Inhibition of Influenza A Virus Infection In Vitro by Peptides Designed In Silico

Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H⁺ ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity.     

Antiviral ribozymes

Catalytic RNAs are a genetic property not only of some particular viroids or viruses, but also are more common naturally among eukaryotes and even prokaryotes than earlier expected. However, the major interest in ribozymes results from their potential for development of “tailor-made” cDNA constructions designed to be transcribed into catalytic RNAs that will recognize by hybridization and destroy by specific cleavage their cellular or viral RNA targets. The efficiency of an antiviral ribozyme is determined by both the accessibility and sequence conservation of the target region, as well as the design of the ribozyme: its type, size, and composition of flanking sequences; expression rates; and cellular compartment localization. Until now the most frequently selected viral target is the human immunodeficiency virus, where an up to a 10⁴-fold inhibition in its progeny production has been achieved. Although the first generation ribozymes focused on improvements in basic design and expression rates, more recently the efficiency of antiviral catalytic activity has been increased by employing polyribozymes and/or multitarget ribozymes, as well as special constructions to enhance the cellular co-compartmentation of the ribozyme with its viral RNA target.     

宿主防御肽抗病毒感染机制研究进展

宿主防御肽是一类广泛存在于脊椎动物的小分子多肽,具有广谱的抗菌活性以及抗炎、细胞趋化、促进血管生成和修复损伤等免疫调节功能。以往的研究多集中在宿主防御肽抗细菌和真菌感染的研究上。近年来大量研究发现,宿主防御肽也具有广泛的抗病毒活性,在临床各类病毒病的预防和治疗上具有潜在的应用前景。本文围绕宿主防御肽直接杀伤病毒、调节病毒感染过程和参与宿主抗病毒天然免疫调节这3个方面的作用机制进行综述,为宿主防御肽抗病毒相关研究和相关抗病毒生物药物的研发提供参考和借鉴。     

Selection of Antiviral Peptides Against Mink Enteritis Virus Using a Phage Display Peptide Library

Mink enteritis virus (MEV) causes high morbidity and mortality in mink worldwide, and there are no effective treatments. This study used a phage display library to find specific peptides capable of binding MEV and preventing its replication in F81 cells. After three rounds of biopanning, the phage enrichment was 117 times higher than that after the first round. Twelve phage clones that showed threefold higher MEV-binding affinity than controls were selected by ELISA. Following sequence analyses, the peptides RLNNRARIILRA and LAHKSRLYERHM were synthesized and used for antiviral experiments. MTT assays demonstrated that both peptides increased cell viability by >20 % at 100 μg/ml when pre-incubated with MEV. However, no effect was seen if the peptides were added 2 h after viral inoculation of cells, indicating that the antiviral activity is due to inhibition of viral attachment to the cell surface.     

A p7 Ion Channel-derived Peptide Inhibits Hepatitis C Virus Infection in Vitro

Viral infection is an early stage of its life cycle and represents a promising target for antiviral drug development. Here we designed and characterized three peptide inhibitors of hepatitis C virus (HCV) infection based on the structural features of the membrane-associated p7 polypeptide of HCV. The three peptides exhibited low toxicity and high stability while potently inhibiting initial HCV infection and suppressed established HCV infection at non-cytotoxic concentrations in vitro. The most efficient peptide (designated H2-3), which is derived from the H2 helical region of HCV p7 ion channel, inhibited HCV infection by inactivating both intracellular and extracellular viral particles. The H2-3 peptide inactivated free HCV with an EC₅₀ (50% effective concentration) of 82.11 nm, which is >1000-fold lower than the CC₅₀ (50% cytotoxic concentration) of Huh7.5.1 cells. H2-3 peptide also bound to cell membrane and protected host cells from viral infection. The peptide H2-3 did not alter the normal electrophysiological profile of the p7 ion channel or block viral release from Huh7.5.1 cells. Our work highlights a new anti-viral peptide design strategy based on ion channel, giving the possibility that ion channels are potential resources to generate antiviral peptides.     

Physicochemical characterization of a peptide deriving from the glycoprotein gp36 of the feline immunodeficiency virus and its lipoylated analogue in micellar systems

P59 is the Trp-rich 20-mer peptide (⁷⁶⁷L-G⁷⁸⁶), partial sequence of the membrane-proximal external region (MPER) of the FIV gp36. It has potent antiviral activity, possibly due to a mechanism that inhibits the fusion of the virus with the cell membranes. In the hypothesis that a lipophilic tail could enhance the adhesion of P59 to the membrane so improving its antiviral activity, we synthesized its lipoylated analogue lipo-P59. Fluorescence, CD and NMR investigations in membrane mimicking environments (such as SDS and DPC micelles) were aimed to assess the potential of the lipo-P59 lipophilic tail to affect the biophysical and conformational behaviour of the peptide. In vitro inhibitory assays using lymphoid cell cultures to check the antiviral activity of peptides were also performed. The data show that the biophysical properties and the conformational preferences of the peptides are not dramatically affected by the hydrophobic tail, suggesting that the lipopeptide is capable of preserving all the biophysical peculiarities. Similarly, antiviral experimental data show that the membrane-anchored lipo-P59 peptide is also effective in inhibiting virus replication. Moreover, the lipophilic tail allows P59 to preserve its antiviral activity even in conditions in which the non lipoylated peptide is devoid of activity. In accordance with the unusual high Trp presence, the peptides confirm the preference to be positioned on the membrane interface. Furthermore, the data point out a peculiarity of interaction of the peptides with SDS as compared with DPC.     

DNA immunization with a plasmid carrying the gene of hepatitis C virus protein 5A (NS5A) induces an effective cellular immune response

In spite of extensive research, no effective vaccine against hepatitis C virus (HCV) has been developed so far. DNA immunization is a potent technique of vaccine design strongly promoting the cellular arm of immune response. The genes encoding nonstructural HCV proteins (NS2-NS5B) are promising candidates for vaccine development. NS5A is a protein involved in viral pathogenesis, in the induction of immune response, and probably in viral resistance to interferon treatment. The objective of this study was to construct a DNA vaccine encoding NS5A protein and evaluate its immunogenicity. A plasmid encoding a full-size NS5A protein was produced using the pcDNA3.1 (+) vector for eukaryotic expression system. The expression of the NS5A gene was confirmed by immunoperoxidase staining of the transfected eukaryotic cells with anti- NS5A monoclonal antibodies. Triple immunization of mice with the plasmid vaccine induced a pronounced cellular immune response against a broad spectrum of NS5A epitopes as assessed by T-cell proliferation and secretion of antiviral cytokines IFN-γ and IL-2. In T-cell stimulation in vitro experiments, NS5A-derived antigens were modeled by synthetic peptides, recombinant proteins of various genotypes, and phages carrying exposed NS5A peptides. A novel immunomodulator Immunomax showed high adjuvant activity in DNA immunization. The data obtained indicate that the suggested DNA construct has a strong potential in the development of the gene vaccines against hepatitis C.     

A general strategy to endow natural fusion-protein-derived peptides with potent antiviral activity

Fusion between the viral and target cell membranes is an obligatory step for the infectivity of all enveloped virus, and blocking this process is a clinically validated therapeutic strategy.Viral fusion is driven by specialized proteins which, although specific to each virus, act through a common mechanism, the formation of a complex between two heptad repeat (HR) regions. The HR regions are initially separated in an intermediate termed "prehairpin", which bridges the viral and cell membranes, and then fold onto each other to form a 6-helical bundle (6HB), driving the two membranes to fuse. HR-derived peptides can inhibit viral infectivity by binding to the prehairpin intermediate and preventing its transition to the 6HB.The antiviral activity of HR-derived peptides differs considerably among enveloped viruses. For weak inhibitors, potency can be increased by peptide engineering strategies, but sequence-specific optimization is time-consuming. In seeking ways to increase potency without changing the native sequence, we previously reported that attachment to the HR peptide of a cholesterol group ("cholesterol-tagging") dramatically increases its antiviral potency, and simultaneously increases its half-life in vivo. We show here that antiviral potency may be increased by combining cholesterol-tagging with dimerization of the HR-derived sequence, using as examples human parainfluenza virus, Nipah virus, and HIV-1. Together, cholesterol-tagging and dimerization may represent strategies to boost HR peptide potency to levels that in some cases may be compatible with in vivo use, possibly contributing to emergency responses to outbreaks of existing or novel viruses.     

Potent Inhibition of Late Stages of Hepadnavirus Replication by a Modified Cell Penetrating Peptide

Cationic cell-penetrating peptides (CPPs) and their lipid domain-conjugates (CatLip) are agents for the delivery of (uncharged) biologically active molecules into the cell. Using infection and transfection assays we surprisingly discovered that CatLip peptides were able to inhibit replication of Duck Hepatitis B Virus (DHBV), a reference model for human HBV. Amongst twelve CatLip peptides we identified Deca-(Arg)₈ having a particularly potent antiviral activity, leading to a drastic inhibition of viral particle secretion without detectable toxicity. Inhibition of virion secretion was correlated with a dose-dependent increase in intracellular viral DNA. Deca-(Arg)₈ peptide did neither interfere with DHBV entry, nor with formation of mature nucleocapsids nor with their travelling to the nucleus. Instead, Deca-(Arg)₈ caused envelope protein accumulation in large clusters as revealed by confocal laser scanning microscopy indicating severe structural changes of preS/S. Sucrose gradient analysis of supernatants from Deca-(Arg)₈-treated cells showed unaffected naked viral nucleocapsids release, which was concomitant with a complete arrest of virion and surface protein-containing subviral particle secretion. This is the first report showing that a CPP is able to drastically block hepadnaviral release from infected cells by altering late stages of viral morphogenesis via interference with enveloped particle formation, without affecting naked nucleocapsid egress, thus giving a view inside the mode of inhibition. Deca-(Arg)₈ may be a useful tool for elucidating the hepadnaviral secretory pathway, which is not yet fully understood. Moreover we provide the first evidence that a modified CPP displays a novel antiviral mechanism targeting another step of viral life cycle compared to what has been so far described for other enveloped viruses.     

小干扰RNA抗病毒研究进展

小干扰RNA(siRNA)介导的对同源基因转录后水平的抑制或由此引发的细胞质内同源mRNA降解的转录后基因沉默现象,在抗病毒研究中具有重要意义。由病毒感染引起的各种疾病严重危害着人类的健康,而siRNA在抗病毒感染的研究领域中显示出高度特异性,故受到人们的普遍关注。近年来,siRNA抗病毒方面的研究成为国内外的热点,为临床病毒感染性疾病的防治提供了新的途径。我们就siRNA的机制和特点、siRNA的制备及其抗病毒研究进展做简要综述。     

The pH-sensitive action of cholesterol-conjugated peptide inhibitors of influenza virus

Influenza viruses are major human pathogens, responsible for respiratory diseases affecting millions of people worldwide, with high morbidity and significant mortality. Infections by influenza can be controlled by vaccines and antiviral drugs. However, this virus is constantly under mutations, limiting the effectiveness of these clinical antiviral strategies. It is therefore urgent to develop new ones. Influenza hemagglutinin (HA) is involved in receptor binding and promotes the pH-dependent fusion of viral and cell endocytic membranes. HA-targeted peptides may emerge as a novel antiviral option to block this viral entry step. In this study, we evaluated three HA-derived (lipo)peptides using fluorescence spectroscopy. Peptide membrane interaction assays were performed at neutral and acidic pH to better resemble the natural conditions in which influenza fusion occurs. We found that peptide affinity towards membranes decreases upon the acidification of the environment. Therefore, the released peptides would be able to bind their complementary domain and interfere with the six-helix bundle formation necessary for viral fusion, and thus for the infection of the target cell. Our results provide new insight into molecular interactions between HA-derived peptides and cell membranes, which may contribute to the development of new influenza virus inhibitors.     

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

For rational design of therapeutic vaccines, detailed knowledge about target epitopes that are endogenously processed and truly presented on infected or transformed cells is essential. Many potential target epitopes (viral or mutation‐derived), are presented at low abundance. Therefore, direct detection of these peptides remains a challenge. This study presents a method for the isolation and LC‐MS³‐based targeted detection of low‐abundant human leukocyte antigen (HLA) class‐I‐presented peptides from transformed cells. Human papillomavirus (HPV) was used as a model system, as the HPV oncoproteins E6 and E7 are attractive therapeutic vaccination targets and expressed in all transformed cells, but present at low abundance due to viral immune evasion mechanisms. The presented approach included preselection of target antigen‐derived peptides by in silico predictions and in vitro binding assays. The peptide purification process was tailored to minimize contaminants after immunoprecipitation of HLA‐peptide complexes, while keeping high isolation yields of low‐abundant target peptides. The subsequent targeted LC‐MS³ detection allowed for increased sensitivity, which resulted in successful detection of the known HLA‐A2‐restricted epitope E7_11–19 and ten additional E7‐derived peptides on the surface of HPV16‐transformed cells. T‐cell reactivity was shown for all the 11 detected peptides in ELISpot assays, which shows that detection by our approach has high predictive value for immunogenicity. The presented strategy is suitable for validating even low‐abundant candidate epitopes to be true immunotherapy targets.     

Grifonin-1: a small HIV-1 entry inhibitor derived from the algal lectin, Griffithsin

Background

Griffithsin, a 121-residue protein isolated from a red algal Griffithsia sp., binds high mannose N-linked glycans of virus surface glycoproteins with extremely high affinity, a property that allows it to prevent the entry of primary isolates and laboratory strains of T- and M-tropic HIV-1. We used the sequence of a portion of griffithsin''s sequence as a design template to create smaller peptides with antiviral and carbohydrate-binding properties.

Methodology/Results

The new peptides derived from a trio of homologous β-sheet repeats that comprise the motifs responsible for its biological activity. Our most active antiviral peptide, grifonin-1 (GRFN-1), had an EC₅₀ of 190.8±11.0 nM in in vitro TZM-bl assays and an EC₅₀ of 546.6±66.1 nM in p24^gag antigen release assays. GRFN-1 showed considerable structural plasticity, assuming different conformations in solvents that differed in polarity and hydrophobicity. Higher concentrations of GRFN-1 formed oligomers, based on intermolecular β-sheet interactions. Like its parent protein, GRFN-1 bound viral glycoproteins gp41 and gp120 via the N-linked glycans on their surface.

Conclusion

Its substantial antiviral activity and low toxicity in vitro suggest that GRFN-1 and/or its derivatives may have therapeutic potential as topical and/or systemic agents directed against HIV-1.     

Discovery of the pan-genotypic hepatitis C virus NS3/4A protease inhibitor voxilaprevir (GS-9857): A component of Vosevi®

Treatment of hepatitis C virus (HCV) infection has been historically challenging due the high viral genetic complexity wherein there are eight distinct genotypes and at least 86 viral subtypes. While HCV NS3/4A protease inhibitors are an established treatment option for genotype 1 infection, limited coverage of genotypes 2 and/or 3 combined with serum alanine transaminase (ALT) elevations for some compounds has limited the broad utility of this therapeutic class. Our discovery efforts were focused on identifying an NS3/4A protease inhibitor with pan-genotypic antiviral activity, improved coverage of resistance associated substitutions, and a decreased risk of hepatotoxicity. Towards this goal, distinct interactions with the conserved catalytic triad of the NS3/4A protease were identified that improved genotype 3 antiviral activity. We further discovered that protein adduct formation strongly correlated with clinical ALT elevation for this therapeutic class. Improving metabolic stability and decreasing protein adduct formation through structural modifications ultimately resulted in voxilaprevir. Voxilaprevir, in combination with sofosbuvir and velpatasvir, has demonstrated pan-genotypic antiviral clinical activity. Furthermore, hepatotoxicity was not observed in Phase 3 clinical trials with voxilaprevir, consistent with our design strategy. Vosevi^® (sofosbuvir, velpatasvir, and voxilaprevir) is now an approved pan-genotypic treatment option for the most difficult-to-cure individuals who have previously failed direct acting antiviral therapy.     

Phosphatidylinositol inhibits respiratory syncytial virus infection

Respiratory syncytial virus (RSV) infects nearly all children under age 2, and reinfection occurs throughout life, seriously impacting adults with chronic pulmonary diseases. Recent data demonstrate that the anionic pulmonary surfactant lipid phosphatidylglycerol (PG) exerts a potent antiviral effect against RSV in vitro and in vivo. Phosphatidylinositol (PI) is also an anionic pulmonary surfactant phospholipid, and we tested its antiviral activity. PI liposomes completely suppress interleukin-8 production from BEAS2B epithelial cells challenged with RSV. The presence of PI during viral challenge in vitro reduces infection by a factor of >10³. PI binds RSV with high affinity, preventing virus attachment to epithelial cells. Intranasal inoculation with PI along with RSV in mice reduces the viral burden 30-fold, eliminates the influx of inflammatory cells, and reduces tissue histopathology. Pharmacological doses of PI persist for >6 h in mouse lung. Pretreatment of mice with PI at 2 h prior to viral infection effectively suppresses inflammation and reduces the viral burden by 85%. These data demonstrate that PI has potent antiviral properties, a long residence time in the extracellular bronchoalveolar compartment, and a significant prophylaxis window. The findings demonstrate PG and PI have complementary roles as intrinsic, innate immune antiviral mediators in the lung.     

Company profile: Spider stories

SpiderBiotech is a biotech company that has carried out extensive research and development on peptide-based anti-infectives, with five people involved in R&D activities and a strong network of industrial and academic partners experienced in the field of anti-infectives. SpiderBiotech has also created a proprietary library of bioactive peptides and lipopeptides (both linear and dendrimeric) active against bacterial and viral infections. At the moment they have two ongoing projects: the most advanced is focused on the development of a panel of peptide based antibiotics. The second project is related to novel antiviral drugs to treat acyclovir resistant Herpes virus infections.     

2’-5’寡聚腺苷酸合成酶（OAS）及其临床意义的研究进展

干扰素作用于靶细胞膜表面的受体后,通过信号转导系统诱导一系列抗病毒蛋白产生,干扰病毒复制以达到抗病毒目的。2’-5’寡聚腺苷酸合成酶（2’．5’oligoadenylatesynthetase,OAS）是干扰素作用于细胞后产生的一种重要的抗病毒蛋白,几十年来,国内外学者对OAS家族及其抗病毒机制进行了大量研究并取得了一定的进展,OAS被dsRNA激活后,催化生成2-5A,2-5A激活核酸内切酶RNaseL,降解病毒RNA,阻断病毒蛋白合成,从而发挥抗病毒作用。体内外研究表明,OAS的表达量或活性的变化可用于评价机体对干扰素的反应,反映干扰素抗病毒效果,另外,它还可作为系统性红斑狼疮的病情活动度的一种检测指标。因此,OAS具有重要的临床应用价值。本文就OAS家族及其抗病毒机制,其测定方法与对于病毒性肝炎和系统性红斑狼疮疾病的临床意义展开综述,以期对OAS的研究和应用提供参考。OAS是典型的干扰素诱导产物,可反映机体内干扰素的抗病毒水平,具有广阔的应用前景。     

Interferon-induced transmembrane protein 3 (IFITM3) and its antiviral activity

Infections caused by enveloped viruses require fusion with cellular membranes for viral genome entry. Viral entry occurs following an interaction of viral and cellular membranes allowing the formation of fusion pores, by which the virus accesses the cytoplasm. Here, we focus on interferon-induced transmembrane protein 3 (IFITM3) and its antiviral activity. IFITM3 is predicted to block or stall viral fusion at an intermediate state, causing viral propagation to fail. After introducing IFITM3, we describe the generalized lipid membrane fusion pathway and how it can be stalled, particularly with respect to IFITM3, and current questions regarding IFITM3's topology, with specific emphasis on IFITM3's amphipathic α-helix (AAH) ₅₉V-₆₈M, which is necessary for the antiviral activity. We report new hydrophobicity and hydrophobic moment calculations for this peptide and a variety of active site peptides from known membrane-remodeling proteins. Finally, we discuss the effects of posttranslational modifications and localization, how IFITM3's AAH may block viral fusion, and possible ramifications of membrane composition.     

Insects antiviral and anticancer peptides: new leads for the future?

Insect produce wide range of protein and peptides as a first fast defense line against pathogen infection. These agents act in different ways including insect immune system activation or by direct impact on the target tumor cells or viruses. It has been shown that some of the insect peptides suppress viral gene and protein expression, rybosilate DNA, whereas others cause membrane lysis, induce apoptosis or arrest cell cycle. Several of the purified and characterized peptides of insect origin are very promising in treating of serious human diseases like human immunodeficiency virus (HIV), herpex simplex virus (HSV) or leukaemia. However, some obstacles need to be overcome. Cytotoxic activity of peptides, susceptibility to proteases or high cost of production remain still unsolved problems. Reports on the peptides antiviral and antitumour mechanisms are scanty. Thus, in this review we present characteristic, mode of action and potential medical applications of insects origin peptides with the antiviral and antitumour activity.