Construction of an Infectious Clone for Mosquito-Derived Tembusu Virus Prototypical Strain

AbstractCoat protein (CP) is required for potyviruses to move and establish a systemic infection in plants. π-stackings formed by aromatic residues play critical roles in maintaining protein stability and functions. As we know, many aromatic residues located in the core region of potyvirus CPs are conserved. However, their roles in potyvirus infection remain largely unknown. Here, through analysis of the three-dimensional model of the tobacco vein banding mosaic virus (TVBMV; genus Potyvirus) CP, 16 aromatic residues were predicated to form π-stackings. The results of transient expression experiments demonstrated that deletion of any of these 16 aromatic residues reduced CP accumulation. Infectivity assays showed that deletion of any of these aromatic residues in the TVBMV infectious clone abolished cell-to-cell movement and reduced replication of the virus. Substitution of Y105 and Y147 individually with non-aromatic residues alanine or glycine reduced CP accumulation, virus replication, and abolished the ability of TVBMV to move intercellularly, while substitution of these two residues individually with aromatic residues phenylalanine or tryptophan, had no or little effect on CP accumulation and TVBMV systemic movement and replication. Similar results were obtained from the CP mutants of watermelon mosaic virus (WMV, genus Potyvirus). Taken together, our results demonstrate that multiple aromatic residues in CP are involved in potyvirus movement by forming π-stackings to maintain CP accumulation.

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Interaction of Pestiviral E1 and E2 Sequences in Dimer Formation and Intracellular Retention

International Journal of Molecular Sciences ◽

10.3390/ijms22147285 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7285

Author(s):

Yu Mu ◽

Birke Andrea Tews ◽

Christine Luttermann ◽

Gregor Meyers

Keyword(s):

Life Cycle ◽

Cell Surface ◽

Double Mutant ◽

Essential Role ◽

Infectious Clone ◽

Intracellular Localization ◽

Dimer Formation ◽

Covalent Linkage ◽

Crucial Step ◽

Retention Signal

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.

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Engineering viable foot-and-mouth disease viruses with increased acid stability facilitate the development of improved vaccines

Applied Microbiology and Biotechnology ◽

10.1007/s00253-019-10280-9 ◽

2020 ◽

Vol 104 (4) ◽

pp. 1683-1694 ◽

Cited By ~ 2

Author(s):

Hong Yuan ◽

Pinghua Li ◽

Huifang Bao ◽

Pu Sun ◽

Xingwen Bai ◽

...

Keyword(s):

Structural Integrity ◽

Infectious Clone ◽

Foot And Mouth Disease ◽

Acid Resistance ◽

Mouth Disease ◽

Acid Stability ◽

Intact Virion ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Negative Effect

AbstractFoot-and-mouth disease virus (FMDV), the most acid-unstable virus among picornaviruses, tends to disassemble into pentamers at pH values slightly below neutrality. However, the structural integrity of intact virion is one of the most important factors that influence the induction of a protective antibody response. Thus, improving the acid stability of FMDV is required for the efficacy of vaccine preparations. According to the previous studies, a single substitution or double amino acid substitutions (VP1 N17D, VP2 H145Y, VP2 D86H, VP3 H142D, VP3 H142G, and VP1 N17D + VP2 H145Y) in the capsid were introduced into the full-length infectious clone of type O FMDV vaccine strain O/HN/CHN/93 to develop seed FMDV with improved acid stability. After the transfection into BSR/T7 cells of constructed plasmids, substitution VP1 N17D or VP2 D86H resulted in viable and genetically stable FMDVs, respectively. However, substitution VP2 H145Y or VP1 N17D + VP2 H145Y showed reverse mutation and additional mutations, and substitution VP3 H141G or VP3 H141D prevented viral viability. We found that substitution VP1 N17D or VP2 D86H could confer increased acid resistance, alkali stability, and thermostability on FMDV O/HN/CHN/93, whereas substitution VP1 N17D was observed to lead to a decreased replication ability in BHK-21 cells and mildly impaired virulence in suckling mice. In contrast, substitution VP2 D86H had no negative effect on viral infectivity. These results indicated that the mutant rD86H carrying substitution VP2 D86H firstly reported by us could be more adequate for the development of inactivated FMD vaccines with enhanced acid stability.

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Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽

10.3390/v13040632 ◽

2021 ◽

Vol 13 (4) ◽

pp. 632

Author(s):

Yingyun Cai ◽

Shuiqing Yu ◽

Ying Fang ◽

Laura Bollinger ◽

Yanhua Li ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Cell Lines ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Infectious Clone ◽

Hemorrhagic Fever ◽

Simian Hemorrhagic Fever Virus ◽

Hemorrhagic Fever Virus ◽

Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

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Molecular identification of duck DDX3X and its potential role in response to Tembusu virus

Developmental & Comparative Immunology ◽

10.1016/j.dci.2019.103599 ◽

2020 ◽

Vol 106 ◽

pp. 103599 ◽

Cited By ~ 1

Author(s):

Ning Li ◽

Shengnan Jiang ◽

Jun Zhao ◽

Yudong Yang ◽

Kai Deng ◽

...

Keyword(s):

Molecular Identification ◽

Potential Role ◽

Tembusu Virus

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In VitroAssessment of Combinations of Enterovirus Inhibitors against Enterovirus 71

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01073-16 ◽

2016 ◽

Vol 60 (9) ◽

pp. 5357-5367 ◽

Cited By ~ 9

Author(s):

Yizhuo Wang ◽

Guiming Li ◽

Shilin Yuan ◽

Qianqian Gao ◽

Ke Lan ◽

...

Keyword(s):

Infectious Clone ◽

Enterovirus 71 ◽

Foot And Mouth Disease ◽

Antiviral Effect ◽

Mechanisms Of Action ◽

Rational Basis ◽

Inhibitory Mechanism ◽

Antiviral Therapies ◽

Resistant Phenotype ◽

Combination Regimens

ABSTRACTEnterovirus 71 (EV-A71) is a major causative pathogen of hand, foot, and mouth disease (HFMD) epidemics. No antiviral therapies are currently available for treating EV-A71 infections. Here, we selected five reported enterovirus inhibitors (suramin, itraconazole [ITZ], GW5074, rupintrivir, and favipiravir) with different mechanisms of action to test their abilities to inhibit EV-A71 replication alone and in combination. All selected compounds have anti-EV-A71 activities in cell culture. The combination of rupintrivir and ITZ or favipiravir was synergistic, while the combination of rupintrivir and suramin was additive. The combination of suramin and favipiravir exerted a strong synergistic antiviral effect. The observed synergy was not due to cytotoxicity, as there was no significant increase in cytotoxicity when compounds were used in combinations at the tested doses. To investigate the potential inhibitory mechanism of favipiravir against enterovirus, two favipiravir-resistant EV-A71 variants were independently selected, and both of them carried an S121N mutation in the finger subdomain of the 3D polymerase. Reverse engineering of this 3D S121N mutation into an infectious clone of EV-A71 confirmed the resistant phenotype. Moreover, viruses resistant to ITZ or favipiravir remained susceptible to other inhibitors. Most notably, combined with ITZ, rupintrivir prevented the development of ITZ-resistant variants. Taken together, these results provide a rational basis for the design of combination regimens for use in the treatment of EV-A71 infections.

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Duck Tembusu virus exhibits neurovirulence in BALB/c mice

Virology Journal ◽

10.1186/1743-422x-10-260 ◽

2013 ◽

Vol 10 (1) ◽

pp. 260 ◽

Cited By ~ 38

Author(s):

Shuang Li ◽

Xiaoxia Li ◽

Lijiao Zhang ◽

Yongyue Wang ◽

Xiuling Yu ◽

...

Keyword(s):

Duck Tembusu Virus ◽

Tembusu Virus

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Binding of Hepatitis C Virus-Like Particles Derived from Infectious Clone H77C to Defined Human Cell Lines

Journal of Virology ◽

10.1128/jvi.76.3.1181-1193.2002 ◽

2002 ◽

Vol 76 (3) ◽

pp. 1181-1193 ◽

Cited By ~ 74

Author(s):

Sabine Wellnitz ◽

Bettina Klumpp ◽

Heidi Barth ◽

Susumu Ito ◽

Erik Depla ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Lines ◽

Human Cell ◽

Envelope Glycoprotein ◽

Infectious Clone ◽

Cell Interactions ◽

Target Cells ◽

Human Cell Lines ◽

Glycoprotein E2

ABSTRACT Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of viral entry and infection has been hampered by the inability to efficiently propagate the virus in cultured cells and the lack of a small-animal model. Recent studies have shown that in insect cells, the HCV structural proteins assemble into HCV-like particles (HCV-LPs) with morphological, biophysical, and antigenic properties similar to those of putative virions isolated from HCV-infected humans. In this study, we used HCV-LPs derived from infectious clone H77C as a tool to examine virus-cell interactions. The binding of partially purified particles to human cell lines was analyzed by fluorescence-activated cell sorting with defined monoclonal antibodies to envelope glycoprotein E2. HCV-LPs demonstrated dose-dependent and saturable binding to defined human lymphoma and hepatoma cell lines but not to mouse cell lines. Binding could be inhibited by monoclonal anti-E2 antibodies, indicating that the HCV-LP-cell interaction was mediated by envelope glycoprotein E2. Binding appeared to be CD81 independent and did not correlate with low-density lipoprotein receptor expression. Heat denaturation of HCV-LPs drastically reduced binding, indicating that the interaction of HCV-LPs with target cells was dependent on the proper conformation of the particles. In conclusion, our data demonstrate that insect cell-derived HCV-LPs bind specifically to defined human cell lines. Since the envelope proteins of HCV-LPs are presumably presented in a virion-like conformation, the binding of HCV-LPs to target cells may allow the study of virus-host cell interactions, including the isolation of HCV receptor candidates and antibody-mediated neutralization of binding.

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