scholarly journals Life cycle process dependencies of positive-sense RNA viruses suggest strategies for inhibiting productive cellular infection

2021 ◽  
Vol 18 (184) ◽  
Author(s):  
Harsh Chhajer ◽  
Vaseef A. Rizvi ◽  
Rahul Roy
Keyword(s):  
Life Cycle ◽  
Early Life ◽  
Rna Viruses ◽  
Virus Assembly ◽  
Rna Virus ◽  
Rna Degradation ◽  
Stochastic Simulations ◽  
Virus Growth ◽  
Cytoplasmic Rna ◽  
Positive Strand Rna

Life cycle processes of positive-strand (+)RNA viruses are broadly conserved across families, yet they employ different strategies to grow in the cell. Using a generalized dynamical model for intracellular (+)RNA virus growth, we decipher these life cycle determinants and their dependencies for several viruses and parse the effects of viral mutations, drugs and host cell permissivity. We show that poliovirus employs rapid replication and virus assembly, whereas the Japanese encephalitis virus leverages its higher rate of translation and efficient cellular reorganization compared to the hepatitis C virus. Stochastic simulations demonstrate infection extinction if all seeding (inoculating) viral RNA degrade before establishing robust replication critical for infection. The probability of this productive cellular infection, ‘cellular infectivity’, is affected by virus–host processes and defined by early life cycle events and viral seeding. An increase in cytoplasmic RNA degradation and delay in vesicular compartment formation reduces infectivity, more so when combined. Synergy among these parameters in limiting (+)RNA virus infection as predicted by our model suggests new avenues for inhibiting infections by targeting the early life cycle bottlenecks.

scholarly journals Life cycle process dependencies of positive-sense RNA viruses suggest strategies for inhibiting productive cellular infection

2020 ◽  
Author(s):  
Harsh Chhajer ◽  
Vaseef A. Rizvi ◽  
Rahul Roy
Keyword(s):  
Life Cycle ◽  
Early Life ◽  
Viral Infections ◽  
Rna Viruses ◽  
Virus Assembly ◽  
Stochastic Simulations ◽  
Virus Growth ◽  
Ssrna Virus ◽  
Positive Strand Rna ◽  
Life Cycle Process

AbstractLife cycle processes of positive-strand (+)RNA viruses are broadly conserved across families, yet they employ different life cycle strategies to grow in the cell. Using a generalized dynamical model for intracellular (+)ssRNA virus growth, we decipher these life cycle determinants and their dependencies for several viruses and parse the effect of viral mutations and host cell permissivity. We show that Poliovirus employs rapid replication and virus assembly whereas Japanese Encephalitis virus leverages its higher rate of translation and efficient cellular reorganization compared to Hepatitis C virus. Stochastic simulations of the model demonstrate infection extinction if all seeding viral RNA degrade before establishing robust replication. The probability of productive cellular infection is affected by virus-host processes, defined by early life cycle events and viral seeding. Synergy among these parameters in limiting infection suggests new avenues for inhibiting viral infections by targeting early life cycle bottlenecks.

scholarly journals Palmitoylation of the envelope membrane proteins GP5 and M of porcine reproductive and respiratory syndrome virus is essential for virus growth

2021 ◽  
Vol 17 (4) ◽  
pp. e1009554
Author(s):  
Minze Zhang ◽  
Xiaoliang Han ◽  
Klaus Osterrieder ◽  
Michael Veit
Keyword(s):  
Membrane Proteins ◽  
Virus Assembly ◽  
Rna Virus ◽  
Respiratory Syndrome Virus ◽  
Virus Growth ◽  
Close Proximity ◽  
Infected Cells ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna ◽  
Exocytic Pathway

Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped positive-strand RNA virus in the Arteiviridae family, is a major pathogen affecting pigs worldwide. The membrane (glyco)proteins GP5 and M form a disulfide-linked dimer, which is a major component of virions. GP5/M are required for virus budding, which occurs at membranes of the exocytic pathway. Both GP5 and M feature a short ectodomain, three transmembrane regions, and a long cytoplasmic tail, which contains three and two conserved cysteines, respectively, in close proximity to the transmembrane span. We report here that GP5 and M of PRRSV-1 and -2 strains are palmitoylated at the cysteines, regardless of whether the proteins are expressed individually or in PRRSV-infected cells. To completely prevent S-acylation, all cysteines in GP5 and M have to be exchanged. If individual cysteines in GP5 or M were substituted, palmitoylation was reduced, and some cysteines proved more important for efficient palmitoylation than others. Neither infectious virus nor genome-containing particles could be rescued if all three cysteines present in GP5 or both present in M were replaced in a PRRSV-2 strain, indicating that acylation is essential for virus growth. Viruses lacking one or two acylation sites in M or GP5 could be rescued but grew to significantly lower titers. GP5 and M lacking acylation sites form dimers and GP5 acquires Endo-H resistant carbohydrates in the Golgi apparatus suggesting that trafficking of the membrane proteins to budding sites is not disturbed. Likewise, GP5 lacking two acylation sites is efficiently incorporated into virus particles and these viruses exhibit no reduction in cell entry. We speculate that multiple fatty acids attached to GP5 and M in the endoplasmic reticulum are required for clustering of GP5/M dimers at Golgi membranes and constitute an essential prerequisite for virus assembly.

scholarly journals Morphogenesis of Endoplasmic Reticulum Membrane-Invaginated Vesicles during Beet Black Scorch Virus Infection: Role of Auxiliary Replication Protein and New Implications of Three-Dimensional Architecture

2015 ◽  
Vol 89 (12) ◽  
pp. 6184-6195 ◽  
Author(s):  
Xiuling Cao ◽  
Xuejiao Jin ◽  
Xiaofeng Zhang ◽  
Ying Li ◽  
Chunyan Wang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Rna Viruses ◽  
Rna Virus ◽  
Three Dimensional ◽  
Replication Protein ◽  
Positive Strand ◽  
Content Type ◽  
Positive Strand Rna ◽  
Tomographic Analysis ◽  
Beet Black Scorch Virus

ABSTRACTAll well-characterized positive-strand RNA viruses[(+)RNA viruses] induce the formation of host membrane-bound viral replication complexes (VRCs), yet the underlying mechanism and machinery for VRC formation remain elusive. We report here the biogenesis and topology of theBeet black scorch virus(BBSV) replication complex. Distinct cytopathological changes typical of endoplasmic reticulum (ER) aggregation and vesiculation were observed in BBSV-infectedNicotiana benthamianacells. Immunogold labeling of the auxiliary replication protein p23 and double-stranded RNA (dsRNA) revealed that the ER-derived membranous spherules provide the site for BBSV replication. Further studies indicated that p23 plays a crucial role in mediating the ER rearrangement. Three-dimensional electron tomographic analysis revealed the formation of multiple ER-originated vesicle packets. Each vesicle packet enclosed a few to hundreds of independent spherules that were invaginations of the ER membranes into the lumen. Strikingly, these vesicle packets were connected to each other via tubules, a rearrangement event that is rare among other virus-induced membrane reorganizations. Fibrillar contents within the spherules were also reconstructed by electron tomography, which showed diverse structures. Our results provide the first, to our knowledge, three-dimensional ultrastructural analysis of membrane-bound VRCs of a plant (+)RNA virus and should help to achieve a better mechanistic understanding of the organization and microenvironment of plant (+)RNA virus replication complexes.IMPORTANCEAssembly of virus replication complexes for all known positive-strand RNA viruses depends on the extensive remodeling of host intracellular membranes.Beet black scorch virus, a necrovirus in the familyTombusviridae, invaginates the endoplasmic reticulum (ER) membranes to form spherules in infected cells. Double-stranded RNAs, the viral replication intermediate, and the viral auxiliary replication protein p23 are all localized within such viral spherules, indicating that these are the sites for generating progeny viral RNAs. Furthermore, the BBSV p23 protein could to some extent reorganize the ER when transiently expressed inN. benthamiana. Electron tomographic analysis resolves the three-dimensional (3D) architecture of such spherules, which are connected to the cytoplasm via a neck-like structure. Strikingly, different numbers of spherules are enclosed in ER-originated vesicle packets that are connected to each other via tubule-like structures. Our results have significant implications for further understanding the mechanisms underlying the replication of positive-strand RNA viruses.

scholarly journals Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-Dependent RNA Polymerases

2014 ◽  
Vol 89 (1) ◽  
pp. 275-286 ◽  
Author(s):  
Grace Campagnola ◽  
Seth McDonald ◽  
Stéphanie Beaucourt ◽  
Marco Vignuzzi ◽  
Olve B. Peersen
Keyword(s):  
Rna Viruses ◽  
Viral Rna ◽  
Mutation Rates ◽  
Rapid Adaptation ◽  
Replication Fidelity ◽  
Virus Growth ◽  
Polymerase Structure ◽  
Positive Strand Rna

ABSTRACTViral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates andin vivofidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects onin vitronucleotide discrimination, and these effects are strongly correlated with elongation rates andin vivomutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity.IMPORTANCEPositive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. In this work, we use a structure-based approach to engineer mutations in viral polymerases and study their effects onin vitronucleotide discrimination as well as virus growth and genome replication fidelity. These results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growthin vivo. These findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness.

Efficient RNA virus targeting via CRISPR-CasRx in fish

2021 ◽  
Author(s):  
Qing Wang ◽  
Yun Liu ◽  
Chong Han ◽  
Min Yang ◽  
Fengqi Huang ◽  
...  
Keyword(s):  
Interference Effect ◽  
Rna Viruses ◽  
Rna Virus ◽  
Economic Losses ◽  
Nervous Necrosis Virus ◽  
Necrosis Virus ◽  
Viral Pathogens ◽  
Positive Strand Rna ◽  
Rna Knockdown

The emergence of the CRISPR-Cas system as a technology has transformed our ability to modify nucleic acids, and the CRISPR-Cas13 system has been used to target RNA. CasRx is a small sized type VI-D effector (Cas13d) with RNA knockdown efficiency that may have an interference effect on RNA viruses. However, the RNA virus-targeting activity of CasRx still needs to be verified in vivo in vertebrates. In this study, we successfully engineered a highly effective CasRx system for fish virus interference. We designed synthetic mRNA coding for CasRx and used CRISPR RNAs to guide it to target the grouper nervous necrosis virus (RGNNV). This technique resulted in significant interference with virus infections both in vitro and in vivo . These results indicate that CRISPR/CasRx can be used to engineer interference against RNA viruses in fish, which provides a potential novel mechanism for RNA-guided immunity against other RNA viruses in vertebrates. Importance RNA viruses are most important viral pathogens infecting vertebrates and mammals. RNA virus populations are highly dynamic due to short generation times, large population sizes, and high mutation frequencies. Therefore, it is difficult to find a widely effective ways to inhibit RNA viruses. Therefore, we urgently need to develop effective antiviral methods. CasRx is a small sized type VI-D effector (Cas13d) with RNA knockdown efficiency that can have an interference effect on RNA viruses. Nervous necrosis virus (NNV), a non-enveloped positive-strand RNA virus, is one of the most serious viral pathogens infecting more than 40 cultured fish species resulting in huge economic losses worldwide. Here, we establish a novel efective CasRx system for RNA virus interference using NNV and grouper (Epinephelus coioices) as model. Our data show that CasRx have the most robust for RNA virus interference applications in fish and demonstrate its suitability for studying key questions relating to virus biology.

scholarly journals Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in Drosophila Cells

2001 ◽  
Vol 75 (23) ◽  
pp. 11664-11676 ◽  
Author(s):  
David J. Miller ◽  
Michael D. Schwartz ◽  
Paul Ahlquist
Keyword(s):  
Rna Viruses ◽  
Rna Virus ◽  
Protein A ◽  
Rna Replication ◽  
Yeast Cells ◽  
Flock House Virus ◽  
Mitochondrial Membranes ◽  
Positive Strand ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna

ABSTRACT The identification and characterization of host cell membranes essential for positive-strand RNA virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents. The alphanodavirus flock house virus (FHV) is a positive-strand RNA virus with one of the smallest known genomes among animal RNA viruses, and it can replicate in insect, plant, mammalian, and yeast cells. To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication. We detected protein A within 4 h after infection ofDrosophila DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other positive-strand RNA viruses. Confocal immunofluorescence microscopy and virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes. Electron microscopy revealed 40- to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombusvirus- and togavirus-induced membrane structures. We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.

scholarly journals A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses

2008 ◽  
Vol 83 (4) ◽  
pp. 1981-1991 ◽  
Author(s):  
Huiquan Liu ◽  
Yanping Fu ◽  
Daohong Jiang ◽  
Guoqing Li ◽  
Jun Xie ◽  
...  
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Hepatitis E Virus ◽  
Rna Viruses ◽  
Rna Virus ◽  
Hepatitis E ◽  
Genomic Rna ◽  
Human Virus ◽  
Positive Strand ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna

ABSTRACT Previously, we reported that three double-stranded RNA (dsRNA) segments, designated L-, M-, and S-dsRNAs, were detected in Sclerotinia sclerotiorum strain Ep-1PN. Of these, the M-dsRNA segment was derived from the genomic RNA of a potexvirus-like positive-strand RNA virus, Sclerotinia sclerotiorum debilitation-associated RNA virus. Here, we present the complete nucleotide sequence of the L-dsRNA, which is 6,043 nucleotides in length, excluding the poly(A) tail. Sequence analysis revealed the presence of a single open reading frame (nucleotide positions 42 to 5936) that encodes a protein with significant similarity to the replicases of the “alphavirus-like” supergroup of positive-strand RNA viruses. A sequence comparison of the L-dsRNA-encoded putative replicase protein containing conserved methyltransferase, helicase, and RNA-dependent RNA polymerase motifs showed that it has significant sequence similarity to the replicase of Hepatitis E virus, a virus infecting humans. Furthermore, we present convincing evidence that the virus-like L-dsRNA could replicate independently with only a slight impact on growth and virulence of its host. Our results suggest that the L-dsRNA from strain Ep-1PN is derived from the genomic RNA of a positive-strand RNA virus, which we named Sclerotinia sclerotiorum RNA virus L (SsRV-L). As far as we know, this is the first report of a positive-strand RNA mycovirus that is related to a human virus. Phylogenetic and sequence analyses of the conserved motifs of the RNA replicase of SsRV-L showed that it clustered with the rubi-like viruses and that it is related to the plant clostero-, beny- and tobamoviruses and to the insect omegatetraviruses. Considering the fact that these related alphavirus-like positive-strand RNA viruses infect a wide variety of organisms, these findings suggest that the ancestral positive-strand RNA viruses might be of ancient origin and/or they might have radiated horizontally among vertebrates, insects, plants, and fungi.

scholarly journals A decade after the generation of a negative-sense RNA virus from cloned cDNA – what have we learned?

2002 ◽  
Vol 83 (11) ◽  
pp. 2635-2662 ◽  
Author(s):  
Gabriele Neumann ◽  
Michael A. Whitt ◽  
Yoshihiro Kawaoka
Keyword(s):  
Reverse Genetics ◽  
De Novo ◽  
Rna Viruses ◽  
Virus Assembly ◽  
Rna Virus ◽  
Viral Proteins ◽  
Laboratory Method ◽  
Cell Immune Response ◽  
Cloned Cdna ◽  
Negative Sense

Since the first generation of a negative-sense RNA virus entirely from cloned cDNA in 1994, similar reverse genetics systems have been established for members of most genera of the Rhabdo- and Paramyxoviridae families, as well as for Ebola virus (Filoviridae). The generation of segmented negative-sense RNA viruses was technically more challenging and has lagged behind the recovery of nonsegmented viruses, primarily because of the difficulty of providing more than one genomic RNA segment. A member of the Bunyaviridae family (whose genome is composed of three RNA segments) was first generated from cloned cDNA in 1996, followed in 1999 by the production of influenza virus, which contains eight RNA segments. Thus, reverse genetics, or the de novo synthesis of negative-sense RNA viruses from cloned cDNA, has become a reliable laboratory method that can be used to study this large group of medically and economically important viruses. It provides a powerful tool for dissecting the virus life cycle, virus assembly, the role of viral proteins in pathogenicity and the interplay of viral proteins with components of the host cell immune response. Finally, reverse genetics has opened the way to develop live attenuated virus vaccines and vaccine vectors.

scholarly journals Coronavirus RNA Synthesis Takes Place within Membrane-Bound Sites

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2540
Author(s):  
Nicole Doyle ◽  
Jennifer Simpson ◽  
Philippa C. Hawes ◽  
Helena J. Maier
Keyword(s):  
Life Cycle ◽  
Infectious Bronchitis Virus ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Viral Rna ◽  
Host Cells ◽  
Infectious Bronchitis ◽  
Welfare Issue ◽  
Membrane Bound ◽  
Positive Strand Rna

Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry, as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection, while a proportion is trafficked to the cytoplasm. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of these viruses is imperative in order to effectively find ways to control them.

scholarly journals On the Host Side of the Hepatitis E Virus Life Cycle

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1294 ◽  
Author(s):  
Noémie Oechslin ◽  
Darius Moradpour ◽  
Jérôme Gouttenoire
Keyword(s):  
Life Cycle ◽  
Viral Entry ◽  
Hepatitis E Virus ◽  
Molecular Mechanisms ◽  
Rna Virus ◽  
Hepatitis E ◽  
Host Factors ◽  
Model Systems ◽  
Novel Technologies ◽  
Positive Strand Rna

Hepatitis E virus (HEV) infection is one of the most common causes of acute hepatitis in the world. HEV is an enterically transmitted positive-strand RNA virus found as a non-enveloped particle in bile as well as stool and as a quasi-enveloped particle in blood. Current understanding of the molecular mechanisms and host factors involved in productive HEV infection is incomplete, but recently developed model systems have facilitated rapid progress in this area. Here, we provide an overview of the HEV life cycle with a focus on the host factors required for viral entry, RNA replication, assembly and release. Further developments of HEV model systems and novel technologies should yield a broader picture in the future.

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