The antigenic landscape of human influenza N2 neuraminidases from 2009 until 2017.

Human H3N2 influenza viruses are subject to rapid antigenic evolution which translates into frequent updates of the composition of seasonal influenza vaccines. Despite these updates, the effectiveness of influenza vaccines against H3N2-associated disease is suboptimal. Seasonal influenza vaccines primarily induce hemagglutinin-specific antibody responses. However, antibodies directed against influenza neuraminidase (NA) also contribute to protection. Here, we analysed the antigenic diversity of a panel of N2 NAs derived from human H3N2 viruses that circulated between 2009 and 2017. The antigenic breadth of these NAs was determined based on the NA inhibition (NAI) of a broad panel of ferret and mouse immune sera that were raised by infection and recombinant N2 NA immunisation. This assessment allowed us to distinguish at least four antigenic groups in the N2 NAs derived from human H3N2 viruses that circulated between 2009 and 2017. Computational analysis further revealed that the amino acid residues in N2 NA that have a major impact on susceptibility to NAI by immune sera are in proximity of the catalytic site. Finally, a machine learning method was developed that allowed to accurately predict the impact of mutations that are present in our N2 NA panel on NAI. These findings have important implications for the renewed interest to develop improved influenza vaccines based on the inclusion of a protective NA antigen formulation.

Two proteins, the hemagglutinin and the neuraminidase, protrude from the surface of the influenza virus. Their detection by the immune system allows the host organism to mount defences against the viral threat. The virus evolves in response to this pressure, which manifests as changes in the appearance of its hemagglutinin and neuraminidase. This process, known as antigenic drift, leads to the proteins evading detection. It is also why flu vaccines require frequent updates, as they rely on 'training' the immune system to recognise the most important strains in circulation ­ primarily by exposing it to appropriate versions of hemagglutinin. While the antigenic drift of hemagglutinin has been extensively studied, much less is known about how the neuraminidase accumulates mutations, and how these affect the immune response. To investigate this question, Catani et al. selected 43 genetically distant neuraminidases from human viral samples isolated between 2009 and 2017. Statistical analyses were applied to define their relatedness, revealing that a group of closely related neuraminidases predominated from 2009 to 2015, before they were being taken over by a second group. A third group, which was identified in viruses isolated in 2013, was remarkably close to the neuraminidase of strains that circulated in the late 1990s. The fourth and final group of neuraminidases was derived from influenza viruses that normally circulate in pigs but can also occasionally infect humans. Next, Catani et al. examined the immune response that these 43 neuraminidases could elicit in mice, as well as in ferrets ­ the animal most traditionally used in influenza research. This allowed them to pinpoint which changes in the neuraminidase sequences were important to escape recognition by the host. Data obtained from the two model species were comparable, suggesting that these experiments could be conducted on mice going forward, which are easier to work with than ferrets. Finally, Catani et al. used machine learning to build a computational model that could predict how strongly the immune system would respond to a specific neuraminidase variant. These findings could help guide the development of new vaccines that include neuraminidases tailored to best prime and train the immune system against a larger variety of strains. This may aid the development of 'supra-seasonal' vaccines that protect against a broad range of influenza viruses, reducing the need for yearly updates.

Anti-neuraminidase and anti-hemagglutinin immune serum can confer inter-lineage cross protection against recent influenza B.

Influenza B viruses (IBV) are responsible for a considerable part of the burden caused by influenza virus infections. Since their emergence in the 1980s, the Yamagata and Victoria antigenic lineages of influenza B circulate in alternate patterns across the globe. Furthermore, their evolutionary divergence and the appearance of new IBV subclades complicates the prediction of future influenza vaccines compositions. It has been proposed that the addition of the neuraminidase (NA) antigen could potentially induce a broader protection and compensate for hemagglutinin (HA) mismatches in the current vaccines. Here we show that anti-NA and -HA sera against both Victoria and Yamagata lineages have limited inter-lineage cross-reactivity. When transferred to mice prior to infection with a panel of IBVs, anti-NA sera were as potent as anti-HA sera in conferring protection against homologous challenge and, in some cases, conferred superior protection against challenge with heterologous IBV strains.

Antibodies directed towards neuraminidase restrict influenza virus replication in primary human bronchial epithelial cells.

Influenza neuraminidase (NA) is implicated in various aspects of the virus replication cycle and therefore is an attractive target for vaccination and antiviral strategies. Here we investigated the potential for NA-specific antibodies to interfere with A(H1N1)pdm09 replication in primary human airway epithelial (HAE) cells. Mouse polyclonal anti-NA sera and a monoclonal antibody could block initial viral entry into HAE cells as well as egress from the cell surface. NA-specific polyclonal serum also reduced virus replication across multiple rounds of infection. Restriction of virus entry correlated with the ability of the serum or monoclonal antibody to mediate neuraminidase inhibition (NI). Finally, human sera with NI activity against the N1 of A(H1N1)pdm09 could decrease H6N1 virus infection of HAE cells, highlighting the potential contribution of anti-NA antibodies in the control of influenza virus infection in humans.

Pre-existing antibodies directed against a tetramerizing domain enhance the immune response against artificially stabilized soluble tetrameric influenza neuraminidase.

The neuraminidase (NA) is an abundant antigen at the surface of influenza virions. Recent studies have highlighted the immune-protective potential of NA against influenza and defined anti-NA antibodies as an independent correlate of protection. Even though NA head domain changes at a slightly slower pace than hemagglutinin (HA), NA is still subject to antigenic drift, and therefore an NA-based influenza vaccine antigen may have to be updated regularly and thus repeatedly administered. NA is a tetrameric type II membrane protein, which readily dissociates into dimers and monomers when expressed in a soluble form. By using a tetramerizing zipper, such as the tetrabrachion (TB) from Staphylothermus marinus, it is possible to stabilize soluble NA in its active tetrameric conformation, an imperative for the optimal induction of protective NA inhibitory antibodies. The impact of repetitive immunizations with TB-stabilized antigens on the immunogenicity of soluble TB-stabilized NA is unknown. We demonstrate that TB is immunogenic in mice. Interestingly, preexisting anti-TB antibodies enhance the anti-NA antibody response induced by immunization with TB-stabilized NA. This immune-enhancing effect was transferable by serum and operated independently of activating Fcγ receptors. We also demonstrate that priming with TB-stabilized NA antigens, enhances the NA inhibitory antibody responses against a heterosubtypic TB-stabilized NA. These findings have implications for the clinical development of oligomeric vaccine antigens that are stabilized by a heterologous oligomerizing domain.

Reverse engineering synthetic antiviral amyloids.

Human amyloids have been shown to interact with viruses and interfere with viral replication. Based on this observation, we employed a synthetic biology approach in which we engineered virus-specific amyloids against influenza A and Zika proteins. Each amyloid shares a homologous aggregation-prone fragment with a specific viral target protein. For influenza we demonstrate that a designer amyloid against PB2 accumulates in influenza A-infected tissue in vivo. Moreover, this amyloid acts specifically against influenza A and its common PB2 polymorphisms, but not influenza B, which lacks the homologous fragment. Our model amyloid demonstrates that the sequence specificity of amyloid interactions has the capacity to tune amyloid-virus interactions while allowing for the flexibility to maintain activity on evolutionary diverging variants.

Bispecific T cell engaging antibody constructs targeting a universally conserved part of the viral M2 ectodomain cure and prevent influenza A virus infection.

The ectodomain of the influenza A matrix protein 2 (M2e) is highly conserved amongst all influenza virus A subtypes. M2e is present on the surface of influenza A virus-infected cells, and therefore a suitable target for broadly protective therapies. We designed bispecific T cell engaging (BiTE®) antibody constructs specific for M2e by genetically fusing a single chain variable fragment (scFv) derived from an M2e-specific murine monoclonal antibody with a CD3ɛ-specific scFv. These so-called FLU BiTE® antibody constructs selectively mediate T cell dependent lysis of M2-expressing and influenza A virus infected cells and protect BALB/c mice against challenge with different influenza A virus subtypes. By humanizing the M2e-binding scFv, we generated human-like FLU BiTE® antibody constructs, with increased in vitro cytotoxic activity and in vivo protective capacity against influenza A virus infection. FLU BiTE® antibody constructs represent a promising new curative and prophylactic treatment option for influenza disease.

Hierarchical and Redundant Roles of Activating FcγRs in Protection against Influenza Disease by M2e-Specific IgG1 and IgG2a Antibodies.

The ectodomain of matrix protein 2 is a universal influenza A virus vaccine candidate that provides protection through antibody-dependent effector mechanisms. Here we compared the functional engagement of Fcγ receptor (FcγR) family members by two M2e-specific monoclonal antibodies (MAbs), MAb 37 (IgG1) and MAb 65 (IgG2a), which recognize a similar epitope in M2e with similar affinities. The binding of MAb 65 to influenza A virus-infected cells triggered all three activating mouse Fcγ receptors in vitro, whereas MAb 37 activated only FcγRIII. The passive transfer of MAb 37 or MAb 65 in wild-type, Fcer1g-/-, Fcgr3-/-, and Fcgr1-/-Fcgr3-/- BALB/c mice revealed the importance of these receptors for protection against influenza A virus challenge, with a clear requirement of FcγRIII for IgG1 MAb 37 being found. We also report that FcγRIV contributes to protection by M2e-specific IgG2a antibodies.IMPORTANCE There is increased awareness that protection by antibodies directed against viral antigens is also mediated by the Fc domain of these antibodies. These Fc-mediated effector functions are often missed in clinical assays, which are used, for example, to define correlates of protection induced by vaccines. The use of antibodies to prevent and treat infectious diseases is on the rise and has proven to be a promising approach in our battle against newly emerging viral infections. It is now also realized that Fcγ receptors significantly enhance the in vivo protective effect of broadly neutralizing antibodies directed against the conserved parts of the influenza virus hemagglutinin. We show here that two M2e-specific monoclonal antibodies with close to identical antigen-binding specificities and affinities have a very different in vivo protective potential that is controlled by their capacity to interact with activating Fcγ receptors.

Long-Lasting Cross-Protection Against Influenza A by Neuraminidase and M2e-based immunization strategies.

There is mounting evidence that in the absence of neutralizing antibodies cross-reactive T cells provide protection against pandemic influenza viruses. Here, we compared protection and CD8+ T cell responses following challenge with H1N1 2009 pandemic and H3N2 viruses of mice that had been immunized with hemagglutinin (HA), neuraminidase (NA) and the extracellular domain of matrix protein 2 (M2e) fused to a virus-like particle (VLP). Mice were challenged a first time with a sublethal dose of H1N1 2009 pandemic virus and, four weeks later, challenged again with an H3N2 virus. Mice that had been vaccinated with HA, NA, NA + M2e-VLP and HA + NA + M2e-VLP were protected against homologous H1N1 virus challenge. Challenged NA and NA + M2e-VLP vaccinated mice mounted CD8+ T cell responses that correlated with protection against secondary H3N2 challenge. HA-vaccinated mice were fully protected against challenge with homologous H1N1 2009 virus, failed to mount cross-reactive CD8+ T cells and succumbed to the second challenge with heterologous H3N2 virus. In summary, NA- and M2e-based immunity can protect against challenge with (homologous) virus without compromising the induction of robust cross-reactive CD8+ T cell responses upon exposure to virus.

A GFP expressing influenza A virus to report in vivo tropism and protection by a matrix protein 2 ectodomain-specific monoclonal antibody.

The severity of influenza-related illness is mediated by many factors, including in vivo cell tropism, timing and magnitude of the immune response, and presence of pre-existing immunity. A direct way to study cell tropism and virus spread in vivo is with an influenza virus expressing a reporter gene. However, reporter gene-expressing influenza viruses are often attenuated in vivo and may be genetically unstable. Here, we describe the generation of an influenza A virus expressing GFP from a tri-cistronic NS segment. To reduce the size of this engineered gene segment, we used a truncated NS1 protein of 73 amino acids combined with a heterologous dimerization domain to increase protein stability. GFP and nuclear export protein coding information were fused in frame with the truncated NS1 open reading frame and separated from each other by 2A self-processing sites. The resulting PR8-NS1(1-73)GFP virus was successfully rescued and replicated as efficiently as the parental PR8 virus in vitro and was slightly attenuated in vivo. Flow cytometry-based monitoring of cells isolated from PR8-NS1(1-73)GFP virus infected BALB/c mice revealed that GFP expression peaked on day two in all cell types tested. In particular respiratory epithelial cells and myeloid cells known to be involved in antigen presentation, including dendritic cells (CD11c+) and inflammatory monocytes (CD11b+ GR1+), became GFP positive following infection. Prophylactic treatment with anti-M2e monoclonal antibody or oseltamivir reduced GFP expression in all cell types studied, demonstrating the usefulness of this reporter virus to analyze the efficacy of antiviral treatments in vivo. Finally, deep sequencing analysis, serial in vitro passages and ex vivo analysis of PR8-NS1(1-73)GFP virus, indicate that this virus is genetically and phenotypically stable.

Single-domain antibodies targeting neuraminidase protect against an H5N1 influenza virus challenge.

UNLABELLED: Influenza virus neuraminidase (NA) is an interesting target of small-molecule antiviral drugs. We isolated a set of H5N1 NA-specific single-domain antibodies (N1-VHHm) and evaluated their in vitro and in vivo antiviral potential. Two of them inhibited the NA activity and in vitro replication of clade 1 and 2 H5N1 viruses. We then generated bivalent derivatives of N1-VHHm by two methods. First, we made N1-VHHb by genetically joining two N1-VHHm moieties with a flexible linker. Second, bivalent N1-VHH-Fc proteins were obtained by genetic fusion of the N1-VHHm moiety with the crystallizable region of mouse IgG2a (Fc). The in vitro antiviral potency against H5N1 of both bivalent N1-VHHb formats was 30- to 240-fold higher than that of their monovalent counterparts, with 50% inhibitory concentrations in the low nanomolar range. Moreover, single-dose prophylactic treatment with bivalent N1-VHHb or N1-VHH-Fc protected BALB/c mice against a lethal challenge with H5N1 virus, including an oseltamivir-resistant H5N1 variant. Surprisingly, an N1-VHH-Fc fusion without in vitro NA-inhibitory or antiviral activity also protected mice against an H5N1 challenge. Virus escape selection experiments indicated that one amino acid residue close to the catalytic site is required for N1-VHHm binding. We conclude that single-domain antibodies directed against influenza virus NA protect against H5N1 virus infection, and when engineered with a conventional Fc domain, they can do so in the absence of detectable NA-inhibitory activity. IMPORTANCE: Highly pathogenic H5N1 viruses are a zoonotic threat. Outbreaks of avian influenza caused by these viruses occur in many parts of the world and are associated with tremendous economic loss, and these viruses can cause very severe disease in humans. In such cases, small-molecule inhibitors of the viral NA are among the few treatment options for patients. However, treatment with such drugs often results in the emergence of resistant viruses. Here we show that single-domain antibody fragments that are specific for NA can bind and inhibit H5N1 viruses in vitro and can protect laboratory mice against a challenge with an H5N1 virus, including an oseltamivir-resistant virus. In addition, plant-produced VHH fused to a conventional Fc domain can protect in vivo even in the absence of NA-inhibitory activity. Thus, NA of influenza virus can be effectively targeted by single-domain antibody fragments, which are amenable to further engineering.

Universal vaccine based on ectodomain of matrix protein 2 of influenza A: Fc receptors and alveolar macrophages mediate protection.

The ectodomain of matrix protein 2 (M2e) of influenza A virus is an attractive target for a universal influenza A vaccine: the M2e sequence is highly conserved across influenza virus subtypes, and induced humoral anti-M2e immunity protects against a lethal influenza virus challenge in animal models. Clinical phase I studies with M2e vaccine candidates have been completed. However, the in vivo mechanism of immune protection induced by M2e-carrier vaccination is unclear. Using passive immunization experiments in wild-type, FcRγ(-/-), FcγRI(-/-), FcγRIII(-/-), and (FcγRI, FcγRIII)(-/-) mice, we report in this study that Fc receptors are essential for anti-M2e IgG-mediated immune protection. M2e-specific IgG1 isotype Abs are shown to require functional FcγRIII for in vivo immune protection but other anti-M2e IgG isotypes can rescue FcγRIII(-/-) mice from a lethal challenge. Using a conditional cell depletion protocol, we also demonstrate that alveolar macrophages (AM) play a crucial role in humoral M2e-specific immune protection. Additionally, we show that adoptive transfer of wild-type AM into (FcγRI, FcγRIII)(-/-) mice restores protection by passively transferred anti-M2e IgG. We conclude that AM and Fc receptor-dependent elimination of influenza A virus-infected cells are essential for protection by anti-M2e IgG.

Universal influenza A M2e-HBc vaccine protects against disease even in the presence of pre-existing anti-HBc antibodies.

The extracellular domain of influenza A virus matrix protein 2 (M2e) is strongly conserved. Therefore, vaccines based on M2e can induce broad-spectrum immunity against influenza. We have mainly used recombinant virus-like particles derived from Hepatitis B virus core (HBc) as carrier for efficacious presentation of the M2e antigen. Here, we address whether pre-existing HBc-specific immunity interferes with the protective immune response obtained by M2e-HBc vaccination. Anti-HBc antibodies were induced by immunizing mice with unsubstituted HBc virus-like particles in the presence of two different adjuvants. We demonstrate that pre-existing HBc-specific antibodies affect neither the induction of M2e-specific antibody responses to vaccination with M2e-HBc particles, nor the protective efficacy of the resulting response. These results suggest that vaccination with M2e-HBc can induce protective anti-M2e antibodies even in anti-HBc positive individuals. The implications of these findings are discussed in the context of the clinical development of an M2e-based universal influenza vaccine, which recently successfully completed a Phase I trial.

E7 properties of mucosal human papillomavirus types 26, 53 and 66 correlate with their intermediate risk for cervical cancer development.

Epidemiological studies have demonstrated that 15 different mucosal human papillomavirus (HPV) types of the genus alpha of the HPV phylogetic tree are classified as high risk for cervical cancer development. Three additional HPV types of the same genus, HPV26, 53 and 66, are classified as probable high-risk types. In this study, we have characterized the biological properties of the E7 oncoproteins from these three HPV types. All of the corresponding E7 proteins were able to associate with retinoblastoma protein (pRb) and up-regulated the expression of several positive cell cycle regulators, i.e. CDK2, cyclin A and cylin E. However, HPV26 E7 appears to be more efficient than HPV53 and 66 E7 in up-regulating the transcription of cyclin A. Unlike E7 from the high-risk type HPV16 protein, HPV26, 53 and 66 did not efficiently promote pRb degradation. In addition, E7 from these viruses was able to promote proliferation of primary human keratinocytes and circumvent G1 arrest imposed by overexpression of p16(INK4a), but with less efficiency than the high-risk HPV16 E7. Together, our data show that in vitro properties of these E7 proteins correlate with the epidemiological classification of HPV26, 53 and 66 as HPV types with an intermediate risk for cervical cancer development.

Skin human papillomavirus type 38 alters p53 functions by accumulation of deltaNp73.

The E6 and E7 of the cutaneous human papillomavirus (HPV) type 38 immortalize primary human keratinocytes, an event normally associated with the inactivation of pathways controlled by the tumour suppressor p53. Here, we show for the first time that HPV38 alters p53 functions. Expression of HPV38 E6 and E7 in human keratinocytes or in the skin of transgenic mice induces stabilization of wild-type p53. This selectively activates the transcription of deltaNp73, an isoform of the p53-related protein p73, which in turn inhibits the capacity of p53 to induce the transcription of genes involved in growth suppression and apoptosis. DeltaNp73 downregulation by an antisense oligonucleotide leads to transcriptional re-activation of p53-regulated genes and apoptosis. Our findings illustrate a novel mechanism of the alteration of p53 function that is mediated by a cutaneous HPV type and support the role of HPV38 and deltaNp73 in human carcinogenesis.

Papillomavirus infection in the conjunctiva of individuals with and without AIDS: an autopsy series from Uganda.

HIV and genus beta human papillomavirus (HPV) types have been associated with squamous cell carcinoma of the conjunctiva (SCC). To determine whether conjunctival HPV infection is associated with AIDS, we analysed 136 lesion-free eye biopsies and tested for genera alpha, beta and gamma HPV types. Only infections with genera beta and gamma HPV types was found. After adjustment for age and gender, no excess of genera beta or gamma HPV infection was found in individuals who had died of or with AIDS compared to those who had died of other infectious diseases [relative risk (RR)=1.3; 95% confidence interval (CI): 0.4-4.8], or chronic diseases or trauma (RR=0.9; 95% CI: 0.3-2.9). Our findings suggest that infection with genera beta or gamma HPV types in lesion-free conjunctivas is common, but not greatly enhanced by the presence of AIDS.

Human papillomavirus type 16 E6 promotes retinoblastoma protein phosphorylation and cell cycle progression.

We show that E6 proteins from benign human papillomavirus type 1 (HPV1) and oncogenic HPV16 have the ability to alter the regulation of the G(1)/S transition of the cell cycle in primary human fibroblasts. Overexpression of both viral proteins induces cellular proliferation, retinoblastoma (pRb) phosphorylation, and accumulation of products of genes that are negatively regulated by pRb, such as p16(INK4a), CDC2, E2F-1, and cyclin A. Hyperphosphorylated forms of pRb are present in E6-expressing cells even in the presence of ectopic levels of p16(INK4a). The E6 proteins strongly increased the cyclin A/cyclin-dependent kinase 2 (CDK2) activity, which is involved in pRb phosphorylation. In addition, mRNA and protein levels of the CDK2 inhibitor p21(WAF1/CIP1) were strongly down-regulated in cells expressing E6 proteins. The down-regulation of the p21(WAF1/CIP1) gene appears to be independent of p53 inactivation, since HPV1 E6 and an HPV16 E6 mutant unable to target p53 were fully competent in decreasing p21(WAF1/CIP1) levels. E6 from HPV1 and HPV16 also enabled cells to overcome the G(1) arrest imposed by oncogenic ras. Immunofluorescence staining of cells coexpressing ras and E6 from either HPV16 or HPV1 revealed that antiproliferative (p16(INK4a)) and proliferative (Ki67) markers were coexpressed in the same cells. Together, these data underline a novel activity of E6 that is not mediated by inactivation of p53.

TP53 mutations in squamous-cell carcinomas of the conjunctiva: evidence for UV-induced mutagenesis.

Squamous cell carcinoma of the conjunctiva is associated with sun exposure and often occurs in HIV-positive individuals. We have analysed TP53 mutations in 21 cases of squamous cell carcinoma and 22 controls with benign conjunctival lesions from a region (Uganda, Africa) with a high prevalence of heavy sun exposure and HIV infection. TP53 mutations were detected in 11 cases (52%) and 3 controls (14%). Seven of the mutations (6 in cases and 1 in controls) were CC-->TT transitions, a molecular signature of mutagenesis by solar UV rays. A similar prevalence (56%) of TP53 mutations was found in 18 squamous cell carcinoma cases positive for epidermodysplasia verruciformis human papillomavirus types. The prevalence of CC-->TT transitions reported here is the highest observed in any cancer type and matches that of skin cancers in subjects with xeroderma pigmentosum, an inherited disease with hypersensitivity to UV damage. These results confirm at the molecular level the causal role of solar UV rays in the aetiology of squamous cell carcinoma of the conjunctiva and suggest that infection with epidermodysplasia verruciformis types of human papillomavirus may act as a cofactor to increase the sensitivity of conjunctiva cells to UV-induced mutagenesis.

Generation of a DNA microarray for determination of E6 natural variants of human papillomavirus type 16.

Infection with high-risk types of human papillomavirus (HPV) is necessary for the development of cervical cancer. However, the majority of the HPV infections are efficiently cleared by the immune system and only a minority persist and induce the development of malignant lesions. Several studies provided evidence that intratype genetic variations are implicated in determining the clinical outcome of HPV infections. In this study, we describe a DNA chip based on arrayed primer extension (APEX) for the analysis of the natural variants of HPV16, the most frequently detected type in cervical cancer world-wide. We show that HPV16 E6 variants are detected efficiently by APEX. In addition, APEX is a valid assay for the simultaneous detection of different HPV16 variants. Thus, it could be used for clinical studies aiming to analyse multiple infections with different HPV16 subtypes. Finally, since APEX combines the advantages of dideoxy DNA sequencing with the high-throughput potential of the microarray, it is suitable for the simultaneous analysis of a number of variable regions of HPV16 genome in large-scale studies.

The role of TP53 in Cervical carcinogenesis.

Functional loss of the tumor suppressor p53 by alterations in its TP53 gene is a frequent event in cancers of different anatomical regions. Cervical cancer is strongly linked to infection by high-risk human papillomavirus (HPV) types. The viral oncoprotein E6 has the ability to associate with and neutralize the function of p53. E6 interacts with a 100-kDa cellular protein, termed E6 associated protein (E6AP; also called ubiquitin-protein ligase E3A or UBE3A), which functions as an ubiquitin protein ligase. The dimeric complex then binds p53 and E6AP catalyzes multi-ubiquitination and degradation of p53. The ability to promote p53 degradation is an exclusive property of E6 from the high-risk HPV types. Indeed, the low-risk E6 proteins lack this activity, although they can bind p53. Consistent with the E6 function of the high-risk HPV types, the majority of cervical cancer cells have a wild-type p53 gene, but the protein levels are strongly decreased. Several independent studies have shown that in a small percentage of cervical tumors the p53 gene is mutated. However, this event appears to be unrelated to the presence or absence of HPV infection and the nature of the tumor.