SARS-CoV-2 exploits cellular RAD51 to promote viral propagation: implication of RAD51 inhibitor as a potential drug candidate against COVID-19.

IMPORTANCE: Viruses are constantly evolving to promote propagation in the host. Here, we show that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes host RAD51 for replication. Silencing of RAD51 impaired SARS-CoV-2 propagation. Viral RNA colocalized with RAD51 in the cytoplasm of SARS-CoV-2-infected cells, suggesting that both viral RNA and RAD51 may form a replication complex. We, therefore, evaluated RAD51 inhibitors as possible therapeutic agents against SARS-CoV-2. Indeed, RAD51 inhibitors exerted antiviral activities against not only Wuhan but also variants of SARS-CoV-2. Molecular docking model shows that RAD51 inhibitors impede SARS-CoV-2 propagation by interfering with dimerization of RAD51. These data suggest that RAD51 may represent a novel host-based drug target for coronavirus disease 2019 treatment.

Polygalic acid inhibits african swine fever virus polymerase activity: findings from machine learning and in vitro testing.

African swine fever virus (ASFV), an extremely contagious virus with high mortality rates, causes severe hemorrhagic viral disease in both domestic and wild pigs. Fortunately, ASFV cannot be transmitted from pigs to humans. However, ongoing ASFV outbreaks could have severe economic consequences for global food security. Although ASFV was discovered several years ago, no vaccines or treatments are commercially available yet; therefore, the identification of new anti-ASFV drugs is urgently warranted. Using molecular docking and machine learning, we have previously identified pentagastrin, cangrelor, and fostamatinib as potential antiviral drugs against ASFV. Here, using machine learning combined with docking simulations, we identified natural products with a high affinity for AsfvPolX proteins. We selected five natural products (NPs) that are located close in chemical space to the six known natural flavonoids that possess anti-ASFV activity. Polygalic acid markedly reduced AsfvPolX polymerase activity in a dose-dependent manner. We propose an efficient protocol for identifying NPs as potential antiviral drugs by identifying chemical spaces containing high-affinity binders against ASFV in NP databases.

Non-invasive administration of AAV to target lung parenchymal cells and develop SARS-CoV-2-susceptible mice.

Adeno-associated virus (AAV)-mediated gene delivery holds great promise for gene therapy. However, the non-invasive delivery of AAV for lung tissues has not been adequately established. Here, we revealed that the intratracheal administration of an appropriate amount of AAV2/8 predominantly targets lung tissue. AAV-mediated gene delivery that we used in this study induced the expression of the desired protein in lung parenchymal cells, including alveolar type II cells. We harnessed the technique to develop severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-susceptible mice. Three kinds of immune function-relevant gene knockout (KO) mice were transduced with AAV encoding human angiotensin-converting enzyme 2 (hACE2) and then injected with SARS-CoV-2. Among these mice, type I interferon receptor (IFNAR) KO mice showed increased viral titer in the lungs compared to that in the other KO mice. Moreover, nucleocapsid protein of SARS-CoV-2 and multiple lesions in the trachea and lung were observed in AAV-hACE2-transduced, SARS-CoV-2-infected IFNAR KO mice, indicating the involvement of type I interferon signaling in the protection of SARS-CoV-2. In this study, we demonstrate the ease and rapidness of the intratracheal administration of AAV for targeting lung tissue in mice, and this can be used to study diverse pulmonary diseases.

PACSIN2 Interacts with Nonstructural Protein 5A and Regulates Hepatitis C Virus Assembly.

Hepatitis C virus (HCV) is a major etiologic agent of chronic liver diseases. HCV is highly dependent on cellular machinery for viral propagation. Using protein microarray analysis, we previously identified 90 cellular proteins as nonstructural 5A (NS5A) interacting partners. Of these, protein kinase C and casein kinase substrate in neurons protein 2 (PACSIN2) was selected for further study. PACSIN2 belongs to the PACSIN family, which is involved in the formation of caveolae. Protein interaction between NS5A and PACSIN2 was confirmed by pulldown assay and further verified by both coimmunoprecipitation and immunofluorescence assays. We showed that PACSIN2 interacted with domain I of NS5A and the Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) region of PACSIN2. Interestingly, NS5A specifically attenuated protein kinase C alpha (PKCα)-mediated phosphorylation of PACSIN2 at serine 313 by interrupting PACSIN2 and PKCα interaction. In fact, mutation of the serine 313 to alanine (S313A) of PACSIN2 increased protein interaction with NS5A. Silencing of PACSIN2 decreased both viral RNA and protein expression levels of HCV. Ectopic expression of the small interfering RNA (siRNA)-resistant PACSIN2 recovered the viral infectivity, suggesting that PACSIN2 was specifically required for HCV propagation. PACSIN2 was involved in viral assembly without affecting other steps of the HCV life cycle. Indeed, overexpression of PACSIN2 promoted NS5A and core protein (core) interaction. We further showed that inhibition of PKCα increased NS5A and core interaction, suggesting that phosphorylation of PACSIN2 might influence HCV assembly. Moreover, PACSIN2 was required for lipid droplet formation via modulating extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Taken together, these data indicate that HCV modulates PACSIN2 via NS5A to promote virion assembly.IMPORTANCE PACSIN2 is a lipid-binding protein that triggers the tubulation of the phosphatidic acid-containing membranes. The functional involvement of PACSIN2 in the virus life cycle has not yet been demonstrated. We showed that phosphorylation of PACSIN2 displayed a negative effect on NS5A and core interaction. The most significant finding is that NS5A prevents PKCα from binding to PACSIN2. Therefore, the phosphorylation level of PACSIN2 is decreased in HCV-infected cells. We showed that HCV NS5A interrupted PKCα-mediated PACSIN2 phosphorylation at serine 313, thereby promoting NS5A-PACSIN2 interaction. We further demonstrated that PACSIN2 modulated lipid droplet formation through ERK1/2 phosphorylation. These data provide evidence that PACSIN2 is a proviral cellular factor required for viral propagation.

Cortactin Interacts with Hepatitis C Virus Core and NS5A Proteins: Implications for Virion Assembly.

Hepatitis C virus (HCV) exploits cellular proteins to facilitate viral propagation. To identify the cellular factors involved in the HCV life cycle, we previously performed protein microarray assays using either HCV nonstructural 5A (NS5A) protein or core protein as a probe. Interestingly, cellular cortactin strongly interacted with both NS5A and core. Cortactin is an actin-binding protein critically involved in tumor progression by regulating the migration and invasion of cancerous cells. Protein interaction between cortactin and NS5A or core was confirmed by coimmunoprecipitation and immunofluorescence assays. We showed that cortactin interacted with NS5A and core via the N-terminal acidic domain of cortactin. Cortactin expression levels were not altered by HCV infection. Small interfering RNA (siRNA)-mediated knockdown of cortactin dramatically decreased HCV protein expression and infectivity levels, whereas overexpression of cortactin increased viral propagation. Ectopic expression of the siRNA-resistant cortactin recovered the viral infectivity, suggesting that cortactin was specifically required for HCV propagation. We further showed that cortactin was involved in the assembly step without affecting viral entry, HCV internal ribosome entry site (IRES)-mediated translation, and the replication steps of the HCV life cycle. Of note, silencing of cortactin markedly reduced both NS5A and core protein levels on the lipid droplets (LDs), and this effect was reversed by the overexpression of cortactin. Importantly, NS5A and core promoted cell migration by activating the phosphorylation of cortactin at tyrosine residues 421 and 466. Taken together, these data suggest that cortactin is not only involved in HCV assembly but also plays an important role in the cell migration.IMPORTANCE Cortactin is a cytoskeletal protein that regulates cell migration in response to a number of extracellular stimuli. The functional involvement of cortactin in the virus life cycle is not yet fully understood. The most significant finding is that cortactin strongly interacted with both hepatitis C virus (HCV) core and NS5A. Cortactin is involved in HCV assembly by tethering core and NS5A on the lipid droplets (LDs) with no effect on LD biogenesis. It was noteworthy that HCV NS5A and core activated cortactin by phosphorylation at tyrosines 421 and 466 to regulate cell migration. Collectively, our study shows that cortactin is a novel host factor involved in viral production and HCV-associated pathogenesis.

Identification of African Swine Fever Virus Inhibitors through High Performance Virtual Screening Using Machine Learning.

African swine fever virus (ASFV) is a highly contagious virus that causes severe hemorrhagic viral disease resulting in high mortality in domestic and wild pigs, until few antiviral agents can inhibit ASFV infections. Thus, new anti-ASFV drugs need to be urgently identified. Recently, we identified pentagastrin as a potential antiviral drug against ASFVs using molecular docking and machine learning models. However, the scoring functions are easily influenced by properties of protein pockets, resulting in a scoring bias. Here, we employed the 5'-P binding pocket of AsfvPolX as a potential binding site to identify antiviral drugs and classified 13 AsfvPolX structures into three classes based on pocket parameters calculated by the SiteMap module. We then applied principal component analysis to eliminate this scoring bias, which was effective in making the SP Glide score more balanced between 13 AsfvPolX structures in the dataset. As a result, we identified cangrelor and fostamatinib as potential antiviral drugs against ASFVs. Furthermore, the classification of the pocket properties of AsfvPolX protein can provide an alternative approach to identify novel antiviral drugs by optimizing the scoring function of the docking programs. Here, we report a machine learning-based novel approach to generate high binding affinity compounds that are individually matched to the available classification of the pocket properties of AsfvPolX protein.

Prediction of African Swine Fever Virus Inhibitors by Molecular Docking-Driven Machine Learning Models.

African swine fever virus (ASFV) causes a highly contagious and severe hemorrhagic viral disease with high mortality in domestic pigs of all ages. Although the virus is harmless to humans, the ongoing ASFV epidemic could have severe economic consequences for global food security. Recent studies have found a few antiviral agents that can inhibit ASFV infections. However, currently, there are no vaccines or antiviral drugs. Hence, there is an urgent need to identify new drugs to treat ASFV. Based on the structural information data on the targets of ASFV, we used molecular docking and machine learning models to identify novel antiviral agents. We confirmed that compounds with high affinity present in the region of interest belonged to subsets in the chemical space using principal component analysis and k-means clustering in molecular docking studies of FDA-approved drugs. These methods predicted pentagastrin as a potential antiviral drug against ASFVs. Finally, it was also observed that the compound had an inhibitory effect on AsfvPolX activity. Results from the present study suggest that molecular docking and machine learning models can play an important role in identifying potential antiviral drugs against ASFVs.

Hepatitis C Virus Downregulates Ubiquitin-Conjugating Enzyme E2S Expression To Prevent Proteasomal Degradation of NS5A, Leading to Host Cells More Sensitive to DNA Damage.

Hepatitis C virus (HCV) infection may cause chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV exploits cellular machineries to establish persistent infection. We demonstrate here that ubiquitin-conjugating enzyme E2S (UBE2S), a member of the ubiquitin-conjugating enzyme family (E2s), was downregulated by endoplasmic reticulum stress caused by HCV in Huh7 cells. UBE2S interacted with domain I of HCV NS5A and degraded NS5A protein through the Lys11-linked proteasome-dependent pathway. Overexpression of UBE2S suppressed viral propagation, while depletion of UBE2S expression increased viral infectivity. Enzymatically inactive UBE2S C95A mutant exerted no antiviral activity, suggesting that ubiquitin-conjugating enzymatic activity was required for the suppressive role of UBE2S. Chromatin ubiquitination plays a crucial role in the DNA damage response. We showed that the levels of UBE2S and Lys11 chains bound to the chromatin were markedly decreased in the context of HCV replication, rendering HCV-infected cells more sensitive to DNA damage. These data suggest that HCV counteracts antiviral activity of UBE2S to optimize viral propagation and may contribute to HCV-induced liver pathogenesis.IMPORTANCE Protein homeostasis is essential to normal cell function. HCV infection disturbs the protein homeostasis in the host cells. Therefore, host cells exert an anti-HCV activity in order to maintain normal cellular metabolism. We showed that UBE2S interacted with HCV NS5A and degraded NS5A protein through the Lys11-linked proteasome-dependent pathway. However, HCV has evolved to overcome host antiviral activity. We demonstrated that the UBE2S expression level was suppressed in HCV-infected cells. Since UBE2S is an ubiquitin-conjugating enzyme and this enzyme activity is involved in DNA damage repair, HCV-infected cells are more sensitive to DNA damage, and thus UBE2S may contribute to viral oncogenesis.

Plasma Short-Chain Fatty Acids in Patients With Parkinson's Disease.

BACKGROUND: Short-chain fatty acids are exclusively produced by gut microbiota and are reduced in feces of patients with Parkinson's disease (PD). The objective of this study was to conduct a case-control study on peripheral concentration of short-chain fatty acids based on evidence of pathologic changes in the blood-brain barrier in PD and the possible role of short-chain fatty acids in blood-brain barrier permeability. METHODS: The plasma short-chain fatty acid concentration was measured in 38 PD and 33 normal controls using gas chromatography. The clinical characteristics of patients with PD and controls were evaluated, and dietary information was obtained using a food frequency questionnaire. Short-chain fatty acid concentrations were further compared after adjusting for age, sex, and significant food frequency questionnaire items. RESULTS: The concentrations of acetate, propionate, and butyrate did not differ between patients with PD and controls in unadjusted comparison. Dietary intakes of fibers, carbohydrates, lipids (total and fatty acids), and proteins did not differ between groups. After correction of covariates, acetic acid concentration was higher in patients with PD than in controls (116.47 ± 16.83 vs 108.20 ± 18.37 µmol/L; P = 0.010). In correlation analyses, acetic acid concentration was positively correlated (R = 0.374, P = 0.021) with age, propionic acid concentration was negatively correlated with UPDRS part III score (R = -0.376, P = 0.020) and use of entacapone (R = -0.325, P = 0.047), and butyric acid concentration was correlated with monoamine oxidase inhibitor use (R = 0.382, P = 0.018) and anticholinergic use (R = -0.385, P = 0.024). CONCLUSIONS: Plasma short-chain fatty acids were paradoxically increased in PD and were associated with disease severity and antiparkinsonian medications. Further studies are warranted to elucidate the relationships of gut dysbiosis and inflammation with plasma short-chain fatty acids. © 2020 International Parkinson and Movement Disorder Society.

Nonstructural Protein 5A Impairs DNA Damage Repair: Implications for Hepatitis C Virus-Mediated Hepatocarcinogenesis.

RAD51-associated protein 1 (RAD51AP1) is a member of the multiprotein complexes postulated to carry out RAD51-mediated homologous recombination and DNA repair in mammalian cells. In the present study, we showed that hepatitis C virus (HCV) NS5A directly bound RAD51AP1 and increased the protein level of RAD51AP1 through modulation of the ubiquitin-proteasome pathway. We also demonstrated that RAD51AP1 protein levels were increased in the liver tissues of HCV-infected patients and NS5A-transgenic mice. Importantly, NS5A impaired DNA repair by disrupting the RAD51/RAD51AP1/UAF1 complex and rendered HCV-infected cells more sensitive to DNA damage. Silencing of RAD51AP1 expression resulted in a decrease of viral propagation. We further demonstrated that RAD51AP1 was involved in the assembly step of the HCV life cycle by protecting viral RNA. These data suggest that HCV exploits RAD51AP1 to promote viral propagation and thus that host DNA repair is compromised in HCV-infected cells. Overall, our findings provide mechanistic insight into the pathogenesis of HCV infection.IMPORTANCE Chronic infection with HCV is the leading cause of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying HCV-induced HCC are not fully understood. Here we demonstrate that the HCV NS5A protein physically interacts with RAD51AP1 and increases the RAD51AP1 protein level through modulation of the ubiquitin-proteasome pathway. HCV coopts host RAD51AP1 to protect viral RNA at an assembly step of the HCV life cycle. Note that the RAD51 protein accumulates in the cytoplasm of HCV-infected cells, and thus the RAD51/RAD51AP1/UAF1-mediated DNA damage repair system in the nucleus is compromised in HCV-infected cells. Our data may provide new insight into the molecular mechanisms of HCV-induced pathogenesis.

Hepatitis C Virus-Induced Rab32 Aggregation and Its Implications for Virion Assembly.

Hepatitis C virus (HCV) is highly dependent on cellular factors for viral propagation. Using high-throughput next-generation sequencing, we analyzed the host transcriptomic changes and identified 30 candidate genes which were upregulated in cell culture-grown HCV (HCVcc)-infected cells. Of these candidates, we selected Rab32 for further investigation. Rab32 is a small GTPase that regulates a variety of intracellular membrane-trafficking events in various cell types. In this study, we demonstrated that both mRNA and protein levels of Rab32 were increased in HCV-infected cells. Furthermore, we showed that HCV infection converted the predominantly expressed GTP-bound Rab32 to GDP-bound Rab32, contributing to the aggregation of Rab32 and thus making it less sensitive to cellular degradation machinery. In addition, GDP-bound Rab32 selectively interacted with HCV core protein and deposited core protein into the endoplasmic reticulum (ER)-associated Rab32-derived aggregated structures in the perinuclear region, which were likely to be viral assembly sites. Using RNA interference technology, we demonstrated that Rab32 was required for the assembly step but not for other stages of the HCV life cycle. Taken together, these data suggest that HCV may modulate Rab32 activity to facilitate virion assembly. IMPORTANCE: Rab32, a member of the Ras superfamily of small GTPases, regulates various intracellular membrane-trafficking events in many cell types. In this study, we showed that HCV infection concomitantly increased Rab32 expression at the transcriptional level and altered the balance between GDP- and GTP-bound Rab32 toward production of Rab32-GDP. GDP-bound Rab32 selectively interacted with HCV core protein and enriched core in the ER-associated Rab32-derived aggregated structures that were probably necessary for viral assembly. Indeed, we showed that Rab32 was specifically required for the assembly of HCV. Collectively, our study identifies that Rab32 is a novel host factor essential for HCV particle assembly.

Hepatitis C Virus Nonstructural 5A Protein Interacts with Abelson Interactor 1 and Modulates Epidermal Growth Factor-mediated MEK/ERK Signaling Pathway.

The propagation of hepatitis C virus (HCV) is highly dependent on host cellular factors. To identify the cellular factors involved in HCV propagation, we have previously performed protein microarray assays using the HCV nonstructural 5A (NS5A) protein as a probe. Of ∼9,000 host proteins immobilized in a microarray, ∼90 cellular proteins were identified as HCV NS5A interacting partners. Of these candidates, we selected Abelson interactor 1 (Abi1) for further characterization. Binding of HCV NS5A to Abi1 was verified by both in vitro pulldown and coimmunoprecipitation assays. HCV NS5A interacted with Abi1 through regions I + II of Abi1 and domain I of NS5A. We further demonstrated that Abi1 colocalized with the HCV NS5A protein in the cytoplasm. We showed that NS5A inhibited epidermal growth factor-mediated ERK and Egr1 activations and this inhibitory activity of NS5A was nullified in Abi1-knockdown cells. Moreover, silencing of Abi1 expression impaired HCV replication, whereas overexpression of Abi1 promoted HCV propagation. Collectively, these data indicate that HCV exploits host Abi1 protein via NS5A to modulate MEK/ERK signaling pathway for its own propagation.

Nonstructural 3 Protein of Hepatitis C Virus Modulates the Tribbles Homolog 3/Akt Signaling Pathway for Persistent Viral Infection.

UNLABELLED: Hepatitis C virus (HCV) infection often causes chronic hepatitis, liver cirrhosis, and ultimately hepatocellular carcinoma. However, the mechanisms underlying HCV-induced liver pathogenesis are still not fully understood. By transcriptome sequencing (RNA-Seq) analysis, we recently identified host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these, tribbles homolog 3 (TRIB3) was selected for further characterization. TRIB3 was initially identified as a binding partner of protein kinase B (also known as Akt). TRIB3 blocks the phosphorylation of Akt and induces apoptosis under endoplasmic reticulum (ER) stress conditions. HCV has been shown to enhance Akt phosphorylation for its own propagation. In the present study, we demonstrated that both mRNA and protein levels of TRIB3 were increased in the context of HCV replication. We further showed that promoter activity of TRIB3 was increased by HCV-induced ER stress. Silencing of TRIB3 resulted in increased RNA and protein levels of HCV, whereas overexpression of TRIB3 decreased HCV replication. By employing an HCV pseudoparticle entry assay, we further showed that TRIB3 was a negative host factor involved in HCV entry. Both in vitro binding and immunoprecipitation assays demonstrated that HCV NS3 specifically interacted with TRIB3. Consequently, the association of TRIB3 and Akt was disrupted by HCV NS3, and thus, TRIB3-Akt signaling was impaired in HCV-infected cells. Moreover, HCV modulated TRIB3 to promote extracellular signal-regulated kinase (ERK) phosphorylation, activator protein 1 (AP-1) activity, and cell migration. Collectively, these data indicate that HCV exploits the TRIB3-Akt signaling pathway to promote persistent viral infection and may contribute to HCV-mediated pathogenesis. IMPORTANCE: TRIB3 is a pseudokinase protein that acts as an adaptor in signaling pathways for important cellular processes. So far, the functional involvement of TRIB3 in virus-infected cells has not yet been demonstrated. We showed that both mRNA and protein expression levels of TRIB3 were increased in the context of HCV RNA replication. Gene silencing of TRIB3 increased HCV RNA and protein levels, and thus, overexpression of TRIB3 decreased HCV replication. TRIB3 is known to promote apoptosis by negatively regulating the Akt signaling pathway under ER stress conditions. Most importantly, we demonstrated that the TRIB3-Akt signaling pathway was disrupted by NS3 in HCV-infected cells. These data provide evidence that HCV modulates the TRIB3-Akt signaling pathway to establish persistent viral infection.

Ameliorative effect of dietary genistein on diabetes induced hyper-inflammation and oxidative stress during early stage of wound healing in alloxan induced diabetic mice.

Among the diabetic complications, diabetic foot ulcer due to delayed wound healing is one of the most significant clinical problems. Early inflammatory stage is important for better prognosis during wound healing. Thus, regulation of inflammatory response during early stage of wound healing is main target for complete cutaneous recovery. This study investigated the role of genistein supplementation in inflammation and oxidative stress, which are related to NLRP3 inflammasome, NFκB and Nrf2 activation, during cutaneous wound healing in alloxan-induced diabetic mice. Mice with diabetes with fasting blood glucose (FBG) levels > 250 mg/dl were fed diets with AIN-93G rodent diet containing 0%, 0.025% (LG) or 0.1% (HG) genistein. After 2 weeks of genistein supplementation, excisional wounds were made by biopsy punches (4 mm). Genistein supplementation improved fasting glucose levels and wound closure rate. Moreover, genistein supplementation restored NLRP3 inflammasome (NLRP3, ASC and caspase-1) at the basal level and ameliorated both inflammation (TNFα, iNOS, COX2 and NFκB) and antioxidant defense system (Nrf2, HO-1, GPx, and catalase) during early stage of wound healing in diabetic mice. Taken together, genistein supplementation would be a potential therapeutic nutrient in prevention and treatment of delayed wound healing by modulation of inflammation and oxidative stress during inflammatory stage.

Pim Kinase Interacts with Nonstructural 5A Protein and Regulates Hepatitis C Virus Entry.

UNLABELLED: The life cycle of hepatitis C virus (HCV) is highly dependent on host cellular proteins for virus propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ∼ 9,000 human cellular proteins immobilized in a microarray, approximately 90 cellular proteins were identified as NS5A interactors. Of these candidates, Pim1, a member of serine/threonine kinase family composed of three different isoforms (Pim1, Pim2, and Pim3), was selected for further study. Pim kinases share a consensus sequence which overlaps with kinase activity. Pim kinase activity has been implicated in tumorigenesis. In the present study, we verified the physical interaction between NS5A and Pim1 by both in vitro pulldown and coimmunoprecipitation assays. Pim1 interacted with NS5A through amino acid residues 141 to 180 of Pim1. We demonstrated that protein stability of Pim1 was increased by NS5A protein and this increase was mediated by protein interplay. Small interfering RNA (siRNA)-mediated knockdown or pharmacological inhibition of Pim kinase abrogated HCV propagation. By employing HCV pseudoparticle entry and single-cycle HCV infection assays, we further demonstrated that Pim kinase was involved in HCV entry at a postbinding step. These data suggest that Pim kinase may represent a new host factor for HCV entry. IMPORTANCE: Pim1 is an oncogenic serine/threonine kinase. HCV NS5A protein physically interacts with Pim1 and contributes to Pim1 protein stability. Since Pim1 protein expression level is upregulated in many cancers, NS5A-mediated protein stability may be associated with HCV pathogenesis. Either gene silencing or chemical inhibition of Pim kinase abrogated HCV propagation in HCV-infected cells. We further showed that Pim kinase was specifically required at an early entry step of the HCV life cycle. Thus, we have identified Pim kinase not only as an HCV cell entry factor but also as a new anti-HCV therapeutic target.

Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation.

UNLABELLED: Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for virus propagation. To identify the cellular factors involved in HCV propagation, we recently performed protein microarray assays using the HCV nonstructural 5A (NS5A) protein as a probe. Of 90 cellular protein candidates, we selected the soluble resistance-related calcium-binding protein (sorcin) for further characterization. Sorcin is a calcium-binding protein and is highly expressed in certain cancer cells. We verified that NS5A interacted with sorcin through domain I of NS5A, and phosphorylation of the threonine residue 155 of sorcin played a crucial role in protein interaction. Small interfering RNA (siRNA)-mediated knockdown of sorcin impaired HCV propagation. Silencing of sorcin expression resulted in a decrease of HCV assembly without affecting HCV RNA and protein levels. We further demonstrated that polo-like kinase 1 (PLK1)-mediated phosphorylation of sorcin was increased by NS5A. We showed that both phosphorylation and calcium-binding activity of sorcin were required for HCV propagation. These data indicate that HCV modulates sorcin activity via NS5A protein for its own propagation. IMPORTANCE: Sorcin is a calcium-binding protein and regulates intracellular calcium homeostasis. HCV NS5A interacts with sorcin, and phosphorylation of sorcin is required for protein interaction. Gene silencing of sorcin impaired HCV propagation at the assembly step of the HCV life cycle. Sorcin is phosphorylated by PLK1 via protein interaction. We showed that sorcin interacted with both NS5A and PLK1, and PLK1-mediated phosphorylation of sorcin was increased by NS5A. Moreover, calcium-binding activity of sorcin played a crucial role in HCV propagation. These data provide evidence that HCV regulates host calcium metabolism for virus propagation, and thus manipulation of sorcin activity may represent a novel therapeutic target for HCV.

Stearoyl coenzyme A desaturase 1 is associated with hepatitis C virus replication complex and regulates viral replication.

UNLABELLED: The hepatitis C virus (HCV) life cycle is tightly regulated by lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have recently screened a small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet formation using cell culture-grown HCV (HCVcc)-infected cells. We selected and characterized the gene encoding stearoyl coenzyme A (CoA) desaturase 1 (SCD1). siRNA-mediated knockdown or pharmacological inhibition of SCD1 abrogated HCV replication in both subgenomic replicon and Jc1-infected cells, while exogenous supplementation of either oleate or palmitoleate, products of SCD1 activity, resurrected HCV replication in SCD1 knockdown cells. SCD1 was coimmunoprecipitated with HCV nonstructural proteins and colocalized with both double-stranded RNA (dsRNA) and HCV nonstructural proteins, indicating that SCD1 is associated with HCV replication complex. Moreover, SCD1 was fractionated and enriched with HCV nonstructural proteins at detergent-resistant membrane. Electron microscopy data showed that SCD1 is required for NS4B-mediated intracellular membrane rearrangement. These data further support the idea that SCD1 is associated with HCV replication complex and that its products may contribute to the proper formation and maintenance of membranous web structures in HCV replication complex. Collectively, these data suggest that manipulation of SCD1 activity may represent a novel host-targeted antiviral strategy for the treatment of HCV infection. IMPORTANCE: Stearoyl coenzyme A (CoA) desaturase 1 (SCD1), a liver-specific enzyme, regulates hepatitis C virus (HCV) replication through its enzyme activity. HCV nonstructural proteins are associated with SCD1 at detergent-resistant membranes, and SCD1 is enriched on the lipid raft by HCV infection. Therein, SCD1 supports NS4B-mediated membrane rearrangement to provide a suitable microenvironment for HCV replication. We demonstrated that either genetic or chemical knockdown of SCD1 abrogated HCV replication in both replicon cells and HCV-infected cells. These findings provide novel mechanistic insights into the roles of SCD1 in HCV replication.

Factors that Affect Sugar Sweetened Beverage Intake in Rural, Southern College Students in the US.

The objective of the present study was to investigate factors that affect sugar sweetened beverage (SSB) intake in rural, southern college students in the US. The majority of the participants were male (58 %) and Caucasian (63 %). The average total SSB consumption was 79.4 fl oz/day (2.35 L/d). Results of binary logistic regression analyses of total SSB intake greater than 57.4 fl oz/day (1.8 L/d) versus less than 57.4 fl oz/day showed that factors associated with greater odds for high SSB intake were age greater than 20 years old (odds ratio [OR] = 3.551, 95 % confidence interval [CI] = 1.385 - 9.104, p = 0.008) and being African American (OR = 3.477, 95 % CI = 1.291 - 9.363, p = 0.013). Results of binary logistic regression analyses of total bottled water intake greater than 39.4 fl oz/day (median) versus less than 39.4 fl oz/day showed that consuming alcohol was significantly related to an increased probability of drinking more than 39.4 fl oz (1.17 L/d) of bottled water per day (median; OR = 2.914, 95 % CI = 1.223 - 6.943, p = 0.016). Culturally sensitive strategies are needed to raise awareness for making healthy beverage choices when dining on campus to effectively reduce college student's SSB consumption.

Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein.

Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray, approximately 100 cellular proteins were identified as HCV core-interacting partners. Of these candidates, mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) was selected for further characterization. MAPKAPK3 is a serine/threonine protein kinase that is activated by stress and growth inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA and protein levels of MAPKAPK3 were elevated in both HCV subgenomic replicon cells and cell culture-derived HCV (HCVcc)-infected cells. Silencing of MAPKAPK3 expression resulted in decreases in both protein and HCV infectivity levels but not in the intracellular HCV RNA level. We showed that MAPKAPK3 increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation.

Solanum melongena extract supplementation protected skeletal muscle and brain damage by regulation of BDNF/PGC1α/irisin pathway via brain function-related myokines in high-fat diet induced obese mice.

In this study, we investigated the protective effects of SM on skeletal muscle and brain damage by regulation of BDNF/PGC1α/irisin pathway via brain function related myokines in high-fat diet-induced OB mice. OB was induced by high-fat diet for 6 weeks. SM extract (SME) was administered with 200 mg/kg BW (LSM) and 500 mg/kg BW (HSM) by oral gavage every day for 12 weeks. Behavior tests such as grip strength, Y-maze, and passive avoidance test were conducted to analyze muscle and cognitive function. Histopathological changes in skeletal muscle and brain were examined by hematoxylin and eosin staining and the protein levels of biomarkers related to oxidative stress, inflammation, protein degradation, neuro-plasticity, and cell cycling were measured by western blot. SME regulated morphological changes (muscle cross-sectional area: 1.23%, 1.40%; density of neurons in hippocampus:1.74%, 1.73%) in T2DM mice. Importantly, SME supplementation significantly increased several muscle-derived myokines which might influence the expression of neuronal markers in OB mice (FGF21: 1.27%, 1.34%; PGC1α: 1.0%, 1.32%; IRISIN: 1.9%, 1.08%; BDNF: 1.35%, 1.23%). Accordingly, SME activated hippocampal neurotrophic factors including BDNF (1.0%, 1.2%) and its associated PGC1α/irisin pathway (PGC1α :1.1%, 1.1%; IRISIN:1.1%, 0.9%) significantly. This study demonstrated the possibliy that protective myokines increased by SME supplementation may contribute to neuro-protection in OB mice. Taken together, the current study suggests that SME can be used to prevent skeletal muscle and brain damage in OB by protecting against oxidative stress and inflammatin via modulation of the BDNF/PGC1α/irisin pathway in the therapeutic approach of obese patients.