-
Genome Biology Dec 2019CRISPR-Cas systems endow bacterial and archaeal species with adaptive immunity mechanisms to fend off invading phages and foreign genetic elements. CRISPR-Cas9 has been...
BACKGROUND
CRISPR-Cas systems endow bacterial and archaeal species with adaptive immunity mechanisms to fend off invading phages and foreign genetic elements. CRISPR-Cas9 has been harnessed to confer virus interference against DNA viruses in eukaryotes, including plants. In addition, CRISPR-Cas13 systems have been used to target RNA viruses and the transcriptome in mammalian and plant cells. Recently, CRISPR-Cas13a has been shown to confer modest interference against RNA viruses. Here, we characterized a set of different Cas13 variants to identify those with the most efficient, robust, and specific interference activities against RNA viruses in planta using Nicotiana benthamiana.
RESULTS
Our data show that LwaCas13a, PspCas13b, and CasRx variants mediate high interference activities against RNA viruses in transient assays. Moreover, CasRx mediated robust interference in both transient and stable overexpression assays when compared to the other variants tested. CasRx targets either one virus alone or two RNA viruses simultaneously, with robust interference efficiencies. In addition, CasRx exhibits strong specificity against the target virus and does not exhibit collateral activity in planta.
CONCLUSIONS
Our data establish CasRx as the most robust Cas13 variant for RNA virus interference applications in planta and demonstrate its suitability for studying key questions relating to virus biology.
Topics: CRISPR-Cas Systems; Host-Pathogen Interactions; Plant Immunity; Plant Viruses; RNA Viruses; Nicotiana
PubMed: 31791381
DOI: 10.1186/s13059-019-1881-2 -
Nature Reviews. Genetics Jul 2020A study reports on the suitability of the yeast as a platform for the assembly and maintenance of diverse RNA virus genomes, including SARS-CoV-2.
A study reports on the suitability of the yeast as a platform for the assembly and maintenance of diverse RNA virus genomes, including SARS-CoV-2.
Topics: Betacoronavirus; COVID-19; Cloning, Molecular; Coronavirus Infections; Genomics; Humans; Pandemics; Pneumonia, Viral; RNA Viruses; Severe acute respiratory syndrome-related coronavirus; SARS-CoV-2
PubMed: 32404960
DOI: 10.1038/s41576-020-0246-8 -
Viruses Jan 2021Liquid-liquid phase separation (LLPS) represents a major physiochemical principle to organize intracellular membrane-less structures. Studies with non-segmented... (Review)
Review
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.
Topics: Animals; Host-Pathogen Interactions; Humans; Liquid-Liquid Extraction; RNA Virus Infections; RNA Viruses; RNA, Viral; Viral Proteins; Virus Physiological Phenomena; Virus Replication
PubMed: 33477448
DOI: 10.3390/v13010126 -
Infectious Agents and Disease Aug 1994The experimental evidence available for animal and plant RNA viruses, as well as other RNA genetic elements (viroids, satellites, retroelements, etc.), reinforces the... (Review)
Review
The experimental evidence available for animal and plant RNA viruses, as well as other RNA genetic elements (viroids, satellites, retroelements, etc.), reinforces the view that many different types of genetic alterations may occur during RNA genome replication. This is fundamentally because of infidelity of genome replication and large population sizes. Homologous and heterologous recombination, as well as gene reassortments occur frequently during replication of retroviruses and most riboviruses, especially those that use enzymes with limited processivity. Following the generation of variant genomes, selection, which is dependent on environmental parameters in ways that are poorly understood, sorts out those genome fits enough to generate viable quasispecies. Chance events can also be destabilizing, as illustrated by recent results on fitness loss and other phenotypic changes accompanying bottleneck transmission. Variation, selection, and random sampling of genomes occur continuously and unavoidably during virus evolution. Evolution of RNA viruses is largely unpredictable because of the stochastic nature of mutation and recombination events, as well as the subtle effects of chance transmission events and host/environmental factors. Among environmental factors, alterations resulting from human intervention (deforestation, agricultural activities, global climatic changes, etc.) may alter dispersal patterns and provide new adaptive possibilities to viral quasispecies. Current understanding of RNA virus evolution suggests several strategies to control and diagnose viral diseases. The new generation of chemically defined vaccines and diagnostic reagents (monoclonal antibodies, peptide antigens, oligonucleotides for polymerase chain reaction amplification, etc.) may be adequate to prevent disease and detect some or even most of the circulating quasispecies of any given RNA pathogen. However, the dynamics of viral quasispecies mandate careful consideration of those reagents to be incorporated into diagnostic kits. Broadening diagnosis without jeopardizing specificity of detection will be challenging. There is a finite probability (impossible to quantify at present) that a defined vaccine may promote selection of escape mutants or a particular diagnostic kit may fail to detect a viral pathogen. Of particular concern are the potential long-term effects of weak selective pressures that may initially go unnoticed. Variant viruses resulting from evolutionary pressure imposed by vaccines or drugs may insidiously and gradually replace previous quasispecies. The great potential for variation and phenotypic diversity of some important RNA virus pathogens (human immunodeficiency virus, the hepatitis viruses, the newly recognized human hantaviruses, etc.) has become clear. Prevention and therapy should rely on multicomponent vaccines and antiviral agents to address the complexity of RNA quasispecies mutant spectra.(ABSTRACT TRUNCATED AT 400 WORDS)
Topics: Biological Evolution; RNA Viruses; Viral Vaccines; Virus Diseases; Virus Replication
PubMed: 7827789
DOI: No ID Found -
Current Opinion in Virology Apr 2014Negative strand RNA virus (NSV) genomes are never free, but always found assembled with multiple copies of their nucleoprotein, as RNPs. A flurry of papers describing... (Review)
Review
Negative strand RNA virus (NSV) genomes are never free, but always found assembled with multiple copies of their nucleoprotein, as RNPs. A flurry of papers describing the X-ray crystal structures of several segmented NSV nucleoproteins have recently appeared. The most significant feature of these various structures is that the arms that are used to oligomerize the nucleoproteins on their genome RNAs are highly flexible, permitting these RNPs to assume virtually unlimited geometries. The structural flexibility of segmented NSV RNPs is undoubtedly important in all aspects of their biology, including genome replication and circularization, and the selection of one copy of each segment for packaging into virus particles.
Topics: Genome, Viral; Models, Molecular; Nucleoproteins; RNA Viruses; RNA, Viral
PubMed: 24486721
DOI: 10.1016/j.coviro.2014.01.003 -
Virus Research Jun 2017Real-time and visual tracking of viral infection is crucial for elucidating the infectious and pathogenesis mechanisms. To track the virus successfully, an efficient... (Review)
Review
Real-time and visual tracking of viral infection is crucial for elucidating the infectious and pathogenesis mechanisms. To track the virus successfully, an efficient labeling method is necessary. In this review, we first discuss the practical labeling techniques for virus tracking in live cells. We then describe the current knowledge of interactions between RNA viruses (especially influenza viruses, immunodeficiency viruses, and Flaviviruses) and host cellular structures, obtained using single particle labeling techniques combined with real-time fluorescence microscopy. Single particle labeling provides an easy system for understanding the RNA virus life cycle.
Topics: Host-Pathogen Interactions; Microscopy, Fluorescence; RNA Viruses; Staining and Labeling; Virology; Virus Replication
PubMed: 28506790
DOI: 10.1016/j.virusres.2017.05.007 -
Expert Review of Vaccines Feb 2015The advent of reverse genetic approaches to manipulate the genomes of both positive (+) and negative (-) sense RNA viruses allowed researchers to harness these genomes...
The advent of reverse genetic approaches to manipulate the genomes of both positive (+) and negative (-) sense RNA viruses allowed researchers to harness these genomes for basic research. Manipulation of positive sense RNA virus genomes occurred first largely because infectious RNA could be transcribed directly from cDNA versions of the RNA genomes. Manipulation of negative strand RNA virus genomes rapidly followed as more sophisticated approaches to provide RNA-dependent RNA polymerase complexes coupled with negative-strand RNA templates were developed. These advances have driven an explosion of RNA virus vaccine vector development. That is, development of approaches to exploit the basic replication and expression strategies of RNA viruses to produce vaccine antigens that have been engineered into their genomes. This study has led to significant preclinical testing of many RNA virus vectors against a wide range of pathogens as well as cancer targets. Multiple RNA virus vectors have advanced through preclinical testing to human clinical evaluation. This review will focus on RNA virus vectors designed to express heterologous genes that are packaged into viral particles and have progressed to clinical testing.
Topics: Genetic Therapy; Genetic Vectors; Humans; RNA Viruses; RNA, Viral; Reverse Genetics; Virion
PubMed: 25382613
DOI: 10.1586/14760584.2015.979798 -
Journal of Molecular Biology Jul 2010
Review
Topics: Adaptation, Biological; Evolution, Molecular; Mutation; Polymorphism, Genetic; RNA Viruses; Selection, Genetic; Virus Replication
PubMed: 20493194
DOI: 10.1016/j.jmb.2010.05.032 -
Current Opinion in Virology Nov 2011In addition to the essential function of the viral RNA as a template during replication of positive-stranded (+)RNA viruses, the RNA also has crucial non-template... (Review)
Review
In addition to the essential function of the viral RNA as a template during replication of positive-stranded (+)RNA viruses, the RNA also has crucial non-template functions. These functions during replication include the recruitment of the viral RNA to the site of replication and assembly of the functional viral replicase complex. The RNA recruitment elements are specifically recognized by the viral replication protein, but also affected by host factors such as elongation factor 1A or P-body proteins. The cis-elements for replicase assembly can partially overlap with RNA recruitment elements and they may provide a platform for the assembly of the replicase complex consisting of viral and host proteins. This review focuses on our current knowledge obtained with tombusviruses and other plant viruses. Altogether, understanding of the non-template functions of the viral RNA during viral replication provides new insights into virus-host interactions.
Topics: DNA Replication; RNA Viruses; RNA, Viral; Templates, Genetic; Tombusvirus; Virus Replication
PubMed: 22440835
DOI: 10.1016/j.coviro.2011.09.011 -
Viruses Feb 2020In this work, we describe the first -infecting leishbunyavirus-the first virus other than (LRV) found in trypanosomatid parasites. Its host is , a human pathogen...
In this work, we describe the first -infecting leishbunyavirus-the first virus other than (LRV) found in trypanosomatid parasites. Its host is , a human pathogen causing infections with a wide range of manifestations from asymptomatic to severe visceral disease. This virus (LBV1) possesses many characteristic features of leishbunyaviruses, such as tripartite organization of its RNA genome, with ORFs encoding RNA-dependent RNA polymerase, surface glycoprotein, and nucleoprotein on L, M, and S segments, respectively. Our phylogenetic analyses suggest that LBV1 originated from leishbunyaviruses of monoxenous trypanosomatids and, probably, is a result of genomic re-assortment. The LBV1 facilitates parasites' infectivity in vitro in primary murine macrophages model. The discovery of a virus in poses the question of whether it influences pathogenicity of this parasite in vivo, similarly to the LRV in other species.
Topics: Animals; Genome, Viral; High-Throughput Nucleotide Sequencing; Leishmania; Macrophages; Mice; Open Reading Frames; Phylogeny; RNA Viruses; RNA-Dependent RNA Polymerase; Reassortant Viruses
PubMed: 32024293
DOI: 10.3390/v12020168