scholarly journals Reduced Nucleoprotein availability impairs negative-sense RNA virus replication and promotes host recognition

2021 ◽  
Author(s):  
Benjamin E. Nilsson-Payant ◽  
Daniel Blanco-Melo ◽  
Skyler Uhl ◽  
Beatriz Escudero-Pérez ◽  
Silke Olschewski ◽  
...  
Keyword(s):  
Host Response ◽  
Measles Virus ◽  
Influenza A ◽  
Ebola Virus ◽  
Rna Viruses ◽  
Viral Rna ◽  
Genome Replication ◽  
Replication Machinery ◽  
Molecular Patterns ◽  
Negative Sense

Negative-sense RNA viruses (NSVs) rely on prepackaged viral RNA-dependent RNA polymerases (RdRp) to replicate and transcribe their viral genomes. Their replication machinery consists of an RdRp bound to viral RNA which is wound around a nucleoprotein (NP) scaffold, forming a viral ribonucleoprotein complex. NSV NP is known to regulate transcription and replication of genomic RNA, however its role in maintaining and protecting the viral genetic material is unknown. Here, we exploited host microRNA expression to target NP of influenza A virus and Sendai virus to ascertain how this would impact genomic levels and the host response to infection. We find that in addition to inducing a drastic decrease in genome replication, the antiviral host response in the absence of NP is dramatically enhanced. Additionally, our data shows that insufficient levels of NP prevent the replication machinery of these NSVs to process full-length genomes, resulting in aberrant replication products which form pathogen-associated molecular patterns in the process. These dynamics facilitate immune recognition by cellular pattern recognition receptors leading to a strong host antiviral response. Moreover, we observe that the consequences of limiting NP levels are universal amongst NSVs including Ebola virus, Lassa virus and Measles virus. Overall, these results provide new insights into viral genome replication of negative-sense RNA viruses and highlight novel avenues towards developing effective antiviral strategies, adjuvants, and/or live-attenuated vaccines. IMPORTANCE Negative-sense RNA viruses comprise some of the most important known human pathogens, including influenza A virus, measles virus and Ebola virus. These viruses possess RNA genomes that are unreadable to the host as they require specific viral RNA dependent RNA polymerases in conjunction with other viral proteins such as nucleoprotein to be replicated and transcribed. As this process generates a significant amount of pathogen-associated molecular patterns, this phylum of viruses can result in a robust induction of the intrinsic host cellular response. To circumvent these defenses, these viruses form tightly regulated ribonucleoprotein replication complexes in order to protect their genomes from detection and to prevent excessive aberrant replication. Here we demonstrate the balance that negative-sense RNA viruses must achieve to both replicate efficiently and to avoid induction of the host defenses.

scholarly journals Structure and assembly of double-headed Sendai virus nucleocapsids

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Na Zhang ◽  
Hong Shan ◽  
Mingdong Liu ◽  
Tianhao Li ◽  
Rui Luo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Measles Virus ◽  
Sendai Virus ◽  
Nipah Virus ◽  
Mumps Virus ◽  
Genome Replication ◽  
Direct Visualization ◽  
Closed State ◽  
Cryo Electron Microscopy ◽  
Negative Sense

AbstractParamyxoviruses, including the mumps virus, measles virus, Nipah virus and Sendai virus (SeV), have non-segmented single-stranded negative-sense RNA genomes which are encapsidated by nucleoproteins into helical nucleocapsids. Here, we reported a double-headed SeV nucleocapsid assembled in a tail-to-tail manner, and resolved its helical stems and clam-shaped joint at the respective resolutions of 2.9 and 3.9 Å, via cryo-electron microscopy. Our structures offer important insights into the mechanism of the helical polymerization, in particular via an unnoticed exchange of a N-terminal hole formed by three loops of nucleoproteins, and unveil the clam-shaped joint in a hyper-closed state for nucleocapsid dimerization. Direct visualization of the loop from the disordered C-terminal tail provides structural evidence that C-terminal tail is correlated to the curvature of nucleocapsid and links nucleocapsid condensation and genome replication and transcription with different assembly forms.

scholarly journals Previremic Identification of Ebola or Marburg Virus Infection Using Integrated Host-Transcriptome and Viral Genome Detection

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Emily Speranza ◽  
Ignacio Caballero ◽  
Anna N. Honko ◽  
Joshua C. Johnson ◽  
J. Kyle Bohannon ◽  
...  
Keyword(s):  
Host Response ◽  
Nonhuman Primates ◽  
Ebola Virus ◽  
Clinical Symptoms ◽  
Infectious Agent ◽  
Marburg Virus ◽  
Viral Rna ◽  
Molecular Tests ◽  
Early Replication ◽  
Host Infection

ABSTRACT Outbreaks of filoviruses, such as those caused by the Ebola (EBOV) and Marburg (MARV) virus, are difficult to detect and control. The initial clinical symptoms of these diseases are nonspecific and can mimic other endemic pathogens. This makes confident diagnosis based on clinical symptoms alone impossible. Molecular diagnostics for these diseases that rely on the detection of viral RNA in the blood are only effective after significant disease progression. As an approach to identify these infections earlier in the disease course, we tested the effectiveness of viral RNA detection combined with an assessment of sentinel host mRNAs that are upregulated following filovirus infection. RNAseq analysis of EBOV-infected nonhuman primates identified host RNAs that are upregulated at early stages of infection. NanoString probes that recognized these host-response RNAs were combined with probes that recognized viral RNA and were used to classify viral infection both prior to viremia and postviremia. This approach was highly successful at identifying samples from nonhuman primate subjects and correctly distinguished the causative agent in a previremic stage in 10 EBOV and 5 MARV samples. This work suggests that unified host response/viral fingerprint assays can enable diagnosis of disease earlier than testing for viral nucleic acid alone, which could decrease transmission events and increase therapeutic effectiveness. IMPORTANCE Current molecular tests that identify infection with high-consequence viruses such as Ebola virus and Marburg virus are based on the detection of virus material in the blood. These viruses do not undergo significant early replication in the blood and, instead, replicate in organs such as the liver and spleen. Thus, virus begins to accumulate in the blood only after significant replication has already occurred in those organs, making viremia an indicator of infection only after initial stages have become established. Here, we show that a multianalyte assay can correctly identify the infectious agent in nonhuman primates (NHPs) prior to viremia through tracking host infection response transcripts. This illustrates that a single-tube, sample-to-answer format assay could be used to advance the time at which the type of infection can be determined and thereby improve outcomes.

scholarly journals Expression, purification, crystallization and preliminary X-ray analysis of full-length human RIG-I

2014 ◽  
Vol 70 (2) ◽  
pp. 248-251
Author(s):  
Jane Kwok ◽  
Kenrie P. Y. Hui ◽  
Julien Lescar ◽  
Masayo Kotaka
Keyword(s):  
Pattern Recognition ◽  
Ebola Virus ◽  
Pattern Recognition Receptors ◽  
Full Length ◽  
Viral Rna ◽  
Microbial Pathogens ◽  
Diffusion Method ◽  
X Ray ◽  
Cell Parameters ◽  
Molecular Patterns

The human innate immune system can detect invasion by microbial pathogens through pattern-recognition receptors that recognize structurally conserved pathogen-associated molecular patterns. Retinoic acid-inducible gene I (RIG-I)-like helicases (RLHs) are one of the two major families of pattern-recognition receptors that can detect viral RNA. RIG-I, belonging to the RLH family, is capable of recognizing intracellular viral RNA from RNA viruses, including influenza virus and Ebola virus. Here, full-length human RIG-I (hRIG-I) was cloned inEscherichia coliand expressed in a recombinant form with a His-SUMO tag. The protein was purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.85 Å resolution; the crystal belonged to space groupF23, with unit-cell parametersa = b=c= 216.43 Å, α = β = γ = 90°.

scholarly journals Novel and potent inhibitors targeting DHODH, a rate-limiting enzyme in de novo pyrimidine biosynthesis, are broad-spectrum antiviral against RNA viruses including newly emerged coronavirus SARS-CoV-2

2020 ◽  
Author(s):  
Rui Xiong ◽  
Leike Zhang ◽  
Shiliang Li ◽  
Yuan Sun ◽  
Minyi Ding ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Influenza A ◽  
Ebola Virus ◽  
De Novo ◽  
Rna Viruses ◽  
Direct Acting Antiviral ◽  
Novel Coronavirus ◽  
Direct Acting ◽  
Viral Mutagenesis

AbstractEmerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of coronavirus SARS-CoV-2. Existing direct-acting antiviral (DAA) drugs cannot be applied immediately to new viruses because of virus-specificity, and the development of new DAA drugs from the beginning is not timely for outbreaks. Thus, host-targeting antiviral (HTA) drugs have many advantages to fight against a broad spectrum of viruses, by blocking the viral replication and overcoming the potential viral mutagenesis simultaneously. Herein, we identified two potent inhibitors of DHODH, S312 and S416, with favorable drug-like and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus (H1N1, H3N2, H9N2), Zika virus, Ebola virus, and particularly against the recent novel coronavirus SARS-CoV-2. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knocking-out cells. We also proposed the drug combination of DAA and HTA was a promising strategy for anti-virus treatment and proved that S312 showed more advantageous than Oseltamivir to treat advanced influenza diseases in severely infected animals. Notably, S416 is reported to be the most potent inhibitor with an EC50 of 17nM and SI value >5882 in SARS-CoV-2-infected cells so far. This work demonstrates that both our self-designed candidates and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-repression may have clinical potentials not only to influenza but also to COVID-19 circulating worldwide, no matter such viruses mutate or not.

scholarly journals A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid

2018 ◽  
Vol 92 (8) ◽  
pp. e00146-18 ◽  
Author(s):  
Ryan H. Gumpper ◽  
Weike Li ◽  
Carlos H. Castañeda ◽  
M. José Scuderi ◽  
James K. Bashkin ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Vesicular Stomatitis Virus ◽  
Ebola Virus ◽  
Rna Viruses ◽  
Rna Virus ◽  
Viral Rna ◽  
Negative Strand ◽  
Double Stranded Dna ◽  
Vesicular Stomatitis ◽  
Syncytial Virus

ABSTRACTPolyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, was found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus.IMPORTANCENegative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit viral growth. The target specificity of the polyamide molecule was proved by its inhibition of thermo-release and RNA nuclease digestion of the RNA bound in a model nucleocapsid, and a crystal structure of the polyamide inside the nucleocapsid. This encouraging observation provided the proof-of-concept rationale for designing polyamides as antiviral drugs against NSVs.

scholarly journals Human and Murine IFIT1 Proteins Do Not Restrict Infection of Negative-Sense RNA Viruses of the Orthomyxoviridae, Bunyaviridae, and Filoviridae Families

2015 ◽  
Vol 89 (18) ◽  
pp. 9465-9476 ◽  
Author(s):  
Amelia K. Pinto ◽  
Graham D. Williams ◽  
Kristy J. Szretter ◽  
James P. White ◽  
José Luiz Proença-Módena ◽  
...  
Keyword(s):  
Cell Culture ◽  
Antiviral Activity ◽  
Ebola Virus ◽  
Rna Viruses ◽  
Ectopic Expression ◽  
A549 Cells ◽  
Host Protein ◽  
Interferon Response ◽  
Restriction Factor ◽  
Negative Sense

ABSTRACTInterferon-induced protein with tetratricopeptide repeats 1 (IFIT1) is a host protein with reported cell-intrinsic antiviral activity against several RNA viruses. The proposed basis for the activity against negative-sense RNA viruses is the binding to exposed 5′-triphosphates (5′-ppp) on the genome of viral RNA. However, recent studies reported relatively low binding affinities of IFIT1 for 5′-ppp RNA, suggesting that IFIT1 may not interact efficiently with this moiety under physiological conditions. To evaluate the ability of IFIT1 to have an impact on negative-sense RNA viruses, we infectedIfit1−/−and wild-type control mice and primary cells with four negative-sense RNA viruses (influenza A virus [IAV], La Crosse virus [LACV], Oropouche virus [OROV], and Ebola virus) corresponding to three distinct families. Unexpectedly, a lack ofIfit1gene expression did not result in increased infection by any of these viruses in cell culture. Analogously, morbidity, mortality, and viral burdens in tissues were identical betweenIfit1−/−and control mice after infection with IAV, LACV, or OROV. Finally, deletion of the human IFIT1 protein in A549 cells did not affect IAV replication or infection, and reciprocally, ectopic expression of IFIT1 in HEK293T cells did not inhibit IAV infection. To explain the lack of antiviral activity against IAV, we measured the binding affinity of IFIT1 for RNA oligonucleotides resembling the 5′ ends of IAV gene segments. The affinity for 5′-ppp RNA was approximately 10-fold lower than that for non-2′-O-methylated (cap 0) RNA oligonucleotides. Based on this analysis, we conclude that IFIT1 is not a dominant restriction factor against negative-sense RNA viruses.IMPORTANCENegative-sense RNA viruses, including influenza virus and Ebola virus, have been responsible for some of the most deadly outbreaks in recent history. The host interferon response and induction of antiviral genes contribute to the control of infections by these viruses. IFIT1 is highly induced after virus infection and reportedly has antiviral activity against several RNA and DNA viruses. However, its role in restricting infection by negative-sense RNA viruses remains unclear. In this study, we evaluated the ability of IFIT1 to inhibit negative-sense RNA virus replication and pathogenesis bothin vitroandin vivo. Detailed cell culture and animal studies demonstrated that IFIT1 is not a dominant restriction factor against three different families of negative-sense RNA viruses.

scholarly journals Analysis of viral RNA-host protein interactomes enables rapid antiviral drug discovery

2021 ◽  
Author(s):  
Shaojun Zhang ◽  
Wenze Huang ◽  
Lili Ren ◽  
Xiaohui Ju ◽  
Mingli Gong ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Ebola Virus ◽  
Rna Viruses ◽  
Antiviral Drug ◽  
Antiviral Agent ◽  
Viral Rna ◽  
Host Protein ◽  
Host Responses ◽  
Interaction Patterns ◽  
Global Public Health

RNA viruses including SARS-CoV-2, Ebola virus (EBOV), and Zika virus (ZIKV) constitute a major threat to global public health and society. The interactions between viral genomes and host proteins are essential in the life cycle of RNA viruses and thus provide targets for drug development. However, viral RNA-host protein interactions have remained poorly characterized. Here we applied ChIRP-MS to profile the interactomes of human proteins and the RNA genomes of SARS-CoV-2, EBOV, and ZIKV in infected cells. Integrated interactome analyses revealed interaction patterns that reflect both common and virus-specific host responses, and enabled rapid drug screening to target the vulnerable host factors. We identified Enasidenib as a SARS-CoV-2 specific antiviral agent, and Trifluoperazine and Cepharanthine as broad spectrum antivirals against all three RNA viruses.

Electron microscopy of frozen, hydrated lacrosse virus

1986 ◽  
Vol 44 ◽  
pp. 104-105
Author(s):  
Y. Talmon ◽  
B. V. V. Prasad ◽  
J. P. M. Clerx ◽  
W. Chiu ◽  
M. J. Hewlett
Keyword(s):  
Electron Microscopy ◽  
Ionic Strength ◽  
Nucleocapsid Protein ◽  
Rna Viruses ◽  
High Sensitivity ◽  
Bilayer Membrane ◽  
Viral Rna ◽  
California Serogroup ◽  
Viral Nucleocapsid ◽  
Negative Sense

LaCrosse (LAC) virus is a member of the California serogroup of the Bunyaviridae. The bunyaviruses are all negative-sense, RNA viruses whose genome consists of three segments of single stranded RNA. LAC virions are composed of multiple viral RNA strands complexed with viral nucleocapsid protein surrounded by a bilayer membrane embedded with 2 glycoproteins.Staining and drying of LAC virus samples produce images in which the virions are collapsed and distorted (Figure 1). Fixation prior to staining, also gives disappointing results, probably due to the high sensitivity of the LAC virus to changes in the ionic strength and composition of the solution in which it is suspended.

scholarly journals Model Suggesting that Replication of Influenza Virus Is Regulated by Stabilization of Replicative Intermediates

2004 ◽  
Vol 78 (17) ◽  
pp. 9568-9572 ◽  
Author(s):  
Frank T. Vreede ◽  
Tanis E. Jung ◽  
George G. Brownlee
Keyword(s):  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Mrna Transcription ◽  
Rna Dependent Rna Polymerase ◽  
Replicative Intermediates ◽  
Negative Sense ◽  
Transcription And Replication

ABSTRACT The RNA-dependent RNA polymerase of influenza A virus is responsible for both transcription and replication of negative-sense viral RNA. It is thought that a “switching” mechanism regulates the transition between these activities. We demonstrate that, in the presence of preexisting viral RNA polymerase and nucleoprotein (NP), influenza A virus synthesizes both mRNA (transcription) and cRNA (replication) early in infection. We suggest that there may be no switch regulating the initiation of RNA synthesis and present a model suggesting that nascent cRNA is degraded by host cell nucleases unless it is stabilized by newly synthesized viral RNA polymerase and NP.

scholarly journals Tyr82 Amino Acid Mutation in PB1 Polymerase Induces an Influenza Virus Mutator Phenotype

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Tadasuke Naito ◽  
Kazumasa Shirai ◽  
Kotaro Mori ◽  
Hidetaka Muratsu ◽  
Hiroshi Ushirogawa ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rna Viruses ◽  
Viral Rna ◽  
Wild Type ◽  
Mutator Phenotype ◽  
Polymerase Fidelity

ABSTRACT In various positive-sense single-stranded RNA viruses, a low-fidelity viral RNA-dependent RNA polymerase (RdRp) confers attenuated phenotypes by increasing the mutation frequency. We report a negative-sense single-stranded RNA virus RdRp mutant strain with a mutator phenotype. Based on structural data of RdRp, rational targeting of key residues, and screening of fidelity variants, we isolated a novel low-fidelity mutator strain of influenza virus that harbors a Tyr82-to-Cys (Y82C) single-amino-acid substitution in the PB1 polymerase subunit. The purified PB1-Y82C polymerase indeed showed an increased frequency of misincorporation compared with the wild-type PB1 in an in vitro biochemical assay. To further investigate the effects of position 82 on PB1 polymerase fidelity, we substituted various amino acids at this position. As a result, we isolated various novel mutators other than PB1-Y82C with higher mutation frequencies. The structural model of influenza virus polymerase complex suggested that the Tyr82 residue, which is located at the nucleoside triphosphate entrance tunnel, may influence a fidelity checkpoint. Interestingly, although the PB1-Y82C variant replicated with wild-type PB1-like kinetics in tissue culture, the 50% lethal dose of the PB1-Y82C mutant was 10 times lower than that of wild-type PB1 in embryonated chicken eggs. In conclusion, our data indicate that the Tyr82 residue of PB1 has a crucial role in regulating polymerase fidelity of influenza virus and is closely related to attenuated pathogenic phenotypes in vivo. IMPORTANCE Influenza A virus rapidly acquires antigenic changes and antiviral drug resistance, which limit the effectiveness of vaccines and drug treatments, primarily owing to its high rate of evolution. Virus populations formed by quasispecies can contain resistance mutations even before a selective pressure is applied. To study the effects of the viral mutation spectrum and quasispecies, high- and low-fidelity variants have been isolated for several RNA viruses. Here, we report the discovery of a low-fidelity RdRp variant of influenza A virus that contains a substitution at Tyr82 in PB1. Viruses containing the PB1-Y82C substitution showed growth kinetics and viral RNA synthesis levels similar to those of the wild-type virus in cell culture; however, they had significantly attenuated phenotypes in a chicken egg infection experiment. These data demonstrated that decreased RdRp fidelity attenuates influenza A virus in vivo, which is a desirable feature for the development of safer live attenuated vaccine candidates.

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