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1.
Molecules ; 27(16)2022 Aug 12.
Article in English | MEDLINE | ID: mdl-36014378

ABSTRACT

Multidrug resistance (MDR), having a multifactorial nature, is one of the major clinical problems causing the failure of anticancer therapy. The aim of this study was to examine the antitumour effects of selected pyridinium salts, 1-methyl-3-nitropyridine chloride (MNP) and 3,3,6,6,10-pentamethyl-3,4,6,7-tetrahydro-[1,8(2H,5H)-dion]acridine chloride (MDION), on sensitive leukaemia HL60 cells and resistant topoisomerase II-defective HL60/MX2 cells. Cell growth was determined by the MTT test. Intracellular ROS level was measured with the aid of 2',7'-DCF-DA. The cell cycle distribution was investigated by performing PI staining. DSB formation was examined using the γ-H2AX histone phosphorylation assay. The activity of caspase-3 and caspase-8 was measured with the use of the FLICA test. The assays for examining the lysosome membrane permeabilization were carried out with the aid of LysoTracker Green DND-26. Both studied compounds exerted very similar cytotoxic activities towards sensitive HL60 cells and their MDR counterparts. They modulated the cellular ROS level in a dose-dependent and time-dependent manner and significantly increased the percentage of sensitive HL60 and resistant HL60/MX2 cells with sub-diploid DNA (sub-G1 fraction). However, the induction of DSB formation was not a significant mechanism of action of these pyridinium salts in studied cells. Both examined compounds triggered caspase-3/caspase-8-dependent apoptosis of sensitive HL60 cells and their MDR counterparts. Additionally, the findings of the study indicate that lysosomes may also participate in the programmed death of HL60 as well as HL60/MX2 cells induced by MDION. The data obtained in this work showed that both examined pyridinium salts, MNP and MDION, are able to retain high antileukaemic effects against multidrug resistant topoisomerase II-defective HL60/MX2 cells.


Subject(s)
Antineoplastic Agents/pharmacology , DNA Topoisomerases, Type II , Leukemia , Apoptosis , Caspase 3/metabolism , Caspase 8/metabolism , Chlorides/pharmacology , DNA Topoisomerases, Type II/metabolism , Drug Resistance, Neoplasm , HL-60 Cells , Humans , Myxovirus Resistance Proteins/metabolism , Myxovirus Resistance Proteins/pharmacology , Reactive Oxygen Species/metabolism , Salts/metabolism , Salts/pharmacology
2.
J Virol ; 94(22)2020 10 27.
Article in English | MEDLINE | ID: mdl-32907985

ABSTRACT

Mx proteins are interferon (IFN) type I (α/ß)- and type III (λ)-induced effector proteins with intrinsic antiviral activity. Mammalian Mx proteins show different subcellular localizations and distinct yet partially overlapping viral specificities. However, the precise mechanism(s) of antiviral action are still unresolved. Human MxA accumulates in the cytoplasm and inhibits a wide variety of RNA and DNA viruses, among them influenza A virus (IAV). In contrast, MxB, the second human Mx protein, localizes via its amino (N) terminus to the outer nuclear membrane at or near nuclear pores and inhibits the nuclear import of incoming human immunodeficiency viruses (HIV) and herpesviruses, but not that of IAV. Here, we evaluated whether the antiviral specificity of MxB is determined by its subcellular localization. For this purpose, we redirected MxB to the nucleus or cytoplasm by either attaching a nuclear localization signal to its N terminus or by exchanging the N terminus of MxB with that of MxA. Interestingly, ectopic expression of these MxB variants in the nucleus or in the cytoplasm rendered the host cells resistant to IAV, revealing that the capacity of MxB to block IAV replication critically depends on the site where the protein accumulates in the infected cell. Furthermore, coimmunoprecipitation (co-IP) assays demonstrated that MxB physically interacted with the nucleoprotein (NP) of IAV. Taken together, the data indicate that the subcellular localization of the MxB protein plays a pivotal role in determining its antiviral specificity.IMPORTANCE The interferon system plays a pivotal role in the defense against viral infections. The dynamin-related Mx proteins form a small family of interferon-induced effector proteins with distinct antiviral specificities and subcellular localizations. So far, it is not clear whether the different virus specificities of Mx proteins are the result of distinct mechanisms of action or are due rather to their different subcellular localization. We show here that the human MxB protein, normally localized to the outer membrane of the cell nucleus, acquires antiviral activity against IAV when redirected to the nucleus or cytoplasm, subcellular sites where other members of the Mx protein family efficiently interfere with IAV replication. Our findings thus strongly suggest that Mx proteins act primarily through a common mechanism and that their viral specificity is at least in part determined by their individual subcellular localization.


Subject(s)
Antiviral Agents/metabolism , Influenza A virus/drug effects , Myxovirus Resistance Proteins/metabolism , Active Transport, Cell Nucleus , Antiviral Agents/pharmacology , Cell Line , Cell Nucleus/metabolism , Cytoplasm/metabolism , HEK293 Cells , HeLa Cells , Humans , Influenza A virus/metabolism , Interferons/metabolism , Myxovirus Resistance Proteins/genetics , Myxovirus Resistance Proteins/pharmacology , Nucleoproteins , Virus Replication/drug effects
3.
Viruses ; 10(7)2018 07 06.
Article in English | MEDLINE | ID: mdl-29986463

ABSTRACT

The Tembusu virus (TMUV) is an avian pathogenic flavivirus that causes a highly contagious disease and catastrophic losses to the poultry industry. The myxovirus resistance protein (Mx) of innate immune effectors is a key antiviral “workhorse" of the interferon (IFN) system. Although mammalian Mx resistance against myxovirus and retrovirus was witnessed for decades, whether or not bird Mx has anti-flavivirus activity remains unknown. In this study, we found that the transcription of goose Mx (goMx) was obviously driven by TMUV infection, both in vivo and in vitro, and that the titers and copies of TMUV were significantly reduced by goMx overexpression. In both primary (goose embryo fibroblasts, GEFs) and passaged cells (baby hamster kidney cells, BHK21, and human fetal kidney cells, HEK 293T), it was shown that goMx was mainly located in the cytoplasm, and sporadically distributed in the nucleus. The intracellular localization of this protein is attributed to the predicted bipartite nuclear localization signal (NLS; 30 residues: the 441st­471st amino acids of goMx). Intuitively, it seems that the cells with a higher level of goMx expression tend to have lower TMUV loads in the cytoplasm, as determined by an immunofluorescence assay. To further explore the antiviral determinants, a panel of variants was constructed. Two amino acids at the 125th (Lys) and 145th (Thr) positions in GTP-binding elements, not in the L4 loop (40 residues: the 532nd­572nd amino acids of goMx), were vital for the antiviral function of goMx against TMUV in vitro. These findings will contribute to our understanding of the functional significance of the antiviral system in aquatic birds, and the development of goMx could be a valuable therapeutic agent against TMUV.


Subject(s)
GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/metabolism , Lysine , Myxovirus Resistance Proteins/chemistry , Myxovirus Resistance Proteins/metabolism , Amino Acid Sequence , Animals , Cell Line , Cell Survival , Fibroblasts/metabolism , Flavivirus/drug effects , Flavivirus/physiology , Geese , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Intracellular Space/metabolism , Lysine/chemistry , Myxovirus Resistance Proteins/genetics , Myxovirus Resistance Proteins/pharmacology , Protein Transport , Virus Replication
4.
Viruses ; 9(1)2017 01 10.
Article in English | MEDLINE | ID: mdl-28075421

ABSTRACT

Mx proteins are interferon (IFN)-induced dynamin-like GTPases that are present in all vertebrates and inhibit the replication of myriad viruses. However, the role Mx proteins play in IFN-mediated suppression of Japanese encephalitis virus (JEV) infection is unknown. In this study, we set out to investigate the effects of Mx1 and Mx2 expression on the interferon-α (IFNα) restriction of JEV replication. To evaluate whether the inhibitory activity of IFNα on JEV is dependent on Mx1 or Mx2, we knocked down Mx1 or Mx2 with siRNA in IFNα-treated PK-15 cells and BHK-21 cells, then challenged them with JEV; the production of progeny virus was assessed by plaque assay, RT-qPCR, and Western blotting. Our results demonstrated that depletion of Mx1 or Mx2 did not affect JEV restriction imposed by IFNα, although these two proteins were knocked down 66% and 79%, respectively. Accordingly, expression of exogenous Mx1 or Mx2 did not change the inhibitory activity of IFNα to JEV. In addition, even though virus-induced membranes were damaged by Brefeldin A (BFA), overexpressing porcine Mx1 or Mx2 did not inhibit JEV proliferation. We found that BFA inhibited JEV replication, not maturation, suggesting that BFA could be developed into a novel antiviral reagent. Collectively, our findings demonstrate that IFNα inhibits JEV infection by Mx-independent pathways.


Subject(s)
Antiviral Agents/pharmacology , Encephalitis Virus, Japanese/immunology , Immunologic Factors/pharmacology , Interferon-alpha/pharmacology , Myxovirus Resistance Proteins/pharmacology , Animals , Blotting, Western , Cell Line , Cricetinae , Encephalitis Virus, Japanese/physiology , Swine , Viral Load , Viral Plaque Assay , Virus Replication
5.
EBioMedicine ; 8: 230-236, 2016 Jun.
Article in English | MEDLINE | ID: mdl-27428433

ABSTRACT

Recent studies have identified human myxovirus resistance protein 2 (MxB or Mx2) as an interferon induced inhibitor of HIV-1 replication. However, whether HIV-1 can overcome MxB restriction without compromise of viral fitness has been undefined. Here, we have discovered that naturally occurring capsid (CA) variants can render HIV-1 resistant to the activity of MxB without losing viral infectivity or the ability to escape from interferon induction. Moreover, these MxB resistant HIV-1 variants do not lose MxB recognition. Surprisingly, MxB resistant CA variants are most commonly found in the Clade C HIV-1 that is the most rapidly expanding Clade throughout the world. Accumulation of MxB resistant mutations is also observed during HIV-1 spreading in human populations. These findings support a potential role for MxB as a selective force during HIV-1 transmission and evolution.


Subject(s)
Anti-HIV Agents/pharmacology , Capsid Proteins/genetics , Drug Resistance, Viral , HIV Infections/epidemiology , HIV Infections/virology , HIV-1/drug effects , HIV-1/genetics , Myxovirus Resistance Proteins/pharmacology , Capsid Proteins/chemistry , China/epidemiology , Genetic Fitness , Genetic Variation , Geography , Humans , Models, Molecular , Population Surveillance , Protein Conformation , Virus Replication
6.
J Virol ; 90(14): 6598-6610, 2016 07 15.
Article in English | MEDLINE | ID: mdl-27170750

ABSTRACT

UNLABELLED: Multiple cellular pathways are regulated by small ubiquitin-like modifier (SUMO) modification, including ubiquitin-mediated proteolysis, signal transduction, innate immunity, and antiviral defense. In the study described in this report, we investigated the effects of SUMO on the replication of two members of the Rhabdoviridae family, vesicular stomatitis virus (VSV) and rabies virus (RABV). We show that stable expression of SUMO in human cells confers resistance to VSV infection in an interferon-independent manner. We demonstrate that SUMO expression did not alter VSV entry but blocked primary mRNA synthesis, leading to a reduction of viral protein synthesis and viral production, thus protecting cells from VSV-induced cell lysis. MxA is known to inhibit VSV primary transcription. Interestingly, we found that the MxA protein was highly stabilized in SUMO-expressing cells. Furthermore, extracts from cells stably expressing SUMO exhibited an increase in MxA oligomers, suggesting that SUMO plays a role in protecting MxA from degradation, thus providing a stable intracellular pool of MxA available to combat invading viruses. Importantly, MxA depletion in SUMO-expressing cells abrogated the anti-VSV effect of SUMO. Furthermore, SUMO expression resulted in interferon-regulatory factor 3 (IRF3) SUMOylation, subsequently decreasing RABV-induced IRF3 phosphorylation and interferon synthesis. As expected, this rendered SUMO-expressing cells more sensitive to RABV infection, even though MxA was stabilized in SUMO-expressing cells, since its expression did not confer resistance to RABV. Our findings demonstrate opposing effects of SUMO expression on two viruses of the same family, intrinsically inhibiting VSV infection through MxA stabilization while enhancing RABV infection by decreasing IFN induction. IMPORTANCE: We report that SUMO expression reduces interferon synthesis upon RABV or VSV infection. Therefore, SUMO renders cells more sensitive to RABV but unexpectedly renders cells resistant to VSV by blocking primary mRNA synthesis. Unlike the interferon-mediated innate immune response, intrinsic antiviral resistance is mediated by constitutively expressed restriction factors. Among the various anti-VSV restriction factors, only MxA is known to inhibit VSV primary transcription, and we show here that its expression does not alter RABV infection. Interestingly, MxA depletion abolished the inhibition of VSV by SUMO, demonstrating that MxA mediates SUMO-induced intrinsic VSV resistance. Furthermore, MxA oligomerization is known to be critical for its protein stability, and we show that higher levels of oligomers were formed in cells expressing SUMO than in wild-type cells, suggesting that SUMO may play a role in protecting MxA from degradation, providing a stable intracellular pool of MxA able to protect cells from viral infection.


Subject(s)
Interferon-alpha/pharmacology , Myxovirus Resistance Proteins/pharmacology , Small Ubiquitin-Related Modifier Proteins/pharmacology , Vesicular Stomatitis/prevention & control , Vesicular stomatitis Indiana virus/physiology , Antiviral Agents/pharmacology , Glioblastoma/metabolism , Glioblastoma/pathology , Glioblastoma/virology , HeLa Cells , Humans , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Protein Processing, Post-Translational , Rabies/metabolism , Rabies/prevention & control , Rabies/virology , Rabies virus/physiology , Tumor Cells, Cultured , Vesicular Stomatitis/metabolism , Vesicular Stomatitis/virology
7.
J Virol ; 89(21): 10879-90, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26292322

ABSTRACT

UNLABELLED: The interferon-induced Mx1 gene is an important part of the mammalian defense against influenza viruses. Mus musculus Mx1 inhibits influenza A virus replication and transcription by suppressing the polymerase activity of viral ribonucleoproteins (vRNPs). Here, we compared the anti-influenza virus activity of Mx1 from Mus musculus A2G with that of its ortholog from Mus spretus. We found that the antiviral activity of M. spretus Mx1 was less potent than that of M. musculus Mx1. Comparison of the M. musculus Mx1 sequence with the M. spretus Mx1 sequence revealed 25 amino acid differences, over half of which were present in the GTPase domain and 2 of which were present in loop L4. However, the in vitro GTPase activity of Mx1 from the two mouse species was similar. Replacement of one of the residues in loop L4 in M. spretus Mx1 by the corresponding residue of A2G Mx1 increased its antiviral activity. We also show that deletion of loop L4 prevented the binding of Mx1 to influenza A virus nucleoprotein and, hence, abolished the antiviral activity of mouse Mx1. These results indicate that loop L4 of mouse Mx1 is a determinant of antiviral activity. Our findings suggest that Mx proteins from different mammals use a common mechanism to inhibit influenza A viruses. IMPORTANCE: Mx proteins are evolutionarily conserved in vertebrates and inhibit a wide range of viruses. Still, the exact details of their antiviral mechanisms remain largely unknown. Functional comparison of the Mx genes from two species that diverged relatively recently in evolution can provide novel insights into these mechanisms. We show that both Mus musculus A2G Mx1 and Mus spretus Mx1 target the influenza virus nucleoprotein. We also found that loop L4 in mouse Mx1 is crucial for its antiviral activity, as was recently reported for primate MxA. This indicates that human and mouse Mx proteins, which have diverged by 75 million years of evolution, recognize and inhibit influenza A viruses by a common mechanism.


Subject(s)
Antiviral Agents/immunology , Influenza A virus/drug effects , Myxovirus Resistance Proteins/genetics , Myxovirus Resistance Proteins/immunology , Amino Acid Sequence , Animals , Antiviral Agents/pharmacology , Base Sequence , Flow Cytometry , Genetic Vectors/genetics , HEK293 Cells , Humans , Immunoprecipitation , Mice , Microscopy, Fluorescence , Molecular Sequence Data , Myxovirus Resistance Proteins/pharmacology , Nucleoproteins/metabolism , Protein Binding , Protein Conformation , Regression Analysis , Sequence Analysis, DNA , Species Specificity
8.
Trends Microbiol ; 23(3): 154-63, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25572883

ABSTRACT

The Mx dynamin-like GTPases are key antiviral effector proteins of the type I and type III interferon (IFN) systems. They inhibit several different viruses by blocking early steps of the viral replication cycle. We focus on new structural and functional insights and discuss recent data revealing that human MxA (MX1) provides a safeguard against introduction of avian influenza A viruses (FLUAV) into the human population. The related human MxB (MX2) serves as restriction factor for HIV-1 and other primate lentiviruses.


Subject(s)
Immunity, Innate , Influenza A virus/immunology , Myxovirus Resistance Proteins/chemistry , Myxovirus Resistance Proteins/physiology , Orthomyxoviridae/physiology , Virus Physiological Phenomena , Amino Acid Sequence , Animals , Humans , Models, Molecular , Myxovirus Resistance Proteins/pharmacology , Orthomyxoviridae/growth & development , Orthomyxoviridae/immunology , Phylogeny , Virus Replication , Viruses/immunology
9.
Antiviral Res ; 104: 128-35, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24500530

ABSTRACT

Classical swine fever virus (CSFV) is the causative pathogen of classical swine fever (CSF), a highly contagious disease of swine. Mx proteins are interferon-induced dynamin-like GTPases present in all vertebrates with a wide range of antiviral activities. Although Zhao et al. (2011) have reported that human MxA can inhibit CSFV replication, whether porcine Mx1 (poMx1) has anti-CSFV activity remains unknown. In this study, we generated a cell line designated PK-15/EGFP-poMx1 which expressed porcine Mx1 protein constitutively, and we observed that the proliferation of progeny virus in this cell line was significantly inhibited as measured by virus titration, indirect immune fluorescence assay, Q-PCR and Western blot. Furthermore, when PTD-poMx1 fusion protein expressed in Escherichia coli (Zhang et al., 2013) was used to treat CSFV-infected PK-15 cells, the results showed that PTD-poMx1 inhibited CSFV replication in a dose-dependent manner. Additionally, the proliferation of progeny virus was inhibited as measured by virus titration and Q-PCR. Overall, the results demonstrated that poMx1 effectively inhibited CSFV replication, suggesting that poMx1 may be a valuable therapeutic agent against CSFV infection.


Subject(s)
Classical Swine Fever Virus/physiology , Myxovirus Resistance Proteins/metabolism , Virus Replication , Animals , Antiviral Agents/pharmacology , Cell Line , Classical Swine Fever Virus/drug effects , Gene Expression Regulation, Viral/drug effects , Myxovirus Resistance Proteins/pharmacology , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Swine , Virus Replication/drug effects
10.
Immunogenetics ; 66(1): 25-32, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24232602

ABSTRACT

The objective was to determine if single nucleotide polymorphisms (SNPs) in porcine MX2 gene affect its antiviral potential. MX proteins are known to suppress the multiplication of several viruses, including influenza virus and vesicular stomatitis virus (VSV). In domestic animals possessing highly polymorphic genome, our previous research indicated that a specific SNP in chicken Mx gene was responsible for its antiviral function. However, there still has been no information about SNPs in porcine MX2 gene. In this study, we first conducted polymorphism analysis in 17 pigs of MX2 gene derived from seven breeds. Consequently, a total of 30 SNPs, of which 11 were deduced to cause amino acid variations, were detected, suggesting that the porcine MX2 is very polymorphic. Next, we classified MX2 into eight alleles (A1-A8) and subsequently carried out infectious experiments with recombinant VSVΔG*-G to each allele. In A1-A5 and A8, position 514 amino acid (514 aa) of MX2 was glycine (Gly), which did not inhibit VSV multiplication, whereas in A6 and A7, 514 aa was arginine (Arg), which exhibited the antiviral ability against VSV. These results demonstrate that a SNP at 514 aa (Gly-Arg) of porcine MX2 plays a pivotal role in the antiviral activity as well as that at 631 aa of chicken Mx.


Subject(s)
Antiviral Agents/pharmacology , Myxovirus Resistance Proteins/genetics , Polymorphism, Single Nucleotide/genetics , Vesicular Stomatitis/prevention & control , Vesicular stomatitis Indiana virus/immunology , Animals , BALB 3T3 Cells , Cloning, Molecular , Mice , Myxovirus Resistance Proteins/immunology , Myxovirus Resistance Proteins/pharmacology , Polymorphism, Restriction Fragment Length , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Swine , Vesicular Stomatitis/immunology , Vesicular Stomatitis/virology , Vesicular stomatitis Indiana virus/genetics
11.
Antiviral Res ; 99(2): 149-57, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23727591

ABSTRACT

Vesicular stomatitis virus (VSV) is the causative agent of Vesicular stomatitis (VS), a highly contagious fatal disease of human and pigs. Few effective antiviral drugs are currently available against VSV infection. Mx proteins are interferon (IFN)-induced dynamin-like GTPases present in all vertebrates with a range of antiviral activities. Previous studies have shown that the transfected cell lines expressing either porcine Mx1 or human MxA acquired a high degree of resistance to VSV. To explore the feasibility of taking porcine Mx1 protein expressed in Escherichia coli as an antiviral agent, we applied the pCold system to express this fusion protein (PTD-poMx1), which consisted of an N-terminal HIV-1 Tat protein transduction domain (PTD) and the full-length porcine Mx1, and investigated its effects on the replication of VSV in Vero cells. The results demonstrated that the purified PTD-poMx1 fusion proteins could transduct into cells after incubated for 5h and had no cytotoxic. Furthermore, plaque reduction assay, determination of TCID50, real-time PCR and Western blot analyses were carried out to confirm the antiviral activity of purified fusion proteins in VSV-infected Vero cells. Altogether, these data suggested that PTD-poMx1 fusion proteins might be applicable to inhibit VSV replication as a novel antiviral therapeutic agent.


Subject(s)
Antiviral Agents/pharmacology , Myxovirus Resistance Proteins/pharmacology , Vesicular stomatitis Indiana virus/drug effects , Virus Replication/drug effects , tat Gene Products, Human Immunodeficiency Virus/pharmacology , Animals , Cell Line , Chlorocebus aethiops , Myxovirus Resistance Proteins/genetics , Recombinant Fusion Proteins/pharmacology , Vero Cells , Vesicular stomatitis Indiana virus/physiology , tat Gene Products, Human Immunodeficiency Virus/genetics
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