scholarly journals A Minigenome Study of Hazara Nairovirus Genomic Promoters

2019 ◽  
Vol 93 (6) ◽  
Author(s):  
Yusuke Matsumoto ◽  
Keisuke Ohta ◽  
Daniel Kolakofsky ◽  
Machiko Nishio
Keyword(s):  
Promoter Activity ◽  
Rna Synthesis ◽  
Rna Virus ◽  
Essential Element ◽  
Hemorrhagic Fever ◽  
Genome Replication ◽  
Hemorrhagic Fever Virus ◽  
Single Stranded Region ◽  
Complementary Region ◽  
Minigenome System

ABSTRACTHazara nairovirus (HAZV) is a trisegmented RNA virus most closely related to Crimean-Congo hemorrhagic fever virus (CCHFV) in the orderBunyavirales. The terminal roughly 20 nucleotides (nt) of its genome ends are highly complementary, similar to those of other segmented negative-strand RNA viruses (sNSV), and act as promoters for RNA synthesis. These promoters contain two elements: the extreme termini of both strands (promoter element 1 [PE1]) are conserved and virus specific and are found bound to separate sites on the polymerase surface in crystal structures of promoter-polymerase complexes. The following sequences (PE2) are segment specific, with the potential to form double-stranded RNA (dsRNA), and the latter aspect is also important for promoter activity. Nairovirus genome promoters differ from those of peribunyaviruses and arenaviruses in that they contain a short single-stranded region between the two regions of complementarity. Using a HAZV minigenome system, we found the single-stranded nature of this region, as well as the potential of the following sequence to form dsRNA, is essential for reporter gene expression. Most unexpectedly, the sequence of the PE2 dsRNA appears to be equally important for promoter activity. These differences in sNSV PE2 promoter elements are discussed in light of our current understanding of the initiation of RNA synthesis.IMPORTANCEA minigenome system for HAZV, closely related to CCHFV, was used to study its genome replication. HAZV genome ends, like those of other sNSV, such as peribunyaviruses and arenaviruses, are highly complementary and serve as promoters for genome synthesis. These promoters are composed of two elements: the extreme termini of both 3′ and 5′ strands that are initially bound to separate sites on the polymerase surface in a sequence-specific fashion and the following sequences with the potential to anneal but whose sequence is not important. Nairovirus promoters differ from the other sNSV cited in that they contain a short single-stranded RNA (ssRNA) region between the two elements. The single-stranded nature of this region is an essential element of the promoter, whereas its sequence is unimportant. The sequence of the following complementary region is unexpectedly also important, a possible rare example of sequence-specific dsRNA recognition.

scholarly journals The rocaglate CR-31-B (-) inhibits SARS-CoV-2 replication at non-cytotoxic, low nanomolar concentrations in vitro and ex vivo

2020 ◽  
Author(s):  
Christin Müller ◽  
Wiebke Obermann ◽  
Nadja Karl ◽  
Hans-Guido Wendel ◽  
Gaspar Taroncher-Oldenburg ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Ex Vivo ◽  
Rna Virus ◽  
Mrna Translation ◽  
Hemorrhagic Fever ◽  
Airway Epithelial Cells ◽  
Initiation Factor ◽  
Protein Accumulation ◽  
Hemorrhagic Fever Virus

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a betacoronavirus in the subgenus Sarbecovirus causes a respiratory disease with varying symptoms referred to as coronavirus disease 2019 (COVID-19) and is responsible for a pandemic that started in early 2020. With no vaccines or effective antiviral treatments available, and infection and fatality numbers continuing to increase globally, the quest for novel therapeutic solutions remains an urgent priority. Rocaglates, a class of plant-derived cyclopenta[b]benzofurans, exhibit broad-spectrum antiviral activity against positive- and negative-sense RNA viruses. This compound class inhibits eukaryotic initiation factor 4A (eIF4A)-dependent mRNA translation initiation, resulting in strongly reduced viral RNA translation. The synthetic rocaglate CR-31-B (-) has previously been shown to inhibit the replication of human coronaviruses, such as HCoV-229E and MERS-CoV, as well as Zika-, Lassa-, Crimean Congo hemorrhagic fever virus in primary cells. Here, we assessed the antiviral activity of CR-31-B (-) against SARS-CoV-2 using both in vitro and ex vivo cell culture models. In African green monkey Vero E6 cells, CR-31-B (-) inhibited SARS-CoV-2 replication with an EC50 of ~1.8 nM. In line with this, viral protein accumulation and replication/transcription complex formation were found to be strongly reduced by this compound. In an ex vivo infection system using human airway epithelial cells, CR-31-B (-) was found to cause a massive reduction of SARS-CoV-2 titers by about 4 logs to nearly non-detectable levels. The data reveal a potent anti-SARS-CoV-2 activity by CR-31-B (-), corroborating previous results obtained for other coronaviruses and supporting the idea that rocaglates may be used in first-line antiviral intervention strategies against novel and emerging RNA virus outbreaks.

scholarly journals Stable Occupancy of the Crimean-Congo Hemorrhagic Fever Virus-Encoded Deubiquitinase Blocks Viral Infection

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Florine E. M. Scholte ◽  
Brian L. Hua ◽  
Jessica R. Spengler ◽  
John V. Dzimianski ◽  
JoAnn D. Coleman-McCray ◽  
...  
Keyword(s):  
Ovarian Tumor ◽  
Rna Virus ◽  
Hemorrhagic Fever ◽  
Cysteine Proteases ◽  
Fever Virus ◽  
Therapeutic Interventions ◽  
Crimean Congo Hemorrhagic Fever ◽  
Hemorrhagic Fever Virus ◽  
Catalytic Cysteine ◽  
Replicative Cycle

ABSTRACT Crimean-Congo hemorrhagic fever virus (CCHFV) infection can result in a severe hemorrhagic syndrome for which there are no antiviral interventions available to date. Certain RNA viruses, such as CCHFV, encode cysteine proteases of the ovarian tumor (OTU) family that antagonize interferon (IFN) production by deconjugating ubiquitin (Ub). The OTU of CCHFV, a negative-strand RNA virus, is dispensable for replication of the viral genome, despite being part of the large viral RNA polymerase. Here, we show that mutations that prevent binding of the OTU to cellular ubiquitin are required for the generation of recombinant CCHFV containing a mutated catalytic cysteine. Similarly, the high-affinity binding of a synthetic ubiquitin variant (UbV-CC4) to CCHFV OTU strongly inhibits viral growth. UbV-CC4 inhibits CCHFV infection even in the absence of intact IFN signaling, suggesting that its antiviral activity is not due to blocking the OTU’s immunosuppressive function. Instead, the prolonged occupancy of the OTU with UbV-CC4 directly targets viral replication by interfering with CCHFV RNA synthesis. Together, our data provide mechanistic details supporting the development of antivirals targeting viral OTUs. IMPORTANCE Crimean-Congo hemorrhagic fever virus is an important human pathogen with a wide global distribution for which no therapeutic interventions are available. CCHFV encodes a cysteine protease belonging to the ovarian tumor (OTU) family which is involved in host immune suppression. Here we demonstrate that artificially prolonged binding of the OTU to a substrate inhibits virus infection. This provides novel insights into CCHFV OTU function during the viral replicative cycle and highlights the OTU as a potential antiviral target.

scholarly journals RIG-I Mediates an Antiviral Response to Crimean-Congo Hemorrhagic Fever Virus

2015 ◽  
Vol 89 (20) ◽  
pp. 10219-10229 ◽  
Author(s):  
Jessica R. Spengler ◽  
Jenish R. Patel ◽  
Ayan K. Chakrabarti ◽  
Marko Zivcec ◽  
Adolfo García-Sastre ◽  
...  
Keyword(s):  
Public Health ◽  
Rna Virus ◽  
Hemorrhagic Fever ◽  
Antiviral Response ◽  
Fever Virus ◽  
Type I ◽  
Immune Recognition ◽  
Crimean Congo Hemorrhagic Fever ◽  
Hemorrhagic Fever Virus ◽  
Significant Public Health

ABSTRACTIn the cytoplasm, the retinoic acid-inducible gene I (RIG-I) senses the RNA genomes of several RNA viruses. RIG-I binds to viral RNA, eliciting an antiviral response via the cellular adaptor MAVS. Crimean-Congo hemorrhagic fever virus (CCHFV), a negative-sense RNA virus with a 5′-monophosphorylated genome, is a highly pathogenic zoonotic agent with significant public health implications. We found that, during CCHFV infection, RIG-I mediated a type I interferon (IFN) response via MAVS. Interfering with RIG-I signaling reduced IFN production and IFN-stimulated gene expression and increased viral replication. Immunostimulatory RNA was isolated from CCHFV-infected cells and from virion preparations, and RIG-I coimmunoprecipitation of infected cell lysates isolated immunostimulatory CCHFV RNA. This report serves as the first description of a pattern recognition receptor for CCHFV and highlights a critical signaling pathway in the antiviral response to CCHFV.IMPORTANCECCHFV is a tick-borne virus with a significant public health impact. In order for cells to respond to virus infection, they must recognize the virus as foreign and initiate antiviral signaling. To date, the receptors involved in immune recognition of CCHFV are not known. Here, we investigate and identify RIG-I as a receptor involved in initiating an antiviral response to CCHFV. This receptor initially was not expected to play a role in CCHFV recognition because of characteristics of the viral genome. These findings are important in understanding the antiviral response to CCHFV and support continued investigation into the spectrum of potential viruses recognized by RIG-I.

scholarly journals Draft Genome Sequences of Eight Crimean-Congo Hemorrhagic Fever Virus Strains

2017 ◽  
Vol 5 (23) ◽  
Author(s):  
J. W. Koehler ◽  
K. L. Delp ◽  
B. J. Kearney ◽  
T. A. Conrad ◽  
R. J. Schoepp ◽  
...  
Keyword(s):  
Rna Virus ◽  
Draft Genome ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Genomic Diversity ◽  
Crimean Congo Hemorrhagic Fever ◽  
Hemorrhagic Fever Virus ◽  
Assay Design ◽  
High Degree ◽  
Virus Strains

ABSTRACT Crimean-Congo hemorrhagic fever virus (CCHFV) is a geographically widespread RNA virus with a high degree of genomic diversity that complicates sequence-based diagnostics. Here, we sequenced eight CCHFV strains for improved assay design and deposition into FDA-ARGOS, the FDA’s pathogen database for development and verification of next generation sequencing assays.

scholarly journals Mutagenic Analysis of Hazara Nairovirus Nontranslated Regions during Single- and Multistep Growth Identifies both Attenuating and Functionally Critical Sequences for Virus Replication

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Daniele F. Mega ◽  
Jack Fuller ◽  
Beatriz Álvarez-Rodríguez ◽  
Jamel Mankouri ◽  
Roger Hewson ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Rna Viruses ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Adjacent Segment ◽  
Virus Growth ◽  
Crimean Congo Hemorrhagic Fever ◽  
Hemorrhagic Fever Virus ◽  
Multiplication Cycle ◽  
Negative Sense

ABSTRACT Hazara nairovirus (HAZV) is a member of the family Nairoviridae in the order Bunyavirales and closely related to Crimean-Congo hemorrhagic fever virus, which is responsible for severe and fatal human disease. The HAZV genome comprises three segments of negative-sense RNA, named S, M, and L, with nontranslated regions (NTRs) flanking a single open reading frame. NTR sequences regulate RNA synthesis and, by analogy with other segmented negative-sense RNA viruses, may direct activities such as virus assembly and innate immune modulation. The terminal-proximal nucleotides of 3′ and 5′ NTRs exhibit extensive terminal complementarity; the first 11 nucleotides are strictly conserved and form promoter element 1 (PE1), with adjacent segment-specific nucleotides forming PE2. To explore the functionality of NTR nucleotides within the context of the nairovirus multiplication cycle, we designed infectious HAZV mutants bearing successive deletions throughout both S segment NTRs. Fitness of rescued viruses was assessed in single-step and multistep growth, which revealed that the 3′ NTR was highly tolerant to change, whereas several deletions of centrally located nucleotides in the 5′ NTR led to significantly reduced growth, indicative of functional disruption. Deletions that encroached upon PE1 and PE2 ablated virus growth and identified additional adjacent nucleotides critical for viability. Mutational analysis of PE2 suggest that its signaling ability relies solely on interterminal base pairing and is an independent cis-acting signaling module. This study represents the first mutagenic analysis of nairoviral NTRs in the context of the infectious cycle, and the mechanistic implications of our findings for nairovirus RNA synthesis are discussed. IMPORTANCE Nairoviruses are a group of RNA viruses that include many serious pathogens of humans and animals, including one of the most serious human pathogens in existence, Crimean-Congo hemorrhagic fever virus. The ability of nairoviruses to multiply and cause disease is controlled in major part by nucleotides that flank the 3′ and 5′ ends of nairoviral genes, called nontranslated regions (NTRs). NTR nucleotides interact with other virus components to perform critical steps of the virus multiplication cycle, such as mRNA transcription and RNA replication, with other roles being likely. To better understand how NTRs work, we performed the first comprehensive investigation of the importance of NTR nucleotides in the context of the entire nairovirus replication cycle. We identified both dispensable and critical NTR nucleotides, as well as highlighting the importance of 3′ and 5′ NTR interactions in virus growth, thus providing the first functional map of the nairovirus NTRs.

scholarly journals Conserved Motifs in a Tombusvirus Polymerase Modulate Genome Replication, Subgenomic Transcription, and Amplification of Defective Interfering RNAs

2015 ◽  
Vol 89 (6) ◽  
pp. 3236-3246 ◽  
Author(s):  
Chaminda D. Gunawardene ◽  
Karolina Jaluba ◽  
K. Andrew White
Keyword(s):  
Rna Synthesis ◽  
Mutational Analysis ◽  
Rna Virus ◽  
Plant Viruses ◽  
Comparative Sequence Analysis ◽  
Terminal Region ◽  
Viral Rna ◽  
Genome Replication ◽  
Conserved Motifs ◽  
Content Type

ABSTRACTThe replication of plus-strand RNA virus genomes is mediated by virally encoded RNA-dependent RNA polymerases (RdRps). We have investigated the role of the C-proximal region in the RdRp of tomato bushy stunt virus (TBSV) in mediating viral RNA synthesis. TBSV is the prototype species in the genusTombusvirus, familyTombusviridae, and its RdRp is responsible for replicating the viral genome, transcribing two subgenomic mRNAs, and supporting replication of defective interfering RNAs. Comparative sequence analysis of the RdRps of tombusvirids identified three highly conserved motifs in their C-proximal regions, and these sequences were subsequently targeted for mutational analysis in TBSV. The results revealed that these motifs are important for (i) synthesizing viral genomic RNA and subgenomic mRNAs, (ii) facilitating plus- and/or minus-strand synthesis, and (iii) modulatingtrans-replication of a defective interfering RNA. These motifs were also found to be conserved in other plant viruses as well as in a fungal and insect virus. The collective findings are discussed in relation to viral RNA synthesis and taxonomy.IMPORTANCELittle is currently known about the structure and function of the viral polymerases that replicate the genomes of RNA plant viruses. Tombusviruses, the prototype of the tombusvirids, have been used as model plus-strand RNA plant viruses for understanding many of the steps in the infectious process; however, their polymerases remain poorly characterized. To help address this issue, the function of the C-terminal region of the polymerase of a tombusvirus was investigated. Three conserved motifs were identified and targeted for mutational analysis. The results revealed that these polymerase motifs are important for determining what type of viral RNA is produced, facilitating different steps in viral RNA production, and amplifying subgenomic RNA replicons. Accordingly, the C-terminal region of the tombusvirus polymerase is needed for a variety of fundamental activities. Furthermore, as these motifs are also present in distantly related viruses, the significance of these results extends beyond tombusvirids.

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