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1.
PLoS One ; 15(9): e0239899, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32986763

RESUMO

Influenza A virus controls replication and transcription of its genome through the tight regulation of interaction between the ribonucleoprotein (RNP) complex subunits. The helical scaffold of RNP is maintained by nucleoprotein (NP). Previous studies have revealed that NP interacts with both PB2 N-terminal and C-terminal regions, with both regions sharing similar affinity to NP as revealed in co-immunoprecipitation assay. Our work here suggests that the interaction between NP and PB2 N-terminal region lies in the cap-binding domain (residue 320-483). By co-immunoprecipitation assay, the interaction was found to involve RNA. On the other hand, the cap-binding activity was not essential in the interaction. As shown by the NHS pull-down assay, a specific RNA sequence was not required. Among the cap-binding domain, residues K331 and R332 of PB2 play a role in RNP function so that polymerase activity was reduced when these residues were mutated, while K331 was found to be more crucial in the NP interaction. Collectively, our findings suggest a new binding mode between NP and PB2 which was mediated by RNA, and such interaction may provide a novel interacting site for influenza drug development.


Assuntos
Interações entre Hospedeiro e Microrganismos/genética , Domínios Proteicos , RNA Replicase/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas Virais/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Imunoprecipitação , Plasmídeos/genética , Ligação Proteica/genética , RNA Replicase/química , RNA Replicase/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Transfecção , Proteínas do Core Viral/genética , Proteínas Virais/química , Proteínas Virais/genética , Replicação Viral/genética
2.
PLoS One ; 15(9): e0238839, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32915862

RESUMO

In patients who are HIV infected, hepatitis B virus (HBV) infection is an important co-morbidity. However, antiretroviral options for HIV/HBV co-infected children are limited and, at the time of this study, only included lamivudine. These children may remain on this regimen for many years until late adolescence. They are at high risk of developing HBV drug resistance and uncontrolled HBV disease. The aim of this study was to characterize HBV infection in HIV/HBV co-infected children. Known HIV-infected/HBsAg-positive children, previously exposed to lamivudine monotherapy against HBV, and their mothers were recruited at the Katutura Hospital paediatric HIV clinic in Windhoek, Namibia. Dried blood spot and serum samples were collected for HBV characterization and serological testing, respectively. Fifteen children and six mothers participated in the study. Eight of the 15 children (53.3%) tested HBV DNA positive; all eight children were on lamivudine-based ART. Lamivudine-associated resistance variants, together with immune escape mutants in the surface gene, were identified in all eight children. Resistance mutations included rtL80I, rtV173L, rtL180M, rtM204I/V and the overlapping sE164D, sW182*, sI195M and sW196LS variants. HBV strains belonged to genotypes E (6/8, 75%) and D3 (2/8, 25%). Further analysis of the HBV core promoter region revealed mutations associated with reduced expression of HBeAg protein and hepatocarcinogenesis. All six mothers, on HBV-active ART containing tenofovir and lamivudine, tested HBV DNA negative. This study confirms the importance of screening HIV-infected children for HBV and ensuring equity of drug access to effective HBV treatment if co-infected.


Assuntos
Coinfecção/epidemiologia , Farmacorresistência Viral/genética , Infecções por HIV/epidemiologia , Hepatite B/epidemiologia , Mutação , Proteínas do Core Viral/genética , Carga Viral , Adolescente , Adulto , Antirretrovirais/uso terapêutico , Criança , Estudos de Coortes , Coinfecção/genética , Coinfecção/virologia , Estudos Transversais , DNA Viral/análise , Feminino , HIV/efeitos dos fármacos , HIV/isolamento & purificação , Infecções por HIV/complicações , Infecções por HIV/genética , Infecções por HIV/virologia , Hepatite B/complicações , Hepatite B/genética , Hepatite B/virologia , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/isolamento & purificação , Humanos , Masculino , Namíbia/epidemiologia , Adulto Jovem
3.
BMC Infect Dis ; 20(1): 480, 2020 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-32631335

RESUMO

BACKGROUND: Influenza A virus (IAV) infection is a serious public health problem not only in South East Asia but also in European and African countries. Scientists are using network biology to dig deep into the essential host factors responsible for regulation of virus infections. Researchers can explore the virus invasion into the host cells by studying the virus-host relationship based on their protein-protein interaction network. METHODS: In this study, we present a comprehensive IAV-host protein-protein interaction network that is obtained based on the literature-curated protein interaction datasets and some important interaction databases. The network is constructed in Cytoscape and analyzed with its plugins including CytoHubba, CytoCluster, MCODE, ClusterViz and ClusterOne. In addition, Gene Ontology and KEGG enrichment analyses are performed on the highly IAV-associated human proteins. We also compare the current results with those from our previous study on Hepatitis C Virus (HCV)-host protein-protein interaction network in order to find out valuable information. RESULTS: We found out 1027 interactions among 829 proteins of which 14 are viral proteins and 815 belong to human proteins. The viral protein NS1 has the highest number of associations with human proteins followed by NP, PB2 and so on. Among human proteins, LNX2, MEOX2, TFCP2, PRKRA and DVL2 have the most interactions with viral proteins. Based on KEGG pathway enrichment analysis of the highly IAV-associated human proteins, we found out that they are enriched in the KEGG pathway of basal cell carcinoma. Similarly, the result of KEGG analysis of the common host factors involved in IAV and HCV infections shows that these factors are enriched in the infection pathways of Hepatitis B Virus (HBV), Viral Carcinoma, measles and certain other viruses. CONCLUSION: It is concluded that the list of proteins we identified might be used as potential drug targets for the drug design against the infectious diseases caused by Influenza A Virus and other viruses.


Assuntos
Interações Hospedeiro-Patógeno/genética , Vírus da Influenza A/metabolismo , Influenza Humana/metabolismo , Mapas de Interação de Proteínas/genética , Biologia de Sistemas/métodos , Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Hepacivirus/metabolismo , Hepatite C/metabolismo , Hepatite C/virologia , Humanos , Influenza Humana/virologia , Fatores de Transcrição/genética , Proteínas do Core Viral/genética , Proteínas não Estruturais Virais/genética , Replicação Viral
4.
J Gen Virol ; 101(4): 385-398, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32553055

RESUMO

The influenza A virus (IAV) ribonucleoprotein (vRNP) complex consists of polymerase subunits, nucleoprotein (NP) and viral RNA and is responsible for RNA transcription and replication. Interactions between the vRNP complex and host factors play important roles in virus replication, pathogenicity and species tropism. In this study, Strep-tag affinity purification coupled with mass spectrometry was used to identify host factors that interact with IAV vRNP complex in infected human cells. We purified vRNP complex from HEK 293T cells infected with a recombinant mouse-adapted IAV (A/Chicken/Hubei/489/2004) containing a Strep-tag PB2 subunit and identified Y-box-binding protein 3 (YBX3) as a negative regulator of IAV replication. Overexpression of YBX3 inhibited the virus replication, viral protein expression and vRNA synthesis. Conversely, RNAi knockdown of YBX3 resulted in significantly increased virus growth rate. Furthermore, knockdown of YBX3 augmented the nuclear accumulation of NP and viral primary transcription in infected cells. Our results suggest that YBX3 restricts IAV replication by interacting with vRNP complex and subsequently imparing its function.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Choque Térmico/metabolismo , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas do Core Viral/metabolismo , Replicação Viral , Células A549 , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Cães , Células HEK293 , Proteínas de Choque Térmico/genética , Interações entre Hospedeiro e Microrganismos , Humanos , Vírus da Influenza A/enzimologia , Vírus da Influenza A/crescimento & desenvolvimento , Células Madin Darby de Rim Canino , Espectrometria de Massas , Camundongos , Ligação Proteica , RNA Interferente Pequeno , RNA Viral/metabolismo , Transcrição Genética , Regulação para Cima , Proteínas do Core Viral/genética , Replicação Viral/fisiologia
5.
J Virol ; 94(15)2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32434887

RESUMO

Hepatitis C virus (HCV) has evolved mechanisms to evade innate immunity that are leading to chronic infections. The immunological function of the HCV frameshift (F) protein, which is a frameshift product of core coding sequences, has not been well characterized. The HCV F protein is produced during natural HCV infections and is found most commonly in genotype 1 HCV. In this study, we investigated whether the F protein plays a role in type I interferon (IFN) induction pathways. We engineered F expression constructs from core coding sequences of 4 genotypes (1a, 2a, 3a, and 4a) of HCV as well as the sequences which would only be able to produce core proteins. The peptide lengths and amino acids sequences of F proteins are highly variable. We hypothesized that F proteins from different genotypes might control the type I IFN production and response differently. We found that both IFN-beta (IFN-ß) promoter activities are significantly higher in genotype 1a F protein (F1a)-expressing cells. Conversely, the IFN-ß promoter activities are lower in genotype 2a F (F2a) protein-expressing cells. We also used real-time PCR to confirm IFN-ß mRNA expression levels. By generating chimera F proteins, we discovered that the effects of F proteins were determined by the amino acid sequence 40 to 57 of genotype 1a. The regulation of type I IFN induction pathway is related but not limited to the activity of F1a to interact with proteasome subunits and to disturb the proteasome activity. Further molecular mechanisms of how F proteins from different genotypes of HCV control these pathways differently remain to be investigated.IMPORTANCE Although naturally present in HCV infection patient serum, the virological or immunological functions of the HCV F protein, which is a frameshift product of core coding sequences, remain unclear. Here, we report the effects of the HCV F protein between genotypes and discuss a potential explanation for the differential responses to type I IFN-based therapy among patients infected with different genotypes of HCV. Our study provides one step forward to understanding the host response during HCV infection and new insights for the prediction of the outcome of IFN-based therapy in HCV patients.


Assuntos
Genótipo , Hepacivirus/metabolismo , Hepatite C/metabolismo , Interferon beta/biossíntese , Transdução de Sinais , Proteínas do Core Viral/metabolismo , Linhagem Celular Tumoral , Hepacivirus/genética , Hepatite C/genética , Humanos , Interferon beta/genética , Proteínas do Core Viral/genética
6.
PLoS Pathog ; 16(3): e1008459, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32226051

RESUMO

Hepatitis B virus (HBV) delivers a partially double-stranded, relaxed circular (RC) DNA genome in complete virions to the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, which establishes and sustains viral infection. An overlength pregenomic RNA (pgRNA) is then transcribed from CCC DNA and packaged into immature nucleocapsids (NCs) by the viral core (HBc) protein. pgRNA is reverse transcribed to produce RC DNA in mature NCs, which are then enveloped and secreted as complete virions, or delivered to the nucleus to replenish the nuclear CCC DNA pool. RC DNA, whether originating from extracellular virions or intracellular mature NCs, must be released upon NC disassembly (uncoating) for CCC DNA formation. HBc is known to undergo dynamic phosphorylation and dephosphorylation at its C-terminal domain (CTD) to facilitate pgRNA packaging and reverse transcription. Here, two putative phosphorylation sites in the HBc N-terminal domain (NTD), S44 and S49, were targeted for genetic and biochemical analysis to assess their potential roles in viral replication. The NTD mutant that mimics the non-phosphorylated state (N2A) was competent in all steps of viral replication tested from capsid assembly, pgRNA packaging, reverse transcription, to virion secretion, except for a decrease in CCC DNA formation. On the other hand, the phosphor-mimetic mutant N2E showed a defect in the early step of pgRNA packaging but enhanced the late step of mature NC uncoating and consequently, increased CCC DNA formation. N2E also enhanced phosphorylation in CTD and possibly elsewhere in HBc. Furthermore, inhibition of the cyclin-dependent kinase 2 (CDK2), which is packaged into viral capsids, could block CCC DNA formation. These results prompted us to propose a model whereby rephosphorylation of HBc at both NTD and CTD by the packaged CDK2, following CTD dephosphorylation during NC maturation, facilitates uncoating and CCC DNA formation by destabilizing mature NCs.


Assuntos
DNA Circular/metabolismo , DNA Viral/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B/metabolismo , Modelos Biológicos , Nucleocapsídeo/metabolismo , Desenvelopamento do Vírus , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , DNA Circular/genética , DNA Viral/genética , Células HEK293 , Células Hep G2 , Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Nucleocapsídeo/genética , Fosforilação , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo
7.
Arch Virol ; 165(5): 1141-1150, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32222822

RESUMO

Pigs are capable of harbouring influenza A viruses of human and avian origin in their respiratory tracts and thus act as an important intermediary host to generate novel influenza viruses with pandemic potential by genetic reassortment between the two viruses. Here, we show that two distinct H1N2 swine influenza viruses contain avian-like or classical swine-like hemagglutinins with polymerase acidic (PA) and nucleoprotein (NP) genes from 2009 pandemic H1N1 influenza viruses that were found to be circulating in Korean pigs in 2018. Swine H1N2 influenza virus containing an avian-like hemagglutinin gene had enhanced pathogenicity, causing severe interstitial pneumonia in infected pigs and mice. The mortality rate of mice infected with swine H1N2 influenza virus containing an avian-like hemagglutinin gene was higher by 100% when compared to that of mice infected with swine H1N2 influenza virus harbouring classical swine-like hemagglutinin. Further, chemokines attracting inflammatory cells were strongly induced in lung tissues of pigs and mice infected by swine H1N2 influenza virus containing an avian-like hemagglutinin gene. In conclusion, it is necessary for the well-being of humans and pigs to closely monitor swine influenza viruses containing avian-like hemagglutinin with PA and NP genes from 2009 pandemic H1N1 influenza viruses.


Assuntos
Vírus da Influenza A Subtipo H1N2/crescimento & desenvolvimento , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/virologia , Fatores de Virulência/genética , Animais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N2/genética , Vírus da Influenza A Subtipo H1N2/isolamento & purificação , Vírus da Influenza A Subtipo H1N2/patogenicidade , Camundongos , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Proteínas de Ligação a RNA/genética , Análise de Sobrevida , Suínos , Doenças dos Suínos/patologia , Proteínas do Core Viral/genética , Virulência
8.
J Biomed Sci ; 27(1): 17, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31906961

RESUMO

BACKGROUND: Influenza A viruses cause epidemics/severe pandemics that pose a great global health threat. Among eight viral RNA segments, the multiple functions of nucleoprotein (NP) play important roles in viral replication and transcription. METHODS: To understand how NP contributes to the virus evolution, we analyzed the NP gene of H3N2 viruses in Taiwan and 14,220 NP sequences collected from Influenza Research Database. The identified genetic variations were further analyzed by mini-genome assay, virus growth assay, viral RNA and protein expression as well as ferret model to analyze their impacts on viral replication properties. RESULTS: The NP genetic analysis by Taiwan and global sequences showed similar evolution pattern that the NP backbones changed through time accompanied with specific residue substitutions from 1999 to 2018. Other than the conserved residues, fifteen sporadic substitutions were observed in which the 31R, 377G and 450S showed higher frequency. We found 31R and 450S decreased polymerase activity while the dominant residues (31 K and 450G) had higher activity. The 31 K and 450G showed better viral translation and replication in vitro and in vivo. CONCLUSIONS: These findings indicated variations identified in evolution have roles in modulating viral replication in vitro and in vivo. This study demonstrates that the interaction between variations of NP during virus evolution deserves future attention.


Assuntos
Evolução Molecular , Variação Genética , Vírus da Influenza A Subtipo H3N2/fisiologia , Biossíntese de Proteínas/genética , Proteínas de Ligação a RNA , Proteínas do Core Viral , Replicação Viral/genética , Células A549 , Animais , Cães , Humanos , Influenza Humana/epidemiologia , Influenza Humana/genética , Influenza Humana/metabolismo , Células Madin Darby de Rim Canino , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Taiwan , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/genética
9.
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
10.
Nat Commun ; 10(1): 5579, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31811120

RESUMO

Although influenza viruses lead to severe illness in high-risk populations, host genetic factors associated with severe disease are largely unknown. As the HLA-A*68:01 allele can be linked to severe pandemic 2009-H1N1 disease, we investigate a potential impairment of HLA-A*68:01-restricted CD8+ T cells to mount robust responses. We elucidate the HLA-A*68:01+CD8+ T cell response directed toward an extended influenza-derived nucleoprotein (NP) peptide and show that only ~35% individuals have immunodominant A68/NP145+CD8+ T cell responses. Dissecting A68/NP145+CD8+ T cells in low vs. medium/high responders reveals that high responding donors have A68/NP145+CD8+ memory T cells with clonally expanded TCRαßs, while low-responders display A68/NP145+CD8+ T cells with predominantly naïve phenotypes and non-expanded TCRαßs. Single-cell index sorting and TCRαß analyses link expansion of A68/NP145+CD8+ T cells to their memory potential. Our study demonstrates the immunodominance potential of influenza-specific CD8+ T cells presented by a risk HLA-A*68:01 molecule and advocates for priming CD8+ T cell compartments in HLA-A*68:01-expressing individuals for establishment of pre-existing protective memory T cell pools.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígenos HLA-A/imunologia , Antígenos HLA-A/metabolismo , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Apresentação do Antígeno , Antígenos Virais/química , Linhagem Celular , Proteção Cruzada , Reações Cruzadas/imunologia , Epitopos de Linfócito T/imunologia , Antígenos HLA-A/química , Antígenos HLA-A/genética , Humanos , Memória Imunológica/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Modelos Moleculares , Nucleoproteínas/química , Orthomyxoviridae/genética , Orthomyxoviridae/imunologia , Fragmentos de Peptídeos/química , Fenótipo , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Proteínas do Core Viral/genética
11.
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
12.
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
13.
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
14.
Br J Biomed Sci ; 76(4): 190-194, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31401936

RESUMO

Background: Chronic hepatitis C is a major public health problem around the world. In monitoring treatment efficacy, although costly and labour-intensive methods of molecular biology are often used, much cheaper and technically easier serological methods evaluating the concentration of HCV core antigen in serum are available. We evaluated HCVcAg quantification as a possible assessment of the treatment efficacy instead of HCV RNA quantification.Methods: We collected 514 serum samples from treated HCV infected patients. Quantitative evaluation of HCV RNA and HCVcAg was carried out before treatment, at the end of treatment, and at least 12 weeks following treatment termination. HCV RNA was determined by automated assay (Roche COBAS) and HCVcAg quantitation with ARCHITECT ci8200 analyser.Results: There was a significant correlation between HCVcAg and HCV RNA concentrations at baseline and follow-up visits, but not at the end of treatment. Among samples collected before the treatment, at the end of treatment and follow-up visit, concordance of HCV RNA and HCVcAg reached level of 98.1%, 98.9% and 98.7%, respectively. Diagnostic sensitivity, specificity, positive and negative predictive values of HCVcAg detection were >97%.Conclusions: HCVcAg measurement could be an alternative for determining HCV treatment efficacy after chemotherapy and could be an option in the diagnosis of HCV infection.


Assuntos
Antivirais/uso terapêutico , Hepacivirus/efeitos dos fármacos , Antígenos da Hepatite C/genética , Hepatite C Crônica/tratamento farmacológico , RNA Viral/genética , Proteínas do Core Viral/genética , Adulto , Feminino , Hepacivirus/genética , Hepacivirus/crescimento & desenvolvimento , Antígenos da Hepatite C/sangue , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/antagonistas & inibidores , RNA Viral/sangue , Resultado do Tratamento , Proteínas do Core Viral/sangue , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
15.
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
16.
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
17.
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
18.
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 , Ligação 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
19.
ACS Infect Dis ; 5(6): 962-973, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-30919621

RESUMO

Phosphatidylinositol-4 kinase III ß (PI4KB) and oxysterol-binding protein (OSBP) family I provide a conserved host pathway required for enterovirus replication. Here, we analyze the role and essentiality of this pathway in enterovirus replication. Phosphatidylinositol 4-phosphate (PI4P) production and cholesterol accumulation in the replication organelle (RO) are severely suppressed in cells infected with a poliovirus (PV) mutant isolated from a PI4KB-knockout cell line (RD[Δ PI4KB]). Major determinants of the mutant for infectivity in RD(Δ PI4KB) cells map to the A5270U(3A-R54W) and U3881C(2B-F17L) mutations. The 3A mutation is required for PI4KB-independent development of RO. The 2B mutation rather sensitizes PV to PI4KB/OSBP inhibitors by itself but confers substantially complete resistance to the inhibitors with the 3A mutation. The 2B mutation also confers hypersensitivity to interferon alpha treatment on PV. These suggest that the PI4KB/OSBP pathway is not necessarily essential for enterovirus replication in vitro. This work supports a two-step resistance model of enterovirus to PI4KB/OSBP inhibitors involving unique recessive epistasis of 3A and 2B and offers insights into a potential evolutionary pathway of enterovirus toward independence from the PI4KB/OSBP pathway.


Assuntos
Evolução Molecular , Mutação , Fosfatidilinositol 4-Fosfato 3-Quinase/genética , Poliovirus/genética , Receptores de Esteroides/genética , Antivirais/farmacologia , Linhagem Celular Tumoral , Epistasia Genética , Técnicas de Inativação de Genes , Humanos , Proteínas de Membrana/genética , Redes e Vias Metabólicas , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol 4-Fosfato 3-Quinase/antagonistas & inibidores , Fosfatidilinositol 4-Fosfato 3-Quinase/metabolismo , Poliovirus/fisiologia , Receptores de Esteroides/antagonistas & inibidores , Receptores de Esteroides/metabolismo , Proteínas do Core Viral/genética , Proteínas não Estruturais Virais/genética , Replicação Viral
20.
Antiviral Res ; 165: 47-54, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30902704

RESUMO

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of an RNA intermediate. The lack of proofreading capacity of the viral DNA polymerase results in a high mutation rate of HBV genome. Under the selective pressure created by the nucleos(t)ide analogue (NA) antiviral drugs, viruses with resistance mutations are selected. However, the replication fitness of NA-resistant mutants is markedly reduced compared to wild-type. Compensatory mutations in HBV polymerase, which restore the viral replication capacity, have been reported to arise under continuous treatment with lamivudine (LMV). We have previously identified a highly replicative LMV-resistant HBV isolate from a chronic hepatitis B patient experiencing acute disease exacerbation. Besides the common YMDD drug-resistant mutations, this isolate possesses multiple additional mutations in polymerase and core regions. The transcomplementation assay demonstrated that the enhanced viral replication is due to the mutations of core protein. Further mutagenesis study revealed that the P5T mutation of core protein plays an important role in the enhanced viral replication through increasing the levels of capsid formation and pregenomic RNA encapsidation. However, the LMV-resistant virus harboring compensatory core mutations remains sensitive to capsid assembly modulators (CpAMs). Taken together, our study suggests that the enhanced HBV nucleocapsid formation resulting from core mutations represents an important viral strategy to surmount the antiviral drug pressure and contribute to viral pathogenesis, and CpAMs hold promise for developing the combinational antiviral therapy for hepatitis B.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Vírus da Hepatite B/genética , Lamivudina/farmacologia , Proteínas do Core Viral/genética , Capsídeo/efeitos dos fármacos , Capsídeo/metabolismo , DNA Viral/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Mutação , Proteínas do Core Viral/efeitos dos fármacos , Montagem de Vírus/efeitos dos fármacos , Montagem de Vírus/fisiologia , Replicação Viral/efeitos dos fármacos
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