scholarly journals A paramyxovirus-like model for Ebola virus bipartite promoters

2020 ◽  
Vol 16 (11) ◽  
pp. e1008972
Author(s):  
Irina Gutsche ◽  
Philippe le Mercier ◽  
Daniel Kolakofsky
Keyword(s):  
Rna Synthesis ◽  
Ebola Virus ◽  
Promoter Element ◽  
Viral Polymerase

Paramyxo- and filovirus nucleocapsids (NCs) have bipartite promoters at their 3′ ends to initiate RNA synthesis. The 2 elements, promoter element 1 (PE1) and promoter element 2 (PE2), are separated by a spacer region that must be exactly a multiple of 6 nucleotides (nt) long. Paramyxovirus NCs have 13 nucleoprotein (NP) subunits/turn, such that PE1 and PE2 are juxtaposed on the same face of the NC helix, for concerted recognition by the viral polymerase. Ebola virus (EBOV) NCs, in contrast, have 25 to 28 subunits/turn, meaning that PE1 and PE2 cannot be juxtaposed. However, there is evidence that the number of subunits/turn at the 3′ end of the EBOV NC is variable. We propose a paramyxovirus-like model for EBOV explaining why there are 8 contiguous copies of the PE2 repeat when 3 are sufficient, why expanding this run to 13 further improves minigenome performance, and why there is a limit to the number of hexa-nt that can be inserted in the spacer region.

scholarly journals Ebola Virus VP35 Interaction with Dynein LC8 Regulates Viral RNA Synthesis

2015 ◽  
Vol 89 (9) ◽  
pp. 5148-5153 ◽  
Author(s):  
Priya Luthra ◽  
David S. Jordan ◽  
Daisy W. Leung ◽  
Gaya K. Amarasinghe ◽  
Christopher F. Basler
Keyword(s):  
Rna Synthesis ◽  
Ebola Virus ◽  
Mutational Analysis ◽  
Cytoplasmic Dynein ◽  
Viral Rna ◽  
Interferon Production ◽  
Dynein Light Chain ◽  
High Affinity ◽  
Functional Consequences ◽  
Oligomerization Domain

Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8.

scholarly journals Sequence in the Influenza A Virus Nucleoprotein Required for Viral Polymerase Binding and RNA Synthesis

2012 ◽  
Vol 86 (13) ◽  
pp. 7292-7297 ◽  
Author(s):  
J. K. Marklund ◽  
Q. Ye ◽  
J. Dong ◽  
Y. J. Tao ◽  
R. M. Krug
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Rna Synthesis ◽  
Viral Polymerase ◽  
Polymerase Binding

scholarly journals Differences in Viral RNA Synthesis but Not Budding or Entry Contribute to the In Vitro Attenuation of Reston Virus Compared to Ebola Virus

2020 ◽  
Vol 8 (8) ◽  
pp. 1215
Author(s):  
Bianca S. Bodmer ◽  
Josephin Greßler ◽  
Marie L. Schmidt ◽  
Julia Holzerland ◽  
Janine Brandt ◽  
...  
Keyword(s):  
Life Cycle ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Severe Disease ◽  
Case Fatality ◽  
Viral Rna ◽  
Pathogenic Potential ◽  
Rnp Complex ◽  
Reston Virus

Most filoviruses cause severe disease in humans. For example, Ebola virus (EBOV) is responsible for the two most extensive outbreaks of filovirus disease to date, with case fatality rates of 66% and 40%, respectively. In contrast, Reston virus (RESTV) is apparently apathogenic in humans, and while transmission of RESTV from domestic pigs to people results in seroconversion, no signs of disease have been reported in such cases. The determinants leading to these differences in pathogenicity are not well understood, but such information is needed in order to better evaluate the risks posed by the repeated spillover of RESTV into the human population and to perform risk assessments for newly emerging filoviruses with unknown pathogenic potential. Interestingly, RESTV and EBOV already show marked differences in their growth in vitro, with RESTV growing slower and reaching lower end titers. In order to understand the basis for this in vitro attenuation of RESTV, we used various life cycle modeling systems mimicking different aspects of the virus life cycle. Our results showed that viral RNA synthesis was markedly slower when using the ribonucleoprotein (RNP) components from RESTV, rather than those for EBOV. In contrast, the kinetics of budding and entry were indistinguishable between these two viruses. These data contribute to our understanding of the molecular basis for filovirus pathogenicity by showing that it is primarily differences in the robustness of RNA synthesis by the viral RNP complex that are responsible for the impaired growth of RESTV in tissue culture.

scholarly journals Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency

2020 ◽  
Vol 295 (20) ◽  
pp. 6785-6797 ◽  
Author(s):  
Calvin J. Gordon ◽  
Egor P. Tchesnokov ◽  
Emma Woolner ◽  
Jason K. Perry ◽  
Joy Y. Feng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Lassa Virus ◽  
Nonstructural Proteins ◽  
Rna Dependent Rna Polymerase ◽  
Direct Acting Antiviral ◽  
Nucleotide Analogue ◽  
Direct Acting

Effective treatments for coronavirus disease 2019 (COVID-19) are urgently needed to control this current pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Replication of SARS-CoV-2 depends on the viral RNA-dependent RNA polymerase (RdRp), which is the likely target of the investigational nucleotide analogue remdesivir (RDV). RDV shows broad-spectrum antiviral activity against RNA viruses, and previous studies with RdRps from Ebola virus and Middle East respiratory syndrome coronavirus (MERS-CoV) have revealed that delayed chain termination is RDV's plausible mechanism of action. Here, we expressed and purified active SARS-CoV-2 RdRp composed of the nonstructural proteins nsp8 and nsp12. Enzyme kinetics indicated that this RdRp efficiently incorporates the active triphosphate form of RDV (RDV-TP) into RNA. Incorporation of RDV-TP at position i caused termination of RNA synthesis at position i+3. We obtained almost identical results with SARS-CoV, MERS-CoV, and SARS-CoV-2 RdRps. A unique property of RDV-TP is its high selectivity over incorporation of its natural nucleotide counterpart ATP. In this regard, the triphosphate forms of 2′-C-methylated compounds, including sofosbuvir, approved for the management of hepatitis C virus infection, and the broad-acting antivirals favipiravir and ribavirin, exhibited significant deficits. Furthermore, we provide evidence for the target specificity of RDV, as RDV-TP was less efficiently incorporated by the distantly related Lassa virus RdRp, and termination of RNA synthesis was not observed. These results collectively provide a unifying, refined mechanism of RDV-mediated RNA synthesis inhibition in coronaviruses and define this nucleotide analogue as a direct-acting antiviral.

scholarly journals Structures of the Mononegavirales Polymerases

2020 ◽  
Vol 94 (22) ◽  
Author(s):  
Bo Liang
Keyword(s):  
Rna Synthesis ◽  
Ebola Virus ◽  
Viral Mrnas ◽  
Future Directions ◽  
Virus Family ◽  
Viral Genomes ◽  
Vesicular Stomatitis ◽  
Syncytial Virus ◽  
Human Parainfluenza Virus ◽  
Negative Sense

ABSTRACT Mononegavirales, known as nonsegmented negative-sense (NNS) RNA viruses, are a class of pathogenic and sometimes deadly viruses that include rabies virus (RABV), human respiratory syncytial virus (HRSV), and Ebola virus (EBOV). Unfortunately, no effective vaccines and antiviral therapeutics against many Mononegavirales are currently available. Viral polymerases have been attractive and major antiviral therapeutic targets. Therefore, Mononegavirales polymerases have been extensively investigated for their structures and functions. Mononegavirales mimic RNA synthesis of their eukaryotic counterparts by utilizing multifunctional RNA polymerases to replicate entire viral genomes and transcribe viral mRNAs from individual viral genes as well as synthesize 5′ methylated cap and 3′ poly(A) tail of the transcribed viral mRNAs. The catalytic subunit large protein (L) and cofactor phosphoprotein (P) constitute the Mononegavirales polymerases. In this review, we discuss the shared and unique features of RNA synthesis, the monomeric multifunctional enzyme L, and the oligomeric multimodular adapter P of Mononegavirales. We outline the structural analyses of the Mononegavirales polymerases since the first structure of the vesicular stomatitis virus (VSV) L protein determined in 2015 and highlight multiple high-resolution cryo-electron microscopy (cryo-EM) structures of the polymerases of Mononegavirales, namely, VSV, RABV, HRSV, human metapneumovirus (HMPV), and human parainfluenza virus (HPIV), that have been reported in recent months (2019 to 2020). We compare the structures of those polymerases grouped by virus family, illustrate the similarities and differences among those polymerases, and reveal the potential RNA synthesis mechanisms and models of highly conserved Mononegavirales. We conclude by the discussion of remaining questions, evolutionary perspectives, and future directions.

scholarly journals The Ebola Virus VP30-NP Interaction Is a Regulator of Viral RNA Synthesis

2016 ◽  
Vol 12 (10) ◽  
pp. e1005937 ◽  
Author(s):  
Robert N. Kirchdoerfer ◽  
Crystal L. Moyer ◽  
Dafna M. Abelson ◽  
Erica Ollmann Saphire
Keyword(s):  
Rna Synthesis ◽  
Ebola Virus ◽  
Viral Rna

scholarly journals Ebola virus inclusion body formation and RNA synthesis are controlled by a novel domain of NP interacting with VP35

2020 ◽  
Author(s):  
Tsuyoshi Miyake ◽  
Charlotte M. Farley ◽  
Benjamin E. Neubauer ◽  
Thomas P. Beddow ◽  
Thomas Hoenen ◽  
...  
Keyword(s):  
Life Cycle ◽  
Inclusion Body ◽  
Inclusion Bodies ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Central Domain ◽  
Body Formation ◽  
Inclusion Body Formation

AbstractEbola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection IBs display dynamic properties regarding their size and location. Also, the contents of IBs must transition prior to further viral maturation, assembly and release, implying additional steps in IB function. Interestingly, expression of the viral nucleoprotein (NP) alone is sufficient for generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30 and the RNA polymerase L. Previously we defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here we show that NP-Ct is absolutely required for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for production of infectious virus-like particles and retention of viral RNA within these particles. Furthermore, co-expression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins only VP35 is able to overcome the defect in IB formation caused by deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself, and which is mediated by a newly identified domain of NP, the “central domain” (CD).ImportanceInclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative sense RNA viruses including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA.

scholarly journals Staufen1 Interacts with Multiple Components of the Ebola Virus Ribonucleoprotein and Enhances Viral RNA Synthesis

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Jingru Fang ◽  
Colette Pietzsch ◽  
Palaniappan Ramanathan ◽  
Rodrigo I. Santos ◽  
Philipp A. Ilinykh ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Binding Proteins ◽  
Ebola Virus ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Host Factors ◽  
Viral Rna ◽  
Virion Protein ◽  
Public Health Importance

ABSTRACTEbola virus (EBOV) genome and mRNAs contain long, structured regions that could hijack host RNA-binding proteins to facilitate infection. We performed RNA affinity chromatography coupled with mass spectrometry to identify host proteins that bind to EBOV RNAs and identified four high-confidence proviral host factors, including Staufen1 (STAU1), which specifically binds both 3′ and 5′ extracistronic regions of the EBOV genome. We confirmed that EBOV infection rate and production of infectious particles were significantly reduced in STAU1-depleted cells. STAU1 was recruited to sites of EBOV RNA synthesis upon infection and enhanced viral RNA synthesis. Furthermore, STAU1 interacts with EBOV nucleoprotein (NP), virion protein 30 (VP30), and VP35; the latter two bridge the viral polymerase to the NP-coated genome, forming the viral ribonucleoprotein (RNP) complex. Our data indicate that STAU1 plays a critical role in EBOV replication by coordinating interactions between the viral genome and RNA synthesis machinery.IMPORTANCEEbola virus (EBOV) is a negative-strand RNA virus with significant public health importance. Currently, no therapeutics are available for Ebola, which imposes an urgent need for a better understanding of EBOV biology. Here we dissected the virus-host interplay between EBOV and host RNA-binding proteins. We identified novel EBOV host factors, including Staufen1, which interacts with multiple viral factors and is required for efficient viral RNA synthesis.

Remdesivir (GS-5734) for COVID-19 Treatment: Past and Recent Updates

Coronaviruses ◽  
2020 ◽  
Vol 01 ◽  
Author(s):  
Santhosh Arul ◽  
Haripriya Dayalan
Keyword(s):  
Rna Synthesis ◽  
Ebola Virus ◽  
Control Measures ◽  
The Novel ◽  
Off Label Use ◽  
Off Label ◽  
The Past ◽  
Different Strains ◽  
And Control ◽  
Viral Outbreak

Background: SARS-CoV-2 is a pandemic now and several preventive and control measures have been taken by several countries to contain and treat the disease. WHO has been working meticulously and have been providing up to date information and statistics on incidences and death. Several broad spectrum anti-viral drugs are available and have been used in the past to fight against the viral outbreak. Recently Remdesivir, an experimental prodrug from Gilead Sciences have been found potential to be used as a therapy to treat the COVID-19. Objective: Here we have reviewed the previous findings that are available in the literature and report several findings that are crucial and provide up to date information. Results : Remdesivir was initially invented for use against Ebola virus treatment and has proved potential against different strains of Ebola, Nipah, and other strains of coronaviruses. Clinical trials with Remdesivir for COVID-19 patients have begun and several off label use of Remdesivir is reported recently. Currently the drug seems to have effect against the SARS-CoV-2 virus with side effects among few patients. The results although are not conclusive are partly promising. This review provides past and recent updates on the use of Remdesivir. Conclusion: From the review we conclude that the drug Remdesivir is known to exhibit its mechanism of action by terminating the RNA synthesis and it is a potential drug against the novel corona virus.

scholarly journals Mechanism of Inhibition of Ebola Virus RNA-Dependent RNA Polymerase by Remdesivir

Viruses ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 326 ◽  
Author(s):  
Egor Tchesnokov ◽  
Joy Feng ◽  
Danielle Porter ◽  
Matthias Götte
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Virus Disease ◽  
Chain Termination ◽  
Mitochondrial Rna ◽  
Long Distance ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogue ◽  
Nucleotide Incorporation

Remdesivir (GS-5734) is a 1′-cyano-substituted adenosine nucleotide analogue prodrug that shows broad-spectrum antiviral activity against several RNA viruses. This compound is currently under clinical development for the treatment of Ebola virus disease (EVD). While antiviral effects have been demonstrated in cell culture and in non-human primates, the mechanism of action of Ebola virus (EBOV) inhibition for remdesivir remains to be fully elucidated. The EBOV RNA-dependent RNA polymerase (RdRp) complex was recently expressed and purified, enabling biochemical studies with the relevant triphosphate (TP) form of remdesivir and its presumptive target. In this study, we confirmed that remdesivir-TP is able to compete for incorporation with adenosine triphosphate (ATP). Enzyme kinetics revealed that EBOV RdRp and respiratory syncytial virus (RSV) RdRp incorporate ATP and remdesivir-TP with similar efficiencies. The selectivity of ATP against remdesivir-TP is ~4 for EBOV RdRp and ~3 for RSV RdRp. In contrast, purified human mitochondrial RNA polymerase (h-mtRNAP) effectively discriminates against remdesivir-TP with a selectivity value of ~500-fold. For EBOV RdRp, the incorporated inhibitor at position i does not affect the ensuing nucleotide incorporation event at position i+1. For RSV RdRp, we measured a ~6-fold inhibition at position i+1 although RNA synthesis was not terminated. Chain termination was in both cases delayed and was seen predominantly at position i+5. This pattern is specific to remdesivir-TP and its 1′-cyano modification. Compounds with modifications at the 2′-position show different patterns of inhibition. While 2′-C-methyl-ATP is not incorporated, ara-ATP acts as a non-obligate chain terminator and prevents nucleotide incorporation at position i+1. Taken together, our biochemical data indicate that the major contribution to EBOV RNA synthesis inhibition by remdesivir can be ascribed to delayed chain termination. The long distance of five residues between the incorporated nucleotide analogue and its inhibitory effect warrant further investigation.

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