Potent Antiviral Protein Research Articles

The Mechanism of Action of Lactoferrin - Nucleoside Diphosphate Kinase Complex in Combating Biofilm Formation.

The ESKAPE group of pathogens which comprise of multidrug resistant bacteria, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species are the cause of deadly nosocomial infections all over the world. While these pathogens have developed robust strategies to resist most antibiotics, their ability to form biofilms is one of their most combative properties. Hence there is an urgent need to discover new antibacterial agents which could prevent or destroy the biofilms made by these bacteria. Though it has been established that lactoferrin (LF), a potent iron binding antibacterial, antifungal, and antiviral protein displays anti-biofilm properties, its mechanisms of action, in addition to its iron chelation property, still remains unclear. The binding and inhibition studies of LF with the enzyme Nucleoside diphosphate Kinase (NDK) and its elastase cleaved truncated 12 kDa fragment (12-NDK). The characterization studies of NDK and 12-NDK using florescence spectroscopy, dynamic light scattering, size exclusion chromatography and ADP-glo Kinase Assay. Inhibition studies of LF-NDK using ADP-glo kinase assay, Surface Plasmon Resonance and Biofilm inhibition studies. NDK and 12-NDK were cloned, expressed and purified from Acinetobacter baumannii and Pseudomonas aeruginosa. The characterization studies revealed NDK and 12-NDK from both species are stable and functional. The inhibition studies of LF-NDK revealed stable binding and inhibition of kinase activity by LF. The binding and inhibition studies have shown that while LF binds with both the NDK and their truncated forms, it tends to have a higher binding affinity with the truncated 12 kDa fragments, resulting in their decreased kinase activity. This study essentially gives a new direction to the field of inhibition of biofilm formation, as it proves that LF has a novel mechanism of action in other than iron sequestration.

BST2 Suppresses LINE-1 Retrotransposition by Reducing the Promoter Activity of LINE-1 5' UTR.

ABSTRACTEndogenous retrotransposons are considered the “molecular fossils” of ancient retroviral insertions. Several studies have indicated that host factors restrict both retroviruses and retrotransposons through different mechanisms. Type 1 long interspersed elements (LINE-1 or L1) are the only active retroelements that can replicate autonomously in the human genome. A recent study reported that LINE-1 retrotransposition is potently suppressed by BST2, a host restriction factor that prevents viral release mainly by physically tethering enveloped virions (such as HIV) to the surface of producer cells. However, no endoplasmic membrane structure has been associated with LINE-1 replication, suggesting that BST2 may utilize a distinct mechanism to suppress LINE-1. In this study, we showed that BST2 is a potent LINE-1 suppressor. Further investigations suggested that BST2 reduces the promoter activity of LINE-1 5′ untranslated region (UTR) and lowers the levels of LINE-1 RNA, proteins, and events during LINE-1 retrotransposition. Surprisingly, although BST2 apparently uses different mechanisms against HIV and LINE-1, two membrane-associated domains that are essential for BST2-mediated HIV tethering also proved important for BST2-induced inhibition of LINE-1 5′ UTR. Additionally, by suppressing LINE-1, BST2 prevented LINE-1-induced genomic DNA damage and innate immune activation. Taken together, our data uncovered the mechanism of BST2-mediated LINE-1 suppression and revealed new roles of BST2 as a promoter regulator, genome stabilizer, and innate immune suppressor.IMPORTANCE BST2 is a potent antiviral protein that suppresses the release of several enveloped viruses, mainly by tethering the envelope of newly synthesized virions and restraining them on the surface of producer cells. In mammalian cells, there are numerous DNA elements replicating through reverse transcription, among which LINE-1 is the only retroelement that can replicate autonomously. Although LINE-1 retrotransposition does not involve the participation of a membrane structure, BST2 has been reported as an efficient LINE-1 suppressor, suggesting a different mechanism for BST2-mediated LINE-1 inhibition and a new function for BST2 itself. We found that BST2 specifically represses the promoter activity of LINE-1 5′ UTR, resulting in decreased levels of LINE-1 transcription, translation, and subsequent retrotransposition. Additionally, by suppressing LINE-1 activity, BST2 maintains genome stability and regulates innate immune activation. These findings expand our understanding of BST2 and its biological significance.

SARS-CoV-2 clearance in COVID-19 patients with Novaferon treatment: A randomized, open-label, parallel-group trial

BackgroundThe antiviral effects of Novaferon, a potent antiviral protein drug, on COVID-19 was evaluated in the laboratory, and in a randomized, open-label, parallel-group trial. MethodsIn the laboratory, Novaferon's inhibition of viral replication in cells infected with SARS-CoV-2, and prevention of SARS-CoV-2 entry into healthy cells was determined. Antiviral effects of Novaferon in COVID-19 patients with treatment of Novaferon, Novaferon plus Lopinavir/Ritonavir, or Lopinavir/Ritonavir were evaluated. The primary endpoint was the SARS-CoV-2 clearance rates on day six of treatment, and the secondary endpoint was the time to SARS-CoV-2 clearance. ResultsNovaferon inhibited viral replication (EC50=1.02ng/ml), and prevented viral infection (EC50=0.10ng/ml). Results from the 89 enrolled COVID-19 patients showed that both Novaferon and Novaferon plus Lopinavir/Ritonavir groups had significantly higher viral clearance rates on day six than Lopinavir/Ritonavir group (50.0% vs. 24.1%, p=0.0400, and 60.0% vs. 24.1%, p=0.0053). The median time to viral clearance was six days, six days, and nine days for three groups, respectively, a 3-day reduction in both the Novaferon and Novaferon plus Lopinavir/Ritonavir groups compared with the Lopinavir/Ritonavir group. ConclusionsNovaferon exhibited anti-SARS-CoV-2 effects in vitro and in COVID-19 patients. These data justify further evaluation of Novaferon. Trial registration numberNumber ChiCTR2000029496 at the Chinese Clinical Trial Registry (http://www.chictr.org.cn/).

Targeting UBE4A Revives Viperin Protein in Epithelium to Enhance Host Antiviral Defense.

Mutation and prevalence of pathogenic viruses prompt the development of broad-spectrum antiviral strategies. Viperin is a potent antiviral protein that inhibits a broad range of viruses. Unexpectedly, we found that Viperin protein production in epithelium is defective in response to both viruses and interferons (IFNs). We further revealed that viruses and IFNs stimulate expression of the acetyltransferase HAT1, which induces Lys197-acetylation on Viperin. Viperin acetylation in turn recruits UBE4A that stimulates K6-linked polyubiquitination at Lys206 of Viperin, leading to Viperin protein degradation. Importantly, UBE4A deficiency restores Viperin protein production in epithelium. We then designed interfering peptides (IPs) to inhibit UBE4A binding with Viperin. We found that VIP-IP3 rescues Viperin protein production in epithelium and therefore enhances cellular antiviral activity. VIP-IP3 renders mice more resistant to viral infection. These findings could provide strategies for both enhancing host broad-spectrum antiviral response and improving the efficacy of IFN-based antiviral therapy.

Interferon γ and α Have Differential Effects on SAMHD1, a Potent Antiviral Protein, in Feline Lymphocytes.

Sterile alpha motif and histidine/aspartic domain-containing protein 1 (SAMHD1) is a protein with anti-viral, anti-neoplastic, and anti-inflammatory properties. By degrading cellular dNTPs to constituent deoxynucleoside and free triphosphate, SAMHD1 limits viral DNA synthesis and prevents replication of HIV-1 and some DNA viruses such as HBV, vaccinia, and HSV-1. Recent findings suggest SAMHD1 is broadly active against retroviruses in addition to HIV-1, such as HIV-2, FIV, BIV, and EIAV. Interferons are cytokines produced by lymphocytes and other cells that induce a wide array of antiviral proteins, including some with activity again lentiviruses. Here we evaluated the role of IFNs on SAMHD1 gene expression, transcription, and post-translational modification in a feline CD4+ T cell line (FeTJ) and in primary feline CD4+ T lymphocytes. SAMHD1 mRNA in FetJ cells increased in a dose-related manner in response to IFNγ treatment concurrent with increased nuclear localization and phosphorylation. IFNα treatment induced SAMHD1 mRNA but did not significantly alter SAMHD1 protein detection, phosphorylation, or nuclear translocation. In purified primary feline CD4+ lymphocytes, IL2 supplementation increased SAMHD1 expression, but the addition of IFNγ did not further alter SAMHD1 protein expression or nuclear localization. Thus, the effect of IFNγ on SAMHD1 expression is cell-type dependent, with increased translocation to the nucleus and phosphorylation in FeTJ but not primary CD4+ lymphocytes. These findings imply that while SAMH1 is inducible by IFNγ, overall activity is cell type and compartment specific, which is likely relevant to the establishment of lentiviral reservoirs in quiescent lymphocyte populations.

Rapid interferon independent expression of IFITM3 following T cell activation protects cells from influenza virus infection.

Interferon-induced transmembrane protein 3 (IFITM3) is a potent antiviral protein that enhances cellular resistance to a variety of pathogens, including influenza virus. Classically defined as an interferon-stimulated gene, expression of IFITM3 on cells is rapidly up-regulated in response to type I and II interferon. Here we found that IFITM3 is rapidly up-regulated by T cells following their activation and this occurred independently of type I and II interferon and the interferon regulatory factors 3 and 7. Up-regulation of IFITM3 on effector T cells protected these cells from virus infection and imparted a survival advantage at sites of virus infection. Our results show that IFITM3 expression on effector T cells is crucial for these cells to mediate their effector function and highlights an interferon independent pathway for the induction of IFITM3 which, if targeted, could be an effective approach to harness the activity of IFITM3 for infection prevention.

MTOR inhibitors lower an intrinsic barrier to virus infection mediated by IFITM3.

Rapamycin and its derivatives are specific inhibitors of mammalian target of rapamycin (mTOR) kinase and, as a result, are well-established immunosuppressants and antitumorigenic agents. Additionally, this class of drug promotes gene delivery by facilitating lentiviral vector entry into cells, revealing its potential to improve gene therapy efforts. However, the precise mechanism was unknown. Here, we report that mTOR inhibitor treatment results in down-regulation of the IFN-induced transmembrane (IFITM) proteins. IFITM proteins, especially IFITM3, are potent inhibitors of virus-cell fusion and are broadly active against a range of pathogenic viruses. We found that the effect of rapamycin treatment on lentiviral transduction is diminished upon IFITM silencing or knockout in primary and transformed cells, and the extent of transduction enhancement depends on basal expression of IFITM proteins, with a major contribution from IFITM3. The effect of rapamycin treatment on IFITM3 manifests at the level of protein, but not mRNA, and is selective, as many other endosome-associated transmembrane proteins are unaffected. Rapamycin-mediated degradation of IFITM3 requires endosomal trafficking, ubiquitination, endosomal sorting complex required for transport (ESCRT) machinery, and lysosomal acidification. Since IFITM proteins exhibit broad antiviral activity, we show that mTOR inhibition also promotes infection by another IFITM-sensitive virus, Influenza A virus, but not infection by Sendai virus, which is IFITM-resistant. Our results identify the molecular basis by which mTOR inhibitors enhance virus entry into cells and reveal a previously unrecognized immunosuppressive feature of these clinically important drugs. In addition, this study uncovers a functional convergence between the mTOR pathway and IFITM proteins at endolysosomal membranes.

The Role of Viperin in Antiflavivirus Responses.

Viperin is an interferon (IFN)-stimulated gene product, which is part of the first line of the intracellular response against viral infection. It is a potent antiviral protein, strongly upregulated after IFN-stimulation and virus infection. Viperin is antivirally active against many different viruses from different families and has been shown to inhibit several flaviviruses. Flaviviruses are an important group of arthropod-borne viruses that cause millions of infections annually. In this review, we focus on the recent advances of the antiviral mechanisms of viperin against these flaviviruses, both pointing to similarities and differences between viruses within the same genera.

A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR).

Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. These viruses encode only four proteins to accomplish the viral life cycle, so each arenavirus protein likely plays unappreciated accessory roles during infection. Here we used immunoprecipitation and mass spectrometry to identify human proteins that interact with the nucleoproteins (NPs) of the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) and the New World arenavirus Junín virus (JUNV) strain Candid #1. Bioinformatic analysis of the identified protein partners of NP revealed that host translation appears to be a key biological process engaged during infection. In particular, NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2α. JUNV infection leads to increased expression of PKR as well as its redistribution to viral replication and transcription factories. Further, phosphorylation of PKR, which is a prerequisite for its ability to phosphorylate eIF2α, is readily induced by JUNV. However, JUNV prevents this pool of activated PKR from phosphorylating eIF2α, even following exposure to the synthetic dsRNA poly(I·C), a potent PKR agonist. This blockade of PKR function is highly specific, as LCMV is unable to similarly inhibit eIF2α phosphorylation. JUNV's ability to antagonize the antiviral activity of PKR appears to be complete, as silencing of PKR expression has no impact on viral propagation. In summary, we provide a detailed map of the host machinery engaged by arenavirus NPs and identify an antiviral pathway that is subverted by JUNV.IMPORTANCE Arenaviruses are important human pathogens for which FDA-approved vaccines do not exist and effective antiviral therapeutics are needed. Design of antiviral treatment options and elucidation of the mechanistic basis of disease pathogenesis will depend on an increased basic understanding of these viruses and, in particular, their interactions with the host cell machinery. Identifying host proteins critical for the viral life cycle and/or pathogenesis represents a useful strategy to uncover new drug targets. This study reveals, for the first time, the extensive human protein interactome of arenavirus nucleoproteins and uncovers a potent antiviral host protein that is neutralized during Junín virus infection. In so doing, it shows further insight into the interplay between the virus and the host innate immune response and provides an important data set for the field.

Mitochondrial C11orf83 is a potent Antiviral Protein Independent of interferon production

Mitochondria have a central position in innate immune response via the adaptor protein MAVS in mitochondrial outer membrane to limit viral replication by inducing interferon production. Here, we reported that C11orf83, a component of complex III of electronic transfer chain in mitochondrial inner membrane, was a potent antiviral protein independent of interferon production. C11orf83 expression significantly increased in response to viral infection, and endows cells with stronger capability of inhibiting viral replication. Deletion of C11orf83 permits viral replication easier and cells were more vulnerable to viral killing. These effects mainly were mediated by triggering OAS3-RNase L system. C11orf83 overexpression induced higher transcription of OAS3, and knockdown either OAS3 or RNase L impaired the antiviral capability of C11orf83. Interestingly, the signaling from C11orf83 to OAS3-RNase L was independent of interferon production. Thus, our findings suggested a new antiviral mechanism by bridging cell metabolic machinery component with antiviral effectors.

MARCH8 inhibits HIV-1 infection by reducing virion incorporation of envelope glycoproteins.

Membrane-associated RING-CH 8 (MARCH8) is one of 11 members of the recently discovered MARCH family of RING (really interesting new gene)-finger E3 ubiquitin ligases. MARCH8 downregulates several host transmembrane proteins, including major histocompatibility complex (MHC)-II, CD86, interleukin (IL)-1 receptor accessory protein, TNF-related apoptosis-inducing ligand (TRAIL) receptor 1 and the transferrin receptor. However, its physiological roles remain largely unknown. Here we identify MARCH8 as a novel antiviral factor. The ectopic expression of MARCH8 in virus-producing cells does not affect levels of lentivirus production, but it does markedly reduce viral infectivity. MARCH8 blocks the incorporation of HIV-1 envelope glycoprotein into virus particles by downregulating it from the cell surface, probably through their interaction, resulting in a substantial reduction in the efficiency of viral entry. The inhibitory effect of MARCH8 on vesicular stomatitis virus G-glycoprotein is even more remarkable, suggesting a broad-spectrum inhibition of enveloped viruses by MARCH8. Notably, the endogenous expression of MARCH8 is high in monocyte-derived macrophages and dendritic cells, and MARCH8 knockdown or knockout in macrophages significantly increases the infectivity of virions produced by these cells. Our findings thus indicate that MARCH8 is highly expressed in terminally differentiated myeloid cells, and that it is a potent antiviral protein that targets viral envelope glycoproteins and reduces their incorporation into virions.

Bifunctional fatty acid chemical reporter for analyzing S-palmitoylated membrane protein-protein interactions in mammalian cells.

Studying the functions of S-palmitoylated proteins in cells can be challenging due to the membrane targeting property and dynamic nature of protein S-palmitoylation. New strategies are therefore needed to specifically capture S-palmitoylated protein complexes in cellular membranes for dissecting their functions in vivo. Here we present a bifunctional fatty acid chemical reporter, x-alk-16, which contains an alkyne and a diazirine, for metabolic labeling of S-palmitoylated proteins and photo-cross-linking of their involved protein complexes in mammalian cells. We demonstrate that x-alk-16 can be metabolically incorporated into known S-palmitoylated proteins such as H-Ras and IFITM3, a potent antiviral protein, and induce covalent cross-linking of IFITM3 oligomerization as well as its specific interactions with other membrane proteins upon in-cell photoactivation. Moreover, integration of x-alk-16-induced photo-cross-linking with label-free quantitative proteomics allows identification of new IFITM3 interacting proteins.

Restoration of IRF1-dependent anticancer effects by MEK inhibition in human cancer cells

Interferon regulatory factor (IRF1) is a potent antiviral, antitumor and immune regulatory protein. Recently, we found that activated Ras/MEK inhibits antiviral response by downregulating IRF1 expression and renders cancer cells susceptible to oncolytic viruses. In this study, we sought to determine whether IRF1 downregulation underlies oncogenesis induced by Ras/MEK activation in human cancer cells. Treatment of the MEK inhibitor U0126 promoted IRF1 expression in 7 of 11 cancer cell lines we tested. IRF1 promotion was also observed in human cancer cell lines treated with different MEK inhibitors or with RNAi oligonucleotides against extracellular signal-regulated kinases (ERKs). Restoration of the expression of antitumor genes, p27 and p53 upregulated modulator of apoptosis (PUMA), by MEK inhibition was less in IRF1 shRNA knockdown cancer cells than in vector control cancer cells, suggesting that Ras/MEK targets IRF1 for the downregulation of the antitumor genes. Moreover, apoptosis induction by U0126 was significantly reduced in IRF1 shRNA knockdown cells than vector control cells. This study demonstrates that IRF1 expression is suppressed by activated Ras/MEK in human cancer cells and that IRF1 plays essential roles in apoptosis induced by Ras/MEK inhibition.

Structural insight into HIV-1 capsid recognition by rhesus TRIM5α

Tripartite motif protein isoform 5 alpha (TRIM5α) is a potent antiviral protein that restricts infection by HIV-1 and other retroviruses. TRIM5α recognizes the lattice of the retrovirus capsid through its B30.2 (PRY/SPRY) domain in a species-specific manner. Upon binding, TRIM5α induces premature disassembly of the viral capsid and activates the downstream innate immune response. We have determined the crystal structure of the rhesus TRIM5α PRY/SPRY domain that reveals essential features for capsid binding. Combined cryo-electron microscopy and biochemical data show that the monomeric rhesus TRIM5α PRY/SPRY, but not the human TRIM5α PRY/SPRY, can bind to HIV-1 capsid protein assemblies without causing disruption of the capsid. This suggests that the PRY/SPRY domain alone constitutes an important pattern-sensing component of TRIM5α that is capable of interacting with viral capsids of different curvatures. Our results provide molecular insights into the mechanisms of TRIM5α-mediated retroviral restriction.

Strong Bonds: International Collaboration in Chemistry; A Special 70 pages Insert in Angewandte Chemie from the German Research Foundation (DFG)

Carbohydrates are important mediators of many biological processes that underlie cellular communication and disease mechanisms. Therapeutic agents include carbohydrate-based vaccines and the potent anti-viral protein Cyanovirin-N (CV-N). CV-N acts by specifically binding the carbohydrate structures decorating the cell surface of deadly viruses including human immunodeficiency virus (HI-V) or Ebola. In search for new carbohydrate-binding proteins and the development of sensors that exploit carbohydrate-protein interactions the label-free cantilever array technique can provides a fast, parallel and low-cost approach.

Reduction of N-Tropic Mutant Porcine Endogenous Retrovirus Infectivity by Human Tripartite Motif-Containing 5-Isoform Alpha

In cases of retroviral infection, the host cell deploys antiviral proteins as a type of innate immunity. Tripartite motif-containing 5-isoform alpha (TRIM5α) is a potent antiviral protein. TRIM5α has been reported to restrict human immunodeficiency virus (HIV) 1 infection in rhesus monkey cells by targeting the incoming viral capsid at the postentry or preintegration stage of the viral life cycle. As a consequence, virus replication and reverse transcription are interrupted. TRIM5α of human origin has also been shown to inhibit N-tropic murine leukemia virus infection. To investigate the inhibitory effect of TRIM5α on porcine endogenous retrovirus (PERV) infection in humans, we constructed a 293T cell line stably expressing human TRIM5α (293T-huTRIM5α) and tested the infectivity of vesicular stomatitis virus glycoprotein envelope pseudotyped viruses (wild-type PERV [wt-PERV], N-tropic mutant PERV, N-tropic murine leukemia virus, and MoMLV). Infectivity of N-tropic mutant PERV was reduced by 43.3% in 293T-huTRIM5α cells, a decrease in efficiency that was more than 3-fold greater than that of wt-PERV in 293T-huTRIM5α cells. Human TRIM5α exhibited inhibitory activity against N-tropic MLV and N-tropic mutant PERV, but showed no antiviral activity against Moloney murine leukemia virus or wt-PERV.

Structural Basis of the Anti-HIV Activity of the Cyanobacterial Oscillatoria Agardhii Agglutinin

The cyanobacterial Oscillatory Agardhii agglutinin (OAA) is a recently discovered HIV-inactivating lectin that interacts with high-mannose sugars. Nuclear magnetic resonance (NMR) binding studies between OAA and α3,α6-mannopentaose (Manα(1-3)[Manα(1-3)[Manα(1-6)]Manα(1-6)]Man), the branched core unit of Man-9, revealed two binding sites at opposite ends of the protein, exhibiting essentially identical affinities. Atomic details of the specific protein-sugar contacts in the recognition loops of OAA were delineated in the high-resolution crystal structures of free and glycan-complexed protein. No major changes in the overall protein structure are induced by carbohydrate binding, with essentially identical apo- and sugar-bound conformations in binding site 1. A single peptide bond flip at W77-G78 is seen in binding site 2. Our combined NMR and crystallographic results provide structural insights into the mechanism by which OAA specifically recognizes the branched Man-9 core, distinctly different from the recognition of the D1 and D3 arms at the nonreducing end of high-mannose carbohydrates by other antiviral lectins.

Broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family Coronaviridae.

Viruses of the family Coronaviridae have recently emerged through zoonotic transmission to become serious human pathogens. The pathogenic agent responsible for severe acute respiratory syndrome (SARS), the SARS coronavirus (SARS-CoV), is a member of this large family of positive-strand RNA viruses that cause a spectrum of disease in humans, other mammals, and birds. Since the publicized outbreaks of SARS in China and Canada in 2002-2003, significant efforts successfully identified the causative agent, host cell receptor(s), and many of the pathogenic mechanisms underlying SARS. With this greater understanding of SARS-CoV biology, many researchers have sought to identify agents for the treatment of SARS. Here we report the utility of the potent antiviral protein griffithsin (GRFT) in the prevention of SARS-CoV infection both in vitro and in vivo. We also show that GRFT specifically binds to the SARS-CoV spike glycoprotein and inhibits viral entry. In addition, we report the activity of GRFT against a variety of additional coronaviruses that infect humans, other mammals, and birds. Finally, we show that GRFT treatment has a positive effect on morbidity and mortality in a lethal infection model using a mouse-adapted SARS-CoV and also specifically inhibits deleterious aspects of the host immunological response to SARS infection in mammals.

Ethanol Promotes Thiamine Deficiency‐Induced Neuronal Death: Involvement of Double‐Stranded RNA‐activated Protein Kinase

Heavy alcohol consumption causes cerebellar degeneration, and the underlying mechanism is unclear. Chronic alcoholism is usually associated with thiamine deficiency (TD) which is known to induce selective neurodegeneration in the brain. However, the role of TD in alcohol-induced cerebellar degeneration remains to be elucidated. The double-stranded RNA-activated protein kinase (PKR) is a potent antiviral protein. Viral infection or binding to dsRNA causes PKR autophosphorylation and subsequent phosphorylation of the alpha-subunit of eukaryotic translation factor-2alpha, leading to inhibition of translation or apoptosis. PKR can also be activated by cellular stresses. In this study, we used an in vitro model, cultured cerebellar granule neurons (CGNs), to investigate the interaction between TD and ethanol and evaluate the contribution of their interaction to neuronal loss. TD was induced by treatment with amprolium in association with ethanol. Cell viability was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assay. PKR expression/phosphorylation and subcellular distribution was analyzed with immunoblotting and immunocytochemistry. Thiamine deficiency caused death of CGNs but ethanol did not. However, TD plus ethanol induced a much greater cell loss than TD alone. TD-induced PKR phosphorylation and ethanol exposure significantly promoted TD-induced PKR phosphorylation as well as its nuclear translocation. A selective PKR inhibitor not only protected CGNs against TD toxicity, but also abolished ethanol potentiation of TD-induced loss of CGNs. Ethanol promoted TD-induced PKR activation and neuronal death. PKR may be a convergent protein that mediates the interaction between TD and ethanol.

Expression and regulation of antiviral protein APOBEC3G in human neuronal cells

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) has recently been identified as a potent antiviral protein. Here, we examined the expression and regulation of APOBEC3G in human brain tissues and the cells of central nervous system (CNS). Similar to the immune cells, human brain tissue and the CNS cells expressed APOBEC3G at both mRNA and protein levels. The expression of APOBEC3G could be up-regulated in human neuronal cells (NT2-N) and astrocytes (U87-MG) by interferons (IFN-α, β and γ), interleukin-1 (IL-1), and tumor necrosis factor. Other cytokines (IL-4, IL-6 and transforming growth factor beta1) and CC-chemokines (CCL3, 4 and 5), however, had little impact on the expression of APOBEC3G. In addition, pseudotyped HIV-1 infection and cytokine/chemokine-enriched supernatants from lipopolysaccharide-stimulated macrophage cultures induced APOBEC3G expression in NT2-N cells. APOBEC3G expressed in the neuronal cells and astrocytes was biologically functional, as the suppression of APOBEC3G expression by the specific siRNA led to increase of pseudotyped HIV-1 replication in these cells. These findings provide direct and compelling evidence that there is intracellular expression and regulation of functional APOBEC3G in the neuronal cells, which may be one of innate defense mechanisms involved in the neuronal protection in the CNS.