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1.
Arch Virol ; 165(3): 583-592, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31927635

RESUMO

Interferon lambda was discovered in recent years to be an antiviral agent, and research on different aspects of this antiviral factor in viral infection and investigations of its effectiveness are also progressing. The immunological effects of interferon lambda on different cell populations is not precisely known, which may be due to its use of a heterodimeric receptor consisting of IL-10R2 and IFN-λR1, which are not broadly expressed in all types of cells. In the present study, signaling by interferon lambda and its effect on the expression of hepatitis C virus (HCV) proteins were measured, and the expression pattern of some antiviral proteins and IL-10 levels were investigated in peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from 50 patients with chronic genotype 1a HCV infection and 10 healthy individuals as controls. The PBMCs were treated with various doses of interferon lambda at different times of cultivation. Real-time PCR was used for relative quantification of Mxa, PKR, OAS, ISG15 and HCV core mRNAs. Expression of the NS5A protein was measured by flow cytometry, and IL-10 production was assessed by ELISA. A significant increase in the expression of mRNA encoding antiviral proteins and a decrease in the expression of mRNAs encoding the HCV core protein were observed when cells were treated with interferon lambda in an intermittent manner. The expression of HCV NS5A protein and interleukin 10 levels were also lower than in the control group. It was shown that the maximum antiviral effect of interferon lambda in PBMCs is dependent on the dose and treatment time.


Assuntos
Hepatite C Crônica/imunologia , Interferons/farmacologia , Interleucinas/farmacologia , Leucócitos Mononucleares/imunologia , Proteínas do Core Viral/biossíntese , Proteínas não Estruturais Virais/biossíntese , Adulto , Antivirais/farmacologia , Linhagem Celular , Hepacivirus/genética , Hepatite C Crônica/virologia , Humanos , Interferon-alfa/imunologia , Interferon-alfa/farmacologia , Interferons/imunologia , Interleucina-10/biossíntese , Interleucinas/imunologia , Leucócitos Mononucleares/metabolismo , Pessoa de Meia-Idade , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Proteínas do Core Viral/genética
2.
Arch Virol ; 164(11): 2747-2759, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31502079

RESUMO

RNA silencing is a major antiviral mechanism in plants, which is counteracted by virus-encoded proteins with silencing suppression activity. ORFs encoding putative silencing suppressor proteins that share no structural or sequence homology have been identified in the genomes of four criniviruses. In this study, we investigated the RNA silencing suppression activity of several proteins encoded by the RNA1 (RdRp, p22) and RNA2 (CP, CPm and p26) of cucurbit chlorotic yellows virus (CCYV) using co-agroinfiltration assays on Nicotiana benthamiana plants. Our results indicate that p22 is a suppressor of local RNA silencing that does not interfere with cell-to-cell movement of the RNA silencing signal or with systemic silencing. Furthermore, comparisons of the suppression activity of CCYV p22 with that of two other well-known crinivirus suppressors (CYSDV p25 and ToCV p22) revealed that CCYV p22 is a weaker suppressor of local RNA silencing than the other two proteins. Finally, a comparative sequence analysis of the p22 genes of seven Greek CCYV isolates was performed, revealing a high level of conservation. Taken together, our research advances our knowledge about plant-virus interactions of criniviruses, an emergent group of pathogens that threatens global agriculture.


Assuntos
Crinivirus/genética , Interferência de RNA/fisiologia , RNA Viral/genética , Tabaco/virologia , Proteínas do Core Viral/genética , Genoma Viral/genética , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/virologia
3.
Nucleic Acids Res ; 47(17): 9231-9242, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31396624

RESUMO

Some viruses package dsDNA together with large amounts of positively charged proteins, thought to help condense the genome inside the capsid with no evidence. Further, this role is not clear because these viruses have typically lower packing fractions than viruses encapsidating naked dsDNA. In addition, it has recently been shown that the major adenovirus condensing protein (polypeptide VII) is dispensable for genome encapsidation. Here, we study the morphology and mechanics of adenovirus particles with (Ad5-wt) and without (Ad5-VII-) protein VII. Ad5-VII- particles are stiffer than Ad5-wt, but DNA-counterions revert this difference, indicating that VII screens repulsive DNA-DNA interactions. Consequently, its absence results in increased internal pressure. The core is slightly more ordered in the absence of VII and diffuses faster out of Ad5-VII- than Ad5-wt fractured particles. In Ad5-wt unpacked cores, dsDNA associates in bundles interspersed with VII-DNA clusters. These results indicate that protein VII condenses the adenovirus genome by combining direct clustering and promotion of bridging by other core proteins. This condensation modulates the virion internal pressure and DNA release from disrupted particles, which could be crucial to keep the genome protected inside the semi-disrupted capsid while traveling to the nuclear pore.


Assuntos
Adenoviridae/genética , Proteínas do Capsídeo/genética , DNA Viral/genética , Proteínas do Core Viral/genética , Genoma Viral/genética , Humanos , Proteínas Virais/genética , Vírion/genética , Montagem de Vírus
4.
Virol J ; 16(1): 101, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399103

RESUMO

BACKGROUND: Current HCV treatments are genotype specific although potential pan-genotype treatments have recently been described. Therefore, genotyping is an essential tool for the therapeutic management of HCV infection and a variety of technologies have been developed for HCV genotypes determination. Sequences analysis of HCV sub-genomic regions is considered as gold standard and is widely used for HCV genotyping. Here, we compared HCV genotyping using core and NS5B regions in routine practice in HCV-positive Cameroonian patients. METHODS: All plasma samples received at Centre Pasteur of Cameroon (CPC) in 2016 for HCV genotyping were included. Viral loads were determined using the Abbott Real Time assay. Further, genotyping was based on the amplification and sequencing of core and NS5B regions following by phylogenetic analysis of corresponding sequences. RESULTS: A total of 369 samples were received during the study period with high viral load values (median: 930,952 IU/ml; IQR: 281,833-2,861,179). Positive amplification was obtained in at least one genomic region (core or NS5B) for all the samples with similar amplification rate in the two genomic regions (p = 0.34). Phylogenetic analysis showed that among the 369 samples, 146 (39.6%) were classified as genotype 4, 132 (35.8%) as genotype 1, 89 (24.1%) as genotype 2, in both core and NS5B regions. Interestingly, for two samples (0.54%) discordant genotypes were obtained in both regions with the core region classified as genotype 4 while the NS5B was identified as genotype 1 indicating the presence of putative HCV recombinant virus or multiple infections in these samples. Discrimination of HCV subtypes was most likely possible with NS5B compared to core region. CONCLUSIONS: We found high amplification rates of HCV in both core and NS5B regions, and a good concordance was obtained at genotype level using both regions except for two samples where putative 1-4 recombinants/multiple infections were detected. Therefore, HCV genotyping based on at least two genomic regions could help to identify putative recombinants and improve therapeutic management of HCV infection.


Assuntos
Técnicas de Genotipagem , Hepacivirus/genética , Hepatite C/virologia , Proteínas do Core Viral/genética , Proteínas não Estruturais Virais/genética , Idoso , Camarões , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Análise de Sequência de DNA , Carga Viral
5.
Emerg Microbes Infect ; 8(1): 989-999, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31267843

RESUMO

It has recently been proposed that the Eurasian avian-like H1N1 (EA H1N1) swine influenza virus (SIV) is one of the most likely zoonotic viruses to cause the next influenza pandemic. Two main genotypes EA H1N1 viruses have been recognized to be infected humans in China. Our study finds that one of the genotypes JS1-like viruses are avirulent in mice. However, the other are HuN-like viruses and are virulent in mice. The molecular mechanism underlying this difference shows that the NP gene determines the virulence of the EA H1N1 viruses in mice. In addition, a single substitution, Q357K, in the NP protein of the EA H1N1 viruses alters the virulence phenotype. This substitution is a typical human signature marker, which is prevalent in human viruses but rarely detected in avian influenza viruses. The NP-Q357K substitution is readily to be occurred when avian influenza viruses circulate in pigs, and may facilitate their infection of humans and allow viruses also carrying NP-357K to circulate in humans. Our study demonstrates that the substitution Q357K in the NP protein plays a key role in the virulence phenotype of EA H1N1 SIVs, and provides important information for evaluating the pandemic risk of field influenza strains.


Assuntos
Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/virologia , Mutação de Sentido Incorreto , Infecções por Orthomyxoviridae/veterinária , Proteínas de Ligação a RNA/genética , Doenças dos Suínos/virologia , Proteínas do Core Viral/genética , Animais , China , Feminino , Genótipo , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/virologia , Filogenia , Proteínas de Ligação a RNA/metabolismo , Suínos , Proteínas do Core Viral/metabolismo , Virulência , Replicação Viral
6.
PLoS One ; 14(5): e0217691, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31150476

RESUMO

By comparing and measuring covariations of viral protein sequences from isolates of the 2009 pH1N1 influenza A virus (IAV), specific substitutions that co-occur in the NP-NA pair were identified. To investigate the effect of these co-occurring substitution pairs, the V100I substitution in NP and the D248N substitution in NA were introduced into laboratory-adapted WSN IAVs. The recombinant WSN with the covarying NPV100I-NAD248N pair exhibited enhanced pathogenicity, as characterized by increased viral production, increased death and inflammation of host cells, and high mortality in infected mice. Although direct interactions between the NPV100I and NAD248N proteins were not detected, the RNA-binding ability of NPV100I was increased, which was further strengthened by NAD248N, in expression-plasmid-transfected cells. Additionally, the NAD248N protein was frequently recruited within lipid rafts, indirectly affecting the RNA-binding ability of NP as well as viral release. Altogether, our data indicate that the covarying NPV100I-NAD248N pair obtained from 2009 pH1N1 IAV sequence information function together to synergistically augment viral assembly and release, which may explain the observed enhanced viral pathogenicity.


Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/genética , Proteínas do Core Viral/genética , Proteínas Virais/genética , Animais , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/virologia , Camundongos , Replicação Viral/genética
7.
Nucleic Acids Res ; 47(11): 5837-5851, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31066445

RESUMO

Ebola virus (EBOV) is a non-segmented, negative-sense RNA virus (NNSV) in the family Filoviridae, and is recognized as one of the most lethal pathogens in the planet. For RNA viruses, cellular or virus-encoded RNA helicases play pivotal roles in viral life cycles by remodelling viral RNA structures and/or unwinding viral dsRNA produced during replication. However, no helicase or helicase-like activity has ever been found to associate with any NNSV-encoded proteins, and it is unknown whether the replication of NNSVs requires the participation of any viral or cellular helicase. Here, we show that despite of containing no conserved NTPase/helicase motifs, EBOV VP35 possesses the NTPase and helicase-like activities that can hydrolyse all types of NTPs and unwind RNA helices in an NTP-dependent manner, respectively. Moreover, guanidine hydrochloride, an FDA-approved compound and inhibitor of certain viral helicases, inhibited the NTPase and helicase-like activities of VP35 as well as the replication/transcription of an EBOV minigenome replicon in cells, highlighting the importance of VP35 helicase-like activity during EBOV life cycle. Together, our findings provide the first demonstration of the NTPase/helicase-like activity encoded by EBOV, and would foster our understanding of EBOV and NNSVs.


Assuntos
Ebolavirus/genética , Ebolavirus/metabolismo , Doença pelo Vírus Ebola/virologia , Nucleoproteínas/fisiologia , RNA de Cadeia Dupla/química , Proteínas do Core Viral/fisiologia , Trifosfato de Adenosina/química , Motivos de Aminoácidos , Células Cultivadas , DNA Helicases/metabolismo , Humanos , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Nucleosídeo-Trifosfatase/genética , Ligação Proteica , RNA Helicases/metabolismo , RNA Viral/metabolismo , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
8.
FEBS Open Bio ; 9(6): 1042-1051, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31021526

RESUMO

Viral suppressors of RNA silencing (VSRSs) are a diverse group of viral proteins that have evolved to disrupt eukaryotic RNA silencing pathways, thereby contributing to viral pathogenicity. The p19 protein is a VSRS that selectively binds to short interfering RNAs (siRNAs) over microRNAs (miRNAs). Mutational analysis has identified single amino acid substitutions that reverse this selectivity through new high-affinity interactions with human miR-122. Herein, we report crystal structures of complexed p19-T111S (2.6 Å), p19-T111H (2.3 Å) and wild-type p19 protein (2.2 Å) from the Carnation Italian ringspot virus with small interfering RNA (siRNA) ligands. Structural comparisons reveal that these mutations do not lead to major changes in p19 architecture, but instead promote subtle rearrangement of residues and solvent molecules along the p19 midline. These observations suggest p19 uses many small interactions to distinguish siRNAs from miRNAs and perturbing these interactions can create p19 variants with novel RNA-recognition properties. DATABASE: Model data are deposited in the PDB database under the accession numbers 6BJG, 6BJH and 6BJV.


Assuntos
Proteínas Mutantes/química , Interferência de RNA , RNA Interferente Pequeno/química , Tombusvirus , Proteínas do Core Viral/química , Sítios de Ligação/genética , Células Cultivadas , Cristalização , Cristalografia por Raios X , Escherichia coli/citologia , Humanos , Ligações de Hidrogênio , MicroRNAs/química , Mutação Puntual , Ligação Proteica , Engenharia de Proteínas/métodos , Estrutura Secundária de Proteína , RNA de Cadeia Dupla , Proteínas do Core Viral/genética
9.
J Virol ; 93(9)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30814281

RESUMO

The influenza C virus (ICV) is a human-pathogenic agent, and the infections are frequently identified in children. Compared to influenza A and B viruses, the nucleoprotein of ICV (NPC) has an extended C-terminal region of which the functional significance is ill defined. We observed that the nuclear localization signals (NLSs) found on the nucleoproteins of influenza A and B virus subtypes are absent at corresponding positions on ICV. Instead, we found that a long bipartite nuclear localization signal resides at the extended C-terminal region, spanning from R513 to K549. Our experimental data determined that the KKMK motif within this region plays important roles in both nuclear import and polymerase activity. Similar to the influenza A viruses, NPC also binds to multiple human importin α isoforms. Taken together, our results enhance the understanding of the virus-host interaction of the influenza C virus.IMPORTANCE As a member of the Orthomyxoviridae family, the polymerase complex of the influenza C virus structurally resembles its influenza A and influenza B virus counterparts, but the nucleoprotein differs by possessing an extra C-terminal region. We have characterized this region in view of nuclear import and interaction with the importin α protein family. Our results demonstrate the functional significance of a previously uncharacterized region on Orthomyxoviridae nucleoprotein (NP). Based on this work, we propose that importin α binding to influenza C virus NP is regulated by a long bipartite nuclear localization signal. Since the sequence of influenza D virus NP shares high homology to that of the influenza C virus, this work will also shed light on how influenza D virus NP functions.


Assuntos
Núcleo Celular/metabolismo , Influenzavirus C/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas do Core Viral/metabolismo , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Núcleo Celular/genética , Núcleo Celular/virologia , Células HEK293 , Humanos , Influenzavirus C/genética , Domínios Proteicos , Ribonucleoproteínas/genética , Proteínas do Core Viral/genética , alfa Carioferinas/genética , alfa Carioferinas/metabolismo
10.
World J Gastroenterol ; 25(1): 42-58, 2019 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-30643357

RESUMO

Hepatocellular carcinoma (HCC) is the fifth most common cancer, and hepatitis C virus (HCV) infection plays a major role in HCC development. The molecular mechanisms by which HCV infection leads to HCC are varied. HCV core protein is an important risk factor in HCV-associated liver pathogenesis and can modulate several signaling pathways involved in cell cycle regulation, cell growth promotion, cell proliferation, apoptosis, oxidative stress and lipid metabolism. The dysregulation of signaling pathways such as transforming growth factor ß (TGF-ß), vascular endothelial growth factor (VEGF), Wnt/ß-catenin (WNT), cyclooxygenase-2 (COX-2) and peroxisome proliferator-activated receptor α (PPARα) by HCV core protein is implicated in the development of HCC. Therefore, it has been suggested that this protein be considered a favorable target for further studies in the development of HCC. In addition, considering the axial role of these signaling pathways in HCC, they are considered druggable targets for cancer therapy. Therefore, using strategies to limit the dysregulation effects of core protein on these signaling pathways seems necessary to prevent HCV-related HCC.


Assuntos
Carcinoma Hepatocelular/patologia , Hepacivirus/patogenicidade , Neoplasias Hepáticas/patologia , Transdução de Sinais , Proteínas do Core Viral/metabolismo , Apoptose , Carcinoma Hepatocelular/virologia , Proliferação de Células , Genoma Viral/genética , Hepacivirus/genética , Hepacivirus/metabolismo , Humanos , Fígado/patologia , Fígado/virologia , Neoplasias Hepáticas/virologia , Proteínas do Core Viral/genética
11.
J Vet Med Sci ; 81(3): 383-388, 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30674743

RESUMO

RNA interference (RNAi) can inhibit Influenza A virus (IAV) infection in a gene-specific manner. In this study, we constructed a transgene expressing a short hairpin RNA (shRNA) that targets the noncoding region of the IAV RNA gene encoding nucleoprotein (NP). To investigate the antiviral effects of the shRNA, we generated two transgenic mouse lines with this transgene. Unfortunately, there was no apparent difference in IAV resistance between transgenic and non-transgenic littermates. To further investigate the antiviral effects of the shRNA, we prepared mouse embryonic fibroblasts (MEFs) from transgenic and non-transgenic mice. In experimental infections using these MEFs, virus production of mouse-adapted IAV strain A/Puerto Rico/8/1934 (PR8) in the transgenic MEFs was suppressed by means of the down-regulation of the viral RNA gene transcription in the early stages of infection in comparison with non-transgenic MEFs. These results indicated that expression of the shRNA was able to confer antiviral properties against IAVs to MEFs, although the effects were limited. Our findings suggest that the shRNA targeting the noncoding region of the viral RNA (vRNA) of NP might be a supporting tool in developing influenza-resistant poultry.


Assuntos
Antivirais/uso terapêutico , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Infecções por Orthomyxoviridae/tratamento farmacológico , RNA Interferente Pequeno/uso terapêutico , Animais , Camundongos , Camundongos Transgênicos , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas do Core Viral/antagonistas & inibidores , Proteínas do Core Viral/genética
12.
J Clin Microbiol ; 57(1)2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30381417

RESUMO

Little is known about the effects of virus- and host-related factors on hepatocarcinogenesis in patients who show viral clearance after HCV RNA eradication by direct-acting antivirals (DAAs). The subjects of this retrospective study were 1,922 patients with HCV genotype 1 (HCV-1)- or HCV-2-related chronic liver disease who showed a sustained virological response (SVR; defined as negative results for HCV RNA at 12 weeks after the cessation of all-oral DAAs). All patients were confirmed to be hepatocellular carcinoma (HCC) free before and during DAAs. HCC was diagnosed in 43 patients during the follow-up, with an incidence rate per 1,000 person years of 9.44. The cumulative HCC rates were 1.2, 2.0, and 3.1% at the end of 1, 2, and 3 years, respectively. The annual rate of HCC during the first 3 years was 1.0%. The incidence rate was significantly higher in patients infected with the HCV-1b core amino acid (aa) 70 mutant than in those infected with HCV-2a/2b, and the rate in patients infected with the HCV-1b core aa 70 wild type tended to be higher than that in patients infected with HCV-2a/2b. The rate in patients infected with the HCV-1b NS5A aa 93 mutant was significantly higher than that in patients infected with HCV-2a/2b. However, the rate was not different between patients infected with the IL28B rs8099917 TT genotype and patients infected with the non-TT genotype. Multivariate analysis identified a Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA+M2BP) cutoff index (COI) of ≥2.5 and infection with the HCV-1b core aa 70 mutant subgroup to be pretreatment predictors of posttreatment HCC. The same analysis identified an alpha-fetoprotein concentration of ≥5 µg/liter and an WFA+M2BP COI of ≥1.0 to be predictors of HCC at 24 weeks after the end of antiviral therapy. We conclude that both virus- and host-related factors seem to influence the development of HCC after HCV RNA eradication.


Assuntos
Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/virologia , Hepacivirus/patogenicidade , Hepatite C Crônica/complicações , Hepatite C Crônica/virologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antivirais/uso terapêutico , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/epidemiologia , Feminino , Genótipo , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/epidemiologia , Interações entre Hospedeiro e Microrganismos , Humanos , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/epidemiologia , Masculino , Pessoa de Meia-Idade , RNA Viral/sangue , RNA Viral/efeitos dos fármacos , Estudos Retrospectivos , Fatores de Risco , Proteínas do Core Viral/genética , Proteínas não Estruturais Virais/genética , Adulto Jovem
13.
Int Immunol ; 31(4): 199-209, 2019 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-30462215

RESUMO

The induction of a dominant Th2-type response is the main cause of harmful inflammation in respiratory syncytial virus (RSV) vaccine trials. A balanced Th1 versus Th2 immune response is needed for a safe and effective RSV vaccine. In this study, we evaluated the potential of a recombinant protein SBP-FG as a vaccine candidate with the main focus on shifting the harmful Th2 response to a Th1 response. SBP-FG consists of epitopes from RSV fusion (F) and attachment (G) proteins conjugated to the N-terminus of HBsAg-binding protein (SBP). SBP-FG induced significantly stronger immune responses assessed at the level of total IgG, IgA and neutralizing antibodies as compared with formalin-inactivated RSV (FI-RSV) and live RSV. Analysis of IgG isotypes, lung cytokines and T helper cells showed that SBP-FG induced a dominant Th1-type response. Further, SBP-FG immunized mice showed significantly reduced lung eosinophilia, reduced viral multiplication in lungs after challenge infection and provided protection against RSV infection. These results suggest that SBP-FG can be developed into a safe and effective vaccine against RSV. However, more studies are required to further evaluate SBP-FG as a potent vaccine candidate against RSV.


Assuntos
Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/fisiologia , Células Th1/imunologia , Células Th2/imunologia , Proteínas do Core Viral/genética , Proteínas Virais de Fusão/genética , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Células Cultivadas , Citocinas/metabolismo , Antígenos de Superfície da Hepatite B/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética , Equilíbrio Th1-Th2 , Vacinação , Proteínas do Core Viral/imunologia , Proteínas Virais de Fusão/imunologia , Ligação Viral
14.
Nucleic Acids Res ; 47(1): 56-68, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30462330

RESUMO

Hepatitis C virus (HCV) infection is a major cause of human chronic liver disease and hepatocellular carcinoma. G-quadruplex (G4) is an important four-stranded secondary structure of nucleic acids. Recently, we discovered that the core gene of HCV contains a G4 RNA structure; however, the interaction between the HCV core RNA G4 and host cellular proteins, and the roles of the HCV core RNA G4 in HCV infection and pathogenesis remain elusive. Here, we identified a cellular protein, nucleolin (NCL), which bound and stabilized the HCV core RNA G4 structure. We demonstrated the direct interaction and colocalization between NCL and wild-type core RNA G4 at both in vitro and in cell physiological conditions of the alive virus; however no significant interaction was found between NCL and G4-modified core RNA. NCL is also associated with HCV particles. HCV infection induced NCL mRNA and protein expression, while NCL suppressed wild-type viral replication and expression, but not G4-modified virus. Silencing of NCL greatly enhanced viral RNA replication. Our findings provide new insights that NCL may act as a host factor for anti-viral innate immunity, and binding of cellular NCL with the viral core RNA G4 structure is involved in suppressing HCV replication.


Assuntos
Quadruplex G , Fosfoproteínas/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas do Core Viral/química , Regulação Viral da Expressão Gênica/genética , Hepacivirus/genética , Hepacivirus/patogenicidade , Hepatite C/genética , Hepatite C/virologia , Humanos , Fosfoproteínas/química , RNA Viral/química , Proteínas de Ligação a RNA/química , Proteínas do Core Viral/genética , Replicação Viral/genética
15.
Artigo em Inglês | MEDLINE | ID: mdl-30373799

RESUMO

NVR 3-778 is the first capsid assembly modulator (CAM) that has demonstrated antiviral activity in hepatitis B virus (HBV)-infected patients. NVR 3-778 inhibited the generation of infectious HBV DNA-containing virus particles with a mean antiviral 50% effective concentration (EC50) of 0.40 µM in HepG2.2.15 cells. The antiviral profile of NVR 3-778 indicates pan-genotypic antiviral activity and a lack of cross-resistance with nucleos(t)ide inhibitors of HBV replication. The combination of NVR 3-778 with nucleos(t)ide analogs in vitro resulted in additive or synergistic antiviral activity. Mutations within the hydrophobic pocket at the dimer-dimer interface of the core protein could confer resistance to NVR 3-778, which is consistent with the ability of the compound to bind to core and to induce capsid assembly. By targeting core, NVR 3-778 inhibits pregenomic RNA encapsidation, viral replication, and the production of HBV DNA- and HBV RNA-containing particles. NVR 3-778 also inhibited de novo infection and viral replication in primary human hepatocytes with EC50 values of 0.81 µM against HBV DNA and between 3.7 and 4.8 µM against the production of HBV antigens and intracellular HBV RNA. NVR 3-778 showed favorable pharmacokinetics and safety in animal species, allowing serum levels in excess of 100 µM to be achieved in mice and, thus, enabling efficacy studies in vivo The overall preclinical profile of NVR 3-778 predicts antiviral activity in vivo and supports its further evaluation for safety, pharmacokinetics, and antiviral activity in HBV-infected patients.


Assuntos
Antivirais/farmacologia , Benzamidas/farmacologia , Capsídeo/efeitos dos fármacos , DNA Viral/antagonistas & inibidores , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Piperidinas/farmacologia , RNA Viral/antagonistas & inibidores , Animais , Antígenos Virais/genética , Antígenos Virais/metabolismo , Antivirais/sangue , Antivirais/química , Antivirais/farmacocinética , Benzamidas/sangue , Benzamidas/química , Benzamidas/farmacocinética , Capsídeo/química , Capsídeo/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Células Hep G2 , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Piperidinas/sangue , Piperidinas/química , Piperidinas/farmacocinética , Cultura Primária de Células , RNA Viral/genética , RNA Viral/metabolismo , Proteínas do Core Viral/antagonistas & inibidores , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Replicação Viral/efeitos dos fármacos
16.
Methods Mol Biol ; 1911: 209-217, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30593628

RESUMO

The infectious virion of hepatitis C virus (HCV) is made up of the viral nucleocapsid surrounded by an envelope that contains an ER-derived membrane bilayer, cellular lipids, and the viral E1 and E2 glycoproteins. Because the infectious HCV particle contains both protein and lipid layers, selective disruption of these layers and analysis for the presence or absence of resulting virion components can be used to study the virion assembly process. This chapter describes an experimental method to measure HCV virion envelopment, which can reveal the mechanisms of how specific viral protein-protein interactions and host factors contribute to the process of HCV envelopment.


Assuntos
Endopeptidase K/metabolismo , Hepacivirus/fisiologia , Hepatite C/virologia , Proteínas do Core Viral/metabolismo , Vírion/fisiologia , Técnicas de Cultura de Células/métodos , Linhagem Celular , Eletroporação/métodos , Hepacivirus/genética , Humanos , Immunoblotting/métodos , RNA Viral/genética , Proteínas do Core Viral/genética , Vírion/genética , Montagem de Vírus
17.
Iran Biomed J ; 23(1): 57-67, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30056690

RESUMO

Background: Hepatitis C virus (HCV) is a blood-borne pathogen, resulting in liver cirrhosis and liver cancer. Despite of many efforts in development of treatments for HCV, no vaccine has been licensed yet. The purpose of this study was to design and prepare a specific mRNA, without 5' cap and poly (A) tail transcribed in vitro capable of coding core protein and also to determine its functionality. Methods: Candidate mRNA was prepared by in vitro transcription of the designed construct consisting of 5ʹ and 3ʹ untranslated regions of heat shock proteins 70 (hsp70) mRNA, T7 promoter, internal ribosome entry site (IRES) sequences of eIF4G related to human dendritic cells (DCs), and the Core gene of HCV. To design the modified mRNA, the 5' cap and poly (A) tail structures were not considered. DCs were transfected by in vitro-transcribed messenger RNA (IVT-mRNA) and the expressions of green fluorescent protein (GFP), and Core genes were determined by microscopic examination and Western blotting assay. Results: Cell transfection results showed that despite the absence of 5' cap and poly (A) tail, the structure of the mRNA was stable. Moreover, the successful expressions of GFP and Core genes were achieved. Conclusion: Our findings indicated the effectiveness of a designed IVT-mRNA harboring the Core gene of HCV in transfecting and expressing the antigens in DCs. Considering the simple and efficient protocol for the preparation of this IVT-mRNA and its effectiveness in expressing the gene that it carries, this IVT-mRNA could be suitable for development of an RNA vaccine against HCV.


Assuntos
Hepacivirus/genética , Proteínas do Core Viral/genética , Vacinas contra Hepatite Viral/biossíntese , Células Dendríticas/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Monócitos/citologia , Conformação de Ácido Nucleico , Plasmídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Termodinâmica , Transcrição Genética
18.
Virology ; 526: 203-213, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30415131

RESUMO

Hepatitis B virus genotype G possesses a 36-nucleotide (nt) insertion at the 5' end of core gene, adding 12 residues to core protein. The insertion markedly increased core protein level irrespective of viral genotype, with the effect reproducible using CMV-core gene construct. Here we used such expression constructs and transient transfection experiments in Huh7 cells to identify the structural bases. The insertion is predicted to create a stem-loop structure 14nt downstream of core gene AUG. A + 1 or + 2 frameshift into the 36nt mitigated enhancement of core protein level. Point mutations to disrupt or restore the stem-loop had opposite effects on core protein expression. Shifting the translation initiation site downstream or further upstream of the stem-loop rendered it inhibitory or no longer stimulatory of core protein expression. Therefore, both the reading frame and a properly positioned stem-loop structure contribute to marked increase in core protein expression by the 36-nt insertion.


Assuntos
Regulação Viral da Expressão Gênica/genética , Vírus da Hepatite B/genética , Conformação de Ácido Nucleico , Biossíntese de Proteínas/genética , RNA Viral/química , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/química , Sequência de Aminoácidos/genética , Códon de Iniciação , Genótipo , Humanos , Sequências Repetidas Invertidas/genética , Mutação , Fases de Leitura Aberta/genética , RNA Viral/genética , Proteínas do Core Viral/genética , Vírion/metabolismo , Replicação Viral
19.
J Virol ; 93(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30355692

RESUMO

Group A rotaviruses (RVAs) are classified according to a nucleotide sequence-based system that assigns a genotype to each of the 11 double-stranded RNA (dsRNA) genome segments. For the segment encoding the VP1 polymerase, 22 genotypes (R1 to R22) are defined with an 83% nucleotide identity cutoff value. For the segment encoding the VP2 core shell protein, which is a functional VP1-binding partner, 20 genotypes (C1 to C20) are defined with an 84% nucleotide identity cutoff value. However, the extent to which the VP1 and VP2 proteins encoded by these genotypes differ in their sequences or interactions has not been described. Here, we sought to (i) delineate the relationships and sites of variation for VP1 and VP2 proteins belonging to the known RVA genotypes and (ii) correlate intergenotypic sequence diversity with functional VP1-VP2 interaction(s) during dsRNA synthesis. Using bioinformatic approaches, we revealed which VP1 and VP2 genotypes encode divergent proteins and identified the positional locations of amino acid changes in the context of known structural domains/subdomains. We then employed an in vitro dsRNA synthesis assay to test whether genotype R1, R2, R4, and R7 VP1 polymerases could be enzymatically activated by genotype C1, C2, C4, C5, and C7 VP2 core shell proteins. Genotype combinations that were incompatible informed the rational design and in vitro testing of chimeric mutant VP1 and VP2 proteins. The results of this study connect VP1 and VP2 nucleotide-level diversity to protein-level diversity for the first time, and they provide new insights into regions/residues critical for VP1-VP2 interaction(s) during viral genome replication.IMPORTANCE Group A rotaviruses (RVAs) are widespread in nature, infecting numerous mammalian and avian hosts and causing severe gastroenteritis in human children. RVAs are classified using a system that assigns a genotype to each viral gene according to its nucleotide sequence. To date, 22 genotypes have been described for the gene encoding the viral polymerase (VP1), and 20 genotypes have been described for the gene encoding the core shell protein (VP2). Here, we analyzed if/how the VP1 and VP2 proteins encoded by the known RVA genotypes differ from each other in their sequences. We also used a biochemical approach to test whether the intergenotypic sequence differences influenced how VP1 and VP2 functionally engage each other to mediate RNA synthesis in a test tube. This work is important because it increases our understanding of RVA protein-level diversity and raises new ideas about the VP1-VP2 binding interface(s) that is important for viral replication.


Assuntos
Proteínas do Capsídeo/genética , Biologia Computacional/métodos , Rotavirus/classificação , Proteínas do Core Viral/genética , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Evolução Molecular , Variação Genética , Genótipo , Modelos Moleculares , Filogenia , Rotavirus/genética , Rotavirus/metabolismo , Proteínas do Core Viral/química
20.
J Virol ; 93(2)2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30355693

RESUMO

To cross the human species barrier, influenza A viruses (IAV) of avian origin have to overcome the interferon-induced host restriction factor MxA by acquiring distinct mutations in their nucleoprotein (NP). We recently demonstrated that North American classical swine IAV are able to partially escape MxA restriction. Here we investigated whether the Eurasian avian-like swine IAV lineage currently circulating in European swine would likewise evade restriction by human MxA. We found that the NP of the influenza virus isolate A/Swine/Belzig/2/2001 (Belzig-NP) exhibits increased MxA escape, similar in extent to that with human IAV NPs. Mutational analysis revealed that the MxA escape mutations in Belzig-NP differ from the known MxA resistance cluster of the North American classical swine lineage and human-derived IAV NPs. A mouse-adapted avian IAV of the H7N7 subtype encoding Belzig-NP showed significantly greater viral growth in both MxA-expressing cells and MxA-transgenic mice than control viruses lacking the MxA escape mutations. Similarly, the growth of the recombinant Belzig virus was only marginally affected in MxA-expressing cells and MxA-transgenic mice, in contrast to that of Belzig mutant viruses lacking MxA escape mutations in the NP. Phylogenetic analysis of the Eurasian avian-like swine IAV revealed that the NP amino acids required for MxA escape were acquired successively and were maintained after their introduction. Our results suggest that the circulation of IAV in the swine population can result in the selection of NP variants with a high degree of MxA resistance, thereby increasing the zoonotic potential of these viruses. IMPORTANCE The human MxA protein efficiently blocks the replication of IAV from nonhuman species. In rare cases, however, these IAV overcome the species barrier and become pandemic. All known pandemic viruses have acquired and maintained MxA escape mutations in the viral NP and thus are not efficiently controlled by MxA. Intriguingly, partial MxA resistance can also be acquired in other hosts that express antivirally active Mx proteins, such as swine. To perform a risk assessment of IAV circulating in the European swine population, we analyzed the degree of MxA resistance of Eurasian avian-like swine IAV. Our data demonstrate that these viruses carry formerly undescribed Mx resistance mutations in the NP that mediate efficient escape from human MxA. We conclude that Eurasian avian-like swine IAV possess substantial zoonotic potential.


Assuntos
Vírus da Influenza A/crescimento & desenvolvimento , Mutação , Proteínas de Resistência a Myxovirus/genética , Infecções por Orthomyxoviridae/veterinária , Proteínas de Ligação a RNA/genética , Doenças dos Suínos/virologia , Proteínas do Core Viral/genética , Animais , Ásia , Aves , Linhagem Celular , Europa (Continente) , Evolução Molecular , Humanos , Vírus da Influenza A/química , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas de Resistência a Myxovirus/metabolismo , Infecções por Orthomyxoviridae/virologia , Filogenia , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Suínos , Proteínas do Core Viral/química , Proteínas do Core Viral/metabolismo
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