Experimental and mathematical insights on the interactions between poliovirus and a defective interfering genome.
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ABSTRACT:
SUBMITTER: Shirogane Y
PROVIDER: S-EPMC8496841 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Equine arteritis virus (EAV), the type member of the family Arteriviridae, is a single-stranded RNA virus with a positive-stranded genome of approximately 13 kb. EAV uses a discontinuous transcription mechanism to produce a nested set of six subgenomic mRNAs from which its structural genes are expressed. We have generated the first documented arterivirus defective interfering (DI) RNAs by serial undiluted passaging of a wild-type EAV stock in BHK-21 cells. A cDNA copy of the smallest DI RNA (5.6 kb) was cloned. Upon transfection into EAV-infected BHK-21 cells, transcripts derived from this clone (pEDI) were replicated and packaged. Sequencing of pEDI revealed that the DI RNA was composed of three segments of the EAV genome (nucleotides 1 to 1057, 1388 to 1684, and 8530 to 12704) which were fused in frame with respect to the replicase reading frame. Remarkably, this DI RNA has retained all of the sequences encoding the structural proteins. By insertion of the chloramphenicol acetyltransferase reporter gene in the DI RNA genome, we were able to delimitate the sequences required for replication/DI-based transcription and packaging of EAV DI RNAs and to reduce the maximal size of a replication-competent EAV DI RNA to approximately 3 kb.
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Project description:Defective interfering particles (DIPs) are regarded as potent broad-spectrum antivirals. We developed a mathematical model that describes intracellular co-infection dynamics of influenza standard virus (STV) and "OP7", a new type of influenza DIP discovered recently. Based on experimental data from in vitro studies to calibrate the model and confirm its predictions, we deduce OP7's mechanisms of interference, which were yet unknown. Simulations suggest that the "superpromoter" on OP7 genomic viral RNA enhances its replication and results in a depletion of viral proteins. This reduces STV genomic RNA replication, which appears to constitute an antiviral effect. Further, a defective viral protein (M1-OP7) likely causes the deficiency of OP7's replication. It appears unable to bind to genomic viral RNAs to facilitate their nuclear export, a critical step in the viral life cycle. An improved understanding of OP7's antiviral mechanism is crucial toward application in humans as a prospective antiviral treatment strategy.
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Project description:Defective interfering particles (DIPs) are virus-like particles that occur naturally during virus infections. These particles are defective, lacking essential genetic materials for replication, but they can interact with the wild-type virus and potentially be used as therapeutic agents. However, the effect of DIPs on infection spread is still unclear due to complicated stochastic effects and nonlinear spatial dynamics. In this work, we develop a model with a new hybrid method to study the spatial-temporal dynamics of viruses and DIPs co-infections within hosts. We present two different scenarios of virus production and compare the results from deterministic and stochastic models to demonstrate how the stochastic effect is involved in the spatial dynamics of virus transmission. We compare the spread features of the virus in simulations and experiments, including the formation and the speed of virus spread and the emergence of stochastic patchy patterns of virus distribution. Our simulations simultaneously capture observed spatial spread features in the experimental data, including the spread rate of the virus and its patchiness. The results demonstrate that DIPs can slow down the growth of virus particles and make the spread of the virus more patchy.
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Project description:Panicum mosaic virus (PMV) is a recently molecularly characterized RNA virus with the unique feature of supporting the replication of two subviral RNAs in a few species of the family Gramineae. The subviral agents include a satellite RNA (satRNA) that is devoid of a coding region and the unrelated satellite panicum mosaic virus (SPMV) that encodes its own capsid protein. Here we report the association of this complex with a new entity in the RNA world, a defective-interfering RNA (DI) of a satellite virus. The specificity of interactions governing this four-component viral system is illustrated by the ability of the SPMV DIs to strongly interfere with the accumulation of the parental SPMV. The SPMV DIs do not interfere with PMV satRNA, but they do slightly enhance the rate of spread and titer of PMV. The SPMV-derived DIs provide an additional avenue by which to investigate fundamental biological questions, including the evolution and interactions of infectious RNAs.
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Project description:Viruses thrive by exploiting the cells they infect, but in order to replicate and infect other cells they must produce viral proteins. As a result, viruses are also susceptible to exploitation by defective versions of themselves that do not produce such proteins. A defective viral genome with deletions in protein-coding genes could still replicate in cells coinfected with full-length viruses. Such a defective genome could even replicate faster due to its shorter size, interfering with the replication of the virus. We have created a synthetic defective interfering version of SARS-CoV-2, the virus causing the Covid-19 pandemic, assembling parts of the viral genome that do not code for any functional protein but enable the genome to be replicated and packaged. This synthetic defective genome replicates three times faster than SARS-CoV-2 in coinfected cells, and interferes with it, reducing the viral load of infected cells by half in 24 hours. The synthetic genome is transmitted as efficiently as the full-length genome, suggesting the location of the putative packaging signal of SARS-CoV-2. A version of such a synthetic construct could be used as a self-promoting antiviral therapy: by enabling replication of the synthetic genome, the virus would promote its own demise.
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Project description:While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles have referred to studies in vitro, there is some evidence for the presence of DI particles in chronic viral infections in vivo. In this study, short fragments of dengue virus (DENV) RNA containing only key regulatory elements at the 3' and 5' ends of the genome were recovered from the sera of patients infected with any of the four DENV serotypes. Identical RNA fragments were detected in the supernatant from cultures of Aedes mosquito cells that were infected by the addition of sera from dengue patients, suggesting that the sub-genomic RNA might be transmitted between human and mosquito hosts in defective interfering (DI) viral particles. In vitro transcribed sub-genomic RNA corresponding to that detected in vivo could be packaged in virus like particles in the presence of wild type virus and transmitted for at least three passages in cell culture. DENV preparations enriched for these putative DI particles reduced the yield of wild type dengue virus following co-infections of C6-36 cells. This is the first report of DI particles in an acute arboviral infection in nature. The internal genomic deletions described here are the most extensive defects observed in DENV and may be part of a much broader disease attenuating process that is mediated by defective viruses.
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Project description:Most of the circulating vaccine-derived polioviruses (cVDPVs) implicated in poliomyelitis outbreaks in Madagascar have been shown to be recombinants between the type 2 poliovirus (PV) strain of the oral polio vaccine (Sabin 2) and another species C human enterovirus (HEV-C), such as type 17 coxsackie A virus (CA17) in particular. We studied intertypic genetic exchanges between PV and non-PV HEV-C by developing a recombination model, making it possible to rescue defective type 2 PV RNA genomes with a short deletion at the 3' end by the cotransfection of cells with defective or infectious CA17 RNAs. We isolated over 200 different PV/CA17 recombinants, using murine cells expressing the human PV receptor (PVR) and selecting viruses with PV capsids. We found some homologous (H) recombinants and, mostly, nonhomologous (NH) recombinants presenting duplications of parental sequences preferentially located in the regions encoding proteins 2A, 2B, and 3A. Short duplications appeared to be stable, whereas longer duplications were excised during passaging in cultured cells or after multiplication in PVR-transgenic mice, generating H recombinants with diverse sites of recombination. This suggests that NH recombination events may be a transient, intermediate step in the generation and selection of the fittest H recombinants. In addition to the classical copy-choice mechanism of recombination thought to generate mostly H recombinants, there may also be a modular mechanism of recombination, involving NH recombinant precursors, shaping the genomes of recombinant enteroviruses and other picornaviruses. Importance: The multiplication of circulating vaccine-derived polioviruses (cVDPVs) in poorly immunized human populations can render these viruses pathogenic, causing poliomyelitis outbreaks. Most cVDPVs are intertypic recombinants between a poliovirus (PV) strain and another human enterovirus, such as type 17 coxsackie A viruses (CA17). For further studies of the genetic exchanges between PV and CA17, we have developed a model of recombination, making it possible to rescue defective PV RNA genomes with a short deletion by cotransfecting cells with the defective PV genome and CA17 genomic RNA. Numerous recombinants were found, including homologous PV/CA17 recombinants, but mostly nonhomologous recombinants presenting duplications of parental sequences preferentially located in particular regions. Long duplications were excised by passages in cultured cells or in mice, generating diverse homologous recombinants. Recombination leading to nonhomologous recombinants, which evolve into homologous recombinants, may therefore be seen as a model of genetic plasticity in enteroviruses and, possibly, in other RNA viruses.
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Project description:Coronavirus RNA-dependent RNA polymerases produce subgenomic RNAs (sgRNAs) that encode viral structural and accessory proteins. The kinetics and efficiency of sgRNAs production during viral replication in different cell types or sgRNA transcription by individual viral strains or variants are yet to be studied to shed light on fundamental mechanisms necessary for viral replication. User-friendly bioinformatic tools to detect and quantify sgRNA production are urgently needed to study a growing number of next-generation sequencing (NGS) data of SARS-CoV-2. Starting from DI-tector, a bioinformatic tool for the detection of viral defective interfering genomes, here we introduced sgDI-tector to identify and quantify sgRNA in SARS-CoV-2 NGS data. This new tool allowed detection of sgRNA without initial knowledge of the transcription-regulatory sequences. As a proof of principle, we analyzed new data sets and successfully detected the nested set of sgRNAs produced with the ranking M>ORF3a>N>ORF6>ORF7a>ORF8>S>E>ORF7b. Our study also compared, for the first time for SARS-CoV-2, the level of sgRNA production with other types of viral RNA products such as defective interfering viral genomes.
2021-12-28 | GSE180632 | GEO
Project description: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.
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