Discovery of divided RdRp sequences and a hitherto unknown genomic complexity in fungal viruses

10.1101/2020.09.08.288829 ◽

2020 ◽

Author(s):

Yuto Chiba ◽

Takashi Yaguchi ◽

Syun-ichi Urayama ◽

Daisuke Hagiwara

Keyword(s):

Rna Polymerase ◽

Rna Viruses ◽

Current Knowledge ◽

Single Gene ◽

Rna Seq ◽

Rna Dependent Rna Polymerase ◽

Independent Manner ◽

Viral Genomes ◽

True Diversity ◽

Fungal Viruses

AbstractBy identifying variations in viral RNA genomes, cutting-edge metagenome technology has potential to reshape current concepts about the evolution of RNA viruses. This technology, however, cannot process low-homology genomic regions properly, leaving the true diversity of RNA viruses unappreciated. To overcome this technological limitation we applied an advanced method, Fragmented and Primer-Ligated Double-stranded (ds) RNA Sequencing (FLDS), to screen RNA viruses from 155 fungal isolates, which allowed us to obtain complete viral genomes in a homology-independent manner. We created a high-quality catalog of 19 RNA viruses (12 viral species) that infect Aspergillus isolates. Among them, nine viruses were not detectable by the conventional methodology involving agarose gel electrophoresis of dsRNA, a hallmark of RNA virus infections. Segmented genome structures were determined in 42% of the viruses. Some RNA viruses had novel genome architectures; one contained a dual methyltransferase domain and another had a separated RNA-dependent RNA polymerase (RdRp) gene. A virus from a different fungal taxon (Pyricularia) had an RdRp sequence that was separated on different segments, suggesting that a divided RdRp is widely present among fungal viruses, despite the belief that all RNA viruses encode RdRp as a single gene. These findings illustrate the previously hidden diversity and evolution of RNA viruses, and prompt reconsideration of the structural plasticity of RdRp. By highlighting the limitations of conventional surveillance methods for RNA viruses, we showcase the potential of FLDS technology to broaden current knowledge about these viruses.Author SummaryThe development of RNA-seq technology has facilitated the discovery of RNA viruses in all types of biological samples. However, it is technically difficult to detect highly novel viruses using RNA-seq. We successfully reconstructed the genomes of multiple novel fungal RNA viruses by screening host fungi using a new technology, FLDS. Surprisingly, we identified two viral species whose RNA-dependent RNA polymerase (RdRp) proteins were separately encoded on different genome segments, overturning the commonly accepted view of the positional unity of RdRp proteins in viral genomes. This new perspective on divided RdRp proteins should hasten the discovery of viruses with unique RdRp structures that have been overlooked, and further advance current knowledge and understanding of the diversity and evolution of RNA viruses.

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Formation and Function of Liquid-Like Viral Factories in Negative-Sense Single-Stranded RNA Virus Infections

Viruses ◽

10.3390/v13010126 ◽

2021 ◽

Vol 13 (1) ◽

pp. 126

Author(s):

Justin M. Su ◽

Maxwell Z. Wilson ◽

Charles E. Samuel ◽

Dzwokai Ma

Keyword(s):

Rna Viruses ◽

Rna Virus ◽

Host Responses ◽

Future Research ◽

Virus Infections ◽

Infected Cells ◽

And Function ◽

Negative Sense ◽

Liquid Liquid Phase Separation

Liquid–liquid phase separation (LLPS) represents a major physiochemical principle to organize intracellular membrane-less structures. Studies with non-segmented negative-sense (NNS) RNA viruses have uncovered a key role of LLPS in the formation of viral inclusion bodies (IBs), sites of viral protein concentration in the cytoplasm of infected cells. These studies further reveal the structural and functional complexity of viral IB factories and provide a foundation for their future research. Herein, we review the literature leading to the discovery of LLPS-driven formation of IBs in NNS RNA virus-infected cells and the identification of viral scaffold components involved, and then outline important questions and challenges for IB assembly and disassembly. We discuss the functional implications of LLPS in the life cycle of NNS RNA viruses and host responses to infection. Finally, we speculate on the potential mechanisms underlying IB maturation, a phenomenon relevant to many human diseases.

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The Characterization of RNA Viruses in Tropical Seawater Using Targeted PCR and Metagenomics

mBio ◽

10.1128/mbio.01210-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 42

Author(s):

Alexander I. Culley ◽

Jaclyn A. Mueller ◽

Madhi Belcaid ◽

Elisha M. Wood-Charlson ◽

Guylaine Poisson ◽

...

Keyword(s):

Rna Viruses ◽

Rna Virus ◽

Buoyant Density ◽

Global Scale ◽

Limited Data ◽

Viral Genomes ◽

Viral Communities ◽

Seawater Samples ◽

Photosynthesis And Respiration

ABSTRACTViruses have a profound influence on the ecology and evolution of plankton, but our understanding of the composition of the aquatic viral communities is still rudimentary. This is especially true of those viruses having RNA genomes. The limited data that have been published suggest that the RNA virioplankton is dominated by viruses with positive-sense, single-stranded (+ss) genomes that have features in common with those of eukaryote-infecting viruses in the orderPicornavirales(picornavirads). In this study, we investigated the diversity of the RNA virus assemblages in tropical coastal seawater samples using targeted PCR and metagenomics. Amplification of RNA-dependent RNA polymerase (RdRp) genes from fractions of a buoyant density gradient suggested that the distribution of two major subclades of the marine picornavirads was largely congruent with the distribution of total virus-like RNA, a finding consistent with their proposed dominance. Analyses of the RdRp sequences in the library revealed the presence of many diverse phylotypes, most of which were related only distantly to those of cultivated viruses. Phylogenetic analysis suggests that there were hundreds of unique picornavirad-like phylotypes in one 35-liter sample that differed from one another by at least as much as the differences among currently recognized species. Assembly of the sequences in the metagenome resulted in the reconstruction of six essentially complete viral genomes that had features similar to viruses in the familiesBacillarna-,Dicistro-, andMarnaviridae. Comparison of the tropical seawater metagenomes with those from other habitats suggests that +ssRNA viruses are generally the most common types of RNA viruses in aquatic environments, but biases in library preparation remain a possible explanation for this observation.IMPORTANCEMarine plankton account for much of the photosynthesis and respiration on our planet, and they influence the cycling of carbon and the distribution of nutrients on a global scale. Despite the fundamental importance of viruses to plankton ecology and evolution, most of the viruses in the sea, and the identities of their hosts, are unknown. This report is one of very few that delves into the genetic diversity within RNA-containing viruses in the ocean. The data expand the known range of viral diversity and shed new light on the physical properties and genetic composition of RNA viruses in the ocean.

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Replication defective viral genomes exploit a cellular pro-survival mechanism to establish paramyxovirus persistence

10.1101/120766 ◽

2017 ◽

Author(s):

Jie Xu ◽

Yan Sun ◽

Yize Li ◽

Gordon Ruthel ◽

Susan R. Weiss ◽

...

Keyword(s):

Cell Fate ◽

Sendai Virus ◽

Rna Virus ◽

Viral Persistence ◽

Virus Infections ◽

Viral Genomes ◽

Persistent Infections ◽

Infected Cells ◽

Syncytial Virus ◽

Survival Effect

ABSTRACTReplication defective viral genomes (DVGs) generated during virus replication are the primary triggers of antiviral immunity in many RNA virus infections. However, DVGs can also facilitate viral persistence. Why and how these two opposing functions of DVGs are achieved remain unknown. Here we report that during Sendai and respiratory syncytial virus infections DVGs selectively protect a subpopulation of cells from death and promote the establishment of persistent infections. We find that during Sendai virus infection this phenotype results from DVGs stimulating a MAVS-mediated TNF response that drives apoptosis of highly infected cells while extending the survival of cells enriched in DVGs. The pro-survival effect of TNF depends on the activity of the TNFR2/TRAF1 pathway that is regulated by MAVS signaling. These results identify TNF as a pivotal factor in determining cell fate during a viral infection and delineate a MAVS/TNFR2-mediated mechanism that drives the persistence of otherwise acute viruses.

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Viral cross-linking and solid-phase purification enables discovery of ribonucleoprotein complexes on incoming RNA virus genomes

10.1101/2020.04.08.032441 ◽

2020 ◽

Cited By ~ 1

Author(s):

Byungil Kim ◽

Sarah Arcos ◽

Katherine Rothamel ◽

Manuel Ascano

Keyword(s):

Protein Interactions ◽

Rna Viruses ◽

Solid Phase ◽

Rna Virus ◽

Host Protein ◽

Host Cells ◽

Cross Linking ◽

Viral Genomes ◽

Ribonucleoprotein Complexes ◽

Virus Genomes

AbstractThe initial interactions between incoming, pre-replicated RNA virus genomes and host protein factors are important in infection and immunity. Yet there are no current methods to study these crucial events. We established VIR-CLASP (VIRal Cross-Linking And Solid-phase Purification) to identify the primary viral RNA-host protein interactions. First, host cells are infected with 4SU-labeled RNA viruses and irradiated with 365 nm light to crosslink 4SU-labeled viral genomes and interacting proteins from host or virus. The cross-linked RBPs are purified by solid-phase reversible immobilization (SPRI) beads with protein denaturing buffers, and then identified by proteomics. With VIR-CLASP, only the incoming viral genomes are labeled with 4SU, so cross-linking events specifically occur between proteins and pre-replicated viral genomic RNA. Since solid-phase purification under protein-denaturing conditions is used to pull-down total RNA and cross-linked RBPs, this facilitates investigation of potentially all RNA viruses, regardless of RNA sequence. Preparation of 4SU-labeled virus takes ∼7 days and VIR-CLASP takes 1 day.

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Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses

Journal of Virology ◽

10.1128/jvi.01299-15 ◽

2015 ◽

Vol 89 (18) ◽

pp. 9383-9392 ◽

Cited By ~ 59

Author(s):

Kyung-No Son ◽

Zhiguo Liang ◽

Howard L. Lipton

Keyword(s):

Influenza A ◽

Rna Viruses ◽

Rna Virus ◽

Immunofluorescence Staining ◽

Virus Infections ◽

Double Stranded Rna ◽

Negative Strand ◽

Animal Virus ◽

Rna And Dna ◽

Dsrna Species

ABSTRACTEarly biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected thein situformation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation.IMPORTANCEAn effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds of IFN-stimulated genes. The present study demonstrates that infections, including those by ssDNA viruses and positive- and negative-strand RNA viruses, produce dsRNAs detectable by standard immunofluorescence staining. While dsRNA staining was primarily observed in the cytoplasm, nuclear staining was also present in some RNA and DNA virus infections. The nucleus is unlikely to have pathogen-associated molecular pattern (PAMP) receptors for dsRNA because of the presence of host dsRNA molecules. Thus, it is likely that most animal virus infections produce dsRNA species detectable by immunofluorescence staining, which may prove useful in viral discovery as well.

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Nucleosides for the treatment of respiratory RNA virus infections

Antiviral Chemistry and Chemotherapy ◽

10.1177/2040206618764483 ◽

2018 ◽

Vol 26 ◽

pp. 204020661876448 ◽

Cited By ~ 37

Author(s):

Paul C Jordan ◽

Sarah K Stevens ◽

Jerome Deval

Keyword(s):

Respiratory Virus ◽

Respiratory Infections ◽

Rna Viruses ◽

Rna Virus ◽

Nucleoside Analogs ◽

Respiratory Viruses ◽

Influenza Viruses ◽

Virus Infections ◽

Nucleotide Analogs ◽

Tract Infections

Influenza virus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, and rhinoviruses are among the most common viruses causing mild seasonal colds. These RNA viruses can also cause lower respiratory tract infections leading to bronchiolitis and pneumonia. Young children, the elderly, and patients with compromised cardiac, pulmonary, or immune systems are at greatest risk for serious disease associated with these RNA virus respiratory infections. In addition, swine and avian influenza viruses, together with severe acute respiratory syndrome-associated and Middle Eastern respiratory syndrome coronaviruses, represent significant pandemic threats to the general population. In this review, we describe the current medical need resulting from respiratory infections caused by RNA viruses, which justifies drug discovery efforts to identify new therapeutic agents. The RNA polymerase of respiratory viruses represents an attractive target for nucleoside and nucleotide analogs acting as inhibitors of RNA chain synthesis. Here, we present the molecular, biochemical, and structural fundamentals of the polymerase of the four major families of RNA respiratory viruses: Orthomyxoviridae, Pneumoviridae/Paramyxoviridae, Coronaviridae, and Picornaviridae. We summarize past and current efforts to develop nucleoside and nucleotide analogs as antiviral agents against respiratory virus infections. This includes molecules with very broad antiviral spectrum such as ribavirin and T-705 (favipiravir), and others targeting more specifically one or a few virus families. Recent advances in our understanding of the structure(s) and function(s) of respiratory virus polymerases will likely support the discovery and development of novel nucleoside analogs.

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Faculty Opinions recommendation of Single-Cell Analysis of RNA Virus Infection Identifies Multiple Genetically Diverse Viral Genomes within Single Infectious Units.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725850284.793510505 ◽

2015 ◽

Author(s):

Santiago F Elena

Keyword(s):

Virus Infection ◽

Single Cell ◽

Single Cell Analysis ◽

Rna Virus ◽

Cell Analysis ◽

Viral Genomes

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Structure Unveils Relationships between RNA Virus Polymerases

Viruses ◽

10.3390/v13020313 ◽

2021 ◽

Vol 13 (2) ◽

pp. 313

Author(s):

Heli A. M. Mönttinen ◽

Janne J. Ravantti ◽

Minna M. Poranen

Keyword(s):

Phylogenetic Tree ◽

Rna Viruses ◽

Rna Virus ◽

Sequence Similarity ◽

Protein Structures ◽

Structural Similarity ◽

Functional Differentiation ◽

Comparison Method ◽

Homologous Structure ◽

Biological Entities

RNA viruses are the fastest evolving known biological entities. Consequently, the sequence similarity between homologous viral proteins disappears quickly, limiting the usability of traditional sequence-based phylogenetic methods in the reconstruction of relationships and evolutionary history among RNA viruses. Protein structures, however, typically evolve more slowly than sequences, and structural similarity can still be evident, when no sequence similarity can be detected. Here, we used an automated structural comparison method, homologous structure finder, for comprehensive comparisons of viral RNA-dependent RNA polymerases (RdRps). We identified a common structural core of 231 residues for all the structurally characterized viral RdRps, covering segmented and non-segmented negative-sense, positive-sense, and double-stranded RNA viruses infecting both prokaryotic and eukaryotic hosts. The grouping and branching of the viral RdRps in the structure-based phylogenetic tree follow their functional differentiation. The RdRps using protein primer, RNA primer, or self-priming mechanisms have evolved independently of each other, and the RdRps cluster into two large branches based on the used transcription mechanism. The structure-based distance tree presented here follows the recently established RdRp-based RNA virus classification at genus, subfamily, family, order, class and subphylum ranks. However, the topology of our phylogenetic tree suggests an alternative phylum level organization.

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Essential role of IPS-1 in innate immune responses against RNA viruses

Journal of Experimental Medicine ◽

10.1084/jem.20060792 ◽

2006 ◽

Vol 203 (7) ◽

pp. 1795-1803 ◽

Cited By ~ 345

Author(s):

Himanshu Kumar ◽

Taro Kawai ◽

Hiroki Kato ◽

Shintaro Sato ◽

Ken Takahashi ◽

...

Keyword(s):

Rna Viruses ◽

Rna Virus ◽

Critical Role ◽

Nuclear Factor Κb ◽

Type I ◽

Innate Immune Responses ◽

Antiviral Responses ◽

Deficient Mice

IFN-β promoter stimulator (IPS)-1 was recently identified as an adapter for retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5), which recognize distinct RNA viruses. Here we show the critical role of IPS-1 in antiviral responses in vivo. IPS-1–deficient mice showed severe defects in both RIG-I– and Mda5-mediated induction of type I interferon and inflammatory cytokines and were susceptible to RNA virus infection. RNA virus–induced interferon regulatory factor-3 and nuclear factor κB activation was also impaired in IPS-1–deficient cells. IPS-1, however, was not essential for the responses to either DNA virus or double-stranded B-DNA. Thus, IPS-1 is the sole adapter in both RIG-I and Mda5 signaling that mediates effective responses against a variety of RNA viruses.

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