scholarly journals Construction of an Influenza D Virus with an Eight-Segmented Genome

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2166
Author(s):  
Hiroho Ishida ◽  
Shin Murakami ◽  
Haruhiko Kamiki ◽  
Hiromichi Matsugo ◽  
Misa Katayama ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Reverse Genetics ◽  
Silent Mutation ◽  
Acceptor Site ◽  
Ns1 Protein ◽  
Genome Packaging ◽  
Cattle Industry ◽  
Bovine Respiratory Disease Complex ◽  
Rescue System ◽  
Segmented Genome

Influenza D virus (IDV) may cause the bovine respiratory disease complex, which is the most common and costly disease affecting the cattle industry. Previously, we revealed that eight segments could be actively packaged in its single virion, suggesting that IDV with the seven-segmented genome shows an agnostic genome packaging mechanism. Herein, we engineered an eight-segmented recombinant IDV in which the NS1 or NS2 genes were separated from NS segment into independent segments (NS1 or NS2 segments, respectively), leading to monocistronic translation of each NS protein. We constructed two plasmids: one for the viral RNA (vRNA)-synthesis of the NS1 segment with a silent mutation at the splicing acceptor site, which controls NS2 transcription in the NS segment; and another for the RNA synthesis of the NS2 segment, with deletion of the intron in the NS segment. These plasmids and six other vRNA-synthesis plasmids were used to fabricate an infectious eight-segmented IDV via reverse genetics. This system enables analysis of the functions of NS1 or NS2. We tested the requirement of the N-terminal overlapping region (NOR) in these proteins for viral infectivity. We rescued a virus with NOR-deleted NS2 protein, which displayed a growth rate equivalent to that of the eight-segmented virus with intact NS2. Thus, the NOR may not influence viral growth. In contrast, a virus with NOR-deleted NS1 protein could not be rescued. These results indicate that the eight-segmented rescue system of IDV may provide an alternative method to analyze viral proteins at the molecular level.

scholarly journals RNA Origami: Packaging a Segmented Genome in Orbivirus Assembly and Replication

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1841
Author(s):  
Po-Yu Sung ◽  
Polly Roy
Keyword(s):  
Reverse Genetics ◽  
Economic Losses ◽  
Active Components ◽  
Rna Packaging ◽  
Genome Packaging ◽  
Specific Order ◽  
Nonenveloped Virus ◽  
Dsrna Genome ◽  
Rna Interaction ◽  
Segmented Genome

Understanding how viruses with multi-segmented genomes incorporate one copy of each segment into their capsids remains an intriguing question. Here, we review our recent progress and describe the advancements made in understanding the genome packaging mechanism of a model nonenveloped virus, Bluetongue virus (BTV), with a 10-segment (S1–S10) double-strand RNA (dsRNA) genome. BTV (multiple serotypes), a member of the Orbivirus genus in the Reoviridae family, is a notable pathogen for livestock and is responsible for significant economic losses worldwide. This has enabled the creation of an extensive set of reagents and assays, including reverse genetics, cell-free RNA packaging, and bespoke bioinformatics approaches, which can be directed to address the packaging question. Our studies have shown that (i) UTRs enable the conformation of each segment necessary for the next level of RNA–RNA interaction; (ii) a specific order of intersegment interactions leads to a complex RNA network containing all the active components in sorting and packaging; (iii) networked segments are recruited into nascent assembling capsids; and (iv) select capsid proteins might be involved in the packaging process. The key features of genome packaging mechanisms for BTV and related dsRNA viruses are novel and open up new avenues of potential intervention.

scholarly journals Plasmid-only rescue of influenza A virus vaccine candidates

2001 ◽  
Vol 356 (1416) ◽  
pp. 1965-1973 ◽  
Author(s):  
J. H. Schickli ◽  
A. Flandorfer ◽  
T. Nakaya ◽  
L. Martinez-Sobrido ◽  
A. Garcia-Sastre ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Genetic Manipulation ◽  
Influenza Viruses ◽  
High Yield ◽  
Ns1 Protein ◽  
Potential Threat ◽  
Rescue System ◽  
Rapid Generation

The potential threat of another influenza virus pandemic stimulates discussion on how to prepare for such an event. The most reasonable prophylactic approach appears to be the use of effective vaccines. Since influenza and other negative–stranded RNA viruses are amenable to genetic manipulation using transfection by plasmids, it is possible to outline new reverse genetics–based approaches for vaccination against influenza viruses. We suggest three approaches. First, we use a plasmid–only rescue system that allows the rapid generation of high–yield recombinant vaccine strains. Second, we propose developing second–generation live influenza virus vaccines by constructing an attenuated master strain with deletions in the NS1 protein, which acts as an interferon antagonist. Third, we suggest the use of Newcastle disease virus recombinants expressing influenza virus haemagglutinin proteins of pandemic (epizootic) strains as novel vaccine vectors for use in animals and possibly humans.

scholarly journals A Reverse Genetics System for Cypovirus Based on a Bacmid Expressing T7 RNA Polymerase

Viruses ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 314 ◽  
Author(s):  
Gaobo Zhang ◽  
Jian Yang ◽  
Fujun Qin ◽  
Congrui Xu ◽  
Jia Wang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Reverse Genetics ◽  
T7 Rna Polymerase ◽  
Sf9 Cells ◽  
Genomic Segment ◽  
Egfp Gene ◽  
A Genome ◽  
Typical Morphology ◽  
Occlusion Bodies ◽  
Rescue System

Dendrolimus punctatus cypovirus (DpCPV), belonging to the genus Cypovirus within the family Reoviridae, is considered the most destructive pest of pine forests worldwide. DpCPV has a genome consisting of 10 linear double-stranded RNA segments. To establish a reverse genetics system, we cloned cDNAs encoding the 10 genomic segments of DpCPV into three reverse genetics vectors in which each segment was transcribed under the control of a T7 RNA polymerase promoter and terminator tagged with a hepatitis delta virus ribozyme sequence. We also constructed a vp80-knockout Autographa californica multiple nucleopolyhedrovirus bacmid to express a T7 RNA polymerase codon-optimized for Sf9 cells. Following transfection of Sf9 cells with the three vectors and the bacmid, occlusion bodies (OBs) with the typical morphology of cypovirus polyhedra were observed by optical microscopy. The rescue system was verified by incorporation of a HindIII restriction enzyme site null mutant of the 9th genomic segment. Furthermore, when we co-transfected Sf9 cells with the reverse genetics vectors, the bacmid, and an additional vector bearing an egfp gene flanked with the 5′ and 3′ untranslated regions of the 10th genomic segment, aggregated green fluorescence co-localizing with the OBs was observed. The rescued OBs were able to infect Spodopetra exigua larvae, although their infectivity was significantly lower than that of wild-type DpCPV. This reverse genetics system for DpCPV could be used to explore viral replication and pathogenesis and to facilitate the development of novel bio-insecticides and expression systems for exogenous proteins.

scholarly journals Changes in RNA secondary structure affect NS1 protein expression during early stage influenza virus infection

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Irina Baranovskaya ◽  
Mariia Sergeeva ◽  
Artem Fadeev ◽  
Renata Kadirova ◽  
Anna Ivanova ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Secondary Structure ◽  
Protein Expression ◽  
Rna Secondary Structure ◽  
Reverse Genetics ◽  
Early Stage ◽  
Influenza Virus Infection ◽  
Main Function ◽  
Ns1 Protein ◽  
Virus Strains

AbstractRNA secondary structures play a key role in splicing, gene expression, microRNA biogenesis, RNA editing, and other biological processes. The importance of RNA structures has been demonstrated in the life cycle of RNA-containing viruses, including the influenza virus. At least two regions of conserved secondary structure in NS segment (+) RNA are predicted to vary among influenza virus strains with respect to thermodynamic stability; both fall in the NS1 open reading frame. The NS1 protein is involved in multiple virus-host interaction processes, and its main function is to inhibit the cellular immune response to viral infection. Using a reverse genetics approach, four influenza virus strains were constructed featuring mutations that have different effects on RNA secondary structure. Growth curve experiments and ELISA data show that, at least in the first viral replication cycle, mutations G123A and A132G affecting RNA structure in the (82–148) NS RNA region influence NS1 protein expression.

scholarly journals Arterivirus Subgenomic mRNA Synthesis and Virion Biogenesis Depend on the Multifunctional nsp1 Autoprotease

2007 ◽  
Vol 81 (19) ◽  
pp. 10496-10505 ◽  
Author(s):  
Marieke A. Tijms ◽  
Danny D. Nedialkova ◽  
Jessika C. Zevenhoven-Dobbe ◽  
Alexander E. Gorbalenya ◽  
Eric J. Snijder
Keyword(s):  
Life Cycle ◽  
Rna Synthesis ◽  
Reverse Genetics ◽  
Rna Viruses ◽  
Regulatory Protein ◽  
Equine Arteritis Virus ◽  
Structural Protein ◽  
Mrna Synthesis ◽  
Nonstructural Proteins ◽  
Autocatalytic Processing

ABSTRACT Many groups of plus-stranded RNA viruses produce additional, subgenomic mRNAs to regulate the expression of part of their genome. Arteriviruses and coronaviruses (order Nidovirales) are unique among plus-stranded RNA viruses for using a mechanism of discontinuous RNA synthesis to produce a nested set of 5′- and 3′-coterminal subgenomic mRNAs, which serve to express the viral structural protein genes. The discontinuous step presumably occurs during minus-strand synthesis and joins noncontiguous sequences copied from the 3′- and 5′-proximal domains of the genomic template. Nidovirus genome amplification (“replication”) and subgenomic mRNA synthesis (“transcription”) are driven by 13 to 16 nonstructural proteins (nsp's), generated by autocatalytic processing of two large “replicase” polyproteins. Previously, using a replicon system, the N-terminal nsp1 replicase subunit of the arterivirus equine arteritis virus (EAV) was found to be dispensable for replication but crucial for transcription. Using reverse genetics, we have now addressed the role of nsp1 against the background of the complete EAV life cycle. Mutagenesis revealed that nsp1 is in fact a multifunctional regulatory protein. Its papain-like autoprotease domain releases nsp1 from the replicase polyproteins, a cleavage essential for viral RNA synthesis. Several mutations in the putative N-terminal zinc finger domain of nsp1 selectively abolished transcription, while replication was either not affected or even increased. Other nsp1 mutations did not significantly affect either replication or transcription but still dramatically reduced the production of infectious progeny. Thus, nsp1 is involved in at least three consecutive key processes in the EAV life cycle: replicase polyprotein processing, transcription, and virion biogenesis.

scholarly journals A Single Amino Acid in the Polymerase Acidic Protein Determines the Pathogenicity of Influenza B Viruses

2018 ◽  
Vol 92 (13) ◽  
pp. e00259-18 ◽  
Author(s):  
Joon-Yong Bae ◽  
Ilseob Lee ◽  
Jin Il Kim ◽  
Sehee Park ◽  
Kirim Yoo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reverse Genetics ◽  
Single Amino Acid ◽  
Influenza B Virus ◽  
Influenza B ◽  
Ns1 Protein ◽  
Viral Polymerase ◽  
Complex Activity ◽  
Protein Coding ◽  
Polymerase Complex

ABSTRACTInfluenza B virus (IBV) is one of the human respiratory viruses and one of the targets of seasonal vaccination. However, the bifurcation of two antigenically distinct lineages of IBVs makes it difficult to arrange proper medical countermeasures. Moreover, compared with pathogenicity-related molecular markers known for influenza A virus, little has been known for IBVs. To understand pathogenicity caused by IBVs, we investigated the molecular determinants of IBV pathogenicity in animal models. After serial lung-to-lung passages of Victoria lineage B/Brisbane/60/2008 (Vc_BR60) and Yamagata lineage B/Wisconsin/01/2010 (Ym_WI01) viruses in BALB/c mice, we identified the mouse-adapted Vc_BR60 (maVc_BR60) and Ym_WI01 (maYm_WI01) viruses, respectively. To find a molecular clue(s) to the increased pathogenicity of maVc_BR60 and maYm_WI01, we determined their genetic sequences. Several amino acid mutations were identified in the PB2, PB1, PA, BM2, and/or NS1 protein-coding regions, and one concurrent lysine (K)-to-arginine (R) mutation in PA residue 338 (PA K338R) was found in both maVc_BR60 and maYm_WI01 viruses. When analyzed using viruses rescued through reverse genetics, it was shown that PA K338R alone could increase the pathogenicity of both IBVs in mice and viral replication in the respiratory tracts of ferrets. In a subsequent minireplicon assay, the effect of PA K338R was highlighted by the enhancement of viral polymerase complex activity of both Vc_BR60 and Ym_WI01 viruses. These results suggest that the PA K338R mutation may be a molecular determinant of IBV pathogenicity via modulating the viral polymerase function of IBVs.IMPORTANCETo investigate molecular pathogenic determinants of IBVs, which are one of the targets of seasonal influenza vaccines, we adapted both Victoria and Yamagata lineage IBVs independently in mice. The recovered mouse-adapted viruses exhibited increased virulence, and of the various mutations identified from both mouse-adapted viruses, a concurrent amino acid mutation was found in the PA protein-coding region. When analyzed using viruses rescued through reverse genetics, the PA mutation alone appeared to contribute to viral pathogenicity in mice within the compatible genetic constellation between the IBV lineages and to the replication of IBVs in ferrets. Regarding the potential mechanism of increased viral pathogenicity, it was shown that the PA mutation could upregulate the viral polymerase complex activity of both IBV lineages. These results indicate that the PA mutation could be a newly defined molecular pathogenic determinant of IBVs that substantiates our understanding of the viral pathogenicity and public health risks of IBVs.

scholarly journals The Glycoprotein Cytoplasmic Tail of Uukuniemi Virus (Bunyaviridae) Interacts with Ribonucleoproteins and Is Critical for Genome Packaging

2007 ◽  
Vol 81 (7) ◽  
pp. 3198-3205 ◽  
Author(s):  
Anna K. Överby ◽  
Ralf F. Pettersson ◽  
Etienne P. A. Neve
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Mutational Analysis ◽  
Cytoplasmic Tail ◽  
Protein Protein Interactions ◽  
Genome Packaging ◽  
Glycoprotein Precursor ◽  
Glycoprotein G ◽  
Rescue System ◽  
Polymerase I

ABSTRACT We have analyzed the importance of specific amino acids in the cytoplasmic tail of the glycoprotein GN for packaging of ribonucleoproteins (RNPs) into virus-like particles (VLPs) of Uukuniemi virus (UUK virus), a member of the Bunyaviridae family. In order to study packaging, we added the GN/GC glycoprotein precursor (p110) to a polymerase I-driven minigenome rescue system to generate VLPs that are released into the supernatant. These particles can infect new cells, and reporter gene expression can be detected. To determine the role of UUK virus glycoproteins in RNP packaging, we performed an alanine scan of the glycoprotein GN cytoplasmic tail (amino acids 1 to 81). First, we discovered three regions in the tail (amino acids 21 to 25, 46 to 50, and 71 to 81) which are important for minigenome transfer by VLPs. Further mutational analysis identified four amino acids that were important for RNP packaging. These amino acids are essential for the binding of nucleoproteins and RNPs to the glycoprotein without affecting the morphology of the particles. No segment-specific interactions between the RNA and the cytoplasmic tail could be observed. We propose that VLP systems are useful tools for analyzing protein-protein interactions important for packaging of viral genome segments, assembly, and budding of other members of the Bunyaviridae family.

scholarly journals A Naturally Occurring Deletion in Its NS Gene Contributes to the Attenuation of an H5N1 Swine Influenza Virus in Chickens

2007 ◽  
Vol 82 (1) ◽  
pp. 220-228 ◽  
Author(s):  
Qiyun Zhu ◽  
Huanliang Yang ◽  
Weiye Chen ◽  
Wenyan Cao ◽  
Gongxun Zhong ◽  
...  
Keyword(s):  
Recombinant Virus ◽  
Reverse Genetics ◽  
Southern China ◽  
Single Gene ◽  
Swine Influenza Virus ◽  
Swine Influenza ◽  
Host Cells ◽  
Ns1 Protein ◽  
Protein Levels ◽  
Ns Gene

ABSTRACT In 2001 and 2003, we isolated two H5N1 viruses, A/swine/Fujian/1/01 (SW/FJ/01) and A/swine/Fujian/1/03 (SW/FJ/03), from pigs in Fujian Province, southern China. Genetically, these two viruses are similar, although the NS gene of the SW/FJ/03 virus has a 15-nucleotide deletion at coding positions 612 to 626. The SW/FJ/01 virus is highly lethal for chickens, whereas the SW/FJ/03 virus is nonpathogenic for chickens when administrated intravenously or intranasally. To understand the molecular basis for the difference in virulence, we used reverse genetics to create a series of single-gene recombinants of both viruses. We found that a recombinant virus containing the mutated NS gene from the SW/FJ/03 virus in the SW/FJ/01 virus background was completely attenuated in chickens. We also found that viruses expressing the mutant NS1 protein of SW/FJ/03 did not antagonize the induction of interferon (IFN) protein. Conversely, only the recombinant virus containing the wild-type SW/FJ/01 NS gene in the SW/FJ/03 background was lethal in chickens and antagonized IFN protein levels. Further, we proved that the NS1 genes of the two viruses differ in their stabilities in the host cells and in their abilities to interact with the chicken cleavage and polyadenylation specificity factor. These results indicate that the deletion of amino acids 191 to 195 of the NS1 protein is critical for the attenuation of the SW/FJ/03 virus in chickens and that this deletion affects the ability of the virus to antagonize IFN induction in host cells.

scholarly journals ISG15 conjugation system targets the viral NS1 protein in influenza A virus–infected cells

2010 ◽  
Vol 107 (5) ◽  
pp. 2253-2258 ◽  
Author(s):  
Chen Zhao ◽  
Tien-Ying Hsiang ◽  
Rei-Lin Kuo ◽  
Robert M. Krug
Keyword(s):  
Antiviral Activity ◽  
Influenza A Virus ◽  
Viral Protein ◽  
Influenza A ◽  
Rna Binding ◽  
Acceptor Site ◽  
Ns1 Protein ◽  
Conjugation System ◽  
Infected Cells ◽  
Ns1a Protein

ISG15 is an IFN-α/β–induced, ubiquitin-like protein that is conjugated to a wide array of cellular proteins through the sequential action of three conjugation enzymes that are also induced by IFN-α/β. Recent studies showed that ISG15 and/or its conjugates play an important role in protecting cells from infection by several viruses, including influenza A virus. However, the mechanism by which ISG15 modification exerts antiviral activity has not been established. Here we extend the repertoire of ISG15 targets to a viral protein by demonstrating that the NS1 protein of influenza A virus (NS1A protein), an essential, multifunctional protein, is ISG15 modified in virus-infected cells. We demonstrate that the major ISG15 acceptor site in the NS1A protein in infected cells is a critical lysine residue (K41) in the N-terminal RNA-binding domain (RBD). ISG15 modification of K41 disrupts the association of the NS1A RBD domain with importin-α, the protein that mediates nuclear import of the NS1A protein, whereas the RBD retains its double-stranded RNA-binding activity. Most significantly, we show that ISG15 modification of K41 inhibits influenza A virus replication and thus contributes to the antiviral action of IFN-β. We also show that the NS1A protein directly and specifically binds to Herc5, the major E3 ligase for ISG15 conjugation in human cells. These results establish a “loss of function” mechanism for the antiviral activity of the IFN-induced ISG15 conjugation system, namely, that it inhibits viral replication by conjugating ISG15 to a specific viral protein, thereby inhibiting its function.

scholarly journals Glycine 184 in Nonstructural Protein NS1 Determines the Virulence of Influenza A Virus Strain PR8 without Affecting the Host Interferon Response

2010 ◽  
Vol 84 (24) ◽  
pp. 12761-12770 ◽  
Author(s):  
Sabine Steidle ◽  
Luis Martínez-Sobrido ◽  
Markus Mordstein ◽  
Stefan Lienenklaus ◽  
Adolfo García-Sastre ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Interferon Response ◽  
Binding Motif ◽  
Ns1 Protein ◽  
Viral Virulence ◽  
Functional Rna

ABSTRACT The nonstructural protein NS1 of influenza A virus counteracts the interferon (IFN) system and thereby promotes viral replication. NS1 has acquired different mechanisms to limit induction of IFN. It prevents double-stranded RNA (dsRNA) and RIG-I-mediated activation of interferon regulatory factor 3 (IRF3), and it blocks posttranscriptional processing of cellular mRNAs by binding to the cleavage and polyadenylation specificity factor (CPSF). Using a mouse-adapted A/PR/8/34 virus and reverse genetics to introduce specific mutations in NS1 which eliminate one or both functions, we determined the relative contributions of these two activities of NS1 to viral virulence in mice. We found that a functional RNA-binding motif was required for IFN suppression and virulence. Restoration of CPSF binding in the NS1 protein of wild-type A/PR/8/34 virus, which cannot bind CPSF due to mutations in the central binding motif at positions 103 and 106, resulted in enhanced virulence. Surprisingly, if CPSF binding was abolished by substituting glycine for arginine at position 184 in the classical NS1-CPSF binding motif, the mutant virus replicated much more slowly in mice, although the mutated NS1 protein continued to repress the IFN response very efficiently. Our results show that a functional RNA-binding motif is decisive for NS1 of A/PR/8/34 virus to suppress IFN induction. They further demonstrate that in addition to its contribution to CPSF binding, glycine 184 strongly influences viral virulence by an unknown mechanism which does not involve the IFN system.

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