Aloe vera and its Components Inhibit Influenza A Virus-Induced Autophagy and Replication

2019 ◽  
Vol 47 (06) ◽  
pp. 1307-1324 ◽  
Author(s):  
Jang-Gi Choi ◽  
Heeeun Lee ◽  
Young Soo Kim ◽  
Youn-Hwan Hwang ◽  
You-Chang Oh ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Matrix Protein ◽  
Mdck Cells ◽  
Aloe Vera ◽  
Post Treatment ◽  
M2 Protein ◽  
Mrna Synthesis ◽  
Catechin Hydrate

Aloe vera ethanol extract (AVE) reportedly has significant anti-influenza virus activity, but its underlying mechanisms of action and constituents have not yet been completely elucidated. Previously, we have confirmed that AVE treatment significantly reduces the viral replication of green fluorescent protein-labeled influenza A virus in Madin-Darby canine kidney (MDCK) cells. In addition, post-treatment with AVE inhibited viral matrix protein 1 (M1), matrix protein 2 (M2), and hemagglutinin (HA) mRNA synthesis and viral protein (M1, M2, and HA) expressions. In this study, we demonstrated that AVE inhibited autophagy induced by influenza A virus in MDCK cells and also identified quercetin, catechin hydrate, and kaempferol as the active antiviral components of AVE. We also found that post-treatment with quercetin, catechin hydrate, and kaempferol markedly inhibited M2 viral mRNA synthesis and M2 protein expression. A docking simulation suggested that the binding affinity of quercetin, catechin hydrate, and kaempferol for the M2 protein may be higher than that of known M2 protein inhibitors. Thus, the inhibition of autophagy induced by influenza virus may explain the antiviral activity of AVE against H1N1 or H3N2. Aloe vera extract and its constituents may, therefore, be potentially useful for the development of anti-influenza agents.

scholarly journals Deep Mutational Scan of the Highly Conserved Influenza A Virus M1 Matrix Protein Reveals Substantial Intrinsic Mutational Tolerance

2019 ◽  
Vol 93 (13) ◽  
Author(s):  
Nancy Hom ◽  
Lauren Gentles ◽  
Jesse D. Bloom ◽  
Kelly K. Lee
Keyword(s):  
Cell Culture ◽  
Influenza Virus ◽  
Amino Acid ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Influenza A ◽  
Matrix Protein ◽  
Influenza Virus Infection ◽  
Sequence Diversity ◽  
Virus Protein

ABSTRACTInfluenza A virus matrix protein M1 is involved in multiple stages of the viral infectious cycle. Despite its functional importance, our present understanding of this essential viral protein is limited. The roles of a small subset of specific amino acids have been reported, but a more comprehensive understanding of the relationship between M1 sequence, structure, and virus fitness remains elusive. In this study, we used deep mutational scanning to measure the effect of every amino acid substitution in M1 on viral replication in cell culture. The map of amino acid mutational tolerance we have generated allows us to identify sites that are functionally constrained in cell culture as well as sites that are less constrained. Several sites that exhibit low tolerance to mutation have been found to be critical for M1 function and production of viable virions. Surprisingly, significant portions of the M1 sequence, especially in the C-terminal domain, whose structure is undetermined, were found to be highly tolerant of amino acid variation, despite having extremely low levels of sequence diversity among natural influenza virus strains. This unexpected discrepancy indicates that not all sites in M1 that exhibit high sequence conservation in nature are under strong constraint during selection for viral replication in cell culture.IMPORTANCEThe M1 matrix protein is critical for many stages of the influenza virus infection cycle. Currently, we have an incomplete understanding of this highly conserved protein’s function and structure. Key regions of M1, particularly in the C terminus of the protein, remain poorly characterized. In this study, we used deep mutational scanning to determine the extent of M1’s tolerance to mutation. Surprisingly, nearly two-thirds of the M1 sequence exhibits a high tolerance for substitutions, contrary to the extremely low sequence diversity observed across naturally occurring M1 isolates. Sites with low mutational tolerance were also identified, suggesting that they likely play critical functional roles and are under selective pressure. These results reveal the intrinsic mutational tolerance throughout M1 and shape future inquiries probing the functions of this essential influenza A virus protein.

scholarly journals Extending the Cytoplasmic Tail of the Influenza A Virus M2 Protein Leads to Reduced Virus Replication In Vivo but Not In Vitro

2007 ◽  
Vol 82 (2) ◽  
pp. 1059-1063 ◽  
Author(s):  
Wai-Hong Wu ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Cytoplasmic Tail ◽  
M2 Protein ◽  
Coding Region ◽  
Epitope Tag ◽  
Carboxy Terminal

ABSTRACT A carboxy-terminal epitope tag introduced into the coding region of the A/WSN/33 M2 protein resulted in a recombinant virus (rWSN M2myc) which replicated to titers similar to those of the parental virus (rWSN) in MDCK cells. The rWSN M2myc virus was attenuated in its ability to induce mortality and weight loss after the intranasal inoculation of BALB/c mice, indicating that the M2 cytoplasmic tail plays a role in virus virulence. Mice infected with rWSN M2myc were completely protected from subsequent challenge with rWSN, suggesting that epitope tagging of the M2 protein may be a useful way of attenuating influenza A virus strains.

scholarly journals In Vitro System for Modeling Influenza A Virus Resistance under Drug Pressure

2010 ◽  
Vol 54 (8) ◽  
pp. 3442-3450 ◽  
Author(s):  
Ashley N. Brown ◽  
James J. McSharry ◽  
Qingmei Weng ◽  
Elizabeth M. Driebe ◽  
David M. Engelthaler ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Mdck Cells ◽  
Influenza Virus Infection ◽  
Infection Model ◽  
Virus Infections ◽  
Drug Pressure

ABSTRACT One of the biggest challenges in the effort to treat and contain influenza A virus infections is the emergence of resistance during treatment. It is well documented that resistance to amantadine arises rapidly during the course of treatment due to mutations in the gene coding for the M2 protein. To address this problem, it is critical to develop experimental systems that can accurately model the selection of resistance under drug pressure as seen in humans. We used the hollow-fiber infection model (HFIM) system to examine the effect of amantadine on the replication of influenza virus, A/Albany/1/98 (H3N2), grown in MDCK cells. At 24 and 48 h postinfection, virus replication was inhibited in a dose-dependent fashion. At 72 and 96 h postinfection, virus replication was no longer inhibited, suggesting the emergence of amantadine-resistant virus. Sequencing of the M2 gene revealed that mutations appeared at between 48 and 72 h of drug treatment and that the mutations were identical to those identified in the clinic for amantadine-resistant viruses (e.g., V27A, A30T, and S31N). Interestingly, we found that the type of mutation was strongly affected by the dose of the drug. The data suggest that the HFIM is a good model for influenza virus infection and resistance generation in humans. The HFIM has the advantage of being a highly controlled system where multiplicity parameters can be directly and accurately controlled and measured.

scholarly journals MicroRNA-Based Attenuation of Influenza Virus across Susceptible Hosts

2017 ◽  
Vol 92 (2) ◽  
Author(s):  
Barbara M. Waring ◽  
Louisa E. Sjaastad ◽  
Jessica K. Fiege ◽  
Elizabeth J. Fay ◽  
Ismarc Reyes ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Vaccine Safety ◽  
Human Populations ◽  
Significant Morbidity ◽  
Influenza A Viruses ◽  
Live Attenuated Vaccines ◽  
Zoonotic Infections

ABSTRACTInfluenza A virus drives significant morbidity and mortality in humans and livestock. Annual circulation of the virus in livestock and waterfowl contributes to severe economic disruption and increases the risk of zoonotic transmission of novel strains into the human population, where there is no preexisting immunity. Seasonal vaccinations in humans help prevent infection and can reduce symptoms when infection does occur. However, current vaccination regimens available for livestock are limited in part due to safety concerns regarding reassortment/recombination with circulating strains. Therefore, inactivated vaccines are used instead of the more immunostimulatory live attenuated vaccines. MicroRNAs (miRNAs) have been used previously to generate attenuated influenza A viruses for use as a vaccine. Here, we systematically targeted individual influenza gene mRNAs using the same miRNA to determine the segment(s) that yields maximal attenuation potential. This analysis demonstrated that targeting of NP mRNA most efficiently ablates replication. We further increased the plasticity of miRNA-mediated attenuation of influenza A virus by exploiting a miRNA, miR-21, that is ubiquitously expressed across influenza-susceptible hosts. In order to construct this targeted virus, we used CRISPR/Cas9 to eliminate the universally expressed miR-21 from MDCK cells. miR-21-targeted viruses were attenuated in human, mouse, canine, and avian cells and drove protective immunity in mice. This strategy has the potential to enhance the safety of live attenuated vaccines in humans and zoonotic reservoirs.IMPORTANCEInfluenza A virus circulates annually in both avian and human populations, causing significant morbidity, mortality, and economic burden. High incidence of zoonotic infections greatly increases the potential for transmission to humans, where no preexisting immunity or vaccine exists. There is a critical need for new vaccine strategies to combat emerging influenza outbreaks. MicroRNAs were used previously to attenuate influenza A viruses. We propose the development of a novel platform to produce live attenuated vaccines that are highly customizable, efficacious across a broad species range, and exhibit enhanced safety over traditional vaccination methods. This strategy exploits a microRNA that is expressed abundantly in influenza virus-susceptible hosts. By eliminating this ubiquitous microRNA from a cell line, targeted viruses that are attenuated across susceptible strains can be generated. This approach greatly increases the plasticity of the microRNA targeting approach and enhances vaccine safety.

scholarly journals Tyrosines in the Influenza A Virus M2 Protein Cytoplasmic Tail Are Critical for Production of Infectious Virus Particles

2010 ◽  
Vol 84 (17) ◽  
pp. 8765-8776 ◽  
Author(s):  
Michael L. Grantham ◽  
Shaun M. Stewart ◽  
Erin N. Lalime ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Infectious Virus ◽  
Mdck Cells ◽  
Cytoplasmic Tail ◽  
M2 Protein ◽  
Alanine Scanning Mutagenesis ◽  
Particle Assembly ◽  
Virus Particles

ABSTRACT The cytoplasmic tail of the influenza A virus M2 protein is required for the production of infectious virions. In this study, critical residues in the M2 cytoplasmic tail were identified by single-alanine scanning mutagenesis. The tyrosine residue at position 76, which is conserved in >99% of influenza virus strains sequenced to date, was identified as being critical for the formation of infectious virus particles using both reverse genetics and a protein trans-complementation assay. Recombinant viruses encoding M2 with the Y76A mutation demonstrated replication defects in MDCK cells as well as in primary differentiated airway epithelial cell cultures, defects in the formation of filamentous virus particles, and reduced packaging of nucleoprotein into virus particles. These defects could all be overcome by a mutation of serine to tyrosine at position 71 of the M2 cytoplasmic tail, which emerged after blind passage of viruses containing the Y76A mutation. These data confirm and extend our understanding of the significance of the M2 protein for infectious virus particle assembly.

scholarly journals Influenza Virus M2 Protein Ion Channel Activity Helps To Maintain Pandemic 2009 H1N1 Virus Hemagglutinin Fusion Competence during Transport to the Cell Surface

2014 ◽  
Vol 89 (4) ◽  
pp. 1975-1985 ◽  
Author(s):  
Esmeralda Alvarado-Facundo ◽  
Yamei Gao ◽  
Rosa María Ribas-Aparicio ◽  
Alicia Jiménez-Alberto ◽  
Carol D. Weiss ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Cell Surface ◽  
Avian Influenza ◽  
Influenza A Virus ◽  
Conformational Changes ◽  
Influenza A ◽  
Low Ph ◽  
Proton Channel ◽  
Channel Activity ◽  
M2 Protein

ABSTRACTThe influenza virus hemagglutinin (HA) envelope protein mediates virus entry by first binding to cell surface receptors and then fusing viral and endosomal membranes during endocytosis. Cleavage of the HA precursor (HA0) into a surface receptor-binding subunit (HA1) and a fusion-inducing transmembrane subunit (HA2) by host cell enzymes primes HA for fusion competence by repositioning the fusion peptide to the newly created N terminus of HA2. We previously reported that the influenza virus M2 protein enhances pandemic 2009 influenza A virus [(H1N1)pdm09] HA-pseudovirus infectivity, but the mechanism was unclear. In this study, using cell-cell fusion and HA-pseudovirus infectivity assays, we found that the ion channel function of M2 was required for enhancement of HA fusion and HA-pseudovirus infectivity. The M2 activity was needed only during HA biosynthesis, and proteolysis experiments indicated that M2 proton channel activity helped to protect (H1N1)pdm09 HA from premature conformational changes as it traversed low-pH compartments during transport to the cell surface. While M2 has previously been shown to protect avian influenza virus HA proteins of the H5 and H7 subtypes that have polybasic cleavage motifs, this study demonstrates that M2 can protect HA proteins from human H1N1 strains that lack a polybasic cleavage motif. This finding suggests that M2 proton channel activity may play a wider role in preserving HA fusion competence among a variety of HA subtypes, including HA proteins from emerging strains that may have reduced HA stability.IMPORTANCEInfluenza virus infects cells when the hemagglutinin (HA) surface protein undergoes irreversible pH-induced conformational changes after the virus is taken into the cell by endocytosis. HA fusion competence is primed when host cell enzymes cleave the HA precursor. The proton channel function of influenza virus M2 protein has previously been shown to protect avian influenza virus HA proteins that contain a polybasic cleavage site from pH-induced conformational changes during biosynthesis, but this effect is less well understood for human influenza virus HA proteins that lack polybasic cleavage sites. Using assays that focus on HA entry and fusion, we found that the M2 protein also protects (H1N1)pdm09 influenza A virus HA from premature conformational changes as it transits low-pH compartments during biosynthesis. This work suggests that M2 may play a wider role in preserving HA function in a variety of influenza virus subtypes that infect humans and may be especially important for HA proteins that are less stable.

scholarly journals Palmitoylation of the Influenza A Virus M2 Protein Is Not Required for Virus Replication In Vitro but Contributes to Virus Virulence

2009 ◽  
Vol 83 (17) ◽  
pp. 8655-8661 ◽  
Author(s):  
Michael L. Grantham ◽  
Wai-Hong Wu ◽  
Erin N. Lalime ◽  
Maria E. Lorenzo ◽  
Sabra L. Klein ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Mdck Cells ◽  
Cysteine Residue ◽  
M2 Protein ◽  
Recombinant Viruses ◽  
Virus Particles

ABSTRACT The influenza A virus M2 protein has important roles during virus entry and in the assembly of infectious virus particles. The cytoplasmic tail of the protein can be palmitoylated at a cysteine residue, but this residue is not conserved in a number of human influenza A virus isolates. Recombinant viruses encoding M2 proteins with a serine substituted for the cysteine at position 50 were generated in the A/WSN/33 (H1N1) and A/Udorn/72 (H3N2) genetic backgrounds. The recombinant viruses were not attenuated for replication in MDCK cells, Calu-3 cells, or in primary differentiated murine trachea epithelial cell cultures, indicating there was no significant contribution of M2 palmitoylation to virus replication in vitro. The A/WSN/33 M2C50S virus displayed a slightly reduced virulence after infection of mice, suggesting that there may be novel functions for M2 palmitoylation during in vivo infection.

scholarly journals Anti-Influenza Activity of an Ethyl Acetate Fraction of a Rhus verniciflua Ethanol Extract by Neuraminidase Inhibition

2020 ◽  
Vol 2020 ◽  
pp. 1-15
Author(s):  
Young Soo Kim ◽  
Wei Li ◽  
Ji Hye Kim ◽  
Hwan-Suck Chung ◽  
Jang-Gi Choi
Keyword(s):  
Influenza Virus ◽  
Ethyl Acetate ◽  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Influenza Virus Infection ◽  
Ethyl Acetate Fraction ◽  
Influenza Viruses ◽  
Neuraminidase Inhibition ◽  
Rhus Verniciflua

Antigenic mismatch can cause influenza vaccines to be ineffective, and influenza viruses resistant to antiviral drugs are rising. Thus, development of antiviral agents against these viruses is an immediate need. Rhus verniciflua (RVS) has long been used in herbal medicine and as a nutritional supplement. The effect of RVS and its components on influenza virus has not, however, been reported. We found that RVS treatment significantly reduced viral replication when evaluated with green fluorescent protein- (GFP-) tagged virus (influenza A virus, A/PR/8/34-GFP) in Madin–Darby canine kidney (MDCK) cells. RVS showed significant inhibition of neuraminidase from A/PR/8/34. Subsequently, three fractions were prepared from an ethanolic crude extract of RVS. In vitro assays indicated that an ethyl acetate fraction (RVSE) was more potent than H2O and CHCl3 fractions. RVSE significantly suppressed influenza virus infection in MDCK cells via neuraminidase inhibition. Additionally, RVSE treatment inhibited expression of several virus proteins and decreased mortality of mice exposed to influenza A/PR/8/34 by 50% and reduced weight loss by 11.5%. Active components in RVSE were isolated, and 5-deoxyluteolin (5) and sulfuretin (7) demonstrate the highest neuraminidase inhibitory activity against influenza A virus. RVS, RVSE, and their constituents may be useful for the development of anti-influenza agents.

scholarly journals Triple Combination of Oseltamivir, Amantadine, and Ribavirin Displays Synergistic Activity against Multiple Influenza Virus Strains In Vitro

2009 ◽  
Vol 53 (10) ◽  
pp. 4115-4126 ◽  
Author(s):  
Jack T. Nguyen ◽  
Justin D. Hoopes ◽  
Donald F. Smee ◽  
Mark N. Prichard ◽  
Elizabeth M. Driebe ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
New Caledonia ◽  
Mdck Cells ◽  
The United States ◽  
Health Concern ◽  
Synergistic Activity ◽  
Triple Combination ◽  
Virus Strains

ABSTRACT The recurring emergence of influenza virus strains that are resistant to available antiviral medications has become a global health concern, especially in light of the potential for a new influenza virus pandemic. Currently, virtually all circulating strains of influenza A virus in the United States are resistant to either of the two major classes of anti-influenza drugs (adamantanes and neuraminidase inhibitors). Thus, new therapeutic approaches that can be rapidly deployed and that will address the issue of recurring resistance should be developed. We have tested double and triple combinations of the approved anti-influenza drugs oseltamivir and amantadine together with ribavirin against three influenza virus strains using cytopathic effect inhibition assays in MDCK cells. We selected A/New Caledonia/20/99 (H1N1) and A/Sydney/05/97 (H3N2) as representatives of the wild-type versions of the predominant circulating seasonal influenza virus strains and A/Duck/MN/1525/81 (H5N1) as a representative of avian influenza virus strains. Dose-response curves were generated for all drug combinations, and the degree of drug interaction was quantified using a model that calculates the synergy (or antagonism) between the drugs in double and triple combinations. This report demonstrates that a triple combination of antivirals was highly synergistic against influenza A virus. Importantly, the synergy of the triple combination was 2- to 13-fold greater than the synergy of any double combination depending on the influenza virus subtype. These data support the investigation of a novel combination of oseltamivir, amantadine, and ribavirin as an effective treatment for both seasonal and pandemic influenza virus, allowing the efficient use of the existing drug supplies.

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