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Advances in Virus Research 2022Reverse genetics is the prospective analysis of how genotype determines phenotype. In a typical experiment, a researcher alters a viral genome, then observes the...
Reverse genetics is the prospective analysis of how genotype determines phenotype. In a typical experiment, a researcher alters a viral genome, then observes the phenotypic outcome. Among RNA viruses, this approach was first applied to positive-strand RNA viruses in the mid-1970s and over nearly 50 years has become a powerful and widely used approach for dissecting the mechanisms of viral replication and pathogenesis. During this time the global health importance of two virus groups, flaviviruses (genus Flavivirus, family Flaviviridae) and betacoronaviruses (genus Betacoronavirus, subfamily Orthocoronavirinae, family Coronaviridae), have dramatically increased, yet these viruses have genomes that are technically challenging to manipulate. As a result, several new techniques have been developed to overcome these challenges. Here I briefly review key historical aspects of positive-strand RNA virus reverse genetics, describe some recent reverse genetic innovations, particularly as applied to flaviviruses and coronaviruses, and discuss their benefits and limitations within the larger context of rigorous genetic analysis.
Topics: Flavivirus; Genome, Viral; Positive-Strand RNA Viruses; RNA Viruses; Reverse Genetics; Virus Replication
PubMed: 35840179
DOI: 10.1016/bs.aivir.2022.03.001 -
Viruses Jul 2020Negative strand RNA viruses (NSVs) include many important human pathogens, such as influenza virus, Ebola virus, and rabies virus. One of the unique characteristics that... (Review)
Review
Negative strand RNA viruses (NSVs) include many important human pathogens, such as influenza virus, Ebola virus, and rabies virus. One of the unique characteristics that NSVs share is the assembly of the nucleocapsid and its role in viral RNA synthesis. In NSVs, the single strand RNA genome is encapsidated in the linear nucleocapsid throughout the viral replication cycle. Subunits of the nucleocapsid protein are parallelly aligned along the RNA genome that is sandwiched between two domains composed of conserved helix motifs. The viral RNA-dependent-RNA polymerase (vRdRp) must recognize the protein-RNA complex of the nucleocapsid and unveil the protected genomic RNA in order to initiate viral RNA synthesis. In addition, vRdRp must continuously translocate along the protein-RNA complex during elongation in viral RNA synthesis. This unique mechanism of viral RNA synthesis suggests that the nucleocapsid may play a regulatory role during NSV replication.
Topics: Genome, Viral; Models, Molecular; Negative-Sense RNA Viruses; Nucleocapsid; Nucleocapsid Proteins; Protein Conformation; Protein Folding; RNA, Viral; RNA-Dependent RNA Polymerase
PubMed: 32751700
DOI: 10.3390/v12080835 -
Trends in Molecular Medicine Jan 2017microRNAs (miRNAs) are non-coding RNAs that regulate many processes within a cell by manipulating protein levels through direct binding to mRNA and influencing... (Review)
Review
microRNAs (miRNAs) are non-coding RNAs that regulate many processes within a cell by manipulating protein levels through direct binding to mRNA and influencing translation efficiency, or mRNA abundance. Recent evidence demonstrates that miRNAs can also affect RNA virus replication and pathogenesis through direct binding to the RNA virus genome or through virus-mediated changes in the host transcriptome. Here, we review the current knowledge on the interaction between RNA viruses and cellular miRNAs. We also discuss how cell and tissue-specific expression of miRNAs can directly affect viral pathogenesis. Understanding the role of cellular miRNAs during viral infection may lead to the identification of novel mechanisms to block RNA virus replication or cell-specific regulation of viral vector targeting.
Topics: Animals; Genome, Viral; Host-Pathogen Interactions; Humans; MicroRNAs; RNA Virus Infections; RNA Viruses; RNA, Viral; Transcriptome; Virus Replication
PubMed: 27989642
DOI: 10.1016/j.molmed.2016.11.003 -
Viruses Mar 2020Macrodomains, enzymes that remove ADP-ribose from proteins, are encoded by several families of RNA viruses and have recently been shown to counter innate immune... (Review)
Review
Macrodomains, enzymes that remove ADP-ribose from proteins, are encoded by several families of RNA viruses and have recently been shown to counter innate immune responses to virus infection. ADP-ribose is covalently attached to target proteins by poly-ADP-ribose polymerases (PARPs), using nicotinamide adenine dinucleotide (NAD+) as a substrate. This modification can have a wide variety of effects on proteins including alteration of enzyme activity, protein-protein interactions, and protein stability. Several PARPs are induced by interferon (IFN) and are known to have antiviral properties, implicating ADP-ribosylation in the host defense response and suggesting that viral macrodomains may counter this response. Recent studies have demonstrated that viral macrodomains do counter the innate immune response by interfering with PARP-mediated antiviral defenses, stress granule formation, and pro-inflammatory cytokine production. Here, we will describe the known functions of the viral macrodomains and review recent literature demonstrating their roles in countering PARP-mediated antiviral responses.
Topics: ADP-Ribosylation; Adenosine Diphosphate Ribose; Cytoplasmic Granules; Humans; Interferons; Mutation; Poly(ADP-ribose) Polymerases; Protein Domains; RNA Virus Infections; RNA Viruses; Viral Nonstructural Proteins; Virus Replication
PubMed: 32244383
DOI: 10.3390/v12040384 -
Seminars in Cell & Developmental Biology May 2020Autophagy is an evolutionarily conserved process central to host metabolism. Among its major functions are conservation of energy during starvation, recycling... (Review)
Review
Autophagy is an evolutionarily conserved process central to host metabolism. Among its major functions are conservation of energy during starvation, recycling organelles, and turnover of long-lived proteins. Besides, autophagy plays a critical role in removing intracellular pathogens and very likely represents a primordial intrinsic cellular defence mechanism. More recent findings indicate that it has not only retained its ability to degrade intracellular pathogens, but also functions to augment and fine tune antiviral immune responses. Interestingly, viruses have also co-evolved strategies to manipulate this pathway and use it to their advantage. Particularly intriguing is infection-dependent activation of autophagy with positive stranded (+)RNA virus infections, which benefit from the pathway without succumbing to lysosomal degradation. In this review we summarise recent data on viral manipulation of autophagy, with a particular emphasis on +RNA viruses and highlight key unanswered questions in the field that we believe merit further attention.
Topics: Autophagy; Humans; RNA Viruses
PubMed: 31382014
DOI: 10.1016/j.semcdb.2019.07.013 -
Proceedings of the National Academy of... Aug 2023RNA viruses rapidly adapt to selective conditions due to the high intrinsic mutation rates of their RNA-dependent RNA polymerases (RdRps). Insertions and deletions...
RNA viruses rapidly adapt to selective conditions due to the high intrinsic mutation rates of their RNA-dependent RNA polymerases (RdRps). Insertions and deletions (indels) in viral genomes are major contributors to both deleterious mutational load and evolutionary novelty, but remain understudied. To characterize the mechanistic details of their formation and evolutionary dynamics during infection, we developed a hybrid experimental-bioinformatic approach. This approach, called MultiMatch, extracts insertions and deletions from ultradeep sequencing experiments, including those occurring at extremely low frequencies, allowing us to map their genomic distribution and quantify the rates at which they occur. Mapping indel mutations in adapting poliovirus and dengue virus populations, we determine the rates of indel generation and identify mechanistic and functional constraints shaping indel diversity. Using poliovirus RdRp variants of distinct fidelity and genome recombination rates, we demonstrate tradeoffs between fidelity and Indel generation. Additionally, we show that maintaining translation frame and viral RNA structures constrain the Indel landscape and that, due to these significant fitness effects, Indels exert a significant deleterious load on adapting viral populations. Conversely, we uncover positively selected Indels that modulate RNA structure, generate protein variants, and produce defective interfering genomes in viral populations. Together, our analyses establish the kinetic and mechanistic tradeoffs between misincorporation, recombination, and Indel rates and reveal functional principles defining the central role of Indels in virus evolution, emergence, and the regulation of viral infection.
Topics: Evolution, Molecular; Genome; Mutation Rate; INDEL Mutation; RNA, Viral; RNA Viruses
PubMed: 37487061
DOI: 10.1073/pnas.2304667120 -
Current Opinion in Virology Aug 2018Virus assembly, a key stage in any viral life cycle, had long been considered to be primarily driven by protein-protein interactions and nonspecific interactions between... (Review)
Review
Virus assembly, a key stage in any viral life cycle, had long been considered to be primarily driven by protein-protein interactions and nonspecific interactions between genomic RNA and capsid protein. We review here a modelling paradigm for RNA virus assembly that illustrates the crucial roles of multiple dispersed, specific interactions between viral genomes and coat proteins in capsid assembly. The model reveals how multiple sequence-structure motifs in the genomic RNA, termed packaging signals, with a shared coat protein recognition motif enable viruses to overcome a viral assembly-equivalent of Levinthal's Paradox in protein folding. The fitness advantages conferred by this mechanism suggest that it should be widespread in viruses, opening up new perspectives on viral evolution and anti-viral therapy.
Topics: Binding Sites; Capsid Proteins; Evolution, Molecular; Genome, Viral; Models, Molecular; Nucleic Acid Conformation; Protein Binding; RNA Viruses; RNA, Viral; Virus Assembly
PubMed: 30078702
DOI: 10.1016/j.coviro.2018.07.003 -
Cellular and Molecular Life Sciences :... May 2022Gastroenteritis is inflammation of the lining of stomach and intestines and causes significant morbidity and mortality worldwide. Many viruses, especially RNA viruses... (Review)
Review
Gastroenteritis is inflammation of the lining of stomach and intestines and causes significant morbidity and mortality worldwide. Many viruses, especially RNA viruses are the most common cause of enteritis. Innate immunity is the first line of host defense against enteric RNA viruses and virus-induced intestinal inflammation. The first layer of defense against enteric RNA viruses in the intestinal tract is intestinal epithelial cells (IECs), dendritic cells and macrophages under the intestinal epithelium. These innate immune cells express pathogen-recognition receptors (PRRs) for recognizing enteric RNA viruses through sensing viral pathogen-associated molecular patterns (PAMPs). As a result of this recognition type I interferon (IFN), type III IFN and inflammasome activation occurs, which function cooperatively to clear infection and reduce viral-induced intestinal inflammation. In this review, we summarize recent findings about mechanisms involved in enteric RNA virus-induced intestinal inflammation. We will provide an overview of the enteric RNA viruses, their RNA sensing mechanisms by host PRRs, and signaling pathways triggered by host PRRs, which shape the intestinal immune response to maintain intestinal homeostasis.
Topics: Humans; Immunity, Innate; Inflammation; Intestinal Mucosa; Intestines; Pathogen-Associated Molecular Pattern Molecules; RNA Viruses
PubMed: 35604464
DOI: 10.1007/s00018-022-04332-z -
Microbiology and Molecular Biology... Sep 2022Negative-sense RNA virus populations are composed of diverse viral components that interact to form a community and shape the outcome of virus infections. At the genomic... (Review)
Review
Negative-sense RNA virus populations are composed of diverse viral components that interact to form a community and shape the outcome of virus infections. At the genomic level, RNA virus populations consist not only of a homogeneous population of standard viral genomes but also of an extremely large number of genome variants, termed viral quasispecies, and nonstandard viral genomes, which include copy-back viral genomes, deletion viral genomes, mini viral RNAs, and hypermutated RNAs. At the particle level, RNA virus populations are composed of pleomorphic particles, particles missing or having additional genomes, and single particles or particle aggregates. As we continue discovering more about the components of negative-sense RNA virus populations and their crucial functions during virus infection, it will become more important to study RNA virus populations as a whole rather than their individual parts. In this review, we will discuss what is known about the components of negative-sense RNA virus communities, speculate how the components of the virus community interact, and summarize what vaccines and antiviral therapies are being currently developed to target or harness these components.
Topics: Antiviral Agents; Genome, Viral; Negative-Sense RNA Viruses; RNA Viruses; RNA, Viral; Virus Assembly
PubMed: 35658541
DOI: 10.1128/mmbr.00086-21 -
Virus Research Apr 2017Like all positive strand RNA viruses, the picornaviruses replicate their genomes using a virally encoded RNA-dependent RNA polymerase enzyme known as 3D. Over the past... (Review)
Review
Like all positive strand RNA viruses, the picornaviruses replicate their genomes using a virally encoded RNA-dependent RNA polymerase enzyme known as 3D. Over the past decade we have made tremendous advances in our understanding of 3D structure and function, including the discovery of a novel mechanism for closing the active site that allows these viruses to easily fine tune replication fidelity and quasispecies distributions. This review summarizes current knowledge of picornaviral polymerase structure and how the enzyme interacts with RNA and other viral proteins to form stable and processive elongation complexes. The picornaviral RdRPs are among the smallest viral polymerases, but their fundamental molecular mechanism for catalysis appears to be generally applicable as a common feature of all positive strand RNA virus polymerases.
Topics: Catalytic Domain; Picornaviridae; Protein Binding; Protein Conformation; Protein Multimerization; RNA, Viral; RNA-Dependent RNA Polymerase; Transcription, Genetic; Viral Proteins; Virus Replication
PubMed: 28163093
DOI: 10.1016/j.virusres.2017.01.026