inhibitor of HPV16 E1/E2- and E6/E7-mRNAs thereby generating intron-containing E1- and E6-mRNAs, respectively. N- and C-termini of hnRNP D contributed to HPV16 mRNA splicing control differently. HnRNP D interacted with the components of splicing machinery and with HPV16 RNA to exert its inhibitory function. As a result, the cytoplasmic levels of intron-retained HPV16 mRNAs were increased in the presence of hnRNP D. Association of hnRNP D with HPV16 mRNAs in the cytoplasm was observed, and this may correlate with unexpected inhibition of HPV16 E1- and E6-mRNA translation. Notably, hnRNP D40 interacted with HPV16 mRNAs in an HPV16-driven tonsillar cancer cell line and in HPV16-immortalized human keratinocytes. Furthermore, knockdown of hnRNP D in HPV16-driven cervical cancer cells enhanced production of the HPV16 E7 oncoprotein. Our results suggest that hnRNP D plays significant roles in the regulation of HPV gene expression and HPV-associated cancer development.     

Mapping and characterization of the interaction domains of human papillomavirus type 16 E1 and E2 proteins.

The papillomavirus E1 and E2 proteins are both necessary and sufficient in vivo for efficient origin-dependent viral DNA replication. The ability of E1 and E2 to complex with each other appears to be essential for efficient viral DNA replication. In this study, we used the yeast two-hybrid system and in vitro binding assays to map the domains of the human papillomavirus type 16 (HPV16) E1 and E2 proteins required for complex formation. The amino-terminal 190-amino-acid domain of HPV16 E2 was both required and sufficient for E1 binding. The carboxyl-terminal 229 amino acids of E 1 were essential for binding E2, and the amino-terminal 143 amino acids of HPV16 E1 were dispensable. Although the ability of the E1 minimal domain (amino acids [aa] 421 to 649) to interact with E2 was strong at 4 degrees C, it was significantly reduced at temperatures above 25 degrees C. A larger domain of E1 from aa 144 to 649 bound E2 efficiently at any temperature, suggesting that aa 144 to 420 of E1 may play a role in the HPV16 E1-E2 interaction at physiological temperatures.     

Recombinant Antigens E6 and E7 of Human Papilloma Virus Type 16: Preparation,Purification, and Assessment of the Immunogenicity and Antitumor Effects In Vivo

Applied Biochemistry and Microbiology - The recombinant proteins E6-CBD and E7-CBD, which are antigens E6 and E7 of human papillomavirus type 16 (HPV16), were connected by a glycine-serine spacer...     

Activation of cap-dependent translation by mucosal human papillomavirus E6 proteins is dependent on the integrity of the LXXLL binding motif

The human papillomavirus (HPV) type 16 (HPV16) E6 protein can stimulate mechanistic target of rapamycin complex 1 (mTORC1) signaling and cap-dependent translation through activation of the PDK1 and mTORC2 kinases. Here we report that HPV18 E6 also enhances cap-dependent translation. The integrity of LXXLL and PDZ protein binding domains is important for activation of cap-dependent translation by high-risk mucosal HPV E6 proteins. Consistent with this model, low-risk mucosal HPV6b and HPV11 E6 proteins, which do not contain a PDZ protein binding motif, also activate cap-dependent translation and mTORC1, albeit at a lower efficiency than high-risk HPV E6 proteins. In contrast, cutaneous HPV5 and HPV8 E6 proteins, which lack LXXLL and PDZ motif protein binding, do not enhance cap-dependent translation. Mutagenic analyses of low-risk HPV E6 proteins revealed that association with the LXXLL motif containing ubiquitin ligase E6AP (UBE3A) correlates with activation of cap-dependent translation. Hence, activation of mTORC1 and cap-dependent translation may be important for the viral life cycle in specific epithelial tissue types and contribute to cellular transformation in cooperation with other biological activities of high-risk HPV E6-containing proteins.     

Inhibition of growth, transformation, and expression of human papillomavirus type 16 E7 in human keratinocytes by alpha interferons.

We used a model system of normal human keratinocytes (HKc) and HKc immortalized with human papillomavirus type 16 DNA (HKc/HPV16) to investigate the effects of alpha interferons (IFN-alpha) on the growth of HPV16-immortalized human epithelial cells, on HPV16-mediated immortalization of normal HKc, and on HPV16 gene expression. Normal HKc and HKc/HPV16 were treated with several recombinant human IFN-alpha subtypes (IFN-alpha B, IFN-alpha D, and IFN-alpha B/D). These IFN-alpha subtypes inhibited proliferation of both normal HKc and HKc/HPV16 in a dose-dependent fashion; however, although 1,000 to 10,000 U of IFN-alpha per ml were required to inhibit growth of normal HKc, HKc/HPV16 were substantially growth inhibited by 100 U/ml. In addition, 100 U of IFN-alpha B/D per ml inhibited transformation of normal HKc by HPV16 DNA. Northern (RNA) blot analysis showed no effect of IFN-alpha on the mRNA levels of the HPV16 E6 and E7 open reading frames. However, immunofluorescence studies of the HPV16 E6 and E7 proteins with anti-E6 and anti-E7 monoclonal antibodies showed significant inhibition of E7 protein expression in cells treated with IFN-alpha, whereas E6 protein expression was not altered. The inhibition of E7 protein expression in cells treated with IFN-alpha was further confirmed by Western immunoblot analysis. These results suggest that IFN-alpha may inhibit HPV16-mediated transformation of HKc and proliferation of HKc/HPV16 through an inhibition of HPV16 E7 protein expression.     

Antigen processing defects in cervical carcinomas limit the presentation of a CTL epitope from human papillomavirus 16 E6.

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with the development of cervical cancer. The E6 and E7 proteins of HPV are constitutively expressed in cervical carcinoma cells making them attractive targets for CTL-based immunotherapy. However, few studies have addressed whether cervical carcinomas can process and present HPV E6/E7-derived Ags for recognition by CTL. We generated HLA-A*0201-restricted CTL clones against HPV16 E6(29-38) that recognized HPV16 E6 Ags transfected into B lymphoblastoid cells. These CTL were unable to recognize HLA-A*0201(+) HPV16 E6(+) cervical carcinoma cell lines even when the level of endogenous HPV16 E6 in these cells was increased by transfection. This defect in presentation of HPV16 E6(29-38) correlated with low level expression of HLA class I, proteasome subunits low molecular mass protein 2 and 7, and the transporter proteins TAP1 and TAP2 in the cervical carcinoma cell lines. The expression of all of these proteins could be up-regulated by IFN-gamma, but this was insufficient for CTL recognition unless the level of HPV16 E6 Ag was also increased by transfection. CTL recognition of the HPV16 E6(29-38) epitope in 721.174 B cells was dependent on TAP expression but independent of immunoproteasome expression. Collectively, these findings suggest that presentation of the HPV16 E6(29-38) epitope in cervical carcinoma cell lines is limited both by the level of TAP expression and by the low level or availability of the source HPV E6 oncoprotein. These observations place constraints on the use of this, and potentially other, HPV-derived CTL epitopes for the immunotherapy of cervical cancer.     

E6 and E7 fusion immunoglobulin from human papilloma virus 16 induces dendritic cell maturation and antigen specific activation of T helper 1 response

Human papilloma virus (HPV) 16 causes cervical cancer. Induction of oncogenesis by HPV 16 is primarily dependent on the function of E6 and E7 proteins, which inactivate the function of p53 and pRB, respectively. Thus, blocking the activity of the E6 and E7 proteins from HPV 16 is critical to inhibiting oncogenesis during infection. We have expressed and purified soluble HPV 16 E6 and E7 fusion immunoglobulin (Ig), which were combined with the constant region of an Ig heavy chain, in a mammalian system. To assess whether soluble E6 and E7 fusion Igs induce effective cellular immune responses, immature dendritic cells (DCs) were treated with these fusion proteins. Soluble E6 and E7 fusion Igs effectively induced maturation of DCs. Furthermore, immunization with soluble E6 and E7 fusion Igs in mice resulted in antigen-specific activation of T helper 1 (Th1) cells. This is the first comprehensive study to show the molecular basis of how soluble HPV 16 E6 or E7 fusion Igs induces Th1 responses through the maturation of DCs. In addition, we show that DC therapy using soluble HPV E6 and E7 fusion Igs may be a valuable tool for controlling the progress of cervical cancer.     

Induction of pRb degradation by the human papillomavirus type 16 E7 protein is essential to efficiently overcome p16INK4a-imposed G1 cell cycle Arrest

It has previously been shown that the E7 protein from the cutaneous human papillomavirus type 1 (HPV1), which is associated with benign skin lesions, binds the product of the tumor suppressor gene retinoblastoma (pRb) with an efficiency similar to that of the E7 protein from the oncogenic HPV type 16. Despite this ability, HPV1 E7 does not display any activity in transforming primary cells. In addition, the two viral proteins differ in their mechanisms of targeting pRb. HPV16 E7 promotes pRb destabilization, while cells expressing HPV1 E7 do not show any decrease in pRb levels. In this study, we show that HPV1 E7, in contrast to HPV16 E7, has only a weak activity to neutralize the effect of cyclin-dependent kinase inhibitor p16INK4a. By generation of HPV1/16 E7 chimeric proteins, we have identified a central motif in the two E7 proteins, which determines their different abilities to overcome the p16INK4a-mediated cell cycle arrest. This motif is located downstream of the pRb-binding domain and comprises only three amino acids in HPV16 E7. Swapping this central motif in the two viral proteins causes an exchange of their activities involved in circumventing the inhibitory function of p16INK4a. Most importantly, our data show that the efficiency of the E7 proteins in neutralizing the inhibitory effect of p16INK4a correlates with their ability to promote pRb degradation.