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1.
J Med Virol ; 96(4): e29522, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38533889

RESUMO

The tick-borne encephalitis virus (TBEV) serocomplex includes several medically important flavivirus members endemic to Europe, Asia, and North America, which can induce severe neuroinvasive or viscerotropic diseases with unclear mechanisms of pathogenesis. Langat virus (LGTV) shares a high sequence identity with TBEV but exhibits lower pathogenic potential in humans and serves as a model for virus-host interactions. In this study, we demonstrated that LGTV infection inhibits the activation of gp130/JAK/STAT (Janus kinases (JAK) and signal transducer and activator of transcription (STAT)) signaling, which plays a pivotal role in numerous biological processes. Our data show that the LGTV-infected cells had significantly lower phosphorylated STAT3 (pSTAT3) protein upon oncostatin M (OSM) stimulation than the mock-infected control. LGTV infection blocked the nuclear translocation of STAT3 without a significant effect on total STAT3 protein level. LGTV inhibited JAK1 activation and reduced gp130 protein expression in infected cells, with the viral NS5 protein mediating this effect. TBEV infection also reduces gp130 level. On the other hand, pretreatment of Vero cells with OSM significantly reduces LGTV replication, and STAT1/STAT2 knockdown had little effect on OSM-mediated antiviral effect, which suggests it is independent of STAT1/STAT2 and, instead, it is potentially mediated by STAT3 signlaing. These findings shed light on the LGTV and TBEV-cell interactions, offering insights for the future development of antiviral therapeutics and improved vaccines.


Assuntos
Fenômenos Biológicos , Vírus da Encefalite Transmitidos por Carrapatos , Animais , Chlorocebus aethiops , Humanos , Janus Quinases/metabolismo , Células Vero , Receptor gp130 de Citocina/metabolismo , Antivirais/metabolismo
2.
Virol J ; 21(1): 161, 2024 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-39039546

RESUMO

Hepatitis E virus (HEV) is a positive-sense, single-stranded RNA virus and causes primarily acute self-limiting infections. The ORF1 of the HEV genome encodes a polyprotein around 190 kDa, which contains several putative domains, including helicase and RNA-dependent RNA polymerase. The HEV-encoded helicase is a member of the superfamily 1 helicase family and possesses multiple enzymatic functions, such as RNA 5'-triphosphatase, RNA unwinding, and NTPase, which are thought to contribute to viral RNA synthesis. However, the helicase interaction with cellular proteins remains less known. Oxysterol binding protein (OSBP) is a lipid regulator that shuffles between the Golgi apparatus and the endoplasmic reticulum for cholesterol and phosphatidylinositol-4-phosphate exchange and controls the efflux of cholesterol from cells. In this study, the RNAi-mediated silencing of OSBP significantly reduced HEV replication. Further studies indicate that the HEV helicase interacted with OSBP, shown by co-immunoprecipitation and co-localization in co-transfected cells. The presence of helicase blocked OSBP preferential translocation to the Golgi apparatus. These results demonstrate that OSBP contributes to HEV replication and enrich our understanding of the HEV-cell interactions.


Assuntos
Complexo de Golgi , Vírus da Hepatite E , Receptores de Esteroides , Replicação Viral , Vírus da Hepatite E/fisiologia , Vírus da Hepatite E/genética , Receptores de Esteroides/metabolismo , Receptores de Esteroides/genética , Humanos , Complexo de Golgi/metabolismo , Complexo de Golgi/virologia , Interações Hospedeiro-Patógeno , Linhagem Celular , Ligação Proteica , Hepatite E/virologia , Hepatite E/metabolismo
3.
mBio ; : e0230824, 2024 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-39345136

RESUMO

The primary challenge posed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is COVID-19-related mortality, often exacerbated by additional medical complications, such as COVID-19-associated kidney injuries (CAKIs). Up to half of COVID-19 patients experience kidney complications, with those facing acute respiratory failure and kidney injury having the worst overall prognosis. Despite the significant impact of CAKI on COVID-19-related mortality and its enduring effects in long COVID, the underlying causes and molecular mechanisms of CAKI remain elusive. In this study, we identified a functional relationship between the expression of the SARS-CoV-2 ORF3a protein and inflammation-driven apoptotic death of renal tubular epithelial cells in patients with CAKI. We demonstrate in vitro that ORF3a independently induces renal cell-specific apoptotic cell death, as evidenced by the elevation of kidney injury molecule-1 (KIM-1) and the activation of NF-kB-mediated proinflammatory cytokine (TNFα and IL-6) production. By examining kidney tissues of SARS-CoV-2-infected K18-ACE2 transgenic mice, we observed a similar correlation between ORF3a-induced cytopathic changes and kidney injury. This correlation was further validated through reconstitution of the ORF3a effects via direct adenoviral injection into mouse kidneys. Through medicinal analysis, we identified a natural compound, glycyrrhizin (GL4419), which not only blocks viral replication in renal cells, but also mitigates ORF3a-induced renal cell death by inhibiting activation of a high mobility group box 1 (HMGB1) protein, leading to a reduction of KIM-1. Moreover, ORF3a interacts with HMGB1. Overproduction or downregulation of hmgb1 expression results in correlative changes in renal cellular KIM-1 response and respective cytokine production, implicating a crucial role of HMGB1 in ORF3a-inflicted kidney injuries. Our data suggest a direct functional link between ORF3a and kidney injury, highlighting ORF3a as a unique therapeutic target contributing to CAKI. IMPORTANCE: The major challenge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during the pandemic is COVID-19-related mortality, which has tragically claimed millions of lives. COVID-19-associated morbidity and mortality are often exacerbated by pre-existing medical conditions, such as chronic kidney diseases (CKDs), or the development of acute kidney injury (AKI) due to COVID-19, collectively known as COVID-19-associated kidney injuries (CAKIs). Patients who experience acute respiratory failure with CAKI have the poorest clinical outcomes, including increased mortality. Despite these alarming clinical findings, there is a critical gap in our understanding of the underlying causes of CAKI. Our study establishes a direct correlation between the expression of the SARS-CoV-2 viral ORF3a protein and kidney injury induced by ORF3a linking to CAKI. This functional relationship was initially observed in our clinical studies of COVID-19 patients with AKI and was further validated through animal and in vitro cellular studies, either by expressing ORF3a alone or in the context of viral infection. By elucidating this functional relationship and its underlying mechanistic pathways, our research deepens the understanding of COVID-19-associated kidney diseases and presents potential therapeutic avenues to address the healthcare challenges faced by individuals with underlying conditions.

4.
Front Cell Dev Biol ; 10: 1011221, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36506095

RESUMO

The ongoing SARS-CoV-2/COVID-19 pandemic caused a global public health crisis. Yet, everyone's response to SARS-CoV-2 infection varies, and different viral variants confer diverse pathogenicity. Thus, it is imperative to understand how viral determinants contribute to COVID-19. Viral ORF3a protein is one of those viral determinants, as its functions are linked to induction of cell and tissues damages, disease severity and cytokine storm that is a major cause of COVID-19-related death. ORF3a is a membrane-associated protein. Upon synthesis, it is transported from endoplasmic reticulum, Golgi apparatus to plasma membrane and subcellular endomembranes including endosomes and lysosomes. However, how ORF3a is transported intracellularly remains elusive. The goal of this study was to carry out a systematic mutagenesis study to determine the structural relationship of ORF3a protein with its subcellular locations. Single amino acid (aa) and deletion mutations were generated in the putative function-relevant motifs and other regions of interest. Immunofluorescence and ImageJ analyses were used to determine and quantitate subcellular locations of ORF3a mutants in comparison with wildtype ORF3a. The wildtype ORF3a localizes predominantly (Pearson's coefficients about 0.8) on the membranes of endosomes and lysosomes. Consistent with earlier findings, deletion of the YXXΦ motif, which is required for protein export, retained ORF3a in the Golgi apparatus. Interestingly, mutations in a double glycine (diG) region (aa 187-188) displayed a similar phenotype to the YXXΦ deletion, implicating a similar role of the diG motif in intracellular transport. Indeed, interrupting any one of the two glycine residues such as deletion of a single (dG188), both (dG187/dG188) or substitution (G188Y) of these residues led to ORF3a retention in the Golgi apparatus (Pearson's coefficients ≥0.8). Structural analyses further suggest that the diG motif supports a type-II ß-turn between the anti-parallel ß4 and ß5 sheets and connects to the YXXΦ motif via hydrogen bonds between two monomers. The diG- YXXΦ interaction forms a hand-in-hand configuration that could facilitate dimerization. Together, these observations suggest a functional role of the diG motif in intracellular transport of ORF3a.

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