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1.
Viruses ; 14(12)2022 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-36560777

RESUMO

Defective interfering particles (DIPs) are particles containing defective viral genomes (DVGs) generated during viral replication. DIPs have been found in various RNA viruses, especially in influenza viruses. Evidence indicates that DIPs interfere with the replication and encapsulation of wild-type viruses, namely standard viruses (STVs) that contain full-length viral genomes. DIPs may also activate the innate immune response by stimulating interferon synthesis. In this review, the underlying generation mechanisms and characteristics of influenza virus DIPs are summarized. We also discuss the potential impact of DIPs on the immunogenicity of live attenuated influenza vaccines (LAIVs) and development of influenza vaccines based on NS1 gene-defective DIPs. Finally, we review the antiviral strategies based on influenza virus DIPs that have been used against both influenza virus and SARS-CoV-2. This review provides systematic insights into the theory and application of influenza virus DIPs.


Assuntos
COVID-19 , Vacinas contra Influenza , Orthomyxoviridae , Humanos , Antivirais , Vírus Defeituosos Interferentes , Vírus Defeituosos/fisiologia , SARS-CoV-2 , Orthomyxoviridae/genética , Replicação Viral/genética
2.
Commun Biol ; 5(1): 1140, 2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36302891

RESUMO

Defective interfering (DI) particles arise during virus propagation, are conditional on parental virus for replication and packaging, and interfere with viral expansion. There is much interest in developing DIs as anti-viral agents. Here we characterize DI particles that arose following serial passaging of SARS-CoV-2 at high multiplicity of infection. The prominent DIs identified have lost ~84% of the SARS-CoV-2 genome and are capable of attenuating parental viral titers. Synthetic variants of the DI genomes also interfere with infection and can be used as conditional, gene delivery vehicles. In addition, the DI genomes encode an Nsp1-10 fusion protein capable of attenuating viral replication. These results identify naturally selected defective viral genomes that emerged and stably propagated in the presence of parental virus.


Assuntos
COVID-19 , Vírus Defeituosos , Humanos , Vírus Defeituosos/genética , SARS-CoV-2/genética , Vírus Defeituosos Interferentes , RNA Viral/genética
3.
Cells ; 11(2)2022 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-35053418

RESUMO

The novel coronavirus SARS-CoV-2 has caused a pandemic resulting in millions of deaths worldwide. While multiple vaccines have been developed, insufficient vaccination combined with adaptive mutations create uncertainty for the future. Here, we discuss novel strategies to control COVID-19 relying on Defective Interfering Particles (DIPs) and related particles that arise naturally during an infection. Our intention is to encourage and to provide the basis for the implementation of such strategies by multi-disciplinary teams. We therefore provide an overview of SARS-CoV-2 for a multi-disciplinary readership that is specifically tailored to these strategies, we identify potential targets based on the current knowledge of the properties and functions of coronaviruses, and we propose specific strategies to engineer DIPs and other interfering or therapeutic nanoparticles.


Assuntos
COVID-19 , Vírus Defeituosos Interferentes , Nanopartículas/uso terapêutico , Pandemias , SARS-CoV-2/metabolismo , COVID-19/epidemiologia , COVID-19/metabolismo , COVID-19/terapia , Humanos
4.
Viruses ; 14(1)2022 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-35062341

RESUMO

Particles of many paramyxoviruses include small amounts of proteins with a molecular weight of about 20 kDa. These proteins, termed "C", are basic, have low amino acid homology and some secondary structure conservation. C proteins are encoded in alternative reading frames of the phosphoprotein gene. Some viruses express nested sets of C proteins that exert their functions in different locations: In the nucleus, they interfere with cellular transcription factors that elicit innate immune responses; in the cytoplasm, they associate with viral ribonucleocapsids and control polymerase processivity and orderly replication, thereby minimizing the activation of innate immunity. In addition, certain C proteins can directly bind to, and interfere with the function of, several cytoplasmic proteins required for interferon induction, interferon signaling and inflammation. Some C proteins are also required for efficient virus particle assembly and budding. C-deficient viruses can be grown in certain transformed cell lines but are not pathogenic in natural hosts. C proteins affect the same host functions as other phosphoprotein gene-encoded proteins named V but use different strategies for this purpose. Multiple independent systems to counteract host defenses may ensure efficient immune evasion and facilitate virus adaptation to new hosts and tissue environments.


Assuntos
Imunidade Inata/imunologia , Infecções por Paramyxoviridae/imunologia , Paramyxovirinae/fisiologia , Fosfoproteínas/imunologia , Proteínas Virais/imunologia , Replicação Viral/fisiologia , Animais , Vírus Defeituosos Interferentes , Genoma Viral , Humanos , Evasão da Resposta Imune , Inflamassomos , Fases de Leitura Aberta , Paramyxovirinae/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Filogenia , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Montagem de Vírus
5.
Cell ; 184(25): 6037-6051.e14, 2021 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-34852237

RESUMO

RNA viruses generate defective viral genomes (DVGs) that can interfere with replication of the parental wild-type virus. To examine their therapeutic potential, we created a DVG by deleting the capsid-coding region of poliovirus. Strikingly, intraperitoneal or intranasal administration of this genome, which we termed eTIP1, elicits an antiviral response, inhibits replication, and protects mice from several RNA viruses, including enteroviruses, influenza, and SARS-CoV-2. While eTIP1 replication following intranasal administration is limited to the nasal cavity, its antiviral action extends non-cell-autonomously to the lungs. eTIP1 broad-spectrum antiviral effects are mediated by both local and distal type I interferon responses. Importantly, while a single eTIP1 dose protects animals from SARS-CoV-2 infection, it also stimulates production of SARS-CoV-2 neutralizing antibodies that afford long-lasting protection from SARS-CoV-2 reinfection. Thus, eTIP1 is a safe and effective broad-spectrum antiviral generating short- and long-term protection against SARS-CoV-2 and other respiratory infections in animal models.


Assuntos
Proteínas do Capsídeo/genética , Vírus Defeituosos Interferentes/metabolismo , Replicação Viral/efeitos dos fármacos , Administração Intranasal , Animais , Antivirais/farmacologia , Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Amplamente Neutralizantes/farmacologia , COVID-19 , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Vírus Defeituosos Interferentes/patogenicidade , Modelos Animais de Doenças , Genoma Viral/genética , Humanos , Influenza Humana , Interferons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Poliovirus/genética , Poliovirus/metabolismo , Infecções Respiratórias/virologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/patogenicidade
6.
mBio ; 12(6): e0295921, 2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34809454

RESUMO

Deletion-containing viral genomes (DelVGs) are commonly produced during influenza A virus infection and have been implicated in influencing clinical infection outcomes. Despite their ubiquity, the specific molecular mechanisms that govern DelVG formation and their packaging into defective interfering particles (DIPs) remain poorly understood. Here, we utilized next-generation sequencing to analyze DelVGs that form de novo early during infection, prior to packaging. Analysis of these early DelVGs revealed that deletion formation occurs in clearly defined hot spots and is significantly associated with both direct sequence repeats and enrichment of adenosine and uridine bases. By comparing intracellular DelVGs with those packaged into extracellular virions, we discovered that DelVGs face a significant bottleneck during genome packaging relative to wild-type genomic RNAs. Interestingly, packaged DelVGs exhibited signs of enrichment for larger DelVGs suggesting that size is an important determinant of packaging efficiency. Our data provide the first unbiased, high-resolution portrait of the diversity of DelVGs that are generated by the influenza A virus replication machinery and shed light on the mechanisms that underly DelVG formation and packaging. IMPORTANCE Defective interfering particles (DIPs) are commonly produced by RNA viruses and have been implicated in modulating clinical infection outcomes; hence, there is increasing interest in the potential of DIPs as antiviral therapeutics. For influenza viruses, DIPs are formed by the packaging of genomic RNAs harboring internal deletions. Despite decades of study, the mechanisms that drive the formation of these deletion-containing viral genomes (DelVGs) remain elusive. Here, we used a specialized sequencing pipeline to characterize the first wave of DelVGs that form during influenza virus infection. This data set provides an unbiased profile of the deletion-forming preferences of the influenza virus replicase. In addition, by comparing the early intracellular DelVGs to those that get packaged into extracellular virions, we described a significant segment-specific bottleneck that limits DelVG packaging relative to wild-type viral RNAs. Altogether, these findings reveal factors that govern the production of both DelVGs and DIPs during influenza virus infection.


Assuntos
Vírus Defeituosos Interferentes/fisiologia , Genoma Viral , Vírus da Influenza A/fisiologia , Empacotamento do Genoma Viral , Vírion/fisiologia , Vírus Defeituosos Interferentes/genética , Humanos , Vírus da Influenza A/genética , Influenza Humana/virologia , RNA Viral/genética , RNA Viral/metabolismo , Vírion/genética , Replicação Viral
7.
Cell ; 184(25): 6022-6036.e18, 2021 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-34838159

RESUMO

Viral-deletion mutants that conditionally replicate and inhibit the wild-type virus (i.e., defective interfering particles, DIPs) have long been proposed as single-administration interventions with high genetic barriers to resistance. However, theories predict that robust, therapeutic DIPs (i.e., therapeutic interfering particles, TIPs) must conditionally spread between cells with R0 >1. Here, we report engineering of TIPs that conditionally replicate with SARS-CoV-2, exhibit R0 >1, and inhibit viral replication 10- to 100-fold. Inhibition occurs via competition for viral replication machinery, and a single administration of TIP RNA inhibits SARS-CoV-2 sustainably in continuous cultures. Strikingly, TIPs maintain efficacy against neutralization-resistant variants (e.g., B.1.351). In hamsters, both prophylactic and therapeutic intranasal administration of lipid-nanoparticle TIPs durably suppressed SARS-CoV-2 by 100-fold in the lungs, reduced pro-inflammatory cytokine expression, and prevented severe pulmonary edema. These data provide proof of concept for a class of single-administration antivirals that may circumvent current requirements to continually update medical countermeasures against new variants.


Assuntos
Vírus Defeituosos Interferentes/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/farmacologia , COVID-19/metabolismo , Linhagem Celular , Chlorocebus aethiops , Meios de Cultivo Condicionados/farmacologia , Vírus Defeituosos Interferentes/patogenicidade , Sistemas de Liberação de Medicamentos/métodos , Células Epiteliais , Humanos , Masculino , Mesocricetus , Nanopartículas/uso terapêutico , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Células Vero
8.
Sci Rep ; 11(1): 20477, 2021 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-34650149

RESUMO

Influenza A virus (IAV) infection constitutes a significant health threat. Defective interfering particles (DIPs) can arise during IAV infection and inhibit spread of wild type (WT) IAV. DIPs harbor defective RNA segments, termed DI RNAs, that usually contain internal deletions and interfere with replication of WT viral RNA segments. Here, we asked whether DIPs harboring two instead of one DI RNA exert increased antiviral activity. For this, we focused on DI RNAs derived from segments 1 and 3, which encode the polymerase subunits PB2 and PA, respectively. We demonstrate the successful production of DIPs harboring deletions in segments 1 and/or 3, using cell lines that co-express PB2 and PA. Further, we demonstrate that DIPs harboring two instead of one DI RNA do not exhibit increased ability to inhibit replication of a WT RNA segment. Similarly, the presence of two DI RNAs did not augment the induction of the interferon-stimulated gene MxA and the inhibition of IAV infection. Collectively, our findings suggest that the presence of multiple DI RNAs derived from genomic segments encoding polymerase subunits might not result in increased antiviral activity.


Assuntos
Vírus Defeituosos Interferentes/genética , Vírus da Influenza A/genética , RNA Viral , Animais , Antivirais , Vírus Defeituosos , Cães , Células HEK293 , Humanos , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/virologia
9.
Viruses ; 13(6)2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205760

RESUMO

Historically, adeno-associated virus (AAV)-defective interfering particles (DI) were known as abnormal virions arising from natural replication and encapsidation errors. Through single virion genome analysis, we revealed that a major category of DI particles contains a double-stranded DNA genome in a "snapback" configuration. The 5'- snapback genomes (SBGs) include the P5 promoters and partial rep gene sequences. The 3'-SBGs contains the capsid region. The molecular configuration of 5'-SBGs theoretically may allow double-stranded RNA transcription in their dimer configuration. Our studies demonstrated that 5-SBG regulated AAV rep expression and improved AAV packaging. In contrast, 3'-SBGs at its dimer configuration increased levels of cap protein. The generation and accumulation of 5'-SBGs and 3'-SBGs appears to be coordinated to balance the viral gene expression level. Therefore, the functions of 5'-SBGs and 3'-SBGs may help maximize the yield of AAV progenies. We postulate that AAV virus population behaved as a colony and utilizes its subgenomic particles to overcome the size limit of a viral genome and encodes additional essential functions.


Assuntos
Vírus Defeituosos Interferentes/crescimento & desenvolvimento , Vírus Defeituosos Interferentes/genética , Dependovirus/crescimento & desenvolvimento , Dependovirus/genética , Genoma Viral , Estágios do Ciclo de Vida/genética , Proteínas do Capsídeo/genética , Células HEK293 , Humanos , Proteínas Virais/genética , Vírion/metabolismo , Replicação Viral
10.
J Virol ; 95(24): e0117421, 2021 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-34550771

RESUMO

Defective interfering particles (DIPs) of influenza A virus (IAV) are naturally occurring mutants that have an internal deletion in one of their eight viral RNA (vRNA) segments, rendering them propagation-incompetent. Upon coinfection with infectious standard virus (STV), DIPs interfere with STV replication through competitive inhibition. Thus, DIPs are proposed as potent antivirals for treatment of the influenza disease. To select corresponding candidates, we studied de novo generation of DIPs and propagation competition between different defective interfering (DI) vRNAs in an STV coinfection scenario in cell culture. A small-scale two-stage cultivation system that allows long-term semi-continuous propagation of IAV and its DIPs was used. Strong periodic oscillations in virus titers were observed due to the dynamic interaction of DIPs and STVs. Using next-generation sequencing, we detected a predominant formation and accumulation of DI vRNAs on the polymerase-encoding segments. Short DI vRNAs accumulated to higher fractions than longer ones, indicating a replication advantage, yet an optimum fragment length was observed. Some DI vRNAs showed breaking points in a specific part of their bundling signal (belonging to the packaging signal), suggesting its dispensability for DI vRNA propagation. Over a total cultivation time of 21 days, several individual DI vRNAs accumulated to high fractions, while others decreased. Using reverse genetics for IAV, purely clonal DIPs derived from highly replicating DI vRNAs were generated. We confirm that these DIPs exhibit a superior in vitro interfering efficacy compared to DIPs derived from lowly accumulated DI vRNAs and suggest promising candidates for efficacious antiviral treatment. IMPORTANCE Defective interfering particles (DIPs) emerge naturally during viral infection and typically show an internal deletion in the viral genome. Thus, DIPs are propagation-incompetent. Previous research suggests DIPs as potent antiviral compounds for many different virus families due to their ability to interfere with virus replication by competitive inhibition. For instance, the administration of influenza A virus (IAV) DIPs resulted in a rescue of mice from an otherwise lethal IAV dose. Moreover, no apparent toxic effects were observed when only DIPs were administered to mice and ferrets. IAV DIPs show antiviral activity against many different IAV strains, including pandemic and highly pathogenic avian strains, and even against nonhomologous viruses, such as SARS-CoV-2, by stimulation of innate immunity. Here, we used a cultivation/infection system, which exerted selection pressure toward accumulation of highly competitive IAV DIPs. These DIPs showed a superior interfering efficacy in vitro, and we suggest them for effective antiviral therapy.


Assuntos
Antivirais/farmacologia , Desenho de Fármacos/métodos , Vírus da Influenza A , Influenza Humana/virologia , RNA Viral , Animais , Técnicas de Cultura de Células , Linhagem Celular , Vírus Defeituosos Interferentes , Vírus Defeituosos/genética , Cães , Deleção de Genes , Genoma Viral , Humanos , Imunidade Inata/efeitos dos fármacos , Células Madin Darby de Rim Canino , Oscilometria , Reação em Cadeia da Polimerase em Tempo Real , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
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