In vitro reconstitution of active influenza virus ribonucleoprotein complexes using viral proteins purified from infected cells

Polyphenols have been widely studied for their antiviral effect against respiratory virus infections. Among these, resveratrol (RV) has been demonstrated to inhibit influenza virus replication and more recently, it has been tested together with pterostilbene against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In the present work, we evaluated the antiviral activity of polydatin, an RV precursor, and a mixture of polyphenols and other micronutrients, named A5+, against influenza virus and SARS-CoV-2 infections. To this end, we infected Vero E6 cells and analyzed the replication of both respiratory viruses in terms of viral proteins synthesis and viral titration. We demonstrated that A5+ showed a higher efficacy in inhibiting both influenza virus and SARS-CoV-2 infections compared to polydatin treatment alone. Indeed, post infection treatment significantly decreased viral proteins expression and viral release, probably by interfering with any step of virus replicative cycle. Intriguingly, A5+ treatment strongly reduced IL-6 cytokine production in influenza virus-infected cells, suggesting its potential anti-inflammatory properties during the infection. Overall, these results demonstrate the synergic and innovative antiviral efficacy of A5+ mixture, although further studies are needed to clarify the mechanisms underlying its inhibitory effect.

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Attenuation of Influenza A Virus mRNA Levels by Promoter Mutations

Journal of Virology ◽

10.1128/jvi.72.8.6283-6290.1998 ◽

1998 ◽

Vol 72 (8) ◽

pp. 6283-6290 ◽

Cited By ~ 39

Author(s):

Ervin Fodor ◽

Peter Palese ◽

George G. Brownlee ◽

Adolfo García-Sastre

Keyword(s):

Influenza Virus ◽

Transcription Initiation ◽

Influenza A ◽

Reverse Genetics ◽

Mrna Levels ◽

Double Mutation ◽

Ribonucleoprotein Complexes ◽

Virus Rna ◽

Infected Cells

ABSTRACT We have engineered influenza A/WSN/33 viruses which have viral RNA (vRNA) segments with altered base pairs in the conserved double-stranded region of their vRNA promoters. The mutations were introduced into the segment coding for the neuraminidase (NA) by using a reverse genetics system. Two of the rescued viruses which share a C-G→A-U double mutation at positions 11 and 12′ at the 3′ and 5′ ends of the NA-specific vRNA, respectively, showed approximately a 10-fold reduction of NA levels. The mutations did not dramatically affect the NA-specific vRNA levels found in virions or the NA-specific vRNA and cRNA levels in infected cells. In contrast, there was a significant decrease in the steady-state levels of NA-specific mRNAs in infected cells. Transcription studies in vitro with ribonucleoprotein complexes isolated from the two transfectant viruses indicated that transcription initiation of the NA-specific segment was not affected. However, the majority of NA-specific transcripts lacked poly(A) tails, suggesting that mutations in the double-stranded region of the influenza virus vRNA promoter can attenuate polyadenylation of mRNA molecules. This is the first time that a promoter mutation in an engineered influenza virus has shown a differential effect on influenza virus RNA transcription and replication.

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Identification of a Novel Viral Protein Expressed from the PB2 Segment of Influenza A Virus

Journal of Virology ◽

10.1128/jvi.02175-15 ◽

2015 ◽

Vol 90 (1) ◽

pp. 444-456 ◽

Cited By ~ 41

Author(s):

Seiya Yamayoshi ◽

Mariko Watanabe ◽

Hideo Goto ◽

Yoshihiro Kawaoka

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Viral Protein ◽

Influenza A ◽

Viral Infections ◽

Viral Proteins ◽

Wild Type Virus ◽

Infected Cells

ABSTRACTOver the past 2 decades, several novel influenza virus proteins have been identified that modulate viral infectionsin vitroand/orin vivo. The PB2 segment, which is one of the longest influenza A virus segments, is known to encode only one viral protein, PB2. In the present study, we used reverse transcription-PCR (RT-PCR) targeting viral mRNAs transcribed from the PB2 segment to look for novel viral proteins encoded by spliced mRNAs. We identified a new viral protein, PB2-S1, encoded by a novel spliced mRNA in which the region corresponding to nucleotides 1513 to 1894 of the PB2 mRNA is deleted. PB2-S1 was detected in virus-infected cells and in cells transfected with a protein expression plasmid encoding PB2. PB2-S1 localized to mitochondria, inhibited the RIG-I-dependent interferon signaling pathway, and interfered with viral polymerase activity (dependent on its PB1-binding capability). The nucleotide sequences around the splicing donor and acceptor sites for PB2-S1 were highly conserved among pre-2009 human H1N1 viruses but not among human H1N1pdm and H3N2 viruses. PB2-S1-deficient viruses, however, showed growth kinetics in MDCK cells and virulence in mice similar to those of wild-type virus. The biological significance of PB2-S1 to the replication and pathogenicity of seasonal H1N1 influenza A viruses warrants further investigation.IMPORTANCETranscriptome analysis of cells infected with influenza A virus has improved our understanding of the host response to viral infection, because such analysis yields considerable information about bothin vitroandin vivoviral infections. However, little attention has been paid to transcriptomes derived from the viral genome. Here we focused on the splicing of mRNA expressed from the PB2 segment and identified a spliced viral mRNA encoding a novel viral protein. This result suggests that other, as yet unidentified viral proteins encoded by spliced mRNAs could be expressed in virus-infected cells. A viral transcriptome including the viral spliceosome should be evaluated to gain new insights into influenza virus infection.

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Modulation of Nuclear Localization of the Influenza Virus Nucleoprotein through Interaction with Actin Filaments

Journal of Virology ◽

10.1128/jvi.73.3.2222-2231.1999 ◽

1999 ◽

Vol 73 (3) ◽

pp. 2222-2231 ◽

Cited By ~ 82

Author(s):

Paul Digard ◽

Debra Elton ◽

Konrad Bishop ◽

Elizabeth Medcalf ◽

Alan Weeds ◽

...

Keyword(s):

Influenza Virus ◽

Nuclear Localization ◽

Virus Genome ◽

Actin Binding ◽

Filamentous Actin ◽

Nuclear Localization Signals ◽

Infected Cells ◽

Cytoplasmic Accumulation ◽

High Level

ABSTRACT The influenza virus genome is transcribed in the nuclei of infected cells but assembled into progeny virions in the cytoplasm. This is reflected in the cellular distribution of the virus nucleoprotein (NP), a protein which encapsidates genomic RNA to form ribonucleoprotein structures. At early times postinfection NP is found in the nucleus, but at later times it is found predominantly in the cytoplasm. NP contains several sequences proposed to act as nuclear localization signals (NLSs), and it is not clear how these are overridden to allow cytoplasmic accumulation of the protein. We find that NP binds tightly to filamentous actin in vitro and have identified a cluster of residues in NP essential for the interaction. Complexes containing RNA, NP, and actin could be formed, suggesting that viral ribonucleoproteins also bind actin. In cells, exogenously expressed NP when expressed at a high level partitioned to the cytoplasm, where it associated with F-actin stress fibers. In contrast, mutants unable to bind F-actin efficiently were imported into the nucleus even under conditions of high-level expression. Similarly, nuclear import of NLS-deficient NP molecules was restored by concomitant disruption of F-actin binding. We propose that the interaction of NP with F-actin causes the cytoplasmic retention of influenza virus ribonucleoproteins.

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Role of Initiating Nucleoside Triphosphate Concentrations in the Regulation of Influenza Virus Replication and Transcription

Journal of Virology ◽

10.1128/jvi.00627-08 ◽

2008 ◽

Vol 82 (14) ◽

pp. 6902-6910 ◽

Cited By ~ 27

Author(s):

Frank T. Vreede ◽

Hugh Gifford ◽

George G. Brownlee

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Influenza A ◽

De Novo ◽

Nucleoside Triphosphate ◽

Ribonucleoprotein Complexes ◽

Influenza Virus Replication ◽

High Concentrations

ABSTRACT The mechanisms regulating the synthesis of mRNA, cRNA, and viral genomic RNA (vRNA) by the influenza A virus RNA-dependent RNA polymerase are not fully understood. Previous studies in our laboratory have shown that virion-derived viral ribonucleoprotein complexes synthesize both mRNA and cRNA in vitro and early in the infection cycle in vivo. Our continued studies showed that de novo synthesis of cRNA in vitro is more sensitive to the concentrations of ATP, CTP, and GTP than capped-primer-dependent synthesis of mRNA. Using rescued recombinant influenza A/WSN/33 viruses, we now demonstrate that the 3′-terminal sequence of the vRNA promoter dictates the requirement for a high nucleoside triphosphate (NTP) concentration during de novo-initiated replication to cRNA, whereas this is not the case for the extension of capped primers during transcription to mRNA. In contrast to some other viral polymerases, for which only the initiating NTP is required at high concentrations, influenza virus polymerase requires high concentrations of the first three NTPs. In addition, we show that base pair mutations in the vRNA promoter can lead to nontemplated dead-end mutations during replication to cRNA in vivo. Based on our observations, we propose a new model for the de novo initiation of influenza virus replication.

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The aminobisphosphonate pamidronate controls influenza pathogenesis by expanding a γδ T cell population in humanized mice

Journal of Experimental Medicine ◽

10.1084/jem.20110226 ◽

2011 ◽

Vol 208 (7) ◽

pp. 1511-1522 ◽

Cited By ~ 78

Author(s):

Wenwei Tu ◽

Jian Zheng ◽

Yinping Liu ◽

Sin Fun Sia ◽

Ming Liu ◽

...

Keyword(s):

Influenza Virus ◽

T Cell ◽

Viral Replication ◽

Mononuclear Cells ◽

Immunodeficient Mice ◽

H5n1 Influenza ◽

Infected Cells ◽

Peripheral Mononuclear Cells ◽

Vγ9vδ2 T Cells

There are few antiviral drugs for treating influenza, and the emergence of antiviral resistance has further limited the available therapeutic options. Furthermore, antivirals are not invariably effective in severe influenza, such as that caused by H5N1 viruses. Thus, there is an urgent need to develop alternative therapeutic strategies. Here, we show that human Vγ9Vδ2 T cells expanded by the aminobisphosphonate pamidronate (PAM) kill influenza virus–infected cells and inhibit viral replication in vitro. In Rag2−/−γc−/− immunodeficient mice reconstituted with human peripheral mononuclear cells (huPBMCs), PAM reduces disease severity and mortality caused by human seasonal H1N1 and avian H5N1 influenza virus, and controls the lung inflammation and viral replication. PAM has no such effects in influenza virus–infected Rag2−/−γc−/− mice reconstituted with Vγ9Vδ2 T cell–depleted huPBMCs. Our study provides proof-of-concept of a novel therapeutic strategy for treating influenza by targeting the host rather than the virus, thereby reducing the opportunity for the emergence of drug-resistant viruses. As PAM has been commonly used to treat osteoporosis and Paget’s disease, this new application of an old drug potentially offers a safe and readily available option for treating influenza.

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A Novel Sulfonated Derivative of β-Cyclodextrin Effectively Inhibits Influenza A Virus Infection in vitro and in vivo

Acta Naturae ◽

10.32607/20758251-2019-11-3-20-30 ◽

2019 ◽

Vol 11 (3) ◽

pp. 20-30 ◽

Cited By ~ 1

Author(s):

E. P. Goncharova ◽

Y. A. Kostyro ◽

A. V. Ivanov ◽

M. A. Zenkova

Keyword(s):

Influenza Virus ◽

Influenza A ◽

High Efficiency ◽

Influenza Infection ◽

Lethal Infection ◽

Novel Drugs ◽

Infected Cells ◽

Low Toxicity

The development of novel drugs against the influenza virus with high efficiency and low toxicity is an urgent and important task. Previous reports have demonstrated that compounds based on sulfo derivatives of oligo- and polysaccharides possess high antiviral activity. In this study, we have examined the ability of a novel sulfonated derivative of -cyclodextrin (KS-6469) to inhibit the influenza virus A/WSN/33 (H1N1) infection in vitro and in vivo. The antiviral potential of KS-6469 against the influenza virus was evaluated in Madin-Darby Canine Kidney epithelial cells treated with serially diluted KS-6469. We found out that KS-6469 completely inhibited viral reproduction after treatment of the infected cells with the compound for 48 h. Our data show that double intranasal treatment of mice with KS-6469 fully protected the animals from a lethal infection and significantly decreased the viral titers in the lungs of the infected animals. Thus, the novel sulfonated -cyclodextrin derivative KS-6469 is a promising candidate for the development of antiviral drugs for preventing and treating the influenza infection.

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Porcine reproductive and respiratory syndrome virus-infected alveolar macrophages contain no detectable levels of viral proteins in their plasma membrane and are protected against antibody-dependent, complement-mediated cell lysis

Journal of General Virology ◽

10.1099/vir.0.81808-0 ◽

2006 ◽

Vol 87 (8) ◽

pp. 2341-2351 ◽

Cited By ~ 29

Author(s):

Sarah Costers ◽

Peter L. Delputte ◽

Hans J. Nauwynck

Keyword(s):

Plasma Membrane ◽

Immune Evasion ◽

Alveolar Macrophages ◽

Viral Proteins ◽

Cell Lysis ◽

Respiratory Syndrome Virus ◽

Specific Antibodies ◽

Infected Cells

Porcine reproductive and respiratory syndrome virus (PRRSV) can evade the host immune system, which results in prolonged virus replication for several weeks to several months. To date, the mechanisms of PRRSV immune evasion have not been investigated in detail. One possible immune-evasion strategy is to avoid incorporation of viral proteins into the plasma membrane of infected cells, as this prevents recognition by virus-specific antibodies and consequent cell lysis either by the classical complement pathway or by antibody-dependent, cell-mediated cytotoxicity. In this study, viral proteins were not observed in the plasma membrane of in vitro-infected macrophages by using confocal microscopy or flow cytometry. Subsequently, the sensitivity of PRRSV-infected macrophages towards antibody-dependent, complement-mediated cell lysis (ADCML) was determined by using an ADCML assay. A non-significant percentage of PRRSV-infected cells were killed in the assay, showing that in vitro PRRSV-infected macrophages are protected against ADCML. PRRSV proteins were not detected in the plasma membrane of in vivo-infected alveolar macrophages and ADCML was also not observed. Together, these data indicate that viral proteins are not incorporated into the plasma membrane of PRRSV-infected macrophages, which makes infected cells invisible to PRRSV-specific antibodies. This absence of viral proteins on the cell surface could explain the protection against ADCML observed for in vitro and in vivo PRRSV-infected macrophages, and may play a role in virus persistence.

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Membrane Requirements for Uridylylation of the Poliovirus VPg Protein and Viral RNA Synthesis In Vitro

Journal of Virology ◽

10.1128/jvi.77.21.11408-11416.2003 ◽

2003 ◽

Vol 77 (21) ◽

pp. 11408-11416 ◽

Cited By ~ 26

Author(s):

Mark H. Fogg ◽

Natalya L. Teterina ◽

Ellie Ehrenfeld

Keyword(s):

Membrane Trafficking ◽

Rna Synthesis ◽

Rna Replication ◽

Viral Rna ◽

Chain Elongation ◽

Electron Microscopic ◽

Ribonucleoprotein Complexes ◽

Cell Extracts ◽

Infected Cells

ABSTRACT Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation.

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Integrin α4β7 Is Downregulated on the Surfaces of Simian Immunodeficiency Virus SIVmac239-Infected Cells

Journal of Virology ◽

10.1128/jvi.00430-10 ◽

2010 ◽

Vol 84 (13) ◽

pp. 6344-6351 ◽

Cited By ~ 7

Author(s):

Melisa L. Budde ◽

Jennifer J. Lhost ◽

Dawn M. Dudley ◽

Eva G. Rakasz ◽

David H. O'Connor

Keyword(s):

Simian Immunodeficiency Virus ◽

Immune Evasion ◽

Lymphoid Tissue ◽

De Novo ◽

Ex Vivo ◽

Viral Proteins ◽

Flow Cytometry Assay ◽

Immunodeficiency Virus ◽

Infected Cells

ABSTRACT Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) infection results in an early and enduring depletion of intestinal CD4+ T cells. SIV and HIV bind integrin α4β7, thereby facilitating infection of lymphocytes that home to the gut-associated lymphoid tissue (GALT). Using an ex vivo flow cytometry assay, we found that SIVmac239-infected cells expressed significantly lower levels of integrin α4β7 than did uninfected cells. This finding suggested a potential viral effect on integrin α4β7 expression. Using an in vitro model, we confirmed that integrin α4β7 was downregulated on the surfaces of SIVmac239-infected cells. Further, modulation of integrin α4β7 was dependent on de novo synthesis of viral proteins, but neither cell death, the release of a soluble factor, nor a change in activation state was involved. Downregulation of integrin α4β7 may have an unappreciated role in the CD4 depletion of the mucosal-associated lymphoid compartments, susceptibility to superinfection, and/or immune evasion.

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