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1.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360656

RESUMO

Chikungunya virus (CHIKV) is a mosquito-transmitted infectious agent that causes an endemic or epidemic outbreak(s) of Chikungunya fever that is reported in almost all countries. This virus is an intense global threat, due to its high rate of contagion and the lack of effective remedies. In this study, we developed two baculovirus expression vector system (BEVS)-based approaches for the screening of anti-CHIKV drugs in Spodoptera frugiperda insect (Sf21) cells and U-2OS cells. First, structural protein of CHIKV was co-expressed through BEVS and thereby induced cell fusion in Sf21 cells. We used an internal ribosome entry site (IRES) to co-express the green fluorescent protein (EGFP) for identifying these fusion events. The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form syncytia. We identified that ursolic acid has potential anti-CHIKV activity in vitro, by using this approach. Second, BacMam virus-based gene delivery has been successfully applied for the transient expression of non-structural proteins with a subgenomic promoter-EGFP (SP-EGFP) cassette in U-2OS cells to act as an in vitro CHIKV replicon system. Our BacMam-based screening system has identified that the potential effects of baicalin and baicalein phytocompounds can inhibit the replicon activity of CHIKV in U-2OS cells. In conclusion, our results suggested that BEVS can be a potential tool for screening drugs against CHIKV.


Assuntos
Antivirais/farmacologia , Baculoviridae/genética , Fusão Celular , Febre de Chikungunya/tratamento farmacológico , Vírus Chikungunya/efeitos dos fármacos , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Animais , Febre de Chikungunya/virologia , Vetores Genéticos/genética , Proteínas de Fluorescência Verde , Ensaios de Triagem em Larga Escala , Mosquitos Vetores , Células Sf9 , Proteínas do Envelope Viral/genética
2.
Arch Virol ; 166(10): 2895-2899, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34351521

RESUMO

After the 2005-2009 chikungunya epidemic, intermittent outbreaks were reported in many parts of India. The outbreaks were caused by either locally circulating strains or imported viruses. Virus transmission routes can be traced by complete genome sequencing studies. We investigated two outbreaks in 2014 and 2019 in Kerala, India. Chikungunya virus (CHIKV) was isolated from the samples, and whole genomes were sequenced for a 2014 isolate and a 2019 isolate. Phylogenetic analysis revealed that the isolates formed a separate group with a 2019 isolate from Pune, Maharashtra, and belonged to the East/Central/South African (ECSA) genotype, Indian subcontinent sublineage of the Indian Ocean Lineage (IOL). A novel mutation at amino acid position 76 of the E2 gene was observed in the group. The phylogenetic results suggest that the outbreaks might have been caused by a virus that had been circulating in India since 2014. A detailed study is needed to investigate the evolution of CHIKV in India.


Assuntos
Febre de Chikungunya/epidemiologia , Febre de Chikungunya/virologia , Vírus Chikungunya/genética , Surtos de Doenças , Febre de Chikungunya/transmissão , Vírus Chikungunya/classificação , Vírus Chikungunya/isolamento & purificação , Genoma Viral/genética , Genótipo , Humanos , Índia/epidemiologia , Mutação , Filogenia , RNA Viral/genética , Proteínas do Envelope Viral/genética
3.
Arch Virol ; 166(10): 2803-2815, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34374840

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important pathogen in the Korean swine industry. Despite efforts including improved biosecurity and vaccination protocols, the virus continues to circulate and evolve. Based on phylogenetic analysis of open reading frame 5 (ORF5), Korean PRRSVs are known to form not only globally circulating lineages but also country-specific lineages (Lin Kor A, B, and C). To understand the recent epidemiological status of PRRSV in Korea, a total of 1349 ORF5 sequences of Korean PRRSV isolates from 2014 to 2019 were analyzed. Phylogenetic analysis was conducted using the maximum-likelihood method, and temporal changes in the relative prevalence of lineages were investigated. The analysis showed that PRRSV1 and PRRSV2 were both highly prevalent throughout the years examined. Among the PRRSV1 isolates, subgroup A (90.1%) and vaccine-like subgroup C (9.0%) composed most of the population. For PRRSV2 isolates, vaccine-like lineage 5 (36.3%) was dominant, followed by Lin Kor B (25.9%), Kor C (16.6%), lineage 1 (11.6%), and Kor A (9.1%). The PRRSV2 lineage 1 population increased from 2014 (1.8%) to 2019 (29.6%) in Korea due to the continual spread of sublineage 1.8 (NADC30-like) and introduction of sublineage 1.6 into the country. Additional genetic analysis, including analysis of non synonymous and synonymous mutations, revealed evidence of diversification and positive selection in immunologically important regions of the genome, suggesting that current vaccination is failing and promoting immune-mediated selection. Overall, these findings provide insights into the epidemiological and evolutionary dynamics of cocirculating viral lineages, and constant surveillance of PRRSV occurrence is needed.


Assuntos
Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Variação Genética , Genótipo , Filogenia , Síndrome Respiratória e Reprodutiva Suína/epidemiologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Prevalência , República da Coreia/epidemiologia , Suínos , Vacinas Virais/genética
4.
J Gen Virol ; 102(8)2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34342561

RESUMO

Dengue virus (DENV) is the most prevalent pathogen of the Flaviviridae family. Due to the considerable increase in DENV incidence and spread, symptoms such as CNS involvement have increased. Heparan sulphate (HS) was the first molecule identified as an adhesion factor for DENV in mammalian cells. Viral phenotypes with different HS interactions are associated with various clinical symptoms, including neurological alterations. Here, using in silico analyses, in vitro studies, and the in vivo mouse model, we characterized two natural circulating DENV3 genotype I (GI) lineage 1 (L1) in Brazil-DENV3 MG-20 (from Minas Gerais) and DENV3 PV_BR (from Rondônia) that present divergent neurovirulent profiles and sensitivity to sulphated molecules. We identified substitutions at the viral envelope (E) in positions 62 and 123 as likely responsible for the differences in neurovirulence. The E62K and E123Q substitutions in DENV3 MG-20 and DENV3 PV_BR, respectively, greatly influenced in silico electrostatic density and heparin docking results. In vivo, mice inoculated with DENV3 MG-20 died, but not those infected with DENV3 PV_BR. The clinical symptoms, such as paralysis of the lower limbs and meningoencephalitis, and histopathology, also differed between the inoculated groups. In vitro heparin and heparinases assays further demonstrated the biological impact of these substitutions. Other characteristics that have been previously associated with alterations in cell tropism and neurovirulence, such as changes in the size of lysis plaques and differences in cytopathic effects in glioblastoma cells, were also observed.


Assuntos
Vírus da Dengue/classificação , Vírus da Dengue/genética , Dengue/virologia , Genótipo , Heparitina Sulfato/metabolismo , Proteínas do Envelope Viral/química , Animais , Sítios de Ligação , Encéfalo/patologia , Comunicação Celular , Linhagem Celular , Dengue/patologia , Vírus da Dengue/fisiologia , Modelos Animais de Doenças , Feminino , Heparina , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Fenótipo , Filogenia , Conformação Proteica , Proteínas do Envelope Viral/classificação , Proteínas do Envelope Viral/genética , Virulência , Ligação Viral
5.
Nat Commun ; 12(1): 4502, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301937

RESUMO

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Transporte Biológico , Fusão Celular , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Células Gigantes/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Camundongos , RNA-Seq/métodos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Proteínas do Envelope Viral/genética
6.
Arch Virol ; 166(10): 2733-2741, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34322722

RESUMO

Congenital tremor (CT) type A-II in piglets is a worldwide disease caused by an emerging atypical porcine pestivirus (APPV). Preparation and evaluation of vaccines in laboratory animals is an important preliminary step toward prevention and control of the disease. Here, virus-like particles (VLPs) of APPV were prepared and VLPs vaccine was evaluated in BALB/c mice. Purified Erns and E2 proteins expressed in E. coli were allowed to self-assemble into VLPs, which had the appearance of hollow spherical particles with a diameter of about 100 nm by transmission electron microscopy (TEM). The VLPs induced strong antibody responses and reduced the viral load in tissues of BALB/c mice. The data from animal challenge experiments, RT-PCR, and immunohistochemical analysis demonstrated that BALB/c mice are an appropriate laboratory model for APPV. These results suggest the feasibility of using VLPs as a vaccine for the prevention and control of APPV and provide useful information for further study of APPV in laboratory animals.


Assuntos
Infecções por Pestivirus/prevenção & controle , Pestivirus/imunologia , Vacinação/veterinária , Replicação Viral/efeitos dos fármacos , Animais , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Pestivirus/virologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Suínos , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Carga Viral , Vacinas Virais/genética , Vacinas Virais/imunologia
7.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298900

RESUMO

Pestiviruses contain three envelope proteins: Erns, E1, and E2. Expression of HA-tagged E1 or mutants thereof showed that E1 forms homodimers and -trimers. C123 and, to a lesser extent, C171, affected the oligomerization of E1 with a double mutant C123S/C171S preventing oligomerization completely. E1 also establishes disulfide linked heterodimers with E2, which are crucial for the recovery of infectious viruses. Co-expression analyses with the HA-tagged E1 wt/E1 mutants and E2 wt/E2 mutants demonstrated that C123 in E1 and C295 in E2 are the critical sites for E1/E2 heterodimer formation. Introduction of mutations preventing E1/E2 heterodimer formation into the full-length infectious clone of BVDV CP7 prevented the recovery of infectious viruses, proving that C123 in E1 and C295 in E2 play an essential role in the BVDV life cycle, and further support the conclusion that heterodimer formation is the crucial step. Interestingly, we found that the retention signal of E1 is mandatory for intracellular localization of the heterodimer, so that absence of the E1 retention signal directs the heterodimer to the cell surface even though the E2 retention signal is still present. The covalent linkage between E1 and E2 plays an essential role for this process.


Assuntos
Pestivirus/genética , Proteínas do Envelope Viral/genética , Animais , Bovinos , Linhagem Celular , Cricetinae , Dimerização , Mutação/genética , Coelhos , Internalização do Vírus
8.
Viruses ; 13(7)2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209034

RESUMO

Host plasma membrane protein SERINC5 is incorporated into budding retrovirus particles where it blocks subsequent entry into susceptible target cells. Three structurally unrelated proteins encoded by diverse retroviruses, human immunodeficiency virus type 1 (HIV-1) Nef, equine infectious anemia virus (EIAV) S2, and ecotropic murine leukemia virus (MLV) GlycoGag, disrupt SERINC5 antiviral activity by redirecting SERINC5 from the site of virion assembly on the plasma membrane to an internal RAB7+ endosomal compartment. Pseudotyping retroviruses with particular glycoproteins, e.g., vesicular stomatitis virus glycoprotein (VSV G), renders the infectivity of particles resistant to inhibition by virion-associated SERINC5. To better understand viral determinants for SERINC5-sensitivity, the effect of SERINC5 was assessed using HIV-1, MLV, and Mason-Pfizer monkey virus (M-PMV) virion cores, pseudotyped with glycoproteins from Arenavirus, Coronavirus, Filovirus, Rhabdovirus, Paramyxovirus, and Orthomyxovirus genera. SERINC5 restricted virions pseudotyped with glycoproteins from several retroviruses, an orthomyxovirus, a rhabdovirus, a paramyxovirus, and an arenavirus. Infectivity of particles pseudotyped with HIV-1, amphotropic-MLV (A-MLV), or influenza A virus (IAV) glycoproteins, was decreased by SERINC5, whether the core was provided by HIV-1, MLV, or M-PMV. In contrast, particles pseudotyped with glycoproteins from M-PMV, parainfluenza virus 5 (PIV5), or rabies virus (RABV) were sensitive to SERINC5, but only with particular retroviral cores. Resistance to SERINC5 did not correlate with reduced SERINC5 incorporation into particles, route of viral entry, or absolute infectivity of the pseudotyped virions. These findings indicate that some non-retroviruses may be sensitive to SERINC5 and that, in addition to the viral glycoprotein, the retroviral core influences sensitivity to SERINC5.


Assuntos
Interações Hospedeiro-Patógeno , Proteínas de Membrana/genética , Proteínas do Envelope Viral , Vírion/metabolismo , Vírus/metabolismo , Células HEK293 , HIV-1/metabolismo , Humanos , Vírus da Leucemia Murina/metabolismo , Proteínas de Membrana/imunologia , Retroviridae/classificação , Retroviridae/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vírion/genética , Internalização do Vírus , Vírus/química , Vírus/classificação , Vírus/genética
9.
Poult Sci ; 100(7): 101137, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34111609

RESUMO

The avian leukosis virus (ALV) strain DL00766 was isolated from a farm in China. The phylogenetic analysis showed that env had the highest homology with the E subgroup reference strain, ranging from 94.5% to 94.9%, whereas gp85 had the highest homology with the B and E subgroups, which were 89.0% to 91.3% and 91.3% to 91.8%. In addition, point mutation analysis of gp85 showed that a 400 bp long fragment in gp85 of DL00766 had the highest homology with subgroup B, ranging from 90.1% to 97.5%, and only 82.7% to 83.1% with E subgroup. These results indicate, DL00766 may be an AVL subgroup E isolate with a subgroup B-like gp85 region. This is also the first finding that the E subgroup is used as a recombinant subject, and the subgroup B provides a recombinant virus of an exogenous gene.


Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Doenças das Aves Domésticas , Animais , Galinhas , China , Filogenia , Proteínas do Envelope Viral/genética
10.
J Gen Virol ; 102(6)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34106826

RESUMO

White spot syndrome virus (WSSV) is the most virulent pathogen causing high mortality and economic loss in shrimp aquaculture and various crustaceans. Therefore, the understanding of molecular mechanisms of WSSV infection is important to develop effective therapeutics to control the spread of this viral disease. In a previous study, we found that VP37 could bind with shrimp haemocytes through the interaction between its C-terminal domain and heparin-like molecules on the shrimp cells, and this interaction can also be inhibited by sulphated galactan. In this study, we present the crystal structure of C-terminal domain of VP37 from WSSV at a resolution of 2.51 Å. The crystal structure contains an eight-stranded ß-barrel fold with an antiparallel arrangement and reveals a trimeric assembly. Moreover, there are two sulphate binding sites found in the position corresponding to R213 and K257. In order to determine whether these sulphate binding sites are involved in binding of VP37 to heparin, mutagenesis was performed to replace these residues with alanine (R213A and K257A), and the Surface Plasmon Resonance (SPR) system was used to study the interaction of each mutated VP37 with heparin. The results showed that mutants R213A and K257A exhibited a significant loss in heparin binding activity. These findings indicated that the sites of R213 and K257 on the C-terminal domain of envelope protein VP37 are essential for binding to sulphate molecules of heparin. This study provides further insight into the structure of C-terminal domain of VP37 and it is anticipated that the structure of VP37 might be used as a guideline for development of antivirus agent targeting on the VP37 protein.


Assuntos
Heparina/metabolismo , Sulfatos/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Vírus da Síndrome da Mancha Branca 1/química , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Penaeidae/virologia , Ligação Proteica , Conformação Proteica , Conformação Proteica em Folha beta , Domínios Proteicos , Estrutura Quaternária de Proteína , Ressonância de Plasmônio de Superfície , Proteínas do Envelope Viral/genética , Vírus da Síndrome da Mancha Branca 1/genética
11.
Viruses ; 13(5)2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065826

RESUMO

The contribution of virus components to liberation of herpes simplex virus type 2 (HSV-2) progeny virions from the surface of infected cells is poorly understood. We report that the HSV-2 mutant deficient in the expression of a mucin-like membrane-associated glycoprotein G (mgG) exhibited defect in the release of progeny virions from infected cells manifested by ~2 orders of magnitude decreased amount of infectious virus in a culture medium as compared to native HSV-2. Electron microscopy revealed that the mgG deficient virions were produced in infected cells and present at the cell surface. These virions could be forcibly liberated to a nearly native HSV-2 level by the treatment of cells with glycosaminoglycan (GAG)-mimicking oligosaccharides. Comparative assessment of the interaction of mutant and native virions with surface-immobilized chondroitin sulfate GAG chains revealed that while the mutant virions associated with GAGs ~fourfold more extensively, the lateral mobility of bound virions was much poorer than that of native virions. These data indicate that the mgG of HSV-2 balances the virus interaction with GAG chains, a feature critical to prevent trapping of the progeny virions at the surface of infected cells.


Assuntos
Glicoproteínas/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 2/fisiologia , Proteínas do Envelope Viral/metabolismo , Liberação de Vírus , Membrana Celular/metabolismo , Células Cultivadas , Glicoproteínas/genética , Herpesvirus Humano 2/ultraestrutura , Interações Hospedeiro-Patógeno , Humanos , Mutação , Proteínas do Envelope Viral/genética , Vírion/ultraestrutura
12.
Arch Virol ; 166(8): 2255-2261, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34003359

RESUMO

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a severe disease that causes huge economic losses in the swine industry worldwide. In China, CSF has been under control due to extensive vaccination since 1954. However, there are still sporadic CSF outbreaks in China. Here, we isolated 27 CSFV strains from three Chinese provinces (Shaanxi, Gansu, and Ningxia) from 2011 to 2018. Phylogenetic analysis based on the full-length envelope glycoprotein E2 coding region revealed that 25 out of 27 CSFV isolates clustered within subgroups 2.1 and 2.2, while two strains from Gansu belonged to subgroup 1.1. The sequence identity among these 27 isolates varied from 79.3% to 99.8% (nucleotides) and from 83.1% to 99.7% (amino acids). Further analysis based on the E2 amino acid sequences showed that these new isolates have consistent amino acid substitutions, including R31K and N34S.


Assuntos
Substituição de Aminoácidos , Vírus da Febre Suína Clássica/classificação , Peste Suína Clássica/virologia , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , China , Vírus da Febre Suína Clássica/genética , Vírus da Febre Suína Clássica/isolamento & purificação , Evolução Molecular , Genótipo , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Suínos
13.
J Virol ; 95(14): e0042921, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-33952635

RESUMO

Rift Valley fever phlebovirus (RVFV) has a single-stranded, negative-sense RNA genome, consisting of L, M, and S segments. The virion carries two envelope glycoproteins, Gn and Gc, along with ribonucleoprotein complexes (RNPs), composed of encapsidated genomes carrying N protein and the viral polymerase, L protein. A quantitative analysis of the profile of viral RNA segments packaged into RVFV particles showed that all three genomic RNA segments had similar packaging abilities, whereas among antigenomic RNA segments, the antigenomic S RNA, which serves as the template for the transcription of mRNA expressing the RVFV virulence factor, NSs, displayed a significantly higher packaging ability. To delineate the factor(s) governing the packaging of RVFV RNA segments, we characterized the interactions between Gn and viral RNPs in RVFV-infected cells. Coimmunoprecipitation analysis demonstrated the interaction of Gn with N protein, L protein, and viral RNAs in RVFV-infected cells. Furthermore, UV-cross-linking and immunoprecipitation analysis revealed, for the first time in bunyaviruses, the presence of a direct interaction between Gn and all the viral RNA segments in RVFV-infected cells. Notably, analysis of the ability of Gn to bind to RVFV RNA segments indicated a positive correlation with their respective packaging abilities and highlighted a binding preference of Gn for antigenomic S RNA, among the antigenomic RNA segments, suggesting the presence of a selection mechanism for antigenomic S RNA incorporation into infectious RVFV particles. Collectively, the results of our study illuminate the importance of a direct interaction between Gn and viral RNA segments in determining their efficiency of incorporation into RVFV particles. IMPORTANCE Rift Valley fever phlebovirus, a bunyavirus, is a mosquito-borne, segmented RNA virus that can cause severe disease in humans and ruminants. An essential step in RVFV life cycle is the packaging of viral RNA segments to produce infectious virus particles for dissemination to new hosts. However, there are key gaps in knowledge regarding the mechanisms that regulate viral RNA packaging efficiency in bunyaviruses. Our studies investigating the mechanism of RNA packaging in RVFV revealed the presence of a direct interaction between the viral envelope glycoprotein, Gn, and the viral RNA segments in infected cells, for the first time in bunyaviruses. Furthermore, our data strongly indicate a critical role for the direct interaction between Gn and viral RNAs in determining the efficiency of incorporation of viral RNA segments into RVFV particles. Clarifying the fundamental mechanisms of RNA packaging in RVFV would be valuable for the development of antivirals and live-attenuated vaccines.


Assuntos
RNA Viral , Vírus da Febre do Vale do Rift/genética , Empacotamento do Genoma Viral , Sequência de Empacotamento Viral , Vírion/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Ribonucleoproteínas/metabolismo , Células Vero , Proteínas do Envelope Viral/genética
14.
J Virol ; 95(15): e0052121, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34011544

RESUMO

Pestiviruses are members of the family Flaviviridae, a group of enveloped viruses that bud at intracellular membranes. Pestivirus particles contain three glycosylated envelope proteins, Erns, E1, and E2. Among them, E1 is the least characterized concerning both biochemical features and function. E1 from bovine viral diarrhea virus (BVDV) strain CP7 was analyzed with regard to its intracellular localization and membrane topology. Here, it is shown that even in the absence of other viral proteins, E1 is not secreted or expressed at the cell surface but localizes predominantly in the endoplasmic reticulum (ER). Using engineered chimeric transmembrane domains with sequences from E1 and vesicular stomatitis virus G protein, the E1 ER-retention signal could be narrowed down to six fully conserved polar residues in the middle part of the transmembrane domain of E1. Retention was observed even when several of these polar residues were exchanged for alanine. Mutations with a strong impact on E1 retention prevented recovery of infectious viruses when tested in the viral context. Analysis of the membrane topology of E1 before and after the signal peptide cleavage via a selective permeabilization and an in vivo labeling approach revealed that mature E1 is a typical type I transmembrane protein with a single span transmembrane anchor at its C terminus, whereas it adopts a hairpin-like structure with the C terminus located in the ER lumen when the precleavage situation is mimicked by blocking the cleavage site between E1 and E2. IMPORTANCE The shortage of specific antibodies against E1, making detection and further analysis of E1 difficult, resulted in a lack of knowledge on E1 compared to Erns and E2 with regard to biosynthesis, structure, and function. It is known that pestiviruses bud intracellularly. Here, we show that E1 contains its own ER retention signal: six fully conserved polar residues in the middle part of the transmembrane domain are shown to be the determinants for ER retention of E1. Moreover, those six polar residues could serve as a functional group that intensely affect the generation of infectious viral particles. In addition, the membrane topology of E1 has been determined. In this context, we also identified dynamic changes in membrane topology of E1 with the carboxy terminus located on the luminal side of the ER in the precleavage state and relocation of this sequence upon signal peptidase cleavage. Our work provides the first systematic analysis of the pestiviral E1 protein with regard to its biochemical and functional characteristics.


Assuntos
Vírus da Diarreia Viral Bovina/metabolismo , Retículo Endoplasmático/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Membrana Celular/metabolismo , Cricetinae , Vírus da Diarreia Viral Bovina/genética , Glicoproteínas de Membrana/metabolismo , Conformação Proteica , Sinais Direcionadores de Proteínas/genética , Coelhos , Proteínas do Envelope Viral/genética
16.
ACS Infect Dis ; 7(6): 1503-1518, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34048233

RESUMO

The worldwide expansion of chikungunya virus (CHIKV) into tropical and subtropical areas in the last 15 years has posed a currently unmet need for vaccines and therapeutics. The E2-E1 envelope glycoprotein complex binds receptors on the host cell and promotes membrane fusion during CHIKV entry, thus constituting an attractive target for the development of antiviral drugs. In order to identify CHIKV antivirals acting through inhibition of the envelope glycoprotein complex function, our first approach was to search for amenable druggable sites within the E2-E1 heterodimer. We identified a pocket located in the interface between E2 and E1 around the fusion loop. Then, via a structure-based virtual screening approach and in vitro assay of antiviral activity, we identified compound 7 as a specific inhibitor of CHIKV. Through a lead optimization process, we obtained compound 11 that demonstrated increased antiviral activity and low cytotoxicity (EC50 1.6 µM, CC50 56.0 µM). Molecular dynamics simulations were carried out and described a possible interaction pattern of compound 11 and the E1-E2 dimer that could be useful for further optimization. As expected from target site selection, compound 11 inhibited virus internalization during CHIKV entry. In addition, virus populations resistant to compound 11 included mutation E2-P173S, which mapped to the proposed binding pocket, and second site mutation E1-Y24H. Construction of recombinant viruses showed that these mutations conferred antiviral resistance in the parental background. Finally, compound 11 presents acceptable solubility values and is chemically and enzymatically stable in different media. Altogether, these findings uncover a suitable pocket for the design of CHIKV entry inhibitors with promising antiviral activity and pharmacological profiles.


Assuntos
Vírus Chikungunya , Desenho de Fármacos , Proteínas do Envelope Viral/antagonistas & inibidores , Vírus Chikungunya/efeitos dos fármacos , Envelope Viral , Proteínas do Envelope Viral/genética
17.
Medicine (Baltimore) ; 100(18): e25203, 2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-33950918

RESUMO

ABSTRACT: Introduction: Hepatitis C virus (HCV) infection is a major public health issue. HCV genotype identification is clinically important to tailor the dosage and duration of treatment, and recombination in intra-patient populations of HCV may lead to the generation of escape mutants, as previously observed for other RNA viruses. Up to now, there is no study assessing HCV genotypes and subtypes in Heilongjiang Province, China.Methods: To determine genotype and phylogenetic analysis of HCV in Heilongjiang Province is crucial. In this study, we amplified 3 genome regions (5'UTR, E1, and NS5B) of 30 HCV patients in Heilongjiang Province, amplified products were analyzed by bioinformatics.Results: We found that 23 specimens had concordant subtypes in the 3 gene regions (2a and 1b), 7 HCV patients were considered the recombinants, the recombination pattern of the 7 HCV patients in the 5'UTR, E1, and NS5B region as followed: 1b/2a/1b, 2a/2a/1b, 1b/2a/2a, 1b/2a/1b, 1b/2a/1b, 1b/2a/1b, 2a/2a/1b.Conclusions: The findings in the present study showed that a higher recombination rate (23%) than other researches, and the recombination of 2a/1b in the 5'UTR, E1, and NS5B region was only found in the present study up to now.


Assuntos
Hepacivirus/genética , Hepatite C Crônica/virologia , Regiões 5' não Traduzidas/genética , Adulto , Idoso , China/epidemiologia , Biologia Computacional , DNA Viral/genética , Feminino , Técnicas de Genotipagem , Hepacivirus/isolamento & purificação , Hepatite C Crônica/sangue , Hepatite C Crônica/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Filogenia , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Adulto Jovem
18.
BMC Vet Res ; 17(1): 164, 2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33853597

RESUMO

BACKGROUND: Since 2011, numerous highly virulent and antigenic variant viral strains have been reported in pigs that were vaccinated against the swine pseudorabies virus. These infections have led to substantial economic losses in the Chinese swine industry. RESULTS: This study, constructed a novel recombinant vaccine strain with gI/gE deletion (PRV-GD2013-ΔgI/gE) by overlapping PCR and homologous recombination technology. The growth curves and plaque morphology of the recombinant virus were similar to those of the parental strain. However, PRV-GD2013-ΔgI/gE infection was significantly attenuated in mice compared with that of PRV-GD2013. Two-week-old piglets had normal rectal temperatures and displayed no clinical symptoms after being inoculated with 105 TCID50 PRV-GD2013-ΔgI/gE, indicating that the recombinant virus was avirulent in piglets. Piglets were immunized with different doses of PRV-GD2013-ΔgI/gE, or a single dose of Bartha-K61 or DMEM, and infected with PRV-GD2013 at 14 days post-vaccination. Piglets given high doses of PRV-GD2013-ΔgI/gE showed no obvious clinical symptoms, and their antibody levels were higher than those of other groups, indicating that the piglets were completely protected from PRV-GD2013. CONCLUSIONS: The PRV-GD2013-ΔgI/gE vaccine strain could be effective for immunizing Chinese swine herds against the pseudorabies virus (PRV) strain.


Assuntos
Vacinas contra Pseudorraiva/imunologia , Pseudorraiva/prevenção & controle , Doenças dos Suínos/virologia , Animais , Linhagem Celular , Cricetinae , Feminino , Deleção de Genes , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/imunologia , Recombinação Homóloga , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Pseudorraiva/imunologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas Sintéticas/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
19.
J Virol ; 95(14): e0066021, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-33910956

RESUMO

Hepatitis B virus (HBV) transcribes coterminal mRNAs of 0.7 to 3.5 kb from the 3.2-kb covalently closed circular DNA, with the 2.1-kb RNA being most abundant. The 0.7-kb RNA produces HBx protein, a transcriptional transactivator, while the 3.5-kb pregenomic RNA (pgRNA) drives core and P protein translation as well as genome replication. The large (L) and small (S) envelope proteins are translated from the 2.4-kb and 2.1-kb RNAs, respectively, with the majority of the S protein being secreted as noninfectious subviral particles and detected as hepatitis B surface antigen (HBsAg). pgRNA transcription could inhibit transcription of subgenomic RNAs. The present study characterized naturally occurring in-frame deletions in the 3' preS1 region, which not only codes for L protein but also serves as the promoter for 2.1-kb RNA. The human hepatoma cell line Huh7 was transiently transfected with subgenomic expression constructs for envelope (and HBx) proteins, dimeric constructs, or constructs mimicking covalently closed circular DNA. The results confirmed lost 2.1-kb RNA transcription and HBsAg production from many deletion mutants, accompanied by increases in other (especially 2.4-kb) RNAs, intracellular HBx and core proteins, and replicative DNA but impaired virion and L protein secretion. The highest intracellular L protein levels were achieved by mutants that had residual S protein expression or retained the matrix domain in L protein. Site-directed mutagenesis of a high replicating deletion mutant suggested that increased HBx protein expression and blocked virion secretion both contributed to the high replication phenotype. Our findings could help explain why such deletions are selected at a late stage of chronic HBV infection and how they contribute to viral pathogenesis. IMPORTANCE Expression of hepatitis B e antigen (HBeAg) and overproduction of HBsAg by wild-type HBV are implicated in the induction of immune tolerance to achieve chronic infection. How HBV survives the subsequent immune clearance phase remains incompletely understood. Our previous characterization of core promoter mutations to reduce HBeAg production revealed the ability of the 3.5-kb pgRNA to diminish transcription of coterminal RNAs of 2.4 kb, 2.1 kb, and 0.7 kb. The later stage of chronic HBV infection often selects for in-frame deletions in the preS region. Here, we found that many 3' preS1 deletions prevented transcription of the 2.1-kb RNA for HBsAg production, which was often accompanied by increases in intracellular 3.5-, 0.7-, and especially 2.4-kb RNAs, HBx and core proteins, and replicative DNA but lost virion secretion. These findings established the biological consequences of preS1 deletions, thus shedding light on why they are selected and how they contribute to hepatocarcinogenesis.


Assuntos
Genoma Viral , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/genética , Transativadores/biossíntese , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Proteínas Virais Reguladoras e Acessórias/biossíntese , Replicação Viral , Linhagem Celular Tumoral , Deleção de Genes , Regulação Viral da Expressão Gênica , Células Hep G2 , Vírus da Hepatite B/metabolismo , Humanos , Regiões Promotoras Genéticas , RNA Viral/metabolismo , Replicação Viral/genética
20.
Viruses ; 13(3)2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33801849

RESUMO

The pestivirus envelope protein Erns is anchored in membranes via a long amphipathic helix. Despite the unusual membrane topology of the Erns membrane anchor, it is cleaved from the following glycoprotein E1 by cellular signal peptidase. This was proposed to be enabled by a salt bridge-stabilized hairpin structure (so-called charge zipper) formed by conserved charged residues in the membrane anchor. We show here that the exchange of one or several of these charged residues reduces processing at the Erns carboxy-terminus to a variable extend, but reciprocal mutations restoring the possibility to form salt bridges did not necessarily restore processing efficiency. When introduced into an Erns-only expression construct, these mutations enhanced the naturally occurring Erns secretion significantly, but again to varying extents that did not correlate with the number of possible salt bridges. Equivalent effects on both processing and secretion were also observed when the proteins were expressed in avian cells, which points at phylogenetic conservation of the underlying principles. In the viral genome, some of the mutations prevented recovery of infectious viruses or immediately (pseudo)reverted, while others were stable and neutral with regard to virus growth.


Assuntos
Sequência de Aminoácidos/genética , Potenciais da Membrana/genética , Pestivirus/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , Galinhas , Cricetinae , Genoma Viral/genética , Glicosilação , Proteínas de Membrana/metabolismo , Mutação/genética , Pestivirus/genética , Serina Endopeptidases/metabolismo , Carga Viral , Fatores de Virulência/genética
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