scholarly journals Effects of TDP2/VPg Unlinkase Activity on Picornavirus Infections Downstream of Virus Translation

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 166 ◽  
Author(s):  
Autumn C. Holmes ◽  
Guido Zagnoli-Vieira ◽  
Keith W. Caldecott ◽  
Bert L. Semler
Keyword(s):  
Growth Kinetics ◽  
Rna Synthesis ◽  
Cell Model ◽  
Retinal Pigment ◽  
Viral Rna ◽  
Cell Protein ◽  
Growth Defect ◽  
Virus Protein ◽  
Virus Growth ◽  
Positive Strand Rna

In this study, we characterized the role of host cell protein tyrosyl-DNA phosphodiesterase 2 (TDP2) activity, also known as VPg unlinkase, in picornavirus infections in a human cell model of infection. TDP2/VPg unlinkase is used by picornaviruses to remove the small polypeptide, VPg (Virus Protein genome-linked, the primer for viral RNA synthesis), from virus genomic RNA. We utilized a CRISPR/Cas-9-generated TDP2 knock out (KO) human retinal pigment epithelial-1 (hRPE-1) cell line, in addition to the wild type (WT) counterpart for our studies. We determined that in the absence of TDP2, virus growth kinetics for two enteroviruses (poliovirus and coxsackievirus B3) were delayed by about 2 h. Virus titers were reduced by ~2 log10 units for poliovirus and 0.5 log10 units for coxsackievirus at 4 hours post-infection (hpi), and by ~1 log10 unit at 6 hpi for poliovirus. However, virus titers were nearly indistinguishable from those of control cells by the end of the infectious cycle. We determined that this was not the result of an alternative source of VPg unlinkase activity being activated in the absence of TPD2 at late times of infection. Viral protein production in TDP2 KO cells was also substantially reduced at 4 hpi for poliovirus infection, consistent with the observed growth kinetics delay, but reached normal levels by 6 hpi. Interestingly, this result differs somewhat from what has been reported previously for the TDP2 KO mouse cell model, suggesting that either cell type or species-specific differences might be playing a role in the observed phenotype. We also determined that catalytically inactive TDP2 does not rescue the growth defect, confirming that TDP2 5′ phosphodiesterase activity is required for efficient virus replication. Importantly, we show for the first time that polysomes can assemble efficiently on VPg-linked RNA after the initial round of translation in a cell culture model, but both positive and negative strand RNA production is impaired in the absence of TDP2 at mid-times of infection, indicating that the presence of VPg on the viral RNA affects a step in the replication cycle downstream of translation (e.g., RNA synthesis). In agreement with this conclusion, we found that double-stranded RNA production (a marker of viral RNA synthesis) is delayed in TDP2 KO RPE-1 cells. Moreover, we show that premature encapsidation of nascent, VPg-linked RNA is not responsible for the observed virus growth defect. Our studies provide the first lines of evidence to suggest that either negative- or positive-strand RNA synthesis (or both) is a likely candidate for the step that requires the removal of VPg from the RNA for an enterovirus infection to proceed efficiently.

scholarly journals Studies on the Mechanism of Interferon Action

1970 ◽  
Vol 56 (1) ◽  
pp. 149-171 ◽  
Author(s):  
Robert M. Friedman
Keyword(s):  
Protein Synthesis ◽  
Genetic Information ◽  
Rna Synthesis ◽  
Rna Virus ◽  
Viral Rna ◽  
Virus Protein ◽  
Dna Viruses ◽  
Virus Growth ◽  
Rna And Protein Synthesis ◽  
Main Action

Interferon does not inactivate viruses or viral RNA. Virus growth is inhibited in interferon-treated cells, but apart from conferring resistance to virus growth, no other effect of interferon on cells has been definitely shown to take place. Interferon binds to cells even in the cold, but a period of incubation at 37°C is required for development of antiviral activity. Cytoplasmic uptake of interferon has not been unequivocally demonstrated. Studies with antimetabolites indicate that the antiviral action of interferon requires host RNA and protein synthesis. Experiments with 2-mercapto-1(ß-4-pyridethyl) benzimidazole (MPB) suggest that an additional step is required between the binding and the synthesis of macromolecules. Interferon does not affect the adsorption, penetration, or uncoating of RNA or DNA viruses, but viral RNA synthesis is inhibited in cells infected with RNA viruses. The main action of interferon appears to be the inhibition of the translation of virus genetic information probably by inhibiting the initiation of virus protein synthesis.

scholarly journals Cell-Dependent Role for the Poliovirus 3′ Noncoding Region in Positive-Strand RNA Synthesis

2004 ◽  
Vol 78 (3) ◽  
pp. 1344-1351 ◽  
Author(s):  
David M. Brown ◽  
Steven E. Kauder ◽  
Christopher T. Cornell ◽  
Gwendolyn M. Jang ◽  
Vincent R. Racaniello ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Rna Replication ◽  
Mutant Virus ◽  
Noncoding Region ◽  
Viral Rna ◽  
Wild Type Virus ◽  
Genomic Rna ◽  
Virus Growth ◽  
Positive Strand ◽  
Positive Strand Rna

ABSTRACT We previously reported the isolation of a mutant poliovirus lacking the entire genomic RNA 3′ noncoding region. Infection of HeLa cell monolayers with this deletion mutant revealed only a minor defect in the levels of viral RNA replication. To further analyze the consequences of the genomic 3′ noncoding region deletion, we examined viral RNA replication in a neuroblastoma cell line, SK-N-SH cells. The minor genomic RNA replication defect in HeLa cells was significantly exacerbated in the SK-N-SH cells, resulting in a decreased capacity for mutant virus growth. Analysis of the nature of the RNA replication deficiency revealed that deleting the poliovirus genomic 3′ noncoding region resulted in a positive-strand RNA synthesis defect. The RNA replication deficiency in SK-N-SH cells was not due to a major defect in viral translation or viral protein processing. Neurovirulence of the mutant virus was determined in a transgenic mouse line expressing the human poliovirus receptor. Greater than 1,000 times more mutant virus was required to paralyze 50% of inoculated mice, compared to that with wild-type virus. These data suggest that, together with a cellular factor(s) that is limiting in neuronal cells, the poliovirus 3′ noncoding region is involved in positive-strand synthesis during genome replication.

scholarly journals Measles Virus Circumvents the Host Interferon Response by Different Actions of the C and V Proteins

2008 ◽  
Vol 82 (17) ◽  
pp. 8296-8306 ◽  
Author(s):  
Yuichiro Nakatsu ◽  
Makoto Takeda ◽  
Shinji Ohno ◽  
Yuta Shirogane ◽  
Masaharu Iwasaki ◽  
...  
Keyword(s):  
Protein Expression ◽  
Measles Virus ◽  
Rna Synthesis ◽  
Virulent Strain ◽  
Viral Rna ◽  
Interferon Response ◽  
Growth Defect ◽  
High Morbidity ◽  
C Protein ◽  
V Protein

ABSTRACT Measles is an acute febrile infectious disease with high morbidity and mortality. The genome of measles virus (MV), the causative agent, encodes two accessory products, V and C proteins, that play important roles in MV virulence. The V but not the C protein of the IC-B strain (a well-characterized virulent strain of MV) has been shown to block the Jak/Stat signaling pathway and counteract the cellular interferon (IFN) response. We have recently shown that a recombinant IC-B strain that lacks C protein expression replicates poorly in certain cell lines, and its growth defect is related to translational inhibition and strong IFN induction. Here, we show that the V protein of the MV IC-B strain also blocks the IFN induction pathway mediated by the melanoma differentiation-associated gene 5 product, thus actively interfering with the host IFN response at two different steps. On the other hand, the C protein per se possesses no activity to block the IFN induction pathway. Our data indicate that the C protein acts as a regulator of viral RNA synthesis, thereby acting indirectly to suppress IFN induction. Since recombinant MVs with C protein defective in modulating viral RNA synthesis or lacking C protein expression strongly stimulate IFN production, in spite of V protein production, both the C and V proteins must be required for MV to fully circumvent the host IFN response.

scholarly journals The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules

Viruses ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1030 ◽  
Author(s):  
Nicole Doyle ◽  
Philippa C. Hawes ◽  
Jennifer Simpson ◽  
Lorin H. Adams ◽  
Helena J. Maier
Keyword(s):  
Endoplasmic Reticulum ◽  
Rna Synthesis ◽  
Membrane Vesicles ◽  
Viral Rna ◽  
Double Membrane ◽  
Severe Diarrhea ◽  
The Usa ◽  
History Of ◽  
Host Cell Interactions ◽  
Positive Strand Rna

Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2012 from samples taken from pigs in 2009. PDCoV was subsequently identified in the USA in 2014 in pigs with a history of severe diarrhea. The virus has now been detected in pigs in several countries around the world. Following the development of tissue culture adapted strains of PDCoV, it is now possible to address questions regarding virus–host cell interactions for this genera of coronavirus. Here, we presented a detailed study of PDCoV-induced replication organelles. All positive-strand RNA viruses induce the rearrangement of cellular membranes during virus replication to support viral RNA synthesis, forming the replication organelle. Replication organelles for the Alpha-, Beta-, and Gammacoronavirus genera have been characterized. All coronavirus genera induced the formation of double-membrane vesicles (DMVs). In addition, Alpha- and Betacoronaviruses induce the formation of convoluted membranes, while Gammacoronaviruses induce the formation of zippered endoplasmic reticulum (ER) with tethered double-membrane spherules. However, the structures induced by Deltacoronaviruses, particularly the presence of convoluted membranes or double-membrane spherules, are unknown. Initially, the dynamics of PDCoV strain OH-FD22 replication were assessed with the onset of viral RNA synthesis, protein synthesis, and progeny particle release determined. Subsequently, virus-induced membrane rearrangements were identified in infected cells by electron microscopy. As has been observed for all other coronaviruses studied to date, PDCoV replication was found to induce the formation of double-membrane vesicles. Significantly, however, PDCoV replication was also found to induce the formation of regions of zippered endoplasmic reticulum, small associated tethered vesicles, and double-membrane spherules. These structures strongly resemble the replication organelle induced by avian Gammacoronavirus infectious bronchitis virus.

scholarly journals Mutational Analysis of the Rubella Virus Nonstructural Polyprotein and Its Cleavage Products in Virus Replication and RNA Synthesis

2000 ◽  
Vol 74 (11) ◽  
pp. 5133-5141 ◽  
Author(s):  
Yuying Liang ◽  
Shirley Gillam
Keyword(s):  
Virus Replication ◽  
Rubella Virus ◽  
Rna Synthesis ◽  
Rnase Protection Assay ◽  
Viral Rna ◽  
Nonstructural Proteins ◽  
Rnase Protection ◽  
Positive Strand ◽  
Negative Strand ◽  
Positive Strand Rna

ABSTRACT Rubella virus nonstructural proteins, translated from input genomic RNA as a p200 polyprotein and subsequently processed into p150 and p90 by an intrinsic papain-like thiol protease, are responsible for virus replication. To examine the effect of p200 processing on virus replication and to study the roles of nonstructural proteins in viral RNA synthesis, we introduced into a rubella virus infectious cDNA clone a panel of mutations that had variable defective effects on p200 processing. The virus yield and viral RNA synthesis of these mutants were examined. Mutations that completely abolished (C1152S and G1301S) or largely abolished (G1301A) cleavage of p200 resulted in noninfectious virus. Mutations that partially impaired cleavage of p200 (R1299A and G1300A) decreased virus replication. An RNase protection assay revealed that all of the mutants synthesized negative-strand RNA as efficiently as the wild type does but produced lower levels of positive-strand RNA. Our results demonstrated that processing of rubella virus nonstructural protein is crucial for virus replication and that uncleaved p200 could function in negative-strand RNA synthesis, whereas the cleavage products p150 and p90 are required for efficient positive-strand RNA synthesis.

scholarly journals Strand-Specific RNA Synthesis Defects in a Poliovirus with a Mutation in Protein 3A

2003 ◽  
Vol 77 (23) ◽  
pp. 12679-12691 ◽  
Author(s):  
Natalya L. Teterina ◽  
Mario S. Rinaudo ◽  
Ellie Ehrenfeld
Keyword(s):  
Rna Synthesis ◽  
Binding Activity ◽  
Viral Rna ◽  
Wild Type ◽  
Positive Strand ◽  
Polyprotein Processing ◽  
Delayed Onset ◽  
Methionine Residue ◽  
Cell Extracts ◽  
Positive Strand Rna

ABSTRACT Substitution of a methionine residue at position 79 in poliovirus protein 3A with valine or threonine caused defective viral RNA synthesis, manifested as delayed onset and reduced yield of viral RNA, in HeLa cells transfected with a luciferase-containing replicon. Viruses containing these same mutations produced small or minute plaques that generated revertants upon further passage, with either wild-type 3A sequences or additional nearby compensating mutations. Translation and polyprotein processing were not affected by the mutations, and 3AB proteins containing the altered amino acids at position 79 showed no detectable loss of membrane-binding activity. Analysis of individual steps of viral RNA synthesis in HeLa cell extracts that support translation and replication of viral RNA showed that VPg uridylylation and negative-strand RNA synthesis occurred normally from mutant viral RNA; however, positive-strand RNA synthesis was specifically reduced. The data suggest that a function of viral protein 3A is required for positive-strand RNA synthesis but not for production of negative strands.

scholarly journals Murine Hepatitis Virus Nonstructural Protein 4 Regulates Virus-Induced Membrane Modifications and Replication Complex Function

2009 ◽  
Vol 84 (1) ◽  
pp. 280-290 ◽  
Author(s):  
Mark J. Gadlage ◽  
Jennifer S. Sparks ◽  
Dia C. Beachboard ◽  
Reagan G. Cox ◽  
Joshua D. Doyle ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Critical Role ◽  
Complex Function ◽  
Microscopic Analysis ◽  
Murine Hepatitis Virus ◽  
Electron Microscopic ◽  
Virus Growth ◽  
Positive Strand Rna

ABSTRACT Positive-strand RNA viruses induce modifications of cytoplasmic membranes to form replication complexes. For coronaviruses, replicase nonstructural protein 4 (nsp4) has been proposed to function in the formation and organization of replication complexes. Murine hepatitis virus (MHV) nsp4 is glycosylated at residues Asn176 (N176) and N237 during plasmid expression of nsp4 in cells. To test if MHV nsp4 residues N176 and N237 are glycosylated during virus replication and to determine the effects of N176 and N237 on nsp4 function and MHV replication, alanine substitutions of nsp4 N176, N237, or both were engineered into the MHV-A59 genome. The N176A, N237A, and N176A/N237A mutant viruses were viable, and N176 and N237 were glycosylated during infection of wild-type (wt) and mutant viruses. The nsp4 glycosylation mutants exhibited impaired virus growth and RNA synthesis, with the N237A and N176A/N237A mutant viruses demonstrating more profound defects in virus growth and RNA synthesis. Electron microscopic analysis of ultrastructure from infected cells demonstrated that the nsp4 mutants had aberrant morphology of virus-induced double-membrane vesicles (DMVs) compared to those infected with wt virus. The degree of altered DMV morphology directly correlated with the extent of impairment in viral RNA synthesis and virus growth of the nsp4 mutant viruses. The results indicate that nsp4 plays a critical role in the organization and stability of DMVs. The results also support the conclusion that the structure of DMVs is essential for efficient RNA synthesis and optimal replication of coronaviruses.

scholarly journals Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Philip V'kovski ◽  
Markus Gerber ◽  
Jenna Kelly ◽  
Stephanie Pfaender ◽  
Nadine Ebert ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Host Factors ◽  
Viral Rna ◽  
Host Proteins ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
Starting Point ◽  
Positive Strand Rna

Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses. Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention.

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.

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.

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