Ali Zohaib

MicroRNA-15b Modulates Japanese Encephalitis Virus–Mediated Inflammation via Targeting RNF125

Japanese encephalitis virus (JEV) can target CNS and cause neuroinflammation that is characterized by profound neuronal damage and concomitant microgliosis/astrogliosis. Although microRNAs (miRNAs) have emerged as a major regulatory network with profound effects on inflammatory response, it is less clear how they regulate JEV-induced inflammation. In this study, we found that miR-15b is involved in modulating the JEV-induced inflammatory response. The data demonstrate that miR-15b is upregulated during JEV infection of glial cells and mouse brains. In vitro overexpression of miR-15b enhances the JEV-induced inflammatory response, whereas inhibition of miR-15b decreases it. Mechanistically, ring finger protein 125 (RNF125), a negative regulator of RIG-I signaling, is identified as a direct target of miR-15b in the context of JEV infection. Furthermore, inhibition of RNF125 by miR-15b results in an elevation in RIG-I levels, which, in turn, leads to a higher production of proinflammatory cytokines and type I IFN. In vivo knockdown of virus-induced miR-15b by antagomir-15b restores the expression of RNF125, reduces the production of inflammatory cytokines, attenuates glial activation and neuronal damage, decreases viral burden in the brain, and improves survival in the mouse model. Taken together, our results indicate that miR-15b modulates the inflammatory response during JEV infection by negative regulation of RNF125 expression. Therefore, miR-15b targeting may constitute an interesting and promising approach to control viral-induced neuroinflammation.

Etanercept Reduces Neuroinflammation and Lethality in Mouse Model of Japanese Encephalitis

BACKGROUND Japanese encephalitis virus (JEV) is a neurotropic flavivirus that causes Japanese encephalitis (JE), which leads to high fatality rates in human. Tumor necrosis factor alpha (TNF-α) is a key factor that mediates immunopathology in the central nervous system (CNS) during JE. Etanercept is a safe anti-TNF-α drug that has been commonly used in the treatment of various human autoimmune diseases. METHODS The effect of etanercept on JE was investigated with a JEV-infected mouse model. Four groups of mice were assigned to receive injections of phosphate-buffered saline, etanercept, JEV, or JEV plus etanercept. Inflammatory responses in mouse brains and mortality of mice were evaluated within 23 days post infection. RESULTS The in vitro assay with mouse neuron/glia cultures showed that etanercept treatment reduced the inflammatory response induced by JEV infection. In vivo experiments further demonstrated that administration of etanercept protected mice from JEV-induced lethality. Neuronal damage, glial activation, and secretion of proinflammatory cytokines were found to be markedly decreased in JEV-infected mice that received etanercept treatment. Additionally, etanercept treatment restored the integrity of the blood-brain barrier and reduced viral load in mouse brains. CONCLUSIONS Etanercept effectively reduces the inflammation and provides protection against acute encephalitis in a JEV-infected mouse model.

Heat Shock Protein 70 Is Associated with Replicase Complex of Japanese Encephalitis Virus and Positively Regulates Viral Genome Replication

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes the most prevalent viral encephalitis in Asia. The NS5 protein of JEV is a key component of the viral replicase complex, which plays a crucial role in viral pathogenesis. In this study, tandem affinity purification (TAP) followed by mass spectrometry analysis was performed to identify novel host proteins that interact with NS5. Heat shock protein 70 (Hsp70), eukaryotic elongation factor 1-alpha (eEF-1α) and ras-related nuclear protein (Ran) were demonstrated to interact with NS5. In addition to NS5, Hsp70 was also found to interact with NS3 which is another important member of the replicase complex. It was observed that the cytoplasmic Hsp70 partially colocalizes with the components of viral replicase complex including NS3, NS5 and viral dsRNA during JEV infection. Knockdown of Hsp70 resulted in a significantly reduced JEV genome replication. Further analysis reveals that Hsp70 enhances the stability of viral proteins in JEV replicase complex. These results suggest an important role for Hsp70 in regulating JEV replication, which provides a potential target for the development of anti-JEV therapies.

MicroRNA-33a-5p Modulates Japanese Encephalitis Virus Replication by Targeting Eukaryotic Translation Elongation Factor 1A1

ABSTRACT Japanese encephalitis virus (JEV) is a typical mosquito-borne flavivirus responsible for acute encephalitis and meningitis in humans. However, the molecular mechanism for JEV pathogenesis is still unclear. MicroRNAs (miRNAs) are small noncoding RNAs that act as gene regulators. They are directly or indirectly involved in many cellular functions owing to their ability to target mRNAs for degradation or translational repression. However, how cellular miRNAs are regulated and their functions during JEV infection are largely unknown. In the present study, we found that JEV infection downregulated the expression of endogenous cellular miR-33a-5p. Notably, artificially transfecting with miR-33a-5p mimics led to a significant decrease in viral replication, suggesting that miR-33a-5p acts as a negative regulator of JEV replication. A dual-luciferase reporter assay identified eukaryotic translation elongation factor 1A1 (EEF1A1) as one of the miR-33a-5p target genes. Our study further demonstrated that EEF1A1 can interact with the JEV proteins NS3 and NS5 in replicase complex. Through this interaction, EEF1A1 can stabilize the components of viral replicase complex and thus facilitates viral replication during JEV infection. Taken together, these results suggest that miR-33a-5p is downregulated during JEV infection, which contributes to viral replication by increasing the intracellular level of EEF1A1, an interaction partner of JEV NS3 and NS5. This study provides a better understanding of the molecular mechanisms of JEV pathogenesis. IMPORTANCE MiRNAs are critical regulators of gene expression that utilize sequence complementarity to bind to and modulate the stability or translation efficiency of target mRNAs. Accumulating data suggest that miRNAs regulate a wide variety of molecular mechanisms in the host cells during viral infections. JEV, a neurotropic flavivirus, is one of the major causes of acute encephalitis in humans worldwide. The roles of cellular miRNAs during JEV infections are widely unexplored. The present study explores a novel role of miR-33a-5p as a negative regulator of JEV replication. We found EEF1A1 as a direct target of miR-33a-5p. We also demonstrated that EEF1A1 interacts with and stabilize the components of JEV replicase complex, which positively regulates JEV replication. These findings suggest a new insight into the molecular mechanism of JEV pathogenesis and provide a possible therapeutic entry point for viral encephalitis.

MicroRNA profile analysis of Epithelioma papulosum cyprini cell line before and after SVCV infection

MicroRNAs (miRNAs) play significant roles in regulating almost all of the biological processes in eukaryotes. An accumulating body of evidence shows that miRNAs are associated with cellular changes following viral infection. Spring viremia of carp virus (SVCV) is the pathogen of Spring viremia of carp (SVC), which results in heavy losses in the cultured common carp (Cyprinus carpio) industry in many countries. To study the involvement of miRNAs during SVCV infection, we adopted the Solexa sequencing technology to sequence small RNA libraries from the Epithelioma papulosum cyprini (EPC) cell line before and after infection with SVCV. In this study, a total of 161 conserved and 26 novel miRNAs were identified. Subsequently, the expression patterns of these miRNAs were compared between the uninfected (control library, M) and SVCV-infected (infection library, E) libraries. In addition, to verify the Solexa sequencing results, the expression patterns of 14 randomly selected miRNAs were validated by qRT-PCR. The targets of the significantly differentially expressed miRNAs were then predicted, and the miRNAs that could directly target the SVCV genome were also predicted. No miRNA encoded by SVCV itself was detected. To the best of our knowledge, this study presents the first miRNA profiling assessment in association with fish rhabdovirus infection, and the data presented lay a foundation for further investigations to determine the roles of miRNAs in regulating the molecular mechanism during SVCV infection.

Japanese Encephalitis Virus NS5 Inhibits the Type I Interferon Production by Blocking the Nuclear Translocation of IRF3 and NF-κB

ABSTRACT The type I interferon (IFN) response is part of the first-line defense against viral infection. To initiate replication, viruses have developed powerful evasion strategies to counteract host IFN responses. In the present study, we found that the Japanese encephalitis virus (JEV) NS5 protein could inhibit double-stranded RNA (dsRNA)-induced IFN-β expression in a dose-dependent manner. Our data further demonstrated that JEV NS5 suppressed the activation of the IFN transcriptional factors IFN regulatory factor 3 (IRF3) and NF-κB. However, there was no defect in the phosphorylation of IRF3 and degradation of IκB, an upstream inhibitor of NF-κB, upon NS5 expression, indicating a direct inhibition of the nuclear localization of IRF3 and NF-κB by NS5. Mechanistically, NS5 was shown to interact with the nuclear transport proteins KPNA2, KPNA3, and KPNA4, which competitively blocked the interaction of KPNA3 and KPNA4 with their cargo molecules, IRF3 and p65, a subunit of NF-κB, and thus inhibited the nuclear translocation of IRF3 and NF-κB. Furthermore, overexpression of KPNA3 and KPNA4 restored the activity of IRF3 and NF-κB and increased the production of IFN-β in NS5-expressing or JEV-infected cells. Additionally, an upregulated replication level of JEV was shown upon KPNA3 or KPNA4 overexpression. These results suggest that JEV NS5 inhibits the induction of type I IFN by targeting KPNA3 and KPNA4. IMPORTANCE JEV is the major cause of viral encephalitis in South and Southeast Asia, with high mortality. However, the molecular mechanisms contributing to the severe pathogenesis are poorly understood. The ability of JEV to counteract the host innate immune response is potentially one of the mechanisms responsible for JEV virulence. Here we demonstrate the ability of JEV NS5 to interfere with the dsRNA-induced nuclear translocation of IRF3 and NF-κB by competitively inhibiting the interaction of IRF3 and NF-κB with nuclear transport proteins. Via this mechanism, JEV NS5 suppresses the induction of type I IFN and the antiviral response in host cells. These findings reveal a novel strategy for JEV to escape the host innate immune response and provide new insights into the pathogenesis of JEV.

MicroRNA-22 negatively regulates poly(I:C)-triggered type I interferon and inflammatory cytokine production via targeting mitochondrial antiviral signaling protein (MAVS)

MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in regulating the host immune response. Here we found that miR-22 is induced in glial cells upon stimulation with poly(I:C). Overexpression of miR-22 in the cultured cells resulted in decreased activity of interferon regulatory factor-3 and nuclear factor-kappa B, which in turn led to reduced expression of interferon-β and inflammatory cytokines, including tumor necrosis factor-α, interleukin-1β, interleukin-6, and chemokine (C-C motif) ligand 5, upon stimulation with poly(I:C), whereas knockdown of miR-22 had the opposite effect. We used a combination of bioinformatics and experimental techniques to demonstrate that mitochondrial antiviral signaling protein (MAVS), which positively regulates type I interferon production, is a novel target of miR-22. Overexpression of miR-22 decreased the activity of a luciferase reporter containing the MAVS 3′-untranslated region and led to decreased MAVS mRNA and protein levels. In contrast, ectopic expression of miR-22 inhibitor led to elevated MAVS expression. Collectively, our results demonstrate that miR-22 negatively regulates poly(I:C)-induced production of type I interferon and inflammatory cytokines via targeting MAVS.

CRISPR/Cas9 therapeutics: a cure for cancer and other genetic diseases

Cancer is caused by a series of alterations in genome and epigenome mostly resulting in activation of oncogenes or inactivation of cancer suppressor genes. Genetic engineering has become pivotal in the treatment of cancer and other genetic diseases, especially the formerly-niche use of clustered regularly interspaced short palindromic repeats (CRISPR) associated with Cas9. In defining its superior use, we have followed the recent advances that have been made in producing CRISPR/Cas9 as a therapy of choice. We also provide important genetic mutations where CRISPRs can be repurposed to create adaptive immunity to fight carcinomas and edit genetic mutations causing it. Meanwhile, challenges to CRISPR technology are also discussed with emphasis on ability of pathogens to evolve against CRISPRs. We follow the recent developments on the function of CRISPRs with different carriers which can efficiently deliver it to target cells; furthermore, analogous technologies are also discussed along CRISPRs, including zinc-finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs). Moreover, progress in clinical applications of CRISPR therapeutics is reviewed; in effect, patients can have lower morbidity and/or mortality from the therapeutic method with least possible side-effects.

The Yin and Yang of Antiviral Innate Immunity in Central Nervous System.

The innate immune system provides protection against invading neurotropic viruses. It acts as the first line of defense against invading viruses and plays an elementary role in their pathogenesis. The list of viruses capable of infecting human central nervous system (CNS) is quite long, most important of them are Japanese Encephalitis virus (JEV), rabies virus, West Nile virus (WNV), herpes simplex virus (HSV), St. Louis encephalitis virus (SLEV), La Crosse virus, tick borne encephalitis virus (TEBE) and polio virus. Germ line pattern recognition receptors (PRRs) such as Toll like receptors (TLRs), nucleotide binding oligomerization domain (NOD) - like receptors (NLRs), retinoic acid-inducible gene I (RIG-I) -like helicases or RIG-I-like receptors (RLRs) and cytosolic DNA sensors recognize the pathogen associated molecular patterns (PAMPs) and initiate an immune response against invading pathogen. Although PRRs were originally characterized in peripheral immune cells but accumulating evidence also suggest their crucial roles in CNS to combat against neurotropic viruses. In this review, we will highlight the recent developments in our understating of the mechanisms by which PRRs in resident brain cells provide protection against invading neurotropic viruses.