West Nile virus (WNV) belongs to the Flaviviridae family and has emerged as a significant cause of viral encephalitis in birds and animals including humans. WNV replication directly induces neuronal ...injury, followed by neuronal cell death. We previously showed that accumulation of ubiquitinated protein aggregates was involved in neuronal cell death in the WNV-infected mouse brain. In this study, we attempted to elucidate the mechanisms of the accumulation of protein aggregates in the WNV-infected cells. To identify the viral factor inducing the accumulation of ubiquitinated proteins, intracellular accumulation of ubiquitinated proteins was examined in the cells expressing the viral protein. Expression of capsid (C) protein induced the accumulation, while mutations at residues L51 and A52 in C protein abrogated the accumulation. Wild-type (WT) or mutant WNV in which mutations were introduced into the residues was inoculated into human neuroblastoma cells. The expression levels of LC3-II, an autophagy-related protein, and AMP-activated protein kinase (AMPK), an autophagy inducer, were reduced in the cells infected with WT WNV, while the reduction was not observed in the cells infected with WNV with the mutations in C protein. Similarly, ubiquitination and degradation of AMPK were only observed in the cells infected with WT WNV. In the cells expressing C protein, AMPK was co-precipitated with C protein and mutations in L51 and A52 reduced the interaction. Although the viral replication was not affected, the accumulation of ubiquitinated proteins in brain and neurological symptoms were attenuated in the mouse inoculated with WNV with the mutations in C protein as compared with that with WT WNV. Taken together, ubiquitination and degradation of AMPK by C protein resulted in the inhibition of autophagy and the accumulation of protein aggregates, which contributes to the development of neurological disease.
Neurological diseases caused by encephalitic flaviviruses are severe and associated with high levels of mortality. However, little is known about the detailed mechanisms of viral replication and ...pathogenicity in the brain. Previously, we reported that the genomic RNA of tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is transported and replicated in the dendrites of neurons. In the present study, we analyzed the transport mechanism of the viral genome to dendrites. We identified specific sequences of the 5′ untranslated region of TBEV genomic RNA that act as a cis-acting element for RNA transport. Mutated TBEV with impaired RNA transport in dendrites caused a reduction in neurological symptoms in infected mice. We show that neuronal granules, which regulate the transport and local translation of dendritic mRNAs, are involved in TBEV genomic RNA transport. TBEV genomic RNA bound an RNA-binding protein of neuronal granules and disturbed the transport of dendritic mRNAs. These results demonstrated a neuropathogenic virus hijacking the neuronal granule system for the transport of viral genomic RNA in dendrites, resulting in severe neurological disease.
Tick‐borne encephalitis virus (TBEV) is a zoonotic virus that causes encephalitis in humans. Various deletions have been reported in a variable region of the 3′ untranslated region of the TBEV ...genome. This study analyzed the role of a Y‐shaped secondary structure in the pathogenicity of TBEV by using reverse genetics. Deletion of the structure increased the mortality rate of virus‐infected mice but did not affect the virus multiplication in cultured cells and organs. The results indicate that the secondary structure is involved in the regulation of TBEV pathogenesis.
Tick-borne encephalitis virus (TBEV) is a zoonotic virus belonging to the genus Flavivirus, in the family Flaviviridae. The virus, which is endemic in Europe and northern parts of Asia, causes severe ...encephalitis. Tick-borne encephalitis (TBE) has been reported in Mongolia since the 1980s, but details about the biological characteristics of the endemic virus are lacking. In this study, 680 ticks (Ixodes persulcatus) were collected in Selenge aimag, northern Mongolia, in 2012. Nine Mongolian TBEV strains were isolated from tick homogenates. A sequence analysis of the envelope protein gene revealed that all isolates belonged to the Siberian subtype of TBEV. Two strains showed similar growth properties in cultured cells, but their virulence in mice differed. Whole genome sequencing revealed only thirteen amino acid differences between these Mongolian TBEV strains. Our results suggest that these naturally occurring amino acid mutations affected the pathogenicity of Mongolian TBEV. Our results may be an important platform for monitoring TBEV to evaluate the epidemiological risk in TBE endemic areas of Mongolia.
•We examined the functions of the variable region of 3′-UTR of TBEV.•The partial deletion or polyA insertion in the region increased the virulence of TBEV in mice.•The mutations did not affect the ...production of subgenomic flavivirus RNA and IFN responses.•We concluded that the intact structure of the region is involved in the pathogenicity of TBEV.
Tick-borne encephalitis virus (TBEV) is maintained between ticks and mammals in nature and causes severe neurological disease in human. However, the mechanism of viral pathogenicity is unknown. Previously, we showed that the deletion in the variable region of the 3′-untranslated region (UTR) is involved in the pathogenicity of the strains from the Far-Eastern subtype of TBEV. To investigate the detailed function of the variable region, we constructed recombinant TBEV with partial deletions in the region. In a mouse model, the partial deletions drastically increased the virulence of the virus, with no effect on virus multiplication in mouse brain. Furthermore, the mutations did not affect the production of subgenomic flavivirus RNA from the 3′-UTR, and the induction of interferon (IFN) and IFN-stimulated genes. These data suggested that the conformational structure of the variable region is associated with the pathogenicity of the Far-Eastern subtype of TBEV. These findings provide a foundation for further research to identify the pathogenic mechanisms of TBEV.
•We identified host proteins which interact with the 3′-UTR of TBEV.•CSDE1, FMRP, ILF3, and STRBP interacted with the low-virulent strain of TBEV.•Deletion in the variable region of the 3′-UTR ...suppressed the interaction.
Tick-borne encephalitis virus (TBEV) causes severe neurological disease, but the pathogenetic mechanism is unclear. The conformational structure of the 3′-untranslated region (UTR) of TBEV is associated with its virulence. We tried to identify host proteins interacting with the 3′-UTR of TBEV. Cellular proteins of HEK293T cells were co-precipitated with biotinylated RNAs of the 3′-UTR of low- and high-virulence TBEV strains and subjected to mass spectrometry analysis. Fifteen host proteins were found to bind to the 3′-UTR of TBEV, four of which—cold shock domain containing-E1 (CSDE1), spermatid perinuclear RNA binding protein (STRBP), fragile X mental retardation protein (FMRP), and interleukin enhancer binding factor 3 (ILF3)—bound specifically to that of the low-virulence strain. An RNA immunoprecipitation and pull-down assay confirmed the interactions of the complete 3′-UTRs of TBEV genomic RNA with CSDE1, FMRP, and ILF3. Partial deletion of the stem loop (SL) 3 to SL 5 structure of the variable region of the 3′-UTR did not affect interactions with the host proteins, but the interactions were markedly suppressed by deletion of the complete SL 3, 4, and 5 structures, as in the high-virulence TBEV strain. Further analysis of the roles of host proteins in the neurologic pathogenicity of TBEV is warranted.
•A reverse genetics system for WNV based on homologous recombination was developed.•Biological properties of recombinant WNV were same as those of the parent virus.•A chimeric WNV between different ...strains was produced.
Reverse genetics systems facilitate investigation of many aspects of the life cycle and pathogenesis of viruses. However, genetic instability in Escherichia coli has hampered development of a reverse genetics system for West Nile virus (WNV). In this study, we developed a novel reverse genetics system for WNV based on homologous recombination in mammalian cells. Introduction of the DNA fragment coding for the WNV structural protein together with a DNA-based replicon resulted in the release of infectious WNV. The growth rate and plaque size of the recombinant virus were almost identical to those of the parent WNV. Furthermore, chimeric WNV was produced by introducing the DNA fragment coding for the structural protein and replicon plasmid derived from various strains. Here, we report development of a novel system that will facilitate research into WNV infection.
Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in humans. Despite its high medical relevance, no specific antiviral therapy is currently available. Here we ...demonstrate that treatment with a nucleoside analog, 7-deaza-2'-
-methyladenosine (7-deaza-2'-CMA), substantially improved disease outcomes, increased survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2'-CMA, two drug-resistant TBEV clones were generated and characterized. The two clones shared a signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This mutation conferred resistance to various 2'-
-methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'-
-azidocytidine and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187). All-atom molecular dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that coordinates the position of a metal ion cofactor as 2'-
-methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV strain (Oshima-IC) generated from an infectious cDNA clone and into a TBEV replicon that expresses a reporter luciferase gene (Oshima-REP-luc2A). The mutants were replication impaired, showing reduced growth and a small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These results indicate that TBEV resistance to 2'-
-methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of the viral NS5 RdRp and is associated with strong attenuation of the virus.
This study found that the nucleoside analog 7-deaza-2'-
-methyladenosine (7-deaza-2'-CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-deaza-2'-CMA resulted in significantly higher survival rates and reduced the severity of neurological signs of the disease. Thus, this compound shows promise for further development as an anti-TBEV drug. It is important to generate drug-resistant mutants to understand how the drug works and to develop guidelines for patient treatment. We generated TBEV mutants that were resistant not only to 7-deaza-2'-CMA but also to a broad range of other 2'-
-methylated antiviral medications. Our findings suggest that combination therapy may be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.
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