Antiviral Activity in Tissue Culture Systems of bis-Benzimidazoles, Potent Inhibitors of Rhinoviruses

(S,S)-1,2-bis(5-methoxy-2-benzimidazolyl)-1,2-ethanediol showed antiviral activity in monolayer tissue culture systems against 55 strains of rhinovirus, three types of poliovirus, and strains of type A and B coxsackieviruses. Neither the compound nor any of the analogues tested showed virucidal activity. Its antiviral activity was not associated with interference with viral attachment to or penetration into the cell. At a concentration of 0.1 mg/ml, this group of compounds was generally nontoxic to WI-38, primary bovine kidney, and African green monkey kidney cells and had antiviral activity with 100% inhibition of virus-induced cytopathic effects (CPE). At antiviral levels, these compounds prevented CPE of up to 10(6) median tissue culture infective dose units of virus and completely inhibited formation of new infective virions. The compounds showed antiviral activity both prophylactically and therapeutically against rhinoviruses. Infected cultures could be cleared of CPE up to 90 hr after infection.     

The use of liposomal form of phenylimide of cis-aconitic acid in herpes therapy

The results of study of the antiviral activity and pharmacokinetics of phenylimide of cis-aconitic acid (PCAA) is presented. The 20% increase of the antiviral activity of PCAA incorporated into liposomes in comparison with the antiviral activity of the pure substance was shown. Liposomes with PCAA were tropic to lymphocytes and macrophages with maximum fluorescence being observed in the spleen, while empty liposomes were accumulated mainly in the liver. After the treatment with liposomal PCAA the symptoms of herpetic meningoencephalitis became less severe with 100% survival of the experimental animals. In the control group of rabbits 50% of the animals died, and in the surviving animals blindness or paralysis developed.     

猪γ-干扰素在重组杆状病毒中的表达及其抗病毒活性的测定

研究利用Bac-To-Bac杆状病毒表达系统构建含有猪γ-干扰素(porcine interferon-γ,PoIFN-γ)完整开放阅读框的供体质粒pFastBacTM1-PoIFN-γ,转化DH10Bac感受态细胞获得重组穿梭质粒rBacmid-PoIFN-γ,转染sf9昆虫细胞救获表达PoIFN-γ的重组杆状病毒rBac-PoIFN-γ。以抗PoIFN-γ单克隆抗体为一抗进行Western blot、间接免疫荧光(IFA)及间接ELISA检测,结果表明PoIFN-γ在重组杆状病毒rBac-PoIFN-γ感染的昆虫细胞中获得正确表达。抗病毒活性试验显示,重组杆状病毒表达rPoIFN-γ能有效抑制水疱性口炎病毒(VSV)在猪肾细胞系(PK-15)的复制,rBac-PoIFN-γ感染昆虫细胞培养上清抗病毒活性为2×104抑制单位(IU)/mL,其抗病毒活性可以被鼠抗原核表达重组PoIFN-γ免疫血清有效阻断。结果表明rPoIFN-γ在重组杆状病毒rBac-PoIFN-γ在感染昆虫细胞获得有效表达,并具有高效抗病毒活性。     

牛γ-干扰素在重组杆状病毒中的表达及其抗病毒活性的测定

研究利用Bac-To-Bac杆状病毒表达系统构建含有牛γ-干扰素(Bovine interferon-γ,BoIFN-γ)完整开放阅读框的供体质粒pFastBacTM1-BoIFN-γ,转化DH10Bac感受态细胞获得重组穿梭质粒rBacmid-BoIFN-γ,转染sf9昆虫细胞救获表达BoIFN-γ的重组杆状病毒rBac-BoIFN-γ。采用抗BoIFN-γ单克隆抗体作为一抗进行间接免疫荧光(IFA)及间接ELISA检测,表明BoIFN-γ在重组杆状病毒rBac-BoIFN-γ感染的sf9昆虫细胞中获得正确表达。利用VSV*GFP-MDBK细胞系统测定rBoIFN-γ抗病毒活性,重组杆状病毒表达重组BoIFN-γ(rBoIFN-γ)能有效抑制水疱性口炎病毒(VSV)在牛肾细胞(MDBK)上的复制,rBac-BoIFN-γ感染sf9昆虫细胞上清抗病毒活性为2×105IU/mL,而且其抗病毒活性可以被鼠抗原核表达重组BoIFN-γ免疫血清阻断。结果表明:rBoIFN-γ在重组杆状病毒rBac-BoIFN-γ感染的sf9昆虫细胞中获得良好表达,并具有高效抗病毒活性。     

复合干扰素突变体在毕赤酵母中的表达、纯化及活性分析

根据毕赤酵母密码子偏性合成了复合干扰素突变体基因 ,克隆至分泌型酵母表达载体pMEX9K ,将重组载体pMEX CIFNm用SacⅠ线性化后 ,转化毕赤酵母GS115 .转化子经诱导后 ,培养上清有抗病毒活性的蛋白产生 .经过离子交换 ,疏水层析 ,凝胶过滤三步层析纯化 ,得到了纯度大于95 %的重组复合干扰素突变体 ,经N端氨基酸序列分析表明 ,该蛋白N端序列与理论值一致 ,质谱测定分子量为 19 3kD ,与理论值一致 .用细胞病变抑制法测定其活性 ,并结合Lowry法蛋白定量计算其比活性为 6× 10 8IU mg ,与复合干扰素的比活相当 .     

Novel antiviral activity found in the media of Sindbis virus-persistently infected mosquito (Aedes albopictus) cell cultures.

Aedes albopictus (mosquito) cells persistently infected with Sindbis virus for a period of 6 months release into the medium a low-molecular-weight material capable of specifically reducing the yields of Sindbis virus during the "acute phase" of infection in mosquito cells. The antiviral activity was produced in detectable levels at 3 days after infection, and its concentration in the extracellular medium increased thereafter. The antiviral activity was inactivated by treatment with the enzyme protease K and heat. It was not activated by treatment with antibody prepared against extracts of Sindbis virus-infected BHK-21 cells. The antiviral activity differs from interferon produced by vertebrate cells in that it is virus specific as well as cell specific.     

Penetration and Antiviral Activity of Coxsackievirus B3 (Cvb3)-Specific Phosphorothioate Oligodeoxynucleotides (Ps-Odn)

Abstract

The antiviral activity of PS-ODNs, complementary to different regions of the CVB3 genome, was investigated under in vitro conditions. Inhibition of CVB3 replication was detected only after prolonged pretreatment of HeLa cells with antiviral active PS-ODNs, but not when virus and PS-ODN were applicated simultaneously. Results from flow cytometric analysis indicate that a low cellular uptake of anti-CVB3 oligonucleotides into HeLa cells might be a reason for their moderate antiviral activity.     

INDUCTION OF ANTIVIRAL RESPONSE BY ELECTRIC PULSES

Antiviral activity was detected in porcine leukocytes obtained from animals killed in a slaughterhouse. The activity appeared in the supernatant immediately after the addition of Sendai virus, an intended interferon (IFN) inducer. Electroinduction appears to be responsible for the production of antiviral molecules in both porcine and human leukocytes. The macromolecule conferring the antiviral activity is sensitive to heat and pH 2.0 treatment. The preformed or electroinduced antiviral activity originating from porcine leukocytes was not neutralized by either anti-PoIFN-α or anti-PoIFN-γ. The substance involved differs from all known antiviral proteins in that it is not secreted spontaneously into the extracellular space. Its release requires intact and virulent Sendai virus as a trigger signal. Differences in antigenic properties and characteristics indicate that this is a new antiviral substance.     

A functional GTP-binding motif is necessary for antiviral activity of Mx proteins.

Mx proteins are interferon-induced GTPases that inhibit the multiplication of certain negative-stranded RNA viruses. However, it has been unclear whether GTPase activity is necessary for antiviral function. Here, we have introduced mutations into the tripartite GTP-binding consensus elements of the human MxA and mouse Mx1 proteins. The invariant lysine residue of the first consensus motif, which interacts with the beta- and gamma-phosphates of bound GTP in other GTPases, was deleted or replaced by methionine or alanine. These Mx mutants and appropriate controls were then tested for antiviral activity, GTP-binding capacity, and GTPase activity. We found a direct correlation between the GTP-binding capacities and GTP hydrolysis activities of the purified Mx mutants in vitro and their antiviral activities in transfected 3T3 cells, demonstrating that a functional GTP-binding motif is necessary for virus inhibition. Our results, thus, firmly establish antiviral activity as a novel function of a GTPase, emphasizing the enormous functional diversity of GTPase superfamily members.     

An insertion in loop L7 of human eosinophil-derived neurotoxin is crucial for its antiviral activity

The human eosinophil granule ribonuclease, eosinophil‐derived neurotoxin (EDN) has been shown to have antiviral activity against respiratory syncytial virus‐B (RSV‐B). Other closely related and more active RNases such as RNase A, onconase, and RNase k6 do not have any antiviral activity. A remarkable unique feature of EDN is a nine‐residue insertion in its carboxy‐terminal loop, L7 which is not present in RNase A, and differs in sequence from the corresponding loop in another eosinophil RNase, eosinophil cationic protein (ECP). ECP has a much lower antiviral activity as compared to EDN. The current study probed the role of loop L7 of EDN in its antiviral activity. Three residues in loop L7, Arg117, Pro120, and Gln122, which diverge between EDN, ECP, and RNase A, were mutated to alanine alone and in combination to generate single, double, and triple mutants. These mutants, despite having RNase activity had decreased antiviral activity towards RSV suggesting the involvement of loop L7 in the interaction of EDN with RSV. It appears that the mutations in loop L7 disrupt the interaction of protein with the viral capsid, thereby inhibiting its entry into the virions. The study demonstrates that besides the RNase activity, loop L7 is another important determinant for the antiviral activity of EDN. J. Cell. Biochem. 113: 3104–3112, 2012. © 2012 Wiley Periodicals, Inc.     

Synthetic proteins with antiviral properties of alpha-interferons

Consensus sequence 30-36 LKDRHDF of human alpha-interferons is inserted into the C- and N-terminal sequences of the artificial protein albeferon using genetic engineering methods in order to obtain artificial proteins with antiviral activity. Albeferon, obtained by Dolgikh et al. (Protein Eng., 1996, vol. 9, pp. 195-201), already contains the IFN-alpha 2 130-137 fragment and possesses antiproliferative activity comparable with that of IFN-alpha 2. According to CD spectroscopy, both proteins have regular secondary structures similar to that of the precursor protein. They exhibit antiviral activities, and the activity of one of them is comparable with that of IFN-alpha 2. At the same time, their cytotoxic properties are displayed only at relatively high concentrations, which substantially exceed the minimal antiviral concentrations.     

Antiviral Activity of 1-β-D-Arabinofuranosyl-E-5-(2-Bromovinyl)Uracil against Thymidine Kinase Negative Strains of Varicella-Zoster Virus

Mechanism of antiviral activity of 1-β-d -arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) against the YSR strain of varicella-zoster virus (VZV), which is a mutant derived from the wild YS strain and is completely deficient in viral thymidine kinase (TK), was searched in comparison with antiviral activity of other thymidine analogues, guanosine analogue and thymidylate synthase (TS) inhibitor in human embryo lung fibroblast cells. Thymidine analogues, such as BV-araU, 5-iododeoxyuridine (IUDR), 1-β-d -arabinofuranosylthymine (araT), and guanosine analogue, such as 9-(2-hydroxyethoxymethyl)guanine (ACV), showed higher antiviral activity to the YS strain than to the YSR strain. Though, BV-araU also had the antiviral activity of a microgram level against the YSR strain. In contrast to these results, TS inhibitor, 5-fluorodeoxyuridine (FUDR), had higher antiviral activity to the YSR strain than to the YS strain. Highly synergistic antiviral activities of FUDR to the YS strain and the YSR strain were observed in combination with IUDR, araT, or ACV. However, weakly synergistic or additive inhibition to the YSR strain was shown in combination of BV-araU and FUDR, in spite of highly synergistic effect of this combination to the YS strain. The viral and cellular TS activity was partially inhibited by BV-araU monophosphate, but not by BV-araU. These results indicate that BV-araU is converted into BV-araU monophosphate by cellular TK, and the inhibition of TS activity by BV-araU monophosphate in the YSR strain-infected cells results in the suppression of viral replication.     

7SL RNA mediates virion packaging of the antiviral cytidine deaminase APOBEC3G

Cytidine deaminase APOBEC3G (A3G) has broad antiviral activity against diverse retroviruses and/or retrotransposons, and its antiviral functions are believed to rely on its encapsidation into virions in an RNA-dependent fashion. However, the cofactors of A3G virion packaging have not yet been identified. We demonstrate here that A3G selectively interacts with certain polymerase III (Pol III)-derived RNAs, including Y3 and 7SL RNAs. Among A3G-binding Pol III-derived RNAs, 7SL RNA was preferentially packaged into human immunodeficiency virus type 1 (HIV-1) particles. Efficient packaging of 7SL RNA, as well as A3G, was mediated by the RNA-binding nucleocapsid domain of HIV-1 Gag. A3G mutants that had reduced 7SL RNA binding but maintained wild-type levels of mRNA and tRNA binding were packaged poorly and had impaired antiviral activity. Reducing 7SL RNA packaging by overexpression of SRP19 proteins inhibited 7SL RNA and A3G virion packaging and impaired its antiviral function. Thus, 7SL RNA that is encapsidated into diverse retroviruses is a key cofactor of the antiviral A3G. This selective interaction of A3G with certain Pol III-derived RNAs raises the question of whether A3G and its cofactors may have as-yet-unidentified cellular functions.     

Synergism of antiviral activity in cell cultures treated with low concentrations of interferon and interferon-treated lymphocytes

Human T cells treated with low levels of interferon (IFN) (1-10 units/ml), and washed to remove the IFN, transferred the same level of antiviral activity to recipient WISH cells as an equivalent IFN treatment alone could induce in WISH cells. Further, when T cells pretreated with IFN (1-10 units/ml) were cocultivated with WISH cells in the presence of IFN (1-10 units/ml), a 2.5- to 5-fold greater level of protection developed than could be expected from the additive effect of each. Antibody to leukocyte, fibroblast, or immune IFN blocked the antiviral effect of the respective IFN types but had no effect on the transfer of antiviral activity initiated by leukocyte, fibroblast, or immune IFN. Also, treatment of T cells with actinomycin D blocked the transfer of antiviral activity of IFN-treated T cells. Taken together, the data suggest that the increased antiviral activity is not merely an additive effect of the IFN, but represents a synergistic amplification of protection most likely due to the combination of the separate effects of IFN and IFN-induced transfer. Such interactions would be expected to play a major role in early protection against virus infections in vivo when low levels of interferon are present and lymphocytes are migrating into the area.     

含硒抗病毒多肽的半胱氨酸缺陷型表达及鉴定

目的：在大肠杆菌中进行含硒抗病毒多肽的可溶性半胱氨酸缺陷型融合蛋白的表达,并对其纯化蛋白进行生物学活性的初步鉴定。方法：将含硒抗病毒多肽Se-GBVA10基因克隆到GST融合表达载体pGEX-2T中,转化至半胱氨酸缺陷型大肠杆菌BL21 cysE51 后进行半胱氨酸缺陷型表达,通过谷胱甘肽Sepharose 4B 亲和柱纯化目的蛋白质,并对其含硒量、抗病毒活力等性质进行初步鉴定。结果：利用半胱氨酸缺陷型表达法成功地可溶性表达了sjGST-Se-GBVA10和sjGST-GBVA10融合蛋白,并经凝血酶切后获得了纯化的Se-GBVA10和GBVA10抗病毒多肽。测定Se-GBVA10的含硒量为0.974 mol/mol peptide,GPX活力为47.52 U/μmol,其EC₅₀为21.73μmol/L,CC₅₀为849.41μmol/L。结论：Se-GBVA10具有与GBVA10相同的抗病毒活性,并具有一定的抗氧化活力。     

Increased stability and antiviral activity of 2'-O-phosphoglyceryl derivatives of (2'-5')oligo(adenylate)

Metabolically stable analogues of (2'-5')oligo(adenylate), (2'-5')(A)n, might constitute a new class of antiviral agents as they mimic some of the effects of interferons. 2'-O-phosphoglyceryl derivatives of (2'-5')(A)n oligomers, (2'-5')(A)n-PGro have been synthesized by chemical modification of their terminal ribose residue. Such analogues are resistant to degradation by phosphodiesterases but remain sensitive to phosphatase activity, at least in cell-free extracts. In line with its increased stability, (2'-5')(A)n-PGro has a powerful antiviral activity against an RNA virus when microinjected with micropipettes into the cytoplasm of intact cells. This antiviral activity remains transient however, possibly as a consequence of degradation in intact cells. Since (2'-5')(A)n and its derivatives do not easily cross cell membranes, their possible use in antiviral chemotherapy is tightly linked with the development of vectors suitable for their administration in vivo.     

Mode of Action of the Antiviral Activity of Amantadine in Tissue Culture.

Hoffmann, C. E. (E. I. du Pont de Nemours & Co., Inc., Wilmington, Del.), E. M. Neumayer, R. F. Haff, and R. A. Goldsby. Mode of action of the antiviral activity of amantadine in tissue culture. J. Bacteriol. 90:623-628. 1965.-Amantadine hydrochloride has shown antiviral activity in tissue culture, in ovo, and in vivo. Experiments with it during the course of virus proliferation indicate that its antiviral activity is due to inhibition of virus penetration into the host cell. These studies indicate that amantadine hydrochloride is not virucidal at concentrations active in tissue culture. It does not block virus adsorption to host cells, nor does it affect the virus enzyme neuraminidase. In the presence of amantadine hydrochloride, virus adsorbed to susceptible cells remains at the cell surface in an infective state. An attempt to demonstrate high development of resistance to the antiviral action of amantadine hydrochloride in tissue culture has been unsuccessful.     

Human immunodeficiency virus type 1 Vif inhibits packaging and antiviral activity of a degradation-resistant APOBEC3G variant

Human immunodeficiency virus type 1 (HIV-1) Vif counteracts the antiviral activity of the human cytidine deaminase APOBEC3G (APO3G) by inhibiting its incorporation into virions. This has been attributed to the Vif-induced degradation of APO3G by cytoplasmic proteasomes. We recently demonstrated that although APO3G has a natural tendency to form RNA-dependent homo-multimers, multimerization was not essential for encapsidation into HIV-1 virions or antiviral activity. We now demonstrate that a multimerization-defective APO3G variant (APO3G C97A) is able to assemble into RNase-sensitive high-molecular-mass (HMM) complexes, suggesting that homo-multimerization of APO3G and assembly into HMM complexes are unrelated RNA-dependent processes. Interestingly, APO3G C97A was highly resistant to Vif-induced degradation even though the two proteins were found to interact in coimmunoprecipitation experiments and exhibited partial colocalization in transfected HeLa cells. Surprisingly, encapsidation and antiviral activity of APO3G C97A were both inhibited by Vif despite resistance to degradation. These results demonstrate that targeting of APO3G to proteasome degradation and interference with viral encapsidation are distinct functional properties of Vif.     

Interferon-induced transfer of viral resistance by human B and T lymphocytes

Enriched human B lymphocytes cocultivated with mouse L cells produced human leukocyte interferon (IFN-alpha) and shortly thereafter transferred antiviral activity to the recipient cells (99% inhibition of expected virus yield). In contrast, cocultivation of enriched T-cell populations with mouse L cells resulted in no IFN production or transfer of antiviral activity. In addition, both T and B lymphocytes pretreated with exogenous IFN or stimulated in vitro by mitogens could transfer antiviral activity to human WISH cells. The transfer of antiviral activity was not blocked by antibodies to IFN. The data indicate that both T and B cells can be recruited by IFN to transfer antiviral activity. Thus, once cells are recruited by IFN they can transfer antiviral activity in the absence of IFN and protect cells locally or distally from the site of infection.