Minigenome Research Articles

The W195 Residue of the Newcastle Disease Virus V Protein Is Critical for Multiple Aspects of Viral Self-Regulation through Interactions between V and Nucleoproteins.

The transcription and replication of the Newcastle disease virus (NDV) strictly rely on the viral ribonucleoprotein (RNP) complex, which is composed of viral NP, P, L and RNA. However, it is not known whether other viral non-RNP proteins participate in this process for viral self-regulation. In this study, we used a minigenome (MG) system to identify the regulatory role of the viral non-RNP proteins V, M, W, F and HN. Among them, V significantly reduced MG-encoded reporter activity compared with the other proteins and inhibited the synthesis of viral mRNA and cRNA. Further, V interacted with NP. A mutation in residue W195 of V diminished V-NP interaction and inhibited inclusion body (IB) formation in NP-P-L-cotransfected cells. Furthermore, a reverse-genetics system for the highly virulent strain F48E9 was established. The mutant rF48E9-VW195R increased viral replication and apparently enhanced IB formation. In vivo experiments demonstrated that rF48E9-VW195R decreased virulence and retarded time of death. Overall, the results indicate that the V-NP interaction of the W195 mutant V decreased, which regulated viral RNA synthesis, IB formation, viral replication and pathogenicity. This study provides insight into the self-regulation of non-RNP proteins in paramyxoviruses.

Construction and characterization of a reverse genetics system of bovine parainfluenza virus type 3c as a tool for rapid screening of antivirals in vitro.

Bovine parainfluenza virus type 3 (BPIV3) is a key pathogen associated with bovine respiratory disease complex (BRDC). However, its specific pathogenesis mechanisms have not been fully elucidated. Reverse genetics provides a useful method for understanding the pathogenic mechanism of BPIV3. To ensure the functionality of the rescue platforms, we first constructed a minigenome (MG) system of BPIV3 utilizing a 5-plasmid system in this investigation. Then, a full-length infection clone of BPIV3 was obtained from the SX-2021 strain, and different methods were employed to identify the rescued virus. Additionally, we recovered a recombinant BPIV3 using the reverse genetics system that could express enhanced green fluorescence protein (eGFP). Through the growth curve assays, the replicate capability of rBPIV3-SX-EGFP was found to be similar to that of the parental virus. Subsequently, the rBPIV3-SX-EGFP was used to determine the antiviral activity of ribavirin. The results showed that ribavirin had an anti-BPIV3 effect in MDBK cells. In conclusion, the successful development of a reverse genetic system for the SX-2021 strain establishes a foundation for future studies on BPIV3, including investigations into its pathogenic mechanism, gene function, and antiviral screening properties.

Construction and Rescue of a Minigenome System for Lentogenic D58 Strain of Avian orthoavulavirus 1 (Newcastle Disease Virus)

Background: In reverse genetics studies, minigenomes of negative sense RNA viruses are used to model the process of virus replication and evaluate the virus rescue system in the course of construction of recombinant virus. In the current study, a minigenome (MG) rescue system for lentogenic D58 vaccine strain of Avian orthoavulavirus 1 (AOAV-1) (Newcastle disease virus) was developed to establish a reverse genetic system for this strain. Methods: The minigenome was constructed by assembling the trailer and leader regions of AOAV-1 D58 strain flanking the reporter gene, Aequorea coerulescens green fluorescence protein (AcGFP) in a modified transcription vector. Construction of this Minigenome was done using a simple restriction free technique-Splicing by Overlap Extension PCR. The constructed minigenome was evaluated for the reporter gene expression by transfection in T7 RNA polymerase expressing BSR/T7 cell line. Further, the replication and packaging ability of the constructed minigenome were also analyzed. Result: The reporter gene expression was confirmed in BSR/T7 cells which simulates the virus replication process and verifies the virus rescue system. In addition, this proves the applicability of restriction free cloning by Splicing by Overlap Extension (SOE) PCR for precise construction of the minigenome. The developed minigenome system can be used as a potential tool in the reverse genetic rescue of AOAV-1 D58 strain for the development of marker vaccine.

Topoisomerase II as a Novel Antiviral Target against Panarenaviral Diseases.

Although many arenaviruses cause severe diseases with high fatality rates each year, treatment options are limited to off-label use of ribavirin, and a Food and Drug Administration (FDA)-approved vaccine is not available. To identify novel therapeutic candidates against arenaviral diseases, an RNA polymerase I-driven minigenome (MG) expression system for Lassa virus (LASV) was developed and optimized for high-throughput screening (HTS). Using this system, we screened 2595 FDA-approved compounds for inhibitors of LASV genome replication and identified multiple compounds including pixantrone maleate, a topoisomerase II inhibitor, as hits. Other tested topoisomerase II inhibitors also suppressed LASV MG activity. These topoisomerase II inhibitors also inhibited Junin virus (JUNV) MG activity and effectively limited infection by the JUNV Candid #1 strain, and siRNA knockdown of both topoisomerases (IIα and IIβ) restricted JUNV replication. These results suggest that topoisomerases II regulate arenavirus replication and can serve as molecular targets for panarenaviral replication inhibitors.

Development of accelerated high-throughput antiviral screening systems for emerging orthomyxoviruses

Bourbon virus (BRBV) is an emerging tick-borne orthomyxovirus that causes severe febrile illness in humans. There are no specific treatments for BRBV disease currently available. Here, we developed a highly accessible and robust, quantitative fluorescence-based BRBV minigenome (MG) system and applied it to high-throughput antiviral drug screening. We demonstrated that human dihydroorotate dehydrogenase (DHODH) inhibitors, hDHODH–IN–4 and brequinar, efficiently reduced BRBV RNA synthesis, and validated these findings using infectious BRBV in vitro. The DHODH inhibitors also exhibited high potency in inhibiting MG activities of other orthomyxoviruses with emerging zoonotic potential, including bat influenza A virus, swine influenza D virus, and Thogoto virus. Our newly developed MG system is a powerful platform for antiviral drug screening across the Orthomyxoviridae family, enabling rapid development and deployment of antivirals against future emerging orthomyxoviruses.

Screening and identification of Lassa virus endonuclease-targeting inhibitors from a fragment-based drug discovery library

Lassa virus (LASV) belongs to the Old World genus Mammarenavirus, family Arenaviridae, and order Bunyavirales. Arenavirus contains a segmented negative-sense RNA genome, which is in line with the bunyavirus and orthomyxoviruses. The segmented negative-sense RNA viruses utilize a cap-snatching strategy to provide primers cleavaged from the host capped mRNA for viral mRNA transcription. As a similar strategy and the conformational conservation shared with these viruses, the endonuclease (EN) would serve as an attractive target for developing broad-spectrum inhibitors. Using the LASV minigenome (MG) system, we screened a fragment-based drug discovery library and found that two hits, F1204 and F1781, inhibited LASV MG activity. Both hits also inhibited the prototype arenavirus Lymphocytic choriomeningitis virus (LCMV) MG activity. Furthermore, both hits effectively inhibited authentic LCMV and severe fever with thrombocytopenia syndrome virus (SFTSV) infections. Similarly, both hits could inhibit the activity of LASV, LCMV, and SFTSV EN. The combination of either compound with an arenavirus entry inhibitor had significant synergistic antiviral effects. Moreover, both hits were found to be capable of binding to LASV EN with a binding affinity at the micromolar level. These findings provide a basis for developing the hits as potential candidates for the treatment of segmented negative-sense RNA virus infections.

Recent Advances in Bunyavirus Reverse Genetics Research: Systems Development, Applications, and Future Perspectives.

Bunyaviruses are members of the Bunyavirales order, which is the largest group of RNA viruses, comprising 12 families, including a large group of emerging and re-emerging viruses. These viruses can infect a wide variety of species worldwide, such as arthropods, protozoans, plants, animals, and humans, and pose substantial threats to the public. In view of the fact that a better understanding of the life cycle of a highly pathogenic virus is often a precondition for developing vaccines and antivirals, it is urgent to develop powerful tools to unravel the molecular basis of the pathogenesis. However, biosafety level −3 or even −4 containment laboratory is considered as a necessary condition for working with a number of bunyaviruses, which has hampered various studies. Reverse genetics systems, including minigenome (MG), infectious virus-like particle (iVLP), and infectious full-length clone (IFLC) systems, are capable of recapitulating some or all steps of the viral replication cycle; among these, the MG and iVLP systems have been very convenient and effective tools, allowing researchers to manipulate the genome segments of pathogenic viruses at lower biocontainment to investigate the viral genome transcription, replication, virus entry, and budding. The IFLC system is generally developed based on the MG or iVLP systems, which have facilitated the generation of recombinant infectious viruses. The MG, iVLP, and IFLC systems have been successfully developed for some important bunyaviruses and have been widely employed as powerful tools to investigate the viral replication cycle, virus–host interactions, virus pathogenesis, and virus evolutionary process. The majority of bunyaviruses is generally enveloped negative-strand RNA viruses with two to six genome segments, of which the viruses with bipartite and tripartite genome segments have mostly been characterized. This review aimed to summarize current knowledge on reverse genetic studies of representative bunyaviruses causing severe diseases in humans and animals, which will contribute to the better understanding of the bunyavirus replication cycle and provide some hints for developing designed antivirals.

Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection.

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs that adopt non-canonical orientations, as compared to other proline-rich motifs. An affinity tag-purification mass spectrometry approach identified additional PPxPxY-containing host proteins hnRNP L, hnRNPUL1, and PEG10, as VP30 interactors. hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV infection, whereas hnRNPUL1 enhances. RBBP6 and hnRNP L modulate VP30 phosphorylation, increase viral transcription, andexert additive effects on viral RNA synthesis. PEG10 has more modest inhibitory effects on EBOV replication. hnRNPUL1 positively affects viral RNA synthesis but in a VP30-independent manner. Binding studies demonstrate variable capacity of the PPxPxY motifs from these proteins to bind VP30, define PxPPPPxY as an optimal binding motif, and identify the fifth proline and the tyrosine as most critical for interaction. Competition binding and hydrogen-deuterium exchange mass spectrometry studies demonstrate that each protein binds a similar interface on VP30. VP30 therefore presents a novel proline recognition domain that is targeted by multiple host proteins to modulate viral transcription.

The NF-κB inhibitor, SC75741, is a novel antiviral against emerging tick-borne bandaviruses

Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV) cause viral hemorrhagic fever-like illnesses in humans due to an aberrant host inflammatory response, which contributes to pathogenesis. Here, we established two separate minigenome (MG) systems based on the M-segment of SFTSV and HRTV. Following characterization of both systems for SFTSV and HRTV, we used them as a platform to screen potential compounds that inhibit viral RNA synthesis. We demonstrated that the NF-κB inhibitor, SC75741, reduces viral RNA synthesis of SFTSV and HRTV using our MG platform and validated these results using infectious SFTSV and HRTV. These results may lead to the use of MG systems as potential screening systems for the identification of antiviral compounds and yield novel insights into host-factors that could play role in bandavirus transcription and replication.

Inhibiting pyrimidine biosynthesis impairs Ebola virus replication through depletion of nucleoside pools and activation of innate immune responses

Specific host pathways that may be targeted therapeutically to inhibit the replication of Ebola virus (EBOV) and other emerging viruses remain incompletely defined. A screen of 200,000 compounds for inhibition of an EBOV minigenome (MG) assay that measures the function of the viral polymerase complex identified as hits several compounds with an amino-tetrahydrocarbazole scaffold. This scaffold was structurally similar to GSK983, a compound previously described as having broad-spectrum antiviral activity due to its impairing de novo pyrimidine biosynthesis through inhibition of dihydroorotate dehydrogenase (DHODH). We generated compound SW835, the racemic version of GSK983 and demonstrated that SW835 and brequinar, another DHODH inhibitor, potently inhibit the MG assay and the replication of EBOV, vesicular stomatitis virus (VSV) and Zika (ZIKV) in vitro. Nucleoside and deoxynucleoside supplementation studies demonstrated that depletion of pyrimidine pools contributes to antiviral activity of these compounds. As reported for other DHODH inhibitors, SW835 and brequinar also induced expression of interferon stimulated genes (ISGs). ISG induction was demonstrated to occur without production of IFNα/β and independently of the IFNα receptor and was not blocked by EBOV-encoded suppressors of IFN signaling pathways. Furthermore, we demonstrated that transcription factor IRF1 is required for this ISG induction, and that IRF1 induction requires the DNA damage response kinase ATM. Therefore, de novo pyrimidine biosynthesis is critical for the replication of EBOV and other RNA viruses and inhibition of this pathway activates an ATM and IRF1-dependent innate immune response that subverts EBOV immune evasion functions.

Roles of Arenavirus Z Protein in Mediating Virion Budding, Viral Transcription-Inhibition and Interferon-Beta Suppression.

The smallest arenaviral protein is the zinc-finger protein (Z) that belongs to the RING finger protein family. Z serves as a main component required for virus budding from the membrane of the infected cells through self-oligomerization, a process that can be aided by the viral nucleoprotein (NP) to form the viral matrix of progeny virus particles. Z has also been shown to be essential for mediating viral transcriptional repression activity by locking the L polymerase onto the viral promoter in a catalytically inactive state, thus limiting viral replication. The Z protein has also recently been shown to inhibit the type I interferon-induction pathway by directly binding to the intracellular pathogen-sensor proteins RIG-I and MDA5, and thus inhibiting their normal functions. This chapter describes several assays used to examine the important roles of the arenaviral Z protein in mediating virus budding (i.e., either Z self-budding or NP-Z budding activities), viral transcriptional inhibition in a viral minigenome (MG) assay, and type I IFN suppression in an IFN-β promoter-mediated luciferase reporter assay.

Lassa Virus Reverse Genetics.

The Old World (OW) arenavirus Lassa (LASV ) is estimated to infect several hundred thousand people yearly in West Africa, resulting in high numbers of Lassa fever (LF), a viral hemorrhagic fever (HF) disease associated with high morbidity and mortality. To date, no licensed vaccines are available to LASV infections, and anti-LASV drug therapy is limited to an off-label use of ribavirin (Rib) that is only partially effective. The development of reverse genetics has provided investigators with a novel and powerful approach for the investigation of the molecular, cell biology, and pathogenesis of LASV. The use of cell-based LASV minigenome (MG) systems has allowed examining the cis- and trans-acting factors involved in genome replication and gene transcription and the identification of novel drugable LASV targets. Likewise, it is now feasible to rescue infectious recombinant (r)LASV entirely from cloned cDNAs containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of pathogenesis, as well as to facilitate screens to identify antiviral drugs against LASV and the implementation of novel strategies to develop live-attenuated vaccines (LAV). In this chapter we will summarize the state-of-the-art experimental procedures for implementation of LASV reverse genetics. In addition, we will briefly discuss some significant translational research developments that have been made possible upon the development of LASV reverse genetics.

Ribonuclease from Bacillus Acts as an Antiviral Agent against Negative- and Positive-Sense Single Stranded Human Respiratory RNA Viruses.

Bacillus pumilus ribonuclease (binase) was shown to be a promising antiviral agent in animal models and cell cultures. However, the mode of its antiviral action remains unknown. To assess the binase effect on intracellular viral RNA we have selected single stranded negative- and positive-sense RNA viruses, influenza virus, and rhinovirus, respectively, which annually cause respiratory illnesses and are characterized by high contagious nature, mutation rate, and antigen variability. We have shown that binase exerts an antiviral effect on both viruses at the same concentration, which does not alter the spectrum of A549 cellular proteins and expression of housekeeping genes. The titers of influenza A (H1N1pdm) virus and human rhinovirus serotype 1A were reduced by 40% and 65%, respectively. A preincubation of influenza virus with binase before infection significantly reduced viral titer after single-cycle replication of the virus. Using influenza A virus mini genome system we showed that binase reduced GFP reporter signaling indicating a binase action on the expression of viral mRNA. Binase reduced the level of H1N1pdm viral NP mRNA accumulation in A549 cells by 20%. Since the viral mRNA is a possible target for binase this agent could be potentially applied in the antiviral therapy against both negative- and positive-sense RNA viruses.

Recovery of a Paramyxovirus, the Human Metapneumovirus, from Cloned cDNA.

Human metapneumovirus (HMPV), a single-stranded negative-sense RNA virus belonging to the family Paramyxoviridae, is associated with respiratory tract illness, primarily in young children and persons with underlying disease. Based on genetic and antigenic variation, HMPV strains are classified into two serotypes, with isolates NL/1/00 and NL/1/99 as prototypes for serotypes A and B, respectively. The development of plasmid-based reverse genetics systems for both serotypes has resulted in developments of a wide range of vaccine candidates against HMPV infection. The approach to virus rescue of HMPV is similar to that used for other paramyxoviruses, starting with mini-replicon assays for optimizations of the rescue protocols and subsequent replacement of the mini genome with a plasmid expressing the cDNA of the full-length viral RNA genome. Here, we provide detailed information on the reverse genetics systems for HMPV.

The phosphoprotein genes of measles viruses from subacute sclerosing panencephalitis cases encode functional as well as non-functional proteins and display reduced editing

Products expressed from the second (P/V/C) gene are important in replication and abrogating innate immune responses during acute measles virus (MV) infection. Thirteen clone sets were derived from the P/V/C genes of measles virus (MV) RNA extracted from brains of a unique collection of seven cases of subacute sclerosing panencephalitis (SSPE) caused by persistent MV in the central nervous system (CNS). Whether these functions are fully maintained when MV replicates in the CNS has not been previously determined. Co-transcriptional editing of the P mRNAs by non-template insertion of guanine (G) nucleotides, which generates mRNAs encoding the viral V protein, occurs much less frequently (9%) in the SSPE derived samples than during the acute infection (30–50%). Thus it is likely that less V protein, which is involved in combatting the innate immune response, is produced. The P genes in MV from SSPE cases were not altered by biased hypermutation but exhibited a high degree of variation within each case. Most but not all SSPE derived phospho-(P) proteins were functional in mini genome replication/transcription assays. An eight amino acid truncation of the carboxyl-terminus made the P protein non-functional while the insertion of an additional glycine residue by insertion of G nucleotides at the editing site had no effect on protein function.

Novel Arenavirus Entry Inhibitors Discovered by Using a Minigenome Rescue System for High-Throughput Drug Screening.

Certain members of the Arenaviridae family are category A agents capable of causing severe hemorrhagic fevers in humans. Specific antiviral treatments do not exist, and the only commonly used drug, ribavirin, has limited efficacy and can cause severe side effects. The discovery and development of new antivirals are inhibited by the biohazardous nature of the viruses, making them a relatively poorly understood group of human pathogens. We therefore adapted a reverse-genetics minigenome (MG) rescue system based on Junin virus, the causative agent of Argentine hemorrhagic fever, for high-throughput screening (HTS). The MG rescue system recapitulates all stages of the virus life cycle and enables screening of small-molecule libraries under biosafety containment level 2 (BSL2) conditions. The HTS resulted in the identification of four candidate compounds with potent activity against a broad panel of arenaviruses, three of which were completely novel. The target for all 4 compounds was the stage of viral entry, which positions the compounds as potentially important leads for future development. The arenavirus family includes several members that are highly pathogenic, causing acute viral hemorrhagic fevers with high mortality rates. No specific effective treatments exist, and although a vaccine is available for Junin virus, the causative agent of Argentine hemorrhagic fever, it is licensed for use only in areas where Argentine hemorrhagic fever is endemic. For these reasons, it is important to identify specific compounds that could be developed as antivirals against these deadly viruses.

An Intrinsically Disordered Peptide from Ebola Virus VP35 Controls Viral RNA Synthesis by Modulating Nucleoprotein-RNA Interactions.

During viral RNA synthesis, Ebola virus (EBOV) nucleoprotein (NP) alternates between an RNA-template-bound form and a template-free form to provide the viral polymerase access to the RNA template. In addition, newly synthesized NP must be prevented from indiscriminately binding to noncognate RNAs. Here, we investigate the molecular bases for these critical processes. We identify an intrinsically disordered peptide derived from EBOV VP35 (NPBP, residues 20-48) that binds NP with high affinity and specificity, inhibits NP oligomerization, and releases RNA from NP-RNA complexes invitro. The structure of the NPBP/ΔNPNTD complex, solved to3.7Å resolution, reveals how NPBP peptide occludes a large surface area that is important for NP-NP and NP-RNA interactions and for viral RNA synthesis. Together, our results identify a highly conserved viral interface that is important for EBOV replication and can be targeted for therapeutic development.

Functional analysis of the L Protein of Nipah Virus Using Minigenome System

Nipah virus (NiV) was identified in Malaysia in late September 1998 as the etiological agent of an outbreak of acute encephalitis with the high mortality rate in human. NiV polymerase gene (L) encodes RNA-dependent RNA polymerase (RdRp) which is an enzyme required for viral replication. The aim of this study is to investigate the role of the N terminal of polymerase protein in replication of NiV. Based on the extensive deduced amino acid sequence analyses of a number of L proteins of non-segmented negative-strand (NNS) RNA viruses, a cluster of high-homology sequence segments have been identified within the body of the L proteins. The functional characterization of the NiV L polymerase was addressed in this research by generating a series of progressive N- deletions in the cloned gene and testing them in a minigenome assay. Five mutations that delete increasing amounts of the amino terminus of the L protein (20 amino acids) were generated at the amino-terminal. The ability of new recombinants plasmids in viral replication were tested by using minigenome system which is based on an intracellular and plasmid-based replication assay. This system contains an inserted chloramphenicol acetyltransferase (CAT) gene, for readout of the replication of the mini genome directed by wild type or mutant L protein. The results demonstrated that the first 100 amino acids of the NH2-terminal domain spanning a highly conserved motif are directly involved in transcription of the genome RNA. The possible functional significance of the NH2-terminal domain of paramyxovirus L protein is discussed.

Growth kinetics and immune response of chimeric foot-and-mouth disease virus serotype ‘O’ produced through replication competent mini genome of serotype Asia 1, 63/72, in BHK cell lines

Regular vaccinations with potent vaccine, in endemic countries and vaccination to live in non-endemic countries are the methods available to control foot-and-mouth disease. Selection of candidate vaccine strain is not only cumbersome but the candidate should grow well for high potency vaccine preparation. Alternative strategy is to generate an infectious cDNA of a cell culture-adapted virus and use the replicon for development of tailor-made vaccines. We produced a chimeric ‘O’ virus in the backbone of Asia 1 and studied its characteristics. The chimeric virus showed high infectivity titre (>1010) in BHK 21 cell lines, revealed small plaque morphology and there was no cross reactivity with antiserum against Asia 1. The virus multiplies rapidly and reaches peak at 12h post infection. The vaccine prepared with this virus elicited high antibody titres.

Dual Role of the Lymphocytic Choriomeningitis Virus Intergenic Region in Transcription Termination and Virus Propagation

Each genome segment of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), encodes two genes in ambisense orientation, separated by an intergenic region (IGR). The 3' ends of subgenomic viral mRNAs have been mapped to a stem-loop structure within the IGR, suggesting structure-dependent transcription termination. We have studied the role of the LCMV IGR by using a minigenome (MG) rescue system based on RNA analogues of the short genome segment. An ambisense MG coding for chloramphenicol acetyltransferase (CAT) and green fluorescent protein reporter genes instead of the nucleoprotein and glycoprotein open reading frames, respectively, served as a template for synthesis of full-length anti-MG (aMG) replicate and subgenomic size mRNA for reporter gene expression. An analogous MG without IGR was amplified by the virus polymerase with equal efficiency, but subgenomic mRNA was undetectable. Reporter gene expression from IGR-deficient aMG CAT-sense RNA of genomic length was approximately 5-fold less efficient than that from subgenomic CAT mRNA derived from an IGR-containing MG, but at least 100-fold more efficient than that from a T7 RNA polymerase transcript with the same sequence. Therefore, in the absence of IGR-mediated transcription termination, a fraction of full-length aMG RNA appears to behave as bona fide mRNA. Unexpectedly, MGs without IGR were dramatically impaired in their ability to passage reporter gene activity via infectious virus-like particles. These data suggest that the LCMV IGR serves individual functions in transcription termination for enhanced gene expression and in the virus assembly and/or budding, which are required for the efficient propagation of LCMV infectivity.