scholarly journals Ebolavirus polymerase uses an unconventional genome replication mechanism

2019 ◽  
Vol 116 (17) ◽  
pp. 8535-8543 ◽  
Author(s):  
Laure R. Deflubé ◽  
Tessa N. Cressey ◽  
Adam J. Hume ◽  
Judith Olejnik ◽  
Elaine Haddock ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Rna Virus ◽  
Replication Initiation ◽  
Genome Replication ◽  
Terminal Nucleotide ◽  
Nucleotide Addition ◽  
Replication Strategy ◽  
Virus Genomes ◽  
High Degree

Most nonsegmented negative strand (NNS) RNA virus genomes have complementary 3′ and 5′ terminal nucleotides because the promoters at the 3′ ends of the genomes and antigenomes are almost identical to each other. However, according to published sequences, both ends of ebolavirus genomes show a high degree of variability, and the 3′ and 5′ terminal nucleotides are not complementary. If correct, this would distinguish the ebolaviruses from other NNS RNA viruses. Therefore, we investigated the terminal genomic and antigenomic nucleotides of three different ebolavirus species, Ebola (EBOV), Sudan, and Reston viruses. Whereas the 5′ ends of ebolavirus RNAs are highly conserved with the sequence ACAGG-5′, the 3′ termini are variable and are typically 3′-GCCUGU, ACCUGU, or CCUGU. A small fraction of analyzed RNAs had extended 3′ ends. The majority of 3′ terminal sequences are consistent with a mechanism of nucleotide addition by hairpin formation and back-priming. Using single-round replicating EBOV minigenomes, we investigated the effect of the 3′ terminal nucleotide on viral replication and found that the EBOV polymerase initiates replication opposite the 3′-CCUGU motif regardless of the identity of the 3′ terminal nucleotide(s) and of the position of this motif relative to the 3′ end. Deletion or mutation of the first residue of the 3′-CCUGU motif completely abolished replication initiation, suggesting a crucial role of this nucleotide in directing initiation. Together, our data show that ebolaviruses have evolved a unique replication strategy among NNS RNA viruses resulting in 3′ overhangs. This could be a mechanism to avoid antiviral recognition.

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

2006 ◽  
Vol 203 (7) ◽  
pp. 1795-1803 ◽  
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.

scholarly journals Oligomerization of the Vesicular Stomatitis Virus Phosphoprotein Is Dispensable for mRNA Synthesis but Facilitates RNA Replication

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Louis-Marie Bloyet ◽  
Benjamin Morin ◽  
Vesna Brusic ◽  
Erica Gardner ◽  
Robin A. Ross ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rna Viruses ◽  
Rna Virus ◽  
Rna Replication ◽  
Binding Domain ◽  
Genome Replication ◽  
Mrna Synthesis ◽  
Wild Type ◽  
Vesicular Stomatitis ◽  
Oligomerization Domain

ABSTRACT Nonsegmented negative-strand (NNS) RNA viruses possess a ribonucleoprotein template in which the genomic RNA is sequestered within a homopolymer of nucleocapsid protein (N). The viral RNA-dependent RNA polymerase (RdRP) resides within an approximately 250-kDa large protein (L), along with unconventional mRNA capping enzymes: a GDP:polyribonucleotidyltransferase (PRNT) and a dual-specificity mRNA cap methylase (MT). To gain access to the N-RNA template and orchestrate the LRdRP, LPRNT, and LMT, an oligomeric phosphoprotein (P) is required. Vesicular stomatitis virus (VSV) P is dimeric with an oligomerization domain (OD) separating two largely disordered regions followed by a globular C-terminal domain that binds the template. P is also responsible for bringing new N protomers onto the nascent RNA during genome replication. We show VSV P lacking the OD (PΔOD) is monomeric but is indistinguishable from wild-type P in supporting mRNA transcription in vitro. Recombinant virus VSV-PΔOD exhibits a pronounced kinetic delay in progeny virus production. Fluorescence recovery after photobleaching demonstrates that PΔOD diffuses 6-fold more rapidly than the wild type within viral replication compartments. A well-characterized defective interfering particle of VSV (DI-T) that is only competent for RNA replication requires significantly higher levels of N to drive RNA replication in the presence of PΔOD. We conclude P oligomerization is not required for mRNA synthesis but enhances genome replication by facilitating RNA encapsidation. IMPORTANCE All NNS RNA viruses, including the human pathogens rabies, measles, respiratory syncytial virus, Nipah, and Ebola, possess an essential L-protein cofactor, required to access the N-RNA template and coordinate the various enzymatic activities of L. The polymerase cofactors share a similar modular organization of a soluble N-binding domain and a template-binding domain separated by a central oligomerization domain. Using a prototype of NNS RNA virus gene expression, vesicular stomatitis virus (VSV), we determined the importance of P oligomerization. We find that oligomerization of VSV P is not required for any step of viral mRNA synthesis but is required for efficient RNA replication. We present evidence that this likely occurs through the stage of loading soluble N onto the nascent RNA strand as it exits the polymerase during RNA replication. Interfering with the oligomerization of P may represent a general strategy to interfere with NNS RNA virus replication.

scholarly journals Dual Role of a Viral Polymerase in Viral Genome Replication and Particle Self-Assembly

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Xiaoyu Sun ◽  
Serban L. Ilca ◽  
Juha T. Huiskonen ◽  
Minna M. Poranen
Keyword(s):  
Self Assembly ◽  
Viral Genome ◽  
Rna Viruses ◽  
The Self ◽  
Genome Replication ◽  
Double Stranded Rna ◽  
Polymerase Complex ◽  
Viral Genome Replication ◽  
Dsrna Viruses

ABSTRACTDouble-stranded RNA (dsRNA) viruses package several RNA-dependent RNA polymerases (RdRp) together with their dsRNA genome into an icosahedral protein capsid known as the polymerase complex. This structure is highly conserved among dsRNA viruses but is not found in any other virus group. RdRp subunits typically interact directly with the main capsid proteins, close to the 5-fold symmetric axes, and perform viral genome replication and transcription within the icosahedral protein shell. In this study, we utilizedPseudomonasphage Φ6, a well-established virus self-assembly model, to probe the potential roles of the RdRp in dsRNA virus assembly. We demonstrated that Φ6 RdRp accelerates the polymerase complex self-assembly process and contributes to its conformational stability and integrity. We highlight the role of specific amino acid residues on the surface of the RdRp in its incorporation during the self-assembly reaction. Substitutions of these residues reduce RdRp incorporation into the polymerase complex during the self-assembly reaction. Furthermore, we determined that the overall transcription efficiency of the Φ6 polymerase complex increased when the number of RdRp subunits exceeded the number of genome segments. These results suggest a mechanism for RdRp recruitment in the polymerase complex and highlight its novel role in virion assembly, in addition to the canonical RNA transcription and replication functions.IMPORTANCEDouble-stranded RNA viruses infect a wide spectrum of hosts, including animals, plants, fungi, and bacteria. Yet genome replication mechanisms of these viruses are conserved. During the infection cycle, a proteinaceous capsid, the polymerase complex, is formed. An essential component of this capsid is the viral RNA polymerase that replicates and transcribes the enclosed viral genome. The polymerase complex structure is well characterized for many double-stranded RNA viruses. However, much less is known about the hierarchical molecular interactions that take place in building up such complexes. Using the bacteriophage Φ6 self-assembly system, we obtained novel insights into the processes that mediate polymerase subunit incorporation into the polymerase complex for generation of functional structures. The results presented pave the way for the exploitation and engineering of viral self-assembly processes for biomedical and synthetic biology applications. An understanding of viral assembly processes at the molecular level may also facilitate the development of antivirals that target viral capsid assembly.

scholarly journals BPIFB3 interacts with ARFGAP1 and TMED9 to regulate non-canonical autophagy and RNA virus infection

2020 ◽  
pp. jcs.251835
Author(s):  
Azia S. Evans ◽  
Nicholas J. Lennemann ◽  
Carolyn B. Coyne
Keyword(s):  
Mass Spectrometry ◽  
Mammalian Cells ◽  
Viral Infections ◽  
Rna Viruses ◽  
Rna Virus ◽  
Negative Regulator ◽  
Host Factors ◽  
Important Negative Regulator ◽  
Cellular Components

Autophagy is a degradative cellular pathway that targets cytoplasmic contents and organelles for turnover by the lysosome. Various autophagy pathways play key roles in the clearance of viral infections, and many families of viruses have developed unique methods for avoiding degradation. Some positive stranded RNA viruses, such as enteroviruses and flaviviruses, usurp the autophagic pathway to promote their own replication. We previously identified the endoplasmic reticulum-localized protein BPIFB3 as an important negative regulator of non-canonical autophagy that uniquely impacts the replication of enteroviruses and flaviviruses. Here, we find that many components of the canonical autophagy machinery are not required for BPIFB3 depletion induced autophagy and identify the host factors that facilitate its role in the replication of enteroviruses and flaviviruses. Using proximity-dependent biotinylation (BioID) followed by mass spectrometry, we identify ARFGAP1 and TMED9 as two cellular components that interact with BPIFB3 to regulate autophagy and viral replication. Importantly, our data demonstrate that non-canonical autophagy in mammalian cells can be controlled outside of the traditional pathway regulators and define the role of two proteins in BPIFB3 depletion mediated non-canonical autophagy.

scholarly journals Marine RNA Virus Quasispecies Are Distributed throughout the Oceans

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Marli Vlok ◽  
Andrew S. Lang ◽  
Curtis A. Suttle
Keyword(s):  
Amino Acid ◽  
Rna Viruses ◽  
Rna Virus ◽  
Purifying Selection ◽  
Amino Acid Sequences ◽  
Metagenomic Data ◽  
Spatial And Temporal Patterns ◽  
Data Set ◽  
Virus Diversity ◽  
Virus Genomes

ABSTRACTRNA viruses, particularly genetically diverse members of thePicornavirales, are widespread and abundant in the ocean. Gene surveys suggest that there are spatial and temporal patterns in the composition of RNA virus assemblages, but data on their diversity and genetic variability in different oceanographic settings are limited. Here, we show that specific RNA virus genomes have widespread geographic distributions and that the dominant genotypes are under purifying selection. Genomes from three previously unknown picorna-like viruses (BC-1, -2, and -3) assembled from a coastal site in British Columbia, Canada, as well as marine RNA viruses JP-A, JP-B, andHeterosigma akashiwoRNA virus exhibited different biogeographical patterns. Thus, biotic factors such as host specificity and viral life cycle, and not just abiotic processes such as dispersal, affect marine RNA virus distribution. Sequence differences relative to reference genomes imply that virus quasispecies are under purifying selection, with synonymous single-nucleotide variations dominating in genomes from geographically distinct regions resulting in conservation of amino acid sequences. Conversely, sequences from coastal South Africa that mapped to marine RNA virus JP-A exhibited more nonsynonymous mutations, probably representing amino acid changes that accumulated over a longer separation. This biogeographical analysis of marine RNA viruses demonstrates that purifying selection is occurring across oceanographic provinces. These data add to the spectrum of known marine RNA virus genomes, show the importance of dispersal and purifying selection for these viruses, and indicate that closely related RNA viruses are pathogens of eukaryotic microbes across oceans.IMPORTANCEVery little is known about aquatic RNA virus populations and genome evolution. This is the first study that analyzes marine environmental RNA viral assemblages in an evolutionary and broad geographical context. This study contributes the largest marine RNA virus metagenomic data set to date, substantially increasing the sequencing space for RNA viruses and also providing a baseline for comparisons of marine RNA virus diversity. The new viruses discovered in this study are representative of the most abundant family of marine RNA viruses, theMarnaviridae, and expand our view of the diversity of this important group. Overall, our data and analyses provide a foundation for interpreting marine RNA virus diversity and evolution.

scholarly journals Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis

2018 ◽  
Author(s):  
Adrian Viehweger ◽  
Sebastian Krautwurst ◽  
Kevin Lamkiewicz ◽  
Ramakanth Madhugiri ◽  
John Ziebuhr ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Rna Virus ◽  
Consensus Sequence ◽  
Full Length ◽  
Viral Rna ◽  
Genome Replication ◽  
Nanopore Sequencing ◽  
Human Coronavirus ◽  
Viral Rnas ◽  
Virus Genomes

Sequence analyses of RNA virus genomes remain challenging due to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called 'quasispecies'. Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (i) RNA virus genomes and (ii) subgenome-length (sg) RNAs comprised of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. Using DRS, we were able to map the longest (~26 kb) contiguous read to the viral reference genome. By combining Illumina and nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV) 229E genome (27.3 kb). Furthermore, using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by demonstrating the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that direct RNA sequencing may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.

scholarly journals The Surface-Exposed PA51-72-Loop of the Influenza A Virus Polymerase Is Required for Viral Genome Replication

2018 ◽  
Vol 92 (16) ◽  
Author(s):  
Benjamin E. Nilsson-Payant ◽  
Jane Sharps ◽  
Narin Hengrung ◽  
Ervin Fodor
Keyword(s):  
Influenza A Virus ◽  
Viral Genome ◽  
Influenza A ◽  
Replication Initiation ◽  
Genome Replication ◽  
Influenza A Viruses ◽  
Viral Polymerase ◽  
Virus Rna ◽  
Endonuclease Domain

ABSTRACTThe heterotrimeric influenza A virus RNA-dependent RNA polymerase complex, composed of PB1, PB2, and PA subunits, is responsible for transcribing and replicating the viral RNA genome. The N-terminal endonuclease domain of the PA subunit performs endonucleolytic cleavage of capped host RNAs to generate capped RNA primers for viral transcription. A surface-exposed flexible loop (PA51-72-loop) in the PA endonuclease domain has been shown to be dispensable for endonuclease activity. Interestingly, the PA51-72-loop was found to form different intramolecular interactions depending on the conformational arrangement of the polymerase. In this study, we show that a PA subunit lacking the PA51-72-loop assembles into a heterotrimeric polymerase with PB1 and PB2. We demonstrate that in a cellular context, the PA51-72-loop is required for RNA replication but not transcription by the viral polymerase. In agreement, recombinant viral polymerase lacking the PA51-72-loop is able to carry out cap-dependent transcription but is inhibited inde novoreplication initiationin vitro. Furthermore, viral RNA (vRNA) synthesis is also restricted during ApG-primed extension, indicating that the PA51-72-loop is required not only for replication initiation but also for elongation on a cRNA template. We propose that the PA51-72-loop plays a role in the stabilization of the replicase conformation of the polymerase. Together, these results further our understanding of influenza virus RNA genome replication in general and highlight a role of the PA endonuclease domain in polymerase function in particular.IMPORTANCEInfluenza A viruses are a major global health threat, not only causing significant morbidity and mortality every year but also having the potential to cause severe pandemic outbreaks like the 1918 influenza pandemic. The viral polymerase is a protein complex which is responsible for transcription and replication of the viral genome and therefore is an attractive target for antiviral drug development. For that purpose it is important to understand the mechanisms of how the virus replicates its genome and how the viral polymerase works on a molecular level. In this report, we characterize the role of the flexible surface-exposed PA51-72-loop in polymerase function and offer new insights into the replication mechanism of influenza A viruses.

scholarly journals Hepatitis E Virus Replication

Viruses ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 719 ◽  
Author(s):  
Robert LeDesma ◽  
Ila Nimgaonkar ◽  
Alexander Ploss
Keyword(s):  
Hepatitis E Virus ◽  
Rna Virus ◽  
Hypervariable Region ◽  
Hepatitis E ◽  
Open Reading Frames ◽  
Health Concern ◽  
Viral Gene ◽  
Genome Replication ◽  
Replication Strategy ◽  
Viral Gene Product

Hepatitis E virus (HEV) is a small quasi-enveloped, (+)-sense, single-stranded RNA virus belonging to the Hepeviridae family. There are at least 20 million HEV infections annually and 60,000 HEV-related deaths worldwide. HEV can cause up to 30% mortality in pregnant women and progress to liver cirrhosis in immunocompromised individuals and is, therefore, a greatly underestimated public health concern. Although a prophylactic vaccine for HEV has been developed, it is only licensed in China, and there is currently no effective, non-teratogenic treatment. HEV encodes three open reading frames (ORFs). ORF1 is the largest viral gene product, encoding the replicative machinery of the virus including a methyltransferase, RNA helicase, and an RNA-dependent RNA polymerase. ORF1 additionally contains a number of poorly understood domains including a hypervariable region, a putative protease, and the so-called ‘X’ and ‘Y’ domains. ORF2 is the viral capsid essential for formation of infectious particles and ORF3 is a small protein essential for viral release. In this review, we focus on the domains encoded by ORF1, which collectively mediate the virus’ asymmetric genome replication strategy. We summarize what is known, unknown, and hotly debated regarding the coding and non-coding regions of HEV ORF1, and present a model of how HEV replicates its genome.

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

Viruses ◽  
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.

scholarly journals Protein Nucleotidylylation in +ssRNA Viruses

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1549
Author(s):  
Alice-Roza Eruera ◽  
Alice M. McSweeney ◽  
Geena M. McKenzie-Goldsmith ◽  
Vernon K. Ward
Keyword(s):  
Life Cycle ◽  
Rna Polymerase ◽  
Viral Replication ◽  
Viral Protein ◽  
Rna Viruses ◽  
Replication Initiation ◽  
Genome Replication ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotidyl Transferase ◽  
Initiation Of Translation

Nucleotidylylation is a post-transcriptional modification important for replication in the picornavirus supergroup of RNA viruses, including members of the Caliciviridae, Coronaviridae, Picornaviridae and Potyviridae virus families. This modification occurs when the RNA-dependent RNA polymerase (RdRp) attaches one or more nucleotides to a target protein through a nucleotidyl-transferase reaction. The most characterized nucleotidylylation target is VPg (viral protein genome-linked), a protein linked to the 5′ end of the genome in Caliciviridae, Picornaviridae and Potyviridae. The nucleotidylylation of VPg by RdRp is a critical step for the VPg protein to act as a primer for genome replication and, in Caliciviridae and Potyviridae, for the initiation of translation. In contrast, Coronaviridae do not express a VPg protein, but the nucleotidylylation of proteins involved in replication initiation is critical for genome replication. Furthermore, the RdRp proteins of the viruses that perform nucleotidylylation are themselves nucleotidylylated, and in the case of coronavirus, this has been shown to be essential for viral replication. This review focuses on nucleotidylylation within the picornavirus supergroup of viruses, including the proteins that are modified, what is known about the nucleotidylylation process and the roles that these modifications have in the viral life cycle.

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