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Antiviral Research (v.94, #3)


Inhibition of adenovirus multiplication by short interfering RNAs directly or indirectly targeting the viral DNA replication machinery by Doris Kneidinger; Mirza Ibrišimović; Thomas Lion; Reinhard Klein (pp. 195-207).
► SiRNAs inhibit adenovirus multiplication. ► Adenoviral DNA replication is a key target for siRNA-mediated inhibition. ► SiRNAs can delay the death of adenovirus-infected cells.Human adenoviruses are a common threat to immunocompromised patients, e.g., HIV-positive individuals or solid-organ and, in particular, allogeneic stem cell transplant recipients. Antiviral drugs have a limited effect on adenoviruses, and existing treatment modalities often fail to prevent fatal outcome. Silencing of viral genes by short interfering RNAs (siRNAs) holds a great promise in the treatment of viral infections. The aim of the present study was to identify adenoviral candidate targets for RNA interference-mediated inhibition of adenoviral replication. We investigated the impact of silencing of a set of early, middle, and late viral genes on the replication of adenovirus 5 in vitro. Adenovirus replication was inhibited by siRNAs directed against the adenoviral E1A, DNA polymerase, preterminal protein (pTP), IVa2, hexon, and protease genes. Silencing of early and middle genes was more effective in inhibiting adenovirus multiplication than was silencing of late genes. A siRNA directed against the viral DNA polymerase mRNA decreased viral genome copy numbers and infectious virus progeny by several orders of magnitude. Since silencing of any of the early genes directly or indirectly affected viral DNA synthesis, our data suggest that reducing viral genome copy numbers is a more promising strategy for the treatment of adenoviral infections than is reducing the numbers of proteins necessary for capsid generation. Thus, adenoviral DNA replication was identified as a key target for RNAi-mediated inhibition of adenovirus multiplication. In addition, the E1A transcripts emerged as a second important target, because its knockdown markedly improved the viability of cells at late stages of infection.

Keywords: Abbreviations; RNAi; RNAinterference; siRNA; small interfering RNAAdenovirus; Infection; RNA interference; siRNA


Design of modified U1i molecules against HIV-1 RNA by Stefanie A. Knoepfel; Amaya Abad; Xabi Abad; Puri Fortes; Ben Berkhout (pp. 208-216).
► U1 interference (U1i) molecules were designed to inhibit HIV-1 RNA. ► One U1i inhibitor revealed high potency in various transient transfection assays. ► This potency is due to a tandem target site in the HIV-1 RNA. ► U1i combined with a lentiviral gene therapy approach needs optimization.Several gene therapeutic approaches have been proposed to add to current antiretroviral therapy against HIV-1. U1 interference (U1i) is a promising new gene therapy tool that targets mRNAs with modified U1 snRNAs. For efficient inhibition, the 3′-terminal exon of pre-mRNAs must be recognized by the modified U1 snRNA. Subsequent interaction between the U1-associated 70K protein and poly(A) polymerase leads to inhibition of polyadenylation and consequently degradation of the pre-mRNA. We designed 14 new U1i inhibitors against HIV-1 mRNA regions that are 100% complementary to at least 70% of HIV-1 sequences listed in the HIV database. All U1i inhibitors were tested transiently in HIV-1 production assays as well as luciferase reporter experiments and three candidates were examined further in stably lentivirus-transduced T cell lines. We identified U1i-J that targets the region encoding the NF-κB binding sites as the most effective inhibitor that substantially reduced viral protein expression. The potency of J is determined in part by the presence of a duplicated target within the HIV-1 mRNA. The stably transduced SupT1 T cells were challenged with HIV-1 but no antiviral effect was detected. U1i inhibitors can be potent suppressors of HIV-1 production in transient assays but further optimization of this antiviral approach is needed.

Keywords: HIV-1; U1 interference; Modified U1 snRNAs; Gene therapy; Inhibition; RNA degradation


In vitro anti-influenza virus and anti-inflammatory activities of theaflavin derivatives by Mian Zu; Fan Yang; Weiling Zhou; Ailin Liu; Guanhua Du; Lishu Zheng (pp. 217-224).
► Theaflavin extract and its three derivatives displayed anti-influenza viral activity. ► Their action mechanisms are through inhibition of neuraminidase and haemagglutinin. ► They also decreased the expression level of IL-6 during viral infection. ► Theaflavins are potential natural compounds for influenza prevention and treatment. ► These findings will be important for the development of anti-influenza drug.The theaflavins fraction (TF80%, with a purity of 80%) and three theaflavin (TF) derivatives from black tea have been found to exhibit potent inhibitory effects against influenza virus in vitro. They were evaluated with a neuraminidase (NA) activity assay, a hemagglutination (HA) inhibition assay, a real-time quantitative PCR (qPCR) assay for gene expression of hemagglutinin (HA) and a cytopathic effect (CPE) reduction assay. The experimental results showed that they all exerted significant inhibitory effects on the NA of three different subtypes of influenza virus strains [A/PR/8/34(H1N1), A/Sydney/5/97(H3N2) and B/Jiangsu/10/2003] with 50% inhibitory concentration (IC50) values ranging from 9.27 to 36.55μg/mL, and they also displayed an inhibitory effect on HA; these inhibitory effects might constitute two major mechanisms of their antiviral activity. Time-of-addition studies demonstrated that TF derivatives might have a direct effect on viral particle infectivity, which was consistent with the inhibitory effect on HA. Subsequently, the inhibitory effect of TF derivatives on the replication of the viral HA gene as assayed by qPCR and on the nuclear localization of the influenza virus vRNP further demonstrated that they may primarily act during the early stage of infection. Interestingly, besides the activity against functional viral proteins, TF derivatives also decreased the expression level of the inflammatory cytokine IL-6 during viral infection, expression of which may result in serious tissue injury and apoptosis. Our results indicated that TF derivatives are potential compounds with anti-influenza viral replication and anti-inflammatory properties. These findings will provide important information for new drug design and development for the treatment of influenza virus infection.

Keywords: Antiviral; Anti-inflammatory; Influenza virus; Theaflavin derivatives


Correlation between hepatitis B virus protein and microRNA processor Drosha in cells expressing HBV by Min Ren; Dongdong Qin; Kai Li; Jialin Qu; Liying Wang; Zengchan Wang; Ailong Huang; Hua Tang (pp. 225-231).
► We demonstrated that HBV could down regulate one of miRNA producing protein, Drosha expression. ► We examined the mechanism of HBV regulated Drosha expression. ► HBx may have function in regulated Drosha promoter activity. ► Knockdown of SP1 and AP-2α expressions could partially enhance activity of DGCR8 promoter activity.Drosha regulates the biogenesis of microRNAs (miRNAs) and plays an essential role in the regulation of gene expression. Infection with hepatitis B virus (HBV) causes chronic hepatitis and liver cirrhosis. It is also a major risk factor for hepatocellular carcinoma. Emerging evidence suggests that HBV alters miRNA expression profiles, but the mechanisms underlying this process have not yet been fully elucidated. We therefore examined how HBV affected the production of miRNAs. We found that Drosha mRNA and protein expression were downregulated in cells expressing the HBV genome. This was associated with a reduction in the activity of the Drosha gene promoter. Gene silencing of HBx by RNA interference significantly restored the expression of Drosha. In conclusion, our data show that HBV could downregulate Drosha expression by inhibiting promoter activity, and the transcription factors SP1 and AP-2α may be involved in this process. This provides a new understanding of the mechanism of HBV-induced miRNAs dysregulation.

Keywords: Drosha; miRNA; HBV; SP1; AP-2α; Regulation


Profile of stress and toxicity gene expression in human hepatic cells treated with Efavirenz by Leysa J. Gomez-Sucerquia; Ana Blas-Garcia; Miguel Marti-Cabrera; Juan V. Esplugues; Nadezda Apostolova (pp. 232-241).
► Increased expression in oxidative stress-related genes ( MT2A, HSPA6, GDF15, DDIT3). ► Inflammation-associated genes are up- ( SERPINE1), and down-regulated ( CXCL10). ► Regarding metabolic stress, EFV increases the expression of CYP1A1.Hepatic toxicity and metabolic disorders are major adverse effects elicited during the pharmacological treatment of the human immunodeficiency virus (HIV) infection. Efavirenz (EFV), the most widely used non-nucleoside reverse transcriptase inhibitor (NNRTI), has been associated with these events, with recent studies implicating it in stress responses involving mitochondrial dysfunction and oxidative stress in human hepatic cells. To expand these findings, we analyzed the influence of EFV on the expression profile of selected stress and toxicity genes in these cells.Significant up-regulation was observed with Cytochrome P450, family 1, subfamily A, polypeptide 1 ( CYP1A1), which indicated metabolic stress. Several genes directly related to oxidative stress and damage exhibited increased expression, including Methalothionein 2A ( MT2A), Heat shock 70kDa protein 6 ( HSPA6), Growth differentiation factor 15 ( GDF15) and DNA-damage-inducible transcript 3 (DDIT3). In addition, Early growth response protein 1 ( EGR1) was enhanced, whereas mRNA levels of the inflammatory genes Chemokine (C-X-C motif) ligand 10 ( CXCL10) and Serpin peptidase inhibitor (nexin, plasminogen activator inhibitor type 1), member 1 ( SERPINE1) decreased and increased, respectively. This profile of gene expression supports previous data demonstrating altered mitochondrial function and presence of oxidative stress/damage in EFV-treated hepatic cells, and may be of relevance in the search for molecular targets with therapeutic potential to be employed in the prevention, diagnosis and treatment of the hepatic toxicity associated with HIV therapy.

Keywords: Abbreviations; EFV; Efavirenz; HAART; highly active antiretroviral therapy; NRTI; nucleoside reverse transcriptase inhibitor; NNRTI; non-nucleoside reverse transcriptase inhibitor; ROS; reactive oxygen species; Δ; Ψ; m; mitochondrial transmembrane potential; DNA; polymerase gamma (Pol-γ); ER; endoplasmatic reticulum; UPR; unfolded protein responseMitochondria; Hepatotoxicity; HIV; NNRTI; Differential gene expression; Stress response


Alphaviruses: Population genetics and determinants of emergence by Scott C. Weaver; Richard Winegar; Ian D. Manger; Naomi L. Forrester (pp. 242-257).
► Alphaviruses are important causes of emerging diseases as well as biothreat agents. ► Venezuelan equine encephalitis causes equine-amplified epidemics and enzootic spillover. ► Eastern equine encephalitis virus is one of the most virulent viruses for people and equids. ► Chikungunya virus repeatedly emerged from Africa into epidemic urban cycles in Asia and Europe. ► Population genetic studies are needed to identify natural and intentional alphavirus emergences.Alphaviruses are responsible for several medically important emerging diseases and are also significant veterinary pathogens. Due to the aerosol infectivity of some alphaviruses and their ability to cause severe, sometimes fatal neurologic diseases, they are also of biodefense importance. This review discusses the ecology, epidemiology and molecular virology of the alphaviruses, then focuses on three of the most important members of the genus: Venezuelan and eastern equine encephalitis and chikungunya viruses, with emphasis on their genetics and emergence mechanisms, and how current knowledge as well as gaps influence our ability to detect and determine the source of both natural outbreaks and potential use for bioterrorism. This article is one of a series in Antiviral Research on the genetic diversity of emerging viruses.

Keywords: Alphavirus; Arbovirus; Genetics; Ecology; Epidemiology; Biodefense


Quercetin inhibits rhinovirus replication in vitro and in vivo by Shyamala Ganesan; Andrea N. Faris; Adam T. Comstock; Qiong Wang; Suparna Nanua; Marc B. Hershenson; Uma S. Sajjan (pp. 258-271).
► Rhinovirus is responsible for majority of common colds. ► There are no FDA approved drugs are available to treat rhinovirus infection. ► We show that quercetin, a plant flavonoid blocks viral replication in vitro. ► In addition quercetin inhibits virus-stimulated cytokine expression. ► Quercetin also inhibits viral replication and decreasing lung inflammation in vivo.Rhinovirus (RV), which is responsible for the majority of common colds, also causes exacerbations in patients with asthma and chronic obstructive pulmonary disease. So far, there are no drugs available for treatment of rhinovirus infection. We examined the effect of quercetin, a plant flavanol on RV infection in vitro and in vivo. Pretreatment of airway epithelial cells with quercetin decreased Akt phosphosphorylation, viral endocytosis and IL-8 responses. Addition of quercetin 6h after RV infection (after viral endocytosis) reduced viral load, IL-8 and IFN responses in airway epithelial cells. This was associated with decreased levels of negative and positive strand viral RNA, and RV capsid protein, abrogation of RV-induced eIF4GI cleavage and increased phosphorylation of eIF2α. In mice infected with RV, quercetin treatment decreased viral replication as well as expression of chemokines and cytokines. Quercetin treatment also attenuated RV-induced airway cholinergic hyperresponsiveness. Together, our results suggest that quercetin inhibits RV endocytosis and replication in airway epithelial cells at multiple stages of the RV life cycle. Quercetin also decreases expression of pro-inflammatory cytokines and improves lung function in RV-infected mice. Based on these observations, further studies examining the potential benefits of quercetin in the prevention and treatment of RV infection are warranted.

Keywords: Infection; Translation; Transcription; Inflammation


Polymorphisms in ADAR1 gene affect response to interferon alpha based therapy for chronic hepatitis B in Han Chinese by Xiaopan Wu; Zhenhui Xin; Xilin Zhu; Liping Pan; Zhuo Li; Hui Li; Ying Liu (pp. 272-275).
► This retrospective nested case-control study include 548 HBeAgpositive CHB patients. ► We test if SNPs in IFN pathway genes are associated with therapy efficacy of IFNα. ► SNP rs4845384 in ADAR1 gene is strongly associated with therapy efficacy.Host genetic polymorphisms in interferon pathway genes are reported to be associated with response to interferon therapy. Five hundred and forty-eight α interferon treatment-naïve chronic hepatitis B patients were enrolled in the retrospective nested case-control study. All patients received α interferon based treatment and were examined for therapy efficacy. We genotyped 115 polymorphisms from 16 interferon pathway genes using the MassArray system. We identified rs4845384 in ADAR1 gene is strongly associated with the outcome of interferon therapy allele dose-depended ( P=0.0005), with decreased odds ratios of 0.69 and 0.27 for GA and AA genotypes, respectively (95% confidence interval, 0.47–0.99 for GA; 0.11–0.64 for AA). Our study suggested that rs4845384 in ADAR1 associates with treatment-induced clearance of chronic hepatitis B.

Keywords: IFN pathway gene; Chronic hepatitis B; Interferon α therapy; Single nucleotide polymorphism; Sustained virological response


Amino acid derivatives of the (−) enantiomer of gossypol are effective fusion inhibitors of human immunodeficiency virus type 1 by Tai An; Wenjie Ouyang; Wei Pan; Deyin Guo; Jurong Li; Longlong Li; Gang Chen; Jian Yang; Shuwen Wu; Po Tien (pp. 276-287).
► The derivatives of (−) enantiomer of gossypol were synthesized. ► The derivatives of (−) gossypol are fusion inhibitor to HIV-1. ► We explain that why some modify could enhance its activity but others not.T20 and maraviroc are the only two currently available entry inhibitors that have shown efficacy in treating HIV-1-infected individuals who have failed to respond to first-line antiretroviral drugs. Gossypol is a polyphenolic aldehyde extracted from cotton plants. By modifying the (−) enantiomer of gossypol with a series of small molecules, we have found that neutral amino acids with aliphatic group derivatives of (−) gossypol show the strongest inhibitory activity and the lowest cytotoxicity in vitro among all the derivatives tested. Additionally, the selectivity index of the (−) gossypol-neutral amino acid conjugates is increased 100-fold when compared with (−) gossypol alone. It is widely accepted that gossypol and gossypol derivatives inhibit HIV-1 replication by targeting reverse transcriptase. However, from the results of our time-of-addition assay, HIV-1-mediated cell fusion assay and VSV-G pseudotyped virus assay, we demonstrate that the alanine-(−) gossypol derivative ((−)G-Ala) is an effective HIV-1 entry inhibitor. Further mechanistic analysis revealed that (−)G-Ala neither blocks gp120-CD4 binding nor interacts with the HIV-1 co-receptor CXCR4. Results from sandwich ELISA, native-PAGE and circular dichroism (CD) show that (−)G-Ala inhibits the cell fusion-activated gp41 core domain. Moreover, (−)G-Ala binds to the HIV-5-Helix protein and blocking D-peptide (PIE7) binding to the hydrophobic pocket on the surface of the gp41 internal trimeric coiled-coil domain. The contraceptive properties of (−) gossypol and amino acid derivatives of (−) gossypol are also discussed. Collectively, our results indicate that (−)G-Ala may bind to the gp41 hydrophobic pocket and block the formation of the cell fusion-activated gp41 core to inhibit HIV-1-mediated membrane fusion and subsequent viral entry.

Keywords: HIV-1; Amino acid derivatives of the (−) enantiomer of gossypol; Fusion inhibitor; Gp41


A hexapeptide of the receptor-binding domain of SARS corona virus spike protein blocks viral entry into host cells via the human receptor ACE2 by Anna-Winona Struck; Marco Axmann; Susanne Pfefferle; Christian Drosten; Bernd Meyer (pp. 288-296).
Display Omitted► Hexapeptide YKYRYL specifically inhibits SARS infection in cells. ► Assessment of peptide libraries by SPR screening and virus proliferation assays. ► Hexapeptide YKYRYL is minimal binding epitope of SARS spike protein to ACE2. ► Characterization by SPR, STD and molecular dynamics of the binding mode of the hexapeptide. In vitro infection of Vero E6 cells by SARS coronavirus (SARS-CoV) is blocked by hexapeptide Tyr-Lys-Tyr-Arg-Tyr-Leu. The peptide also inhibits proliferation of coronavirus NL63. On human cells both viruses utilize angiotensin-converting enzyme 2 (ACE2) as entry receptor. Blocking the viral entry is specific as alpha virus Sindbis shows no reduction in infectivity. Peptide438YKYRYL443 is part of the receptor-binding domain (RBD) of the spike protein of SARS-CoV. Peptide libraries were screened by surface plasmon resonance (SPR) to identify RBD binding epitopes.438YKYRYL443 carries the dominant binding epitope and binds to ACE2 with KD=46μM. The binding mode was further characterized by saturation transfer difference (STD) NMR spectroscopy and molecular dynamic simulations. Based on this information the peptide can be used as lead structure to design potential entry inhibitors against SARS-CoV and related viruses.

Keywords: SARS coronavirus; Spike protein; ACE2; Entry inhibitor; Virus proliferation assay; SPR screening
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