RESUMO
The degradation of p53 is a hallmark of high-risk human papillomaviruses (HPVs) of the alpha genus and HPV-related carcinogenicity. The oncoprotein E6 forms a ternary complex with the E3 ubiquitin ligase E6-associated protein (E6AP) and tumor suppressor protein p53 targeting p53 for ubiquitination. The extent of p53 degradation by different E6 proteins varies greatly, even for the closely related HPV16 and HPV31. HPV16 E6 and HPV31 E6 display high sequence identity (â¼67%). We report here, for the first time, the structure of HPV31 E6 bound to the LxxLL motif of E6AP. HPV16 E6 and HPV31 E6 are structurally very similar, in agreement with the high sequence conservation. Both E6 proteins bind E6AP and degrade p53. However, the binding affinities of 31 E6 to the LxxLL motif of E6AP and p53, respectively, are reduced 2-fold and 5.4-fold compared to 16 E6. The affinity of E6-E6AP-p53 ternary complex formation parallels the efficacy of the subsequent reaction, namely, degradation of p53. Therefore, closely related E6 proteins addressing the same cellular targets may still diverge in their binding efficiencies, possibly explaining their different phenotypic or pathological impacts.IMPORTANCE Variations of carcinogenicity of human papillomaviruses are related to variations of the E6 and E7 interactome. While different HPV species and genera are known to target distinct host proteins, the fine differences between E6 and E7 of closely related HPVs, supposed to target the same cellular protein pools, remain to be addressed. We compare the oncogenic E6 proteins of the closely related high-risk HPV31 and HPV16 with regard to their structure and their efficiency of ternary complex formation with their cellular targets p53 and E6AP, which results in p53 degradation. We solved the crystal structure of 31 E6 bound to the E6AP LxxLL motif. HPV16 E6 and 31 E6 structures are highly similar, but a few sequence variations lead to different protein contacts within the ternary complex and, as quantified here, an overall lower binding affinity of 31 E6 than 16 E6. These results align with the observed lower p53 degradation potential of 31 E6.
Assuntos
Papillomavirus Humano 31/metabolismo , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Papillomavirus Humano 16/química , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 31/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Especificidade da Espécie , Proteína Supressora de Tumor p53/química , Ubiquitina-Proteína Ligases/químicaRESUMO
Human papillomaviruses (HPV) exhibit constitutive activation of ATM and ATR DNA damage response (DDR) pathways, which are required for productive viral replication. Expression of HPV31 E7 alone is sufficient to activate the DDR through an unknown mechanism. Here, we demonstrate that the E7 Rb binding domain is required to increase levels of many DDR proteins, including ATM, Chk2, Chk1, the MRN components MRE11, Rad50, and NBS1, as well as the homologous recombination repair proteins BRCA1 and Rad51. Interestingly, we have found that the increase in these DNA repair proteins does not occur solely at the level of transcription, but that E7 broadly increases the half-life of these DDR factors, a phenotype that is lost in the E7 Rb binding mutant. These data suggest that HPV-31 upregulates DNA repair factors necessary for replication by increasing protein half-life in a manner requiring the E7 Rb binding domain.
Assuntos
Reparo do DNA , Papillomavirus Humano 31/metabolismo , Proteínas E7 de Papillomavirus/química , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Replicação do DNA , Papillomavirus Humano 31/química , Papillomavirus Humano 31/genética , Humanos , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Domínios Proteicos , Proteína do Retinoblastoma/genética , Replicação ViralRESUMO
Antibodies that specifically recognize the capsid protein (L1) of human papillomavirus (HPV) are an important tool necessary for designing vaccines against HPV infection. In this work, we have predicted and synthesized peptide fragments mimicking B cell epitopes of L1 HPV type 31 (sequences 49-65, 131-145, 172-189, 349-362 and 402-414), and conjugated their to KLH and BSA to generate the L1-31-specific anti-peptide antibodies in mice. Variants of recombinant L1-31, including full-size and mutants with C-terminal single amino acid changes and deletions and full-size L1-16 were produced in the yeast using monitoring with L1 HPV16-specific monoclonal antibody. Testing of anti-peptide antisera in ELISA showed that antibodies to peptides 49-65 and 172-189 were capable to recognize specifically L1-31 protein, but not L1-16 one. Such antibodies may be used for assay of L1-31 production in various expression systems.