Viral RNA structural equilibria and their interactions with host proteins

2019 ◽  
Author(s):  
◽  
Samantha Elizabeth Brady
Keyword(s):  
Reverse Transcription ◽  
Rna Structure ◽  
Drug Targets ◽  
Rna Viruses ◽  
Rna Virus ◽  
Protein Translation ◽  
Viral Rna ◽  
Primer Binding Site ◽  
In Vitro Techniques

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Understanding viral RNA structure and how it functions is crucial in elucidating new drug targets. There are many kinds of viruses that utilize RNA as a critical component of their life cycle, such as retroviruses, single-stranded plus or minus sense RNA viruses, and double-stranded RNA viruses. Two viruses that are studied in this thesis are human immunodeficiency virus (HIV), which is a retrovirus, and hepatitis C virus (HCV), which is a single-stranded plus sense RNA virus. It has been previously reported that a human host factor, RNA helicase A (RHA), is packaged into HIV virions by binding to the primer binding site (PBS) segment of the 5'untranslated region in the HIV genomic RNA. We determined RHA is required for efficient reverse transcription prior to capsid uncoating by utilizing cell based and in vitro techniques. It has also been suggested that RHA plays other roles during HIV infection besides reverse transcription. Utilizing NMR, we demonstrated that RHA binds to the monomeric 5'UTR at the bottom of the TAR hairpin, which is different from how it binds during viral packaging. Next, we employed NMR techniques to probe the 3'end of the HCV genome called 3'X. We determined that the 3'X is in structural equilibrium between two states: an open conformation and a closed conformation. These two conformations have been suggested to play a role in minus sense synthesis and viral protein translation, respectively. Taken together, my thesis work has elucidated how many viruses manipulate and utilize their RNA structure to modulate their outcome.

scholarly journals Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity

2020 ◽  
Vol 22 (1) ◽  
pp. 58
Author(s):  
Thomas Gremminger ◽  
Zhenwei Song ◽  
Juan Ji ◽  
Avery Foster ◽  
Kexin Weng ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Potential Interaction ◽  
Viral Rna ◽  
Primer Binding Site ◽  
Rna Integrity ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Extended Interaction ◽  
Hiv 1

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

scholarly journals Constraints of Viral RNA Synthesis on Codon Usage of Negative-Strand RNA Virus

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
Ryan H. Gumpper ◽  
Weike Li ◽  
Ming Luo
Keyword(s):  
Rna Polymerase ◽  
Codon Usage ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Rna Virus ◽  
Protein Translation ◽  
Viral Rna ◽  
Genomic Rna ◽  
Negative Strand ◽  
Unique Mechanism

ABSTRACTNegative-strand RNA viruses (NSVs) include some of the most pathogenic human viruses known. NSVs completely rely on the host cell for protein translation, but their codon usage bias is often different from that of the host. This discrepancy may have originated from the unique mechanism of NSV RNA synthesis in that the genomic RNA sequestered in the nucleocapsid serves as the template. The stability of the genomic RNA in the nucleocapsid appears to regulate its accessibility to the viral RNA polymerase, thus placing constraints on codon usage to balance viral RNA synthesis. Byin situanalyses of vesicular stomatitis virus RNA synthesis, specific activities of viral RNA synthesis were correlated with the genomic RNA sequence. It was found that by simply altering the sequence and not the amino acid that it encoded, a significant reduction, up to an ∼750-fold reduction, in viral RNA transcripts occurred. Through subsequent sequence analysis and thermal shift assays, it was found that the purine/pyrimidine content modulates the overall stability of the polymerase complex, resulting in alteration of the activity of viral RNA synthesis. The codon usage is therefore constrained by the obligation of the NSV genome for viral RNA synthesis.IMPORTANCENegative-strand RNA viruses (NSVs) include the most pathogenic viruses known. New methods to monitor their evolutionary trends are urgently needed for the development of antivirals and vaccines. The protein translation machinery of the host cell is currently recognized as a main genomic regulator of RNA virus evolution, which works especially well for positive-strand RNA viruses. However, this approach fails for NSVs because it does not consider the unique mechanism of their viral RNA synthesis. For NSVs, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the genome sequestered in the nucleocapsid. Our work suggests a paradigm shift that the interactions between the RNA genome and the nucleocapsid protein regulate the activity of vRdRp, which selects codon usage.

scholarly journals A 212-nt long RNA structure in the Tobacco necrosis virus-D RNA genome is resistant to Xrn degradation

2019 ◽  
Vol 47 (17) ◽  
pp. 9329-9342 ◽  
Author(s):  
Chaminda D Gunawardene ◽  
Laura R Newburn ◽  
K Andrew White
Keyword(s):  
Rna Structure ◽  
Rna Viruses ◽  
Degradation Products ◽  
Rna Degradation ◽  
Viral Rna ◽  
Tobacco Necrosis Virus ◽  
Rna Structures ◽  
Necrosis Virus

Abstract Plus-strand RNA viruses can accumulate viral RNA degradation products during infections. Some of these decay intermediates are generated by the cytosolic 5′-to-3′ exoribonuclease Xrn1 (mammals and yeast) or Xrn4 (plants) and are formed when the enzyme stalls on substrate RNAs upon encountering inhibitory RNA structures. Many Xrn-generated RNAs correspond to 3′-terminal segments within the 3′-UTR of viral genomes and perform important functions during infections. Here we have investigated a 3′-terminal small viral RNA (svRNA) generated by Xrn during infections with Tobacco necrosis virus-D (family Tombusviridae). Our results indicate that (i) unlike known stalling RNA structures that are compact and modular, the TNV-D structure encompasses the entire 212 nt of the svRNA and is not functionally transposable, (ii) at least two tertiary interactions within the RNA structure are required for effective Xrn blocking and (iii) most of the svRNA generated in infections is derived from viral polymerase-generated subgenomic mRNA1. In vitro and in vivo analyses allowed for inferences on roles for the svRNA. Our findings provide a new and distinct addition to the growing list of Xrn-resistant viral RNAs and stalling structures found associated with different plant and animal RNA viruses.

TaffiX® nasal powder spray forms an effective barrier against infectious new variants of SARS-CoV-2 (COVID-19)

2021 ◽  
Author(s):  
Michal Mandelboim ◽  
Ella Mendelson ◽  
Yaron Drori ◽  
Nofar Atari ◽  
Tair Lapidot ◽  
...  
Keyword(s):  
South African ◽  
Rna Virus ◽  
Rna Extraction ◽  
Real Life ◽  
Preclinical Study ◽  
Viral Rna ◽  
Pcr Analysis ◽  
Effective Barrier ◽  
Gel Layer

Abstract Introduction: While vaccination efforts against SARS-CoV-2 around the world are ongoing -, new high-infectious variants of the virus are being detected. The protection of the available vaccines against some of the new variants is weaker, and experts are concerned that newer as yet undescribed variants of this mutated RNA virus will eventually prove stable against the current vaccines. Additional preventive measures will therefore be needed to protect the population until effective vaccinations are widely available.TaffiX® is a personal, anti-viral nasal powder spray comprised of low pH Hypromellose that upon insufflation into the nose creates a thin gel layer covering the nasal mucosa and forming a protective mechanical barrier that prevents viruses from engaging with nasal cells- the main portal of entry for viruses. Taffix is commercially available in many countries across Europe, Asia America and Africa. In a prior preclinical study, TaffiX® was found to be effective against SARS-CoV-2 Hong Kong/VM20001061/2020 in experimental in vitro conditions. A real-life clinical survey demonstrated that TaffiX® nasal spray significantly reduced the SARS-CoV-2 infection rate post mass-gathering event in a highly endemic community.Objective: The current study aimed to test the protective effect of Taffix against new pathogenic, highly infectious SARS-CoV-2 variants in vitro: the “British” B.1.1.7 (hCoV-19/Israel/CVL-46879-ngs/2020) and the “South African” B.1.351 (hCoV-19/Israel/CVL-2557-ngs/2020) variants.Study design: A TaffiX® gel was formed on a nylon filter, using an amount equivalent to a clinical dose of Taffix . Filters were then seeded with SARS-CoV-2 B.1.1.7 (“British”) and B.1.351 (“South African”) variants. After a 10 -minute incubation at room temperature, the bottom of each filter was washed, and the resulting flow-through was collected and seeded into 24 -well plates containing Vero-E6 cells. After 5 days of incubation, a 200 µl sample from each well was taken for viral RNA extraction followed by SARS-CoV 2 RT-PCR analysis.Results: The TaffiX® gel completely blocked SARS-CoV-2 highly infectious variants B.1.1.7 and B.1.351 in vitro, reducing the titer of recoverable infectious virus as well as viral RNA by 100%.Conclusions: Under in vitro conditions, TaffiX® formed an effective protective barrier against SARS-COV-2 variants (British variant and South African Variant). These results are consistent with prior findings demonstrating the in vitro high efficacy of Taffix gel in preventing viruses from reaching cells and infecting them. These results, added to clinical real-life studies performed with Taffix , support its use as an effective barrier against new variants of SARS-CoV-2 in conjunction with other protective measures.

scholarly journals The global and local distribution of RNA structure throughout the SARS-CoV-2 genome

2020 ◽  
Author(s):  
Rafael de Cesaris Araujo Tavares ◽  
Gandhar Mahadeshwar ◽  
Han Wan ◽  
Nicholas C. Huston ◽  
Anna Marie Pyle
Keyword(s):  
In Silico ◽  
Rna Structure ◽  
Drug Targets ◽  
Rna Folding ◽  
Rna Viruses ◽  
Viral Agent ◽  
Rna Structures ◽  
Viral Rnas ◽  
Viral Genomes ◽  
Rna Genome

SARS-CoV-2 is the causative viral agent of COVID-19, the disease at the center of the current global pandemic. While knowledge of highly structured regions is integral for mechanistic insights into the viral infection cycle, very little is known about the location and folding stability of functional elements within the massive, ∼30kb SARS-CoV-2 RNA genome. In this study, we analyze the folding stability of this RNA genome relative to the structural landscape of other well-known viral RNAs. We present an in-silico pipeline to predict regions of high base pair content across long genomes and to pinpoint hotspots of well-defined RNA structures, a method that allows for direct comparisons of RNA structural complexity within the several domains in SARS-CoV-2 genome. We report that the SARS-CoV-2 genomic propensity for stable RNA folding is exceptional among RNA viruses, superseding even that of HCV, one of the most structured viral RNAs in nature. Furthermore, our analysis suggests varying levels of RNA structure across genomic functional regions, with accessory and structural ORFs containing the highest structural density in the viral genome. Finally, we take a step further to examine how individual RNA structures formed by these ORFs are affected by the differences in genomic and subgenomic contexts, which given the technical difficulty of experimentally separating cellular mixtures of sgRNA from gRNA, is a unique advantage of our in-silico pipeline. The resulting findings provide a useful roadmap for planning focused empirical studies of SARS-CoV-2 RNA biology, and a preliminary guide for exploring potential SARS-CoV-2 RNA drug targets. Importance The RNA genome of SARS-CoV-2 is among the largest and most complex viral genomes, and yet its RNA structural features remain relatively unexplored. Since RNA elements guide function in most RNA viruses, and they represent potential drug targets, it is essential to chart the architectural features of SARS-CoV-2 and pinpoint regions that merit focused study. Here we show that RNA folding stability of SARS-CoV-2 genome is exceptional among viral genomes and we develop a method to directly compare levels of predicted secondary structure across SARS-CoV-2 domains. Remarkably, we find that coding regions display the highest structural propensity in the genome, forming motifs that differ between the genomic and subgenomic contexts. Our approach provides an attractive strategy to rapidly screen for candidate structured regions based on base pairing potential and provides a readily interpretable roadmap to guide functional studies of RNA viruses and other pharmacologically relevant RNA transcripts.

scholarly journals Activation of viral transcription by stepwise largescale folding of an RNA virus genome

2020 ◽  
Vol 48 (16) ◽  
pp. 9285-9300
Author(s):  
Tamari Chkuaseli ◽  
K Andrew White
Keyword(s):  
Rna Structure ◽  
Rna Viruses ◽  
Rna Virus ◽  
Initial Step ◽  
Binding Pocket ◽  
Regulatory Elements ◽  
Virus Genome ◽  
Genomic Rna ◽  
Stem Loop ◽  
Rna Complex

Abstract The genomes of RNA viruses contain regulatory elements of varying complexity. Many plus-strand RNA viruses employ largescale intra-genomic RNA-RNA interactions as a means to control viral processes. Here, we describe an elaborate RNA structure formed by multiple distant regions in a tombusvirus genome that activates transcription of a viral subgenomic mRNA. The initial step in assembly of this intramolecular RNA complex involves the folding of a large viral RNA domain, which generates a discontinuous binding pocket. Next, a distally-located protracted stem-loop RNA structure docks, via base-pairing, into the binding site and acts as a linchpin that stabilizes the RNA complex and activates transcription. A multi-step RNA folding pathway is proposed in which rate-limiting steps contribute to a delay in transcription of the capsid protein-encoding viral subgenomic mRNA. This study provides an exceptional example of the complexity of genome-scale viral regulation and offers new insights into the assembly schemes utilized by large intra-genomic RNA structures.

scholarly journals Physical and Functional Analysis of Viral RNA Genomes by SHAPE

2019 ◽  
Vol 6 (1) ◽  
pp. 93-117 ◽  
Author(s):  
Mark A. Boerneke ◽  
Jeffrey E. Ehrhardt ◽  
Kevin M. Weeks
Keyword(s):  
Rna Structure ◽  
Rna Viruses ◽  
Genome Structure ◽  
Regulatory Elements ◽  
Viral Rna ◽  
Host Immune Responses ◽  
Linear Sequence ◽  
Structure Probing ◽  
Shape Structure ◽  
Shape Selective

RNA viruses encode the information required to usurp cellular metabolism and gene regulation and to enable their own replication in two ways: in the linear sequence of their RNA genomes and in higher-order structures that form when the genomic RNA strand folds back on itself. Application of high-resolution SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) structure probing to viral RNA genomes has identified numerous new regulatory elements, defined new principles by which viral RNAs interact with the cellular host and evade host immune responses, and revealed relationships between virus evolution and RNA structure. This review summarizes our current understanding of genome structure-function interrelationships for RNA viruses, as informed by SHAPE structure probing, and outlines opportunities for future studies.

scholarly journals Sequence-Specific Modifications Enhance the Broad-Spectrum Antiviral Response Activated by RIG-I Agonists

2015 ◽  
Vol 89 (15) ◽  
pp. 8011-8025 ◽  
Author(s):  
Cindy Chiang ◽  
Vladimir Beljanski ◽  
Kevin Yin ◽  
David Olagnier ◽  
Fethia Ben Yebdri ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Infection ◽  
Inflammatory Responses ◽  
Rna Viruses ◽  
Rna Virus ◽  
A549 Cells ◽  
Antiviral Response ◽  
Interferon Response

ABSTRACTThe cytosolic RIG-I (retinoic acid-inducible gene I) receptor plays a pivotal role in the initiation of the immune response against RNA virus infection by recognizing short 5′-triphosphate (5′ppp)-containing viral RNA and activating the host antiviral innate response. In the present study, we generated novel 5′ppp RIG-I agonists of varieous lengths, structures, and sequences and evaluated the generation of the antiviral and inflammatory responses in human epithelial A549 cells, human innate immune primary cells, and murine models of influenza and chikungunya viral pathogenesis. A 99-nucleotide, uridine-rich hairpin 5′pppRNA termed M8 stimulated an extensive and robust interferon response compared to other modified 5′pppRNA structures, RIG-I aptamers, or poly(I·C). Interestingly, manipulation of the primary RNA sequence alone was sufficient to modulate antiviral activity and inflammatory response, in a manner dependent exclusively on RIG-I and independent of MDA5 and TLR3. Both prophylactic and therapeutic administration of M8 effectively inhibited influenza virus and dengue virus replicationin vitro. Furthermore, multiple strains of influenza virus that were resistant to oseltamivir, an FDA-approved therapeutic treatment for influenza, were highly sensitive to inhibition by M8. Finally, prophylactic M8 treatmentin vivoprolonged survival and reduced lung viral titers of mice challenged with influenza virus, as well as reducing chikungunya virus-associated foot swelling and viral load. Altogether, these results demonstrate that 5′pppRNA can be rationally designed to achieve a maximal RIG-I-mediated protective antiviral response against human-pathogenic RNA viruses.IMPORTANCEThe development of novel therapeutics to treat human-pathogenic RNA viral infections is an important goal to reduce spread of infection and to improve human health and safety. This study investigated the design of an RNA agonist with enhanced antiviral and inflammatory properties against influenza, dengue, and chikungunya viruses. A novel, sequence-dependent, uridine-rich RIG-I agonist generated a protective antiviral responsein vitroandin vivoand was effective at concentrations 100-fold lower than prototype sequences or other RNA agonists, highlighting the robust activity and potential clinical use of the 5′pppRNA against RNA virus infection. Altogether, the results identify a novel, sequence-specific RIG-I agonist as an attractive therapeutic candidate for the treatment of a broad range of RNA viruses, a pressing issue in which a need for new and more effective options persists.

scholarly journals Suppression of Viral RNA Recombination by a Host Exoribonuclease

2006 ◽  
Vol 80 (6) ◽  
pp. 2631-2640 ◽  
Author(s):  
Chi-Ping Cheng ◽  
Elena Serviene ◽  
Peter D. Nagy
Keyword(s):  
Rna Virus ◽  
Viral Rna ◽  
Rna Turnover ◽  
Rna Recombination ◽  
Viral Rnas ◽  
Yeast Strains ◽  
Genome Wide ◽  
Host Genes

ABSTRACT RNA viruses of humans, animals, and plants evolve rapidly due to mutations and RNA recombination. A previous genome-wide screen in Saccharomyces cerevisiae, a model host, identified five host genes, including XRN1, encoding a 5′-3′ exoribonuclease, whose absence led to an ∼10- to 50-fold enhancement of RNA recombination in Tomato bushy stunt virus (E. Serviene, N. Shapka, C. P. Cheng, T. Panavas, B. Phuangrat, J. Baker, and P. D. Nagy, Proc. Natl. Acad. Sci. USA 102:10545-10550, 2005). In this study, we found abundant 5′-truncated viral RNAs in xrn1Δ mutant strains but not in the parental yeast strains, suggesting that these RNAs might serve as recombination substrates promoting RNA recombination in xrn1Δ mutant yeast. This model is supported by data showing that an enhanced level of viral recombinant accumulation occurred when two different 5′-truncated viral RNAs were expressed in the parental and xrn1Δ mutant yeast strains or electroporated into plant protoplasts. Moreover, we demonstrate that purified Xrn1p can degrade the 5′-truncated viral RNAs in vitro. Based on these findings, we propose that Xrn1p can suppress viral RNA recombination by rapidly removing the 5′-truncated RNAs, the substrates of recombination, and thus reducing the chance for recombination to occur in the parental yeast strain. In addition, we show that the 5′-truncated viral RNAs are generated by host endoribonucleases. Accordingly, overexpression of the Ngl2p endoribonuclease led to an increased accumulation of cleaved viral RNAs in vivo and in vitro. Altogether, this paper establishes that host ribonucleases and host-mediated viral RNA turnover play major roles in RNA virus recombination and evolution.

scholarly journals Role of an Internal and Two 3′-Terminal RNA Elements in Assembly of Tombusvirus Replicase

2005 ◽  
Vol 79 (16) ◽  
pp. 10608-10618 ◽  
Author(s):  
Zivile Panaviene ◽  
Tadas Panavas ◽  
Peter D. Nagy
Keyword(s):  
Rna Virus ◽  
Viral Rna ◽  
Host Cells ◽  
Replication Proteins ◽  
Rna Sequences ◽  
Alternative Structures ◽  
Recognition Element ◽  
Rna Elements

ABSTRACT Plus-strand RNA virus replication requires the assembly of the viral replicase complexes on intracellular membranes in the host cells. The replicase of Cucumber necrosis virus (CNV), a tombusvirus, contains the viral p33 and p92 replication proteins and possible host factors. In addition, the assembly of CNV replicase is stimulated in the presence of plus-stranded viral RNA (Z. Panaviene et al., J. Virol. 78:8254-8263, 2004). To define cis-acting viral RNA sequences that stimulate replicase assembly, we performed a systematic deletion approach with a model tombusvirus replicon RNA in Saccharomyces cerevisiae, which also coexpressed p33 and p92 replication proteins. In vitro replicase assays performed with purified CNV replicase preparations from yeast revealed critical roles for three RNA elements in CNV replicase assembly: the internal p33 recognition element (p33RE), the replication silencer element (RSE), and the 3′-terminal minus-strand initiation promoter (gPR). Deletion or mutagenesis of these elements reduced the activity of the CNV replicase to a minimal level. In addition to the primary sequences of gPR, RSE, and p33RE, formation of two alternative structures among these elements may also play a role in replicase assembly. Altogether, the role of multiple RNA elements in tombusvirus replicase assembly could be an important factor to ensure fidelity of template selection during replication.

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