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1.
J Hepatol ; 67(4): 687-699, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28600137

RESUMO

BACKGROUND & AIMS: Hepatitis B virus (HBV) RNA can undergo alternative splicing, but the relevance of this post-transcriptional regulation remains elusive. The mechanism of HBV alternative splicing regulation and its impact on liver pathogenesis were investigated. METHODS: HBV RNA-interacting proteins were identified by RNA pull-down, combined with mass spectrometry analysis. HBV splicing regulation was investigated in chemically and surgically induced liver damage, in whole HBV genome transgenic mice and in hepatoma cells. Viral and endogenous gene expression were quantified by quantitative reverse transcription polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay. Resident liver immune cells were studied by fluorescence-activated cell sorting. RESULTS: HBV pregenomic RNA-interacting proteins were identified and 15% were directly related to the splicing machinery. Expression of these splicing factors was modulated in HBV transgenic mice with liver injuries and contributed to an increase of the HBV spliced RNA encoding for HBV splicing-generated protein (HBSP). HBSP transgenic mice with chemically induced liver fibrosis exhibited attenuated hepatic damage. The protective effect of HBSP resulted from a decrease of inflammatory monocyte/macrophage recruitment through downregulation of C-C motif chemokine ligand 2 (CCL2) expression in hepatocytes. In human hepatoma cells, the ability of HBSP to control CCL2 expression was confirmed and maintained in a whole HBV context. Finally, viral spliced RNA detection related to a decrease of CCL2 expression in the livers of HBV chronic carriers underscored this mechanism. CONCLUSION: The microenvironment, modified by liver injury, increased HBSP RNA expression through splicing factor regulation, which in turn controlled hepatocyte chemokine synthesis. This feedback mechanism provides a novel insight into liver immunopathogenesis during HBV infection. Lay summary: Hepatitis B virus persists for decades in the liver of chronically infected patients. Immune escape is one of the main mechanisms developed by this virus to survive. Our study highlights how the crosstalk between virus and liver infected cells may contribute to this immune escape.


Assuntos
Processamento Alternativo , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Processamento Alternativo/imunologia , Animais , Quimiocina CCL2/metabolismo , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Humanos , Evasão da Resposta Imune/genética , Fígado/imunologia , Fígado/lesões , Fígado/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Processamento de RNA/metabolismo , RNA Viral/genética , RNA Viral/metabolismo
2.
J Virol ; 89(2): 1329-39, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25392215

RESUMO

UNLABELLED: In order to develop strategies to prevent HIV-1 (human immunodeficiency virus type 1) transmission, it is crucial to better characterize HIV-1 target cells in the female reproductive tract (FRT) mucosae and to identify effective innate responses. Control of HIV-1 infection in the decidua (the uterine mucosa during pregnancy) can serve as a model to study natural mucosal protection. Macrophages are the main HIV-1 target cells in the decidua. Here we report that in vitro, macrophages and T cells are the main HIV-1 targets in the endometrium in nonpregnant women. As reported for decidual macrophages (dM), endometrial macrophages (eM) were found to have an M2-like phenotype (CD68+ CD163+ CD206+ IL-10high). However, eM and dM may belong to different subpopulations, as they differently express certain markers and secrete different amounts of proinflammatory and anti-inflammatory cytokines. We observed strong expression of the SAMHD1 restriction factor and weak expression of its inactive form (pSAMHD1, phosphorylated at residue Thr592) in both eM and dM. Infection of macrophages from both tissues was enhanced in the presence of the viral protein Vpx, suggesting a role for SAMHD1 in the restriction of HIV-1 infection. This study and further comparisons of the decidua with FRT mucosae in nonpregnant women should help to identify mechanisms of mucosal protection against HIV-1 infection. IMPORTANCE: The female reproductive tract mucosae are major portals of HIV-1 entry into the body. The decidua (uterine mucosa during pregnancy) can serve as a model for studying natural mucosal protection against HIV-1 transmission. A comparison of target cells and innate responses in the decidua versus the endometrium in nonpregnant women could help to identify protective mechanisms. Here, we report for the first time that macrophages are one of the main HIV-1 target cells in the endometrium and that infection of macrophages from both the endometrium and the decidua is restricted by SAMHD1. These findings might have implications for the development of vaccines to prevent HIV-1 mucosal transmission.


Assuntos
Endométrio/imunologia , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Macrófagos/imunologia , Macrófagos/virologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Adulto , Antígenos CD/análise , Feminino , Humanos , Imunofenotipagem , Interleucina-10/análise , Macrófagos/química , Pessoa de Meia-Idade , Proteínas Monoméricas de Ligação ao GTP/imunologia , Proteína 1 com Domínio SAM e Domínio HD , Linfócitos T/imunologia , Linfócitos T/virologia , Adulto Jovem
3.
J Hepatol ; 59(2): 285-91, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23542345

RESUMO

BACKGROUND & AIMS: Conflicting results have been reported regarding the impact of hepatitis B virus X protein (HBx) expression on liver regeneration triggered by partial hepatectomy (PH). In the present report we investigated the mechanisms by which HBx protein alters hepatocyte proliferation after PH. METHODS: PH was performed on a transgenic mouse model in which HBx expression is under the control of viral regulatory elements and liver regeneration was monitored. LPS, IL-6 neutralizing antibody, and SB203580 were injected after PH to evaluate IL-6 participation during liver regeneration. RESULTS: Cell cycle progression of hepatocytes was delayed in HBx transgenic mice compared to WT animals. Moreover, HBx induced higher secretion of IL-6 soon after PH. Upregulation of IL-6 was associated with an elevation of STAT3 phosphorylation, SOCS3 transcript accumulation and a decrease in ERK1/2 phosphorylation in the livers of HBx transgenic mice. The involvement of IL-6 overexpression in cell cycle deregulation was confirmed by the inhibition of liver regeneration in control mice after the upregulation of IL-6 expression using LPS. In addition, IL-6 neutralization with antibodies was able to restore liver regeneration in HBx mice. Finally, the direct role of p38 in IL-6 secretion after PH was demonstrated using SB203580, a pharmacological inhibitor. CONCLUSIONS: HBx is able to induce delayed hepatocyte proliferation after PH, and HBx-induced IL-6 overexpression is involved in delayed liver regeneration. By modulating IL-6 expression during liver proliferation induced by stimulation of the cellular microenvironment, HBx may participate in cell cycle deregulation and progression of liver disease.


Assuntos
Interleucina-6/fisiologia , Regeneração Hepática/fisiologia , Transativadores/fisiologia , Animais , Anticorpos Neutralizantes/administração & dosagem , Ciclo Celular , Proliferação de Células , Elementos Facilitadores Genéticos , Hepatectomia , Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/imunologia , Hepatite B Crônica/patologia , Hepatite B Crônica/virologia , Hepatócitos/imunologia , Hepatócitos/patologia , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Humanos , Imidazóis/administração & dosagem , Interleucina-6/antagonistas & inibidores , Regeneração Hepática/genética , Regeneração Hepática/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Regiões Promotoras Genéticas , Piridinas/administração & dosagem , Transativadores/genética , Proteínas Virais Reguladoras e Acessórias
4.
AIDS ; 37(3): 401-411, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36384811

RESUMO

OBJECTIVE: Liver disease is accelerated in people with HIV (PWH) with hepatitis B virus (HBV) coinfection. We hypothesized that liver fibrosis in HIV-HBV is triggered by increased hepatocyte apoptosis, microbial translocation and/or HIV/HBV viral products. DESIGN: Sera from PWH with HBV coinfection versus from those with HBV only or putative mediators were used to examine the pathogenesis of liver disease in HIV-HBV. METHODS: We applied sera from PWH and HBV coinfection versus HBV alone, or putative mediators (including HMGB1), to primary human hepatic stellate cells (hHSC) and examined pro-fibrogenic changes at the single cell level using flow cytometry. High mobility group box 1 (HMGB1) levels in the applied sera were assessed according to donor fibrosis stage. RESULTS: Quantitative flow cytometric assessment of pro-fibrogenic and inflammatory changes at the single cell level revealed an enhanced capacity for sera from PWH with HBV coinfection to activate hHSC. This effect was recapitulated by lipopolysaccharide, HIV-gp120, hepatocyte conditioned-media and the alarmin HMGB1. Induction of hepatocyte cell death increased their pro-fibrogenic potential, an effect blocked by HMGB1 antagonist glycyrrhizic acid. Consistent with a role for this alarmin, HMGB1 levels were elevated in sera from PWH and hepatitis B coinfection compared to HBV alone and higher in those with HIV-HBV with liver fibrosis compared to those without. CONCLUSIONS: Sera from PWH and HBV coinfection have an enhanced capacity to activate primary hHSC. We identified an increase in circulating HMGB1 which, in addition to HIV-gp120 and translocated microbial products, drove pro-fibrogenic changes in hHSC, as mechanisms contributing to accelerated liver disease in HIV-HBV.


Assuntos
Coinfecção , Infecções por HIV , Proteína HMGB1 , Hepatite B , Humanos , Vírus da Hepatite B , Alarminas , Hepatite B/complicações , Cirrose Hepática/patologia
5.
Sci Transl Med ; 14(640): eabi4670, 2022 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-35417187

RESUMO

A better understanding of mechanisms that regulate CD8+T cell responses to therapeutic vaccines is needed to develop approaches to enhance vaccine efficacy for chronic viral infections and cancers. We show here that NK cell depletion enhanced antigen-specific T cell responses to chimp adenoviral vector (ChAdOx) vaccination in a mouse model of chronic HBV infection (CHB). Probing the mechanism underlying this negative regulation, we observed that CHB drove parallel up-regulation of programmed cell death ligand 1 (PD-L1) on liver-resident NK cells and programmed cell death 1 (PD-1) on intrahepatic T cells. PD-L1-expressing liver-resident NK cells suppressed PD-1hiCD8+T cells enriched within the HBV-specific response to therapeutic vaccination. Cytokine activation of NK cells also induced PD-L1, and combining cytokine activation with PD-L1 blockade resulted in conversion of NK cells into efficient helpers that boosted HBV-specific CD8+T cell responses to therapeutic vaccination in mice and to chronic infection in humans. Our findings delineate an immunotherapeutic combination that can boost the response to therapeutic vaccination in CHB and highlight the broader importance of PD-L1-dependent regulation of T cells by cytokine-activated NK cells.


Assuntos
Antígeno B7-H1 , Vacinas , Animais , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos , Citocinas/metabolismo , Células Matadoras Naturais , Camundongos , Receptor de Morte Celular Programada 1/metabolismo
6.
Retrovirology ; 8: 58, 2011 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-21767373

RESUMO

BACKGROUND: Maternofetal transmission (MFT) of HIV-1 is relatively rare during the first trimester of pregnancy despite the permissivity of placental cells for cell-to-cell HIV-1 infection. Invasive placental cells interact directly with decidual cells of the uterine mucosa during the first months of pregnancy, but the role of the decidua in the control of HIV-1 transmission is unknown. RESULTS: We found that decidual mononuclear cells naturally produce low levels of IL-10, IL-12, IL-15, TNF-α, IFN-α, IFN-γ and CXCL-12 (SDF-1), and large amounts of CCL-2 (MCP1), CCL-3 (MIP-1α), CCL-4 (MIP-1ß), CCL-5 (Rantes), CXCL-10 (IP-10), IL-6 and IL-8. CCL-3 and CCL-4 levels were significantly upregulated by in vitro infection with R5 HIV-1 but not X4. Decidual CD14+ antigen presenting cells were the main CCL-3 and CCL-4 producers among decidual leukocytes. R5 and X4 HIV-1 infection was inhibited by decidual cell culture supernatants in vitro. Using HIV-1 pseudotypes, we found that inhibition of the HIV-1 entry step was inhibited by decidual soluble factors. CONCLUSION: Our findings show that decidual innate immunity (soluble factors) is involved in the control of HIV-1 infection at the maternofetal interface. The decidua could thus serve as a mucosal model for identifying correlates of protection against HIV-1 infection.


Assuntos
Citocinas/imunologia , Decídua/imunologia , Infecções por HIV/transmissão , HIV-1/fisiologia , Transmissão Vertical de Doenças Infecciosas , Complicações Infecciosas na Gravidez/imunologia , Citocinas/genética , Decídua/virologia , Feminino , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Gravidez , Complicações Infecciosas na Gravidez/virologia
7.
J Clin Transl Hepatol ; 8(4): 359-370, 2020 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-33447518

RESUMO

Background and Aims: To better understand nonalcoholic steatohepatitis (NASH) disease progression and to evaluate drug targets and compound activity, we undertook the development of an in vitro 3D model to mimic liver architecture and the NASH environment. Methods: We have developed an in vitro preclinical 3D NASH model by coculturing primary human hepatocytes, human stellate cells, liver endothelial cells and Kupffer cells embedded in a hydrogel of rat collagen on a 96-well plate. A NASH-like environment was induced by addition of medium containing free fatty acids and tumor necrosis factor-α. This model was then characterized by biochemical, imaging and transcriptomics analyses. Results: We succeeded in defining suitable culture conditions to maintain the 3D coculture for up to 10 days in vitro, with the lowest level of steatosis and reproducible low level of inflammation and fibrosis. NASH disease was induced with a custom medium mimicking NASH features. The cell model exhibited the key NASH disease phenotypes of hepatocyte injury, steatosis, inflammation, and fibrosis. Hepatocyte injury was highlighted by a decrease of CYP3A4 expression and activity, without loss of viability up to day 10. Moreover, the model was able to stimulate a stable inflammatory and early fibrotic environment, with expression and secretion of several cytokines. A global gene expression analysis confirmed the NASH induction. Conclusions: This is a new in vitro model of NASH disease consisting of four human primary cell-types that exhibits most features of the disease. The 10-day cell viability and cost effectiveness of the model make it suitable for medium throughput drug screening and provide attractive avenues to better understand disease physiology and to identify and characterize new drug targets.

8.
Virology ; 462-463: 273-82, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24999840

RESUMO

The Hepatitis B virus Precore protein, present in the secretory pathway as the HBeAg precursor, can associate in the cytoplasm with the Core protein to form heterocapsids, likely to favor viral persistence. Core and Precore proteins share their primary sequence except for ten additional aminoacids at the N-terminus of Precore. To address the role of this propeptide sequence in the formation of Precore heterocapsids, we designed a Precore mutant in which the two propeptide tryptophans are replaced by glycines. This mutant retains the properties of the wild-type Precore, notably cell trafficking and ability to interact with Core. However, it is not incorporated into heterocapsids and forms stable dimers distinct from the labile HBe dimers and the presumably Core-like dimers assembled into heterocapsids. Our data highlights the essential role of Precore׳s propeptide in switching between different conformations for different functions and pinpoint the propeptide Tryptophan residues as central in these properties.


Assuntos
Sequência Conservada , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Multimerização Proteica , Precursores de Proteínas/metabolismo , Substituição de Aminoácidos , Linhagem Celular , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica , Precursores de Proteínas/genética
9.
Front Microbiol ; 5: 316, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25071732

RESUMO

Maternofetal pathogen transmission is partially controlled at the level of the maternal uterine mucosa at the fetal implantation site (the decidua basalis), where maternal and fetal cells are in close contact. Toll-like receptors (TLRs) may play an important role in initiating rapid immune responses against pathogens in the decidua basalis, however the tolerant microenvironment should be preserved in order to allow fetal development. Here we investigated the expression and functionality of TLRs expressed by decidual macrophages (dMs) and NK cells (dNKs), the major decidual immune cell populations. We report for the first time that both human dMs and dNK cells express mRNAs encoding TLRs 1-9, albeit with a higher expression level in dMs. TLR2, TLR3, and TLR4 protein expression checked by flow cytometry was positive for both dMs and dNK cells. In vitro treatment of primary dMs and dNK cells with specific TLR2, TLR3, TLR4, TLR7/8, and TLR9 agonists enhanced their secretion of pro- and anti-inflammatory cytokines, as well as cytokines and chemokines involved in immune cell crosstalk. Only dNK cells released IFN-γ, whereas only dMs released IL-1ß, IL-10, and IL-12. TLR9 activation of dMs resulted in a distinct pattern of cytokine expression compared to the other TLRs. The cytokine profiles expressed by dMs and dNK cells upon TLR activation are compatible with maintenance of the fetotolerant immune environment during initiation of immune responses to pathogens at the maternofetal interface.

10.
PLoS One ; 7(1): e30017, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22242197

RESUMO

During the first trimester of human pregnancy, Natural Killer (NK) cells of the maternal uterine mucosa (e.g. decidua) have a unique phenotype and are involved in crucial physiological processes during pregnancy. We investigated whether modifications of the NK receptor repertoire occur during the first trimester of pregnancy. We found significantly decreased expression of KIR2DL1/S1 and KIR2DL2/L3/S2 receptors, NKp30 and NKp44 activatory receptors, and the CD85j (ILT-2) inhibitory receptor. We also observed significantly increased expression of the NKG2D activatory receptor at the decidual NK cell surface. By flow cytometry, we further highlighted an evolution of NK subsets between 8 and 12 weeks of gestation, with a shift from the KIR2DL1/S1⁺/KIR2DL2/L3/S2⁺ subset towards the double negative subset, coupled with a decrease of the CD85j⁺/NKG2D⁻ subset in favour of the CD85j⁻/NKG2D⁺ subset. Furthermore, cell surface expression of NK receptor ligands, including CD85j and NKG2D ligands, has been characterized by flow cytometry on decidual immune CD14⁺ and CD3⁺ cells. HLA-G, the high affinity ligand of CD85j, was detected on both cell types. In contrast, NKG2D ligands ULBP-2 ULBP-3 and MICA/B were not expressed on CD14⁺ and CD3⁺ cells, however a variable expression of ULBP-1 was observed. The ligand expression of KIR2DL1/S1 and KIR2DL2/L3/S2 was also analyzed: the HLA-C molecule was expressed at a low level on some CD14⁺ cells whereas it was not detected on CD3⁺ cell surface. NK receptor ligands are known to be also expressed on the invading placental trophoblast cells. Thus, the phenotypic evolutions of decidual NK cells described in this present study may preserve their activation/inhibition balance during the first trimester of pregnancy.


Assuntos
Antígenos CD/metabolismo , Decídua/citologia , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Primeiro Trimestre da Gravidez/imunologia , Receptores Imunológicos/metabolismo , Apresentação de Antígeno/imunologia , Complexo CD3/metabolismo , Membrana Celular/metabolismo , Feminino , Humanos , Células Matadoras Naturais/citologia , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Ligantes , Receptores de Lipopolissacarídeos/metabolismo , Gravidez , Estatísticas não Paramétricas , Linfócitos T/citologia , Linfócitos T/imunologia , Doadores de Tecidos
11.
Virology ; 431(1-2): 21-8, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22664356

RESUMO

Defective hepatitis B virus (dHBV) generated from spliced RNA is detected in the sera of HBV-chronic carriers. Our study was designed to determine whether the proportion of dHBV changed during the course of infection, and to investigate whether dHBV might interfere with HBV replication. To achieve this, HBV wild-type and dHBV levels were determined by Q-PCR in sera from 56 untreated chronic patients and 23 acute patients, in sequential samples from 4 treated-patients and from liver-humanized mice after HBV infection. The proportion of dHBV was higher in patients with severe compared to null/moderate liver disease or with acute infection. Follow-up showed that the proportion of dHBV increased during disease progression. By contrast, a low and stable proportion of dHBV was observed in the humanized-mouse model of HBV infection. Our results highlight a regulation of the proportion of dHBV during liver disease progression that is independent of interference with viral replication.


Assuntos
Vírus Defeituosos/crescimento & desenvolvimento , Vírus da Hepatite B/crescimento & desenvolvimento , Hepatite B Crônica/virologia , Fígado/virologia , Replicação Viral , Adulto , Animais , Vírus Defeituosos/isolamento & purificação , Modelos Animais de Doenças , Feminino , Vírus da Hepatite B/isolamento & purificação , Humanos , Fígado/patologia , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Soro/virologia , Carga Viral
12.
J Biol Chem ; 283(47): 32352-60, 2008 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-18805786

RESUMO

The hepatitis B virus precore protein is closely related to the nucleocapsid core protein but is processed distinctly in the cell and plays a different role in the viral cycle. Precore is addressed to the endoplasmic reticulum (ER) through a signal peptide, and the form present in the ER is the P22 protein. P22 is then cleaved in its C-terminal part to be secreted as HBe antigen. In addition, a cytosolic form of 22 kDa less characterized has been observed. Precore gene was shown to be implicated in viral persistence, but until now, the actual protein species involved has not been determined. Our work focuses on the cytosolic form of precore. Using human cells expressing precore and a convenient fractionation assay, we demonstrated that the cytosolic form is identical to the ER form and retrotransported in the cytoplasm through the ER-associated degradation pathway. This cellular machinery translocates misfolded proteins to the cytoplasm, where they are ubiquitinated on lysine residues and degraded by proteasome. We showed that precore escapes proteasome due to its low lysine content and accumulates in the cytosol. The role of this retrotransport was investigated. In the presence of precore, we found a specific redistribution of the Grp78/BiP chaperone protein to cytosol and demonstrated a specific interaction between precore and Grp78/BiP. Altogether, these data support the idea that the hepatitis B virus develops a strategy to take advantage of the ER-associated degradation pathway, allowing distinct subcellular localization and probably distinct roles for the viral precore protein.


Assuntos
Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas Virais/química , Transporte Biológico , Linhagem Celular , Citoplasma/metabolismo , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/química , Humanos , Modelos Biológicos , Modelos Genéticos , Chaperonas Moleculares/metabolismo , Ligação Proteica , Desnaturação Proteica , Transporte Proteico , Proteínas do Core Viral/química
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