scholarly journals A host dicer is required for defective viral RNA production and recombinant virus vector RNA instability for a positive sense RNA virus

2008 ◽  
Vol 105 (43) ◽  
pp. 16749-16754 ◽  
Author(s):  
X. Zhang ◽  
D. L. Nuss
Keyword(s):  
Recombinant Virus ◽  
Rna Virus ◽  
Virus Vector ◽  
Viral Rna ◽  
Positive Sense

scholarly journals Autophagy is involved in assisting the replication of Bamboo mosaic virus in Nicotiana benthamiana

2019 ◽  
Vol 70 (18) ◽  
pp. 4657-4670 ◽  
Author(s):  
Ying-Ping Huang ◽  
Ying-Wen Huang ◽  
Yung-Jen Hsiao ◽  
Siou-Cen Li ◽  
Yau-Huei Hsu ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Rna Virus ◽  
Critical Role ◽  
Viral Rna ◽  
Autophagosome Formation ◽  
Orange Fluorescent Protein ◽  
Positive Sense ◽  
Trigger Cell Death

Abstract Autophagy plays a critical role in plants under biotic stress, including the response to pathogen infection. We investigated whether autophagy-related genes (ATGs) are involved in infection with Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA virus. Initially, we observed that BaMV infection in Nicotiana benthamiana leaves upregulated the expression of ATGs but did not trigger cell death. The induction of ATGs, which possibly triggers autophagy, increased rather than diminished BaMV accumulation in the leaves, as revealed by gene knockdown and transient expression experiments. Furthermore, the inhibitor 3-methyladenine blocked autophagosome formation and the autophagy inducer rapamycin, which negatively and positively affected BaMV accumulation, respectively. Pull-down experiments with an antibody against orange fluorescent protein (OFP)-NbATG8f, an autophagosome marker protein, showed that both plus- and minus-sense BaMV RNAs could associate with NbATG8f. Confocal microscopy revealed that ATG8f-enriched vesicles possibly derived from chloroplasts contained both the BaMV viral RNA and its replicase. Thus, BaMV infection may induce the expression of ATGs possibly via autophagy to selectively engulf a portion of viral RNA-containing chloroplast. Virus-induced vesicles enriched with ATG8f could provide an alternative site for viral RNA replication or a shelter from the host silencing mechanism.

scholarly journals Comparative analysis of viral RNA signatures on different RIG-I-like receptors

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Raul Y Sanchez David ◽  
Chantal Combredet ◽  
Odile Sismeiro ◽  
Marie-Agnès Dillies ◽  
Bernd Jagla ◽  
...  
Keyword(s):  
Rna Virus ◽  
Affinity Purification ◽  
Viral Rna ◽  
Coding Region ◽  
Chikv Infection ◽  
Rna Molecules ◽  
Positive Sense ◽  
Rna Ligands ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

The RIG-I-like receptors (RLRs) play a major role in sensing RNA virus infection to initiate and modulate antiviral immunity. They interact with particular viral RNAs, most of them being still unknown. To decipher the viral RNA signature on RLRs during viral infection, we tagged RLRs (RIG-I, MDA5, LGP2) and applied tagged protein affinity purification followed by next-generation sequencing (NGS) of associated RNA molecules. Two viruses with negative- and positive-sense RNA genome were used: measles (MV) and chikungunya (CHIKV). NGS analysis revealed that distinct regions of MV genome were specifically recognized by distinct RLRs: RIG-I recognized defective interfering genomes, whereas MDA5 and LGP2 specifically bound MV nucleoprotein-coding region. During CHIKV infection, RIG-I associated specifically to the 3’ untranslated region of viral genome. This study provides the first comparative view of the viral RNA ligands for RIG-I, MDA5 and LGP2 in the presence of infection.

scholarly journals Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH

2021 ◽  
Author(s):  
Elena Rensen ◽  
Stefano Pietropaoli ◽  
Christian Weber ◽  
Sylvie Souquere ◽  
Pierre Isnard ◽  
...  
Keyword(s):  
Human Tissue ◽  
Rna Virus ◽  
Viral Rna ◽  
Initial Application ◽  
Positive Sense ◽  
Therapeutic Molecules ◽  
Infected Cells ◽  
Detailed Protocol ◽  
Transcription And Replication

AbstractThe current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize viral RNA directly in infected cells are critical to analyze its replication cycle, screen for therapeutic molecules or study infections in human tissue. Here, we report the design, validation and initial application of fluorescence in situ hybridization (FISH) probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy (EM). We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening and diagnostics.

scholarly journals Generation of an infectious Negev virus cDNA clone

2014 ◽  
Vol 95 (9) ◽  
pp. 2071-2074 ◽  
Author(s):  
Rodion V. Gorchakov ◽  
Robert B. Tesh ◽  
Scott C. Weaver ◽  
Farooq Nasar
Keyword(s):  
Cdna Clone ◽  
Rna Virus ◽  
Infectious Cdna Clone ◽  
Full Length ◽  
Viral Rna ◽  
Positive Sense ◽  
Virus Cdna

The genus Negevirus consists of insect-only viruses isolated from mosquitoes and sandflies. Here, we report the successful construction of a full-length infectious cDNA clone of Negev virus (NEGV) strain M30957. Viral RNA was transcribed in vitro and virus was readily rescued with or without the use of a cap analogue. These results strongly suggest that NEGV, and likely other members within the genus, is a non-segmented, single-stranded, positive-sense RNA virus.

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 Cotranslational Coat Protein-Mediated Inhibition of Potyviral RNA Translation

2015 ◽  
Vol 89 (8) ◽  
pp. 4237-4248 ◽  
Author(s):  
Jane Besong-Ndika ◽  
Konstantin I. Ivanov ◽  
Anders Hafrèn ◽  
Thierry Michon ◽  
Kristiina Mäkinen
Keyword(s):  
Coat Protein ◽  
Stop Codon ◽  
Rna Virus ◽  
Viral Rna ◽  
Dependent Manner ◽  
Content Type ◽  
Virion Assembly ◽  
Rna Translation ◽  
Intrinsic Capacity

ABSTRACTPotato virus A(PVA) is a single-stranded positive-sense RNA virus and a member of the familyPotyviridae. The PVA coat protein (CP) has an intrinsic capacity to self-assemble into filamentous virus-like particles, but the mechanism responsible for the initiation of viral RNA encapsidationin vivoremains unclear. Apart from virion assembly, PVA CP is also involved in the inhibition of viral RNA translation. In this study, we show that CP inhibits PVA RNA translation in a dose-dependent manner, through a mechanism involving the CP-encoding region. Analysis of this region, however, failed to identify any RNA secondary structure(s) preferentially recognized by CP, suggesting that the inhibition depends on CP-CP rather than CP-RNA interactions. In agreement with this possibility, insertion of an in-frame stop codon upstream of the CP sequence led to a marked decrease in the inhibition of viral RNA translation. Based on these results, we propose a model in which the cotranslational interactions between excess CP accumulating intransand CP translated from viral RNA incisare required to initiate the translational repression. This model suggests a mechanism for how viral RNA can be sequestered from translation and specifically selected for encapsidation at the late stages of viral infection.IMPORTANCEThe main functions of the CP during potyvirus infection are to protect viral RNA from degradation and to transport it locally, systemically, and from host to host. Although virion assembly is a key step in the potyviral infectious cycle, little is known about how it is initiated and how viral RNA is selected for encapsidation. The results presented here suggest that CP-CP rather than CP-RNA interactions are predominantly involved in the sequestration of viral RNA away from translation. We propose that the cotranslational nature of these interactions may represent a mechanism for the selection of viral RNA for encapsidation. A better understanding of the mechanism of virion assembly may lead to development of crops resistant to potyviruses at the level of viral RNA encapsidation, thereby reducing the detrimental effects of potyvirus infections on food production.

scholarly journals Origins and Evolution of the Global RNA Virome

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Yuri I. Wolf ◽  
Darius Kazlauskas ◽  
Jaime Iranzo ◽  
Adriana Lucía-Sanz ◽  
Jens H. Kuhn ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
Rna Viruses ◽  
Rna Virus ◽  
Phylogenomic Analysis ◽  
Rna Helicases ◽  
Sequence Alignments ◽  
Recent Advances ◽  
Wide Range ◽  
Positive Sense ◽  
Dsrna Viruses

ABSTRACTViruses with RNA genomes dominate the eukaryotic virome, reaching enormous diversity in animals and plants. The recent advances of metaviromics prompted us to perform a detailed phylogenomic reconstruction of the evolution of the dramatically expanded global RNA virome. The only universal gene among RNA viruses is the gene encoding the RNA-dependent RNA polymerase (RdRp). We developed an iterative computational procedure that alternates the RdRp phylogenetic tree construction with refinement of the underlying multiple-sequence alignments. The resulting tree encompasses 4,617 RNA virus RdRps and consists of 5 major branches; 2 of the branches include positive-sense RNA viruses, 1 is a mix of positive-sense (+) RNA and double-stranded RNA (dsRNA) viruses, and 2 consist of dsRNA and negative-sense (−) RNA viruses, respectively. This tree topology implies that dsRNA viruses evolved from +RNA viruses on at least two independent occasions, whereas −RNA viruses evolved from dsRNA viruses. Reconstruction of RNA virus evolution using the RdRp tree as the scaffold suggests that the last common ancestors of the major branches of +RNA viruses encoded only the RdRp and a single jelly-roll capsid protein. Subsequent evolution involved independent capture of additional genes, in particular, those encoding distinct RNA helicases, enabling replication of larger RNA genomes and facilitating virus genome expression and virus-host interactions. Phylogenomic analysis reveals extensive gene module exchange among diverse viruses and horizontal virus transfer between distantly related hosts. Although the network of evolutionary relationships within the RNA virome is bound to further expand, the present results call for a thorough reevaluation of the RNA virus taxonomy.IMPORTANCEThe majority of the diverse viruses infecting eukaryotes have RNA genomes, including numerous human, animal, and plant pathogens. Recent advances of metagenomics have led to the discovery of many new groups of RNA viruses in a wide range of hosts. These findings enable a far more complete reconstruction of the evolution of RNA viruses than was attainable previously. This reconstruction reveals the relationships between different Baltimore classes of viruses and indicates extensive transfer of viruses between distantly related hosts, such as plants and animals. These results call for a major revision of the existing taxonomy of RNA viruses.

scholarly journals Generation of infectious and transmissible virions from a GB virus B full-length consensus clone in tamarins

2001 ◽  
Vol 82 (10) ◽  
pp. 2437-2448 ◽  
Author(s):  
Andrea Sbardellati ◽  
Elisa Scarselli ◽  
Ernst Verschoor ◽  
Amedeo De Tomassi ◽  
Domenico Lazzaro ◽  
...  
Keyword(s):  
Immune Response ◽  
Animal Model ◽  
Recombinant Virus ◽  
Secondary Infection ◽  
Viral Rna ◽  
Hepatic Tissue ◽  
Cdna Clones ◽  
Humoral Immune ◽  
B Genome

The strong similarity between GB virus B (GBV-B) and hepatitis C virus (HCV) makes tamarins infected by GBV-B an acceptable surrogate animal model for HCV infection. Even more attractive, for drug discovery purposes, is the idea of constructing chimeric viruses by inserting HCV genes of interest into a GBV-B genome frame. To accomplish this, infectious cDNA clones of both viruses must be available. The characterization of several HCV molecular clones capable of infecting chimpanzees has been published, whereas only one infectious GBV-B clone inducing hepatitis in tamarins has been reported so far. Here we describe the infection of tamarins by intrahepatic injection of RNA transcribed from a genomic GBV-B clone (FL-3) and transmission of the disease from infected to naive tamarins via serum inoculation. The disease resulting from both direct and secondary infection was characterized for viral RNA titre and hepatitis parameters as well as for viral RNA distribution in the hepatic tissue. Host humoral immune response to GBV-B antigens was also monitored. The progression of the disease was compared to that induced by intravenous injection of different amounts of the non-recombinant virus.

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.

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