Development of a New Reverse Genetics System for Ebola Virus

ABSTRACT Ebola virus (EBOV) is a highly pathogenic negative-stranded RNA virus that has caused several deadly endemics in the past decades. EBOV reverse genetics systems are available for studying live viruses under biosafety level 4 (BSL-4) or subviral particles under BSL-2 conditions. However, these systems all require cotransfection of multiple plasmids expressing viral genome and viral proteins essential for EBOV replication, which is technically challenging and unable to naturally mimic virus propagation using the subviral particle. Here, we established a new EBOV reverse genetics system only requiring transfection of a single viral RNA genome into an engineered cell line that stably expresses viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and large (L) proteins and has been fine-tuned for its superior permissiveness for EBOV replication. Using this system, subviral particles expressing viral VP40, glycoprotein (GP), and VP24 could be produced and continuously propagated and eventually infect the entire cell population. We demonstrated the authentic response of the subviral system to antivirals and uncovered that the VP35 amount is critical for optimal virus replication. Furthermore, we showed that fully infectious virions can be efficiently rescued by delivering the full-length EBOV genome into the same supporting cell, and the efficiency is not affected by genome polarity or virus variant specificity. In summary, our work provides a new tool for studying EBOV under different biosafety levels. IMPORTANCE Ebola virus is among the most dangerous viral pathogens, with a case fatality rate of up to 90%. Since 2013, the two largest and most complex Ebola outbreaks in Africa have revealed the lack of investigation on this notorious virus. A reverse genetics system is an important tool for studying viruses by producing mutant viruses or generating safer and convenient model systems. Here, we developed an EBOV life cycle modeling system in which subviral particles can spontaneously propagate in cell culture. In addition, this system can be employed to rescue infectious virions of homologous or heterologous EBOV isolates using either sense or antisense viral RNA genomes. In summary, we developed a new tool for EBOV research.

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

Microorganisms ◽

10.3390/microorganisms8081215 ◽

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.

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

Journal of Virology ◽

10.1128/jvi.00146-18 ◽

2018 ◽

Vol 92 (8) ◽

pp. e00146-18 ◽

Cited By ~ 5

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.

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Vesicular Stomatitis Virus: From Agricultural Pathogen to Vaccine Vector

Pathogens ◽

10.3390/pathogens10091092 ◽

2021 ◽

Vol 10 (9) ◽

pp. 1092

Author(s):

Guodong Liu ◽

Wenguang Cao ◽

Abdjeleel Salawudeen ◽

Wenjun Zhu ◽

Karla Emeterio ◽

...

Keyword(s):

Vesicular Stomatitis Virus ◽

Reverse Genetics ◽

Ebola Virus ◽

Rna Virus ◽

Research Tool ◽

Vaccine Vector ◽

The Family ◽

Vesicular Stomatitis ◽

Negative Sense ◽

Family Rhabdoviridae

Vesicular stomatitis virus (VSV), which belongs to the Vesiculovirus genus of the family Rhabdoviridae, is a well studied livestock pathogen and prototypic non-segmented, negative-sense RNA virus. Although VSV is responsible for causing economically significant outbreaks of vesicular stomatitis in cattle, horses, and swine, the virus also represents a valuable research tool for molecular biologists and virologists. Indeed, the establishment of a reverse genetics system for the recovery of infectious VSV from cDNA transformed the utility of this virus and paved the way for its use as a vaccine vector. A highly effective VSV-based vaccine against Ebola virus recently received clinical approval, and many other VSV-based vaccines have been developed, particularly for high-consequence viruses. This review seeks to provide a holistic but concise overview of VSV, covering the virus’s ascension from perennial agricultural scourge to promising medical countermeasure, with a particular focus on vaccines.

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Intraspecies Host Specificity of a Single-Stranded RNA Virus Infecting a Marine Photosynthetic Protist Is Determined at the Early Steps of Infection

Journal of Virology ◽

10.1128/jvi.01082-06 ◽

2006 ◽

Vol 81 (3) ◽

pp. 1372-1378 ◽

Cited By ~ 28

Author(s):

Hiroyuki Mizumoto ◽

Yuji Tomaru ◽

Yoshitake Takao ◽

Yoko Shirai ◽

Keizo Nagasaki

Keyword(s):

Host Specificity ◽

Rna Virus ◽

Viral Rna ◽

Host Cells ◽

Progeny Virus ◽

Virus Suspension ◽

Virus Propagation ◽

Protein Coding ◽

Transmission Electron ◽

A Cell

ABSTRACT Viruses are extremely abundant in seawater and are believed to be significant pathogens to photosynthetic protists (microalgae). Recently, several novel RNA viruses were found to infect marine photosynthetic protists; one of them is HcRNAV, which infects Heterocapsa circularisquama (Dinophyceae). There are two distinct ecotypes of HcRNAV with complementary intraspecies host ranges. Nucleotide sequence comparison between them revealed remarkable differences in the coat protein coding gene resulting in a high frequency of amino acid substitutions. However, the detailed mechanism supporting this intraspecies host specificity is still unknown. In this study, virus inoculation experiments were conducted with compatible and incompatible host-virus combinations to investigate the mechanism determining intraspecies host specificity. Cells were infected by adding a virus suspension directly to a host culture or by transfecting viral RNA into host cells by particle bombardment. Virus propagation was monitored by Northern blot analysis with a negative-strand-specific RNA probe, transmission electron microscopy, and a cell lysis assay. With compatible host-virus combinations, propagation of infectious progeny occurred regardless of the inoculation method used. When incompatible combinations were used, direct addition of a virus suspension did not even result in viral RNA replication, while in host cells transfected with viral RNA, infective progeny virus particles with a host range encoded by the imported viral RNA were propagated. This indicates that the intraspecies host specificity of HcRNAV is determined by the upstream events of virus infection. This is the first report describing the reproductive steps of an RNA virus infecting a photosynthetic protist at the molecular level.

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A Comprehensive Roadmap Towards Generation of Influenza B Reporter Assay Using a Single DNA Polymerase Based Cloning of Reporter RNA Construct

10.1101/2021.11.19.469260 ◽

2021 ◽

Author(s):

Nandita Kedia ◽

Saptarshi Banerjee ◽

Arindam Mondal

Keyword(s):

Rna Synthesis ◽

Rna Virus ◽

Assay System ◽

Viral Rna ◽

Host Protein ◽

Influenza B Virus ◽

Influenza B ◽

Reporter Assay ◽

Virus Propagation ◽

B Virus

Mini-genome reporter assay is a key tool for conducting RNA virus research. But, procedural complications and lack of adequate literature pose major challenge towards developing these assay systems. Here we present a novel yet generic and simple cloning strategy for construction of influenza B virus reporter RNA template and describe extensive standardization of the reporter RNP/polymerase activity assay for monitoring viral RNA synthesis in infection free setting. Using this assay system, we, for the first time showed the effect of viral protein NS1 and host protein PKC-Delta upon influenza B virus RNA synthesis. Additionally, the assay system showed promising results in evaluating the efficacy of antiviral drugs targeting viral RNA synthesis and virus propagation. Together, this work offers a detailed protocol for standardization of influenza virus mini-genome assay and an excellent tool for screening of host factors and antivirals in a fast, user friendly and high throughput manner.

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The two-stage interaction of Ebola virus VP40 with nucleoprotein results in a switch from viral RNA synthesis to virion assembly/budding

Protein & Cell ◽

10.1007/s13238-020-00764-0 ◽

2020 ◽

Author(s):

Linjuan Wu ◽

Dongning Jin ◽

Dan Wang ◽

Xuping Jing ◽

Peng Gong ◽

...

Keyword(s):

Rna Synthesis ◽

Matrix Protein ◽

Ebola Virus ◽

Virus Assembly ◽

Rna Virus ◽

Critical Role ◽

Viral Rna ◽

Hydrophobic Core ◽

Virion Assembly ◽

N Terminus

AbstractEbola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40–NP interaction within IBs, sequestering VP40 within IBs, reducing VP40–VLP egress, abolishing the incorporation of NC-like structures into VP40–VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding.

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Nucleocapsid Structure of Negative Strand RNA Virus

Viruses ◽

10.3390/v12080835 ◽

2020 ◽

Vol 12 (8) ◽

pp. 835 ◽

Cited By ~ 1

Author(s):

Ming Luo ◽

James Ross Terrell ◽

Shelby Ashlyn Mcmanus

Keyword(s):

Nucleocapsid Protein ◽

Rna Synthesis ◽

Ebola Virus ◽

Rna Virus ◽

Viral Rna ◽

Human Pathogens ◽

Negative Strand ◽

Rna Genome ◽

Rna Complex ◽

Unique Mechanism

Negative strand RNA viruses (NSVs) include many important human pathogens, such as influenza virus, Ebola virus, and rabies virus. One of the unique characteristics that NSVs share is the assembly of the nucleocapsid and its role in viral RNA synthesis. In NSVs, the single strand RNA genome is encapsidated in the linear nucleocapsid throughout the viral replication cycle. Subunits of the nucleocapsid protein are parallelly aligned along the RNA genome that is sandwiched between two domains composed of conserved helix motifs. The viral RNA-dependent-RNA polymerase (vRdRp) must recognize the protein–RNA complex of the nucleocapsid and unveil the protected genomic RNA in order to initiate viral RNA synthesis. In addition, vRdRp must continuously translocate along the protein–RNA complex during elongation in viral RNA synthesis. This unique mechanism of viral RNA synthesis suggests that the nucleocapsid may play a regulatory role during NSV replication.

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RNA Binding of Ebola Virus VP30 Is Essential for Activating Viral Transcription

Journal of Virology ◽

10.1128/jvi.00271-16 ◽

2016 ◽

Vol 90 (16) ◽

pp. 7481-7496 ◽

Cited By ~ 27

Author(s):

Nadine Biedenkopf ◽

Julia Schlereth ◽

Arnold Grünweller ◽

Stephan Becker ◽

Roland K. Hartmann

Keyword(s):

Transcription Factor ◽

Zinc Finger ◽

Ebola Virus ◽

Rna Binding ◽

Case Fatality ◽

Viral Rna ◽

Viral Transcription ◽

Viral Nucleocapsid ◽

Support Activity ◽

Transcription And Replication

ABSTRACTThe template for Ebola virus (EBOV) transcription and replication is the helical viral nucleocapsid composed of the viral negative-sense (−) RNA genome, which is complexed by the nucleoprotein (NP), VP35, polymerase L, VP24, and VP30. While viral replication is exerted by polymerase L and its cofactor VP35, EBOV mRNA synthesis is regulated by the viral nucleocapsid protein VP30, an essential EBOV-specific transcription factor. VP30 is a homohexameric phosphoprotein containing a nonconventional zinc finger. The transcriptional support activity of VP30 is strongly influenced by its phosphorylation state. We studied here how RNA binding contributed to VP30's function in transcriptional activation. Using a novel mobility shift assay and the 3′-terminal 154 nucleotides of the EBOV genome as a standard RNA substrate, we detected that RNA binding of VP30 was severely impaired by VP30 mutations that (i) destroy the protein's capability to form homohexamers, (ii) disrupt the integrity of its zinc finger domain, (iii) mimic its fully phosphorylated state, or (iv) alter the putative RNA binding region. RNA binding of the mutant VP30 proteins correlated strongly with their transcriptional support activity. Furthermore, we showed that the interaction between VP30 and the polymerase cofactor VP35 is RNA dependent, while formation of VP30 homohexamers and VP35 homotetramers is not. Our data indicate that RNA binding of VP30 is essential for its transcriptional support activity and stabilizes complexes of VP35/L polymerase with the (−) RNA template to favor productive transcriptional initiation in the presence of termination-active RNA secondary structures.IMPORTANCEEbola virus causes severe fevers with unusually high case fatality rates. The recent outbreak of Ebola virus in West Africa claimed more than 11,000 lives and threatened to destabilize a whole region because of its dramatic effects on the public health systems. It is currently not completely understood how Ebola virus manages to balance viral transcription and replication in the infected cells. This study shows that transcriptional support activity of the Ebola virus transcription factor VP30 is highly correlated with its ability to bind viral RNA. The interaction between VP30 and VP35, the Ebola virus polymerase cofactor, is dependent on the presence of RNA as well. Our data contribute to the understanding of the dynamic interplay between nucleocapsid proteins and the viral RNA template in order to promote viral RNA synthesis.

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The Integrity of the YxxL Motif of Ebola Virus VP24 Is Important for the Transport of Nucleocapsid-Like Structures and for the Regulation of Viral RNA Synthesis

Journal of Virology ◽

10.1128/jvi.02170-19 ◽

2020 ◽

Vol 94 (9) ◽

Cited By ~ 2

Author(s):

Yuki Takamatsu ◽

Larissa Kolesnikova ◽

Martin Schauflinger ◽

Takeshi Noda ◽

Stephan Becker

Keyword(s):

Intracellular Transport ◽

Rna Synthesis ◽

Ebola Virus ◽

Case Fatality ◽

Viral Rna ◽

Regulatory Function ◽

Failure To Rescue ◽

Independent Manner ◽

Late Domains ◽

Transcription And Replication

ABSTRACT While it is well appreciated that late domains in the viral matrix proteins are crucial to mediate efficient virus budding, little is known about roles of late domains in the viral nucleocapsid proteins. Here, we characterized the functional relevance of a YxxL motif with potential late-domain function in the Ebola virus nucleocapsid protein VP24. Mutations in the YxxL motif had two opposing effects on the functions of VP24. On the one hand, the mutation affected the regulatory function of VP24 in viral RNA transcription and replication, which correlated with an increased incorporation of minigenomes into released transcription- and replication-competent virus-like particles (trVLPs). Consequently, cells infected with those trVLPs showed higher levels of viral transcription. On the other hand, mutations of the YxxL motif greatly impaired the intracellular transport of nucleocapsid-like structures (NCLSs) composed of the viral proteins NP, VP35, and VP24 and the length of released trVLPs. Attempts to rescue recombinant Ebola virus expressing YxxL-deficient VP24 failed, underlining the importance of this motif for the viral life cycle. IMPORTANCE Ebola virus (EBOV) causes a severe fever with high case fatality rates and, so far, no available specific therapy. Understanding the interplay between viral and host proteins is important to identify new therapeutic approaches. VP24 is one of the essential nucleocapsid components and is necessary to regulate viral RNA synthesis and condense viral nucleocapsids before their transport to the plasma membrane. Our functional analyses of the YxxL motif in VP24 suggested that it serves as an interface between nucleocapsid-like structures (NCLSs) and cellular proteins, promoting intracellular transport of NCLSs in an Alix-independent manner. Moreover, the YxxL motif is necessary for the inhibitory function of VP24 in viral RNA synthesis. A failure to rescue EBOV encoding VP24 with a mutated YxxL motif indicated that the integrity of the YxxL motif is essential for EBOV growth. Thus, this motif might represent a potential target for antiviral interference.

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JOINT ESTIMATION OF THE TRANSMISSIBILITY AND SEVERITY OF EBOLA VIRUS DISEASE IN REAL TIME

Journal of Biological System ◽

10.1142/s0218339017400022 ◽

2017 ◽

Vol 25 (04) ◽

pp. 587-603 ◽

Cited By ~ 2

Author(s):

YUSUKE ASAI ◽

HIROSHI NISHIURA

Keyword(s):

Real Time ◽

Ebola Virus ◽

Virus Disease ◽

Critical Role ◽

Estimation Method ◽

Case Fatality ◽

Ascertainment Bias ◽

Joint Estimation ◽

Ebola Virus Disease ◽

Strong Impact

The effective reproduction number [Formula: see text], the average number of secondary cases that are generated by a single primary case at calendar time [Formula: see text], plays a critical role in interpreting the temporal transmission dynamics of an infectious disease epidemic, while the case fatality risk (CFR) is an indispensable measure of the severity of disease. In many instances, [Formula: see text] is estimated using the reported number of cases (i.e., the incidence data), but such report often does not arrive on time, and moreover, the rate of diagnosis could change as a function of time, especially if we handle diseases that involve substantial number of asymptomatic and mild infections and large outbreaks that go beyond the local capacity of reporting. In addition, CFR is well known to be prone to ascertainment bias, often erroneously overestimated. In this paper, we propose a joint estimation method of [Formula: see text] and CFR of Ebola virus disease (EVD), analyzing the early epidemic data of EVD from March to October 2014 and addressing the ascertainment bias in real time. To assess the reliability of the proposed method, coverage probabilities were computed. When ascertainment effort plays a role in interpreting the epidemiological dynamics, it is useful to analyze not only reported (confirmed or suspected) cases, but also the temporal distribution of deceased individuals to avoid any strong impact of time dependent changes in diagnosis and reporting.

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