Development of a functional cell-based assay that probes the specific interaction between influenza A virus NP and its packaging signal sequence RNA

The NS1 protein is a nonstructural protein encoded by the influenza A virus. It is responsible for many alterations produced in the cellular metabolism upon infection by the virus and for modulation of virus virulence. The NS1 protein is able to perform a large variety of functions due to its ability to bind various types of RNA molecules, from both viral and nonviral origin, and to interact with several cell factors. With the aim of exploring whether the binding of NS1 protein to viral RNA (vRNA) could constitute a novel target for the search of anti-influenza drugs, a filter-binding assay measuring the specific interaction between the recombinant His-NS1 protein from influenza A virus and a radiolabeled model vRNA ( 32P-vNSZ) was adapted to a format suitable for screening and easy automation. Flashplate® technology (PerkinElmer, Waltham, MA), either in 96- or 384-well plates, was used. The Flashplate® wells were precoated with the recombinant His-NS1 protein, and the binding of His-NS1 to a 35S-vNSZ probe was measured. A pilot screening of a collection of 27,520 mixtures of synthetic chemical compounds was run for inhibitors of NS1 binding to vRNA. We found 3 compounds in which the inhibition of NS1 binding to vRNA, observed at submicromolar concentrations, was correlated with a reduction of the cytopathic effect during the infection of cell cultures with influenza virus. These results support the hypothesis that the binding of NS1 to vRNA could be a novel target for the development of anti-influenza drugs. ( Journal of Biomolecular Screening 2008:581-590)

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Organization of the Influenza A Virus Genomic RNA in the Viral Replication Cycle—Structure, Interactions, and Implications for the Emergence of New Strains

Pathogens ◽

10.3390/pathogens9110951 ◽

2020 ◽

Vol 9 (11) ◽

pp. 951

Author(s):

Julita Piasecka ◽

Aleksandra Jarmolowicz ◽

Elzbieta Kierzek

Keyword(s):

Viral Replication ◽

Influenza A Virus ◽

Rna Structure ◽

Influenza A ◽

Respiratory Infections ◽

Specific Interaction ◽

Replication Cycle ◽

Genome Packaging ◽

Functional Roles ◽

Interaction Sites

The influenza A virus is a human pathogen causing respiratory infections. The ability of this virus to trigger seasonal epidemics and sporadic pandemics is a result of its high genetic variability, leading to the ineffectiveness of vaccinations and current therapies. The source of this variability is the accumulation of mutations in viral genes and reassortment enabled by its segmented genome. The latter process can induce major changes and the production of new strains with pandemic potential. However, not all genetic combinations are tolerated and lead to the assembly of complete infectious virions. Reports have shown that viral RNA segments co-segregate in particular circumstances. This tendency is a consequence of the complex and selective genome packaging process, which takes place in the final stages of the viral replication cycle. It has been shown that genome packaging is governed by RNA–RNA interactions. Intersegment contacts create a network, characterized by the presence of common and strain-specific interaction sites. Recent studies have revealed certain RNA regions, and conserved secondary structure motifs within them, which may play functional roles in virion assembly. Growing knowledge on RNA structure and interactions facilitates our understanding of the appearance of new genome variants, and may allow for the prediction of potential reassortment outcomes and the emergence of new strains in the future.

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vRNA-vRNA interactions in influenza A virus HA vRNA packaging

10.1101/2020.01.15.907295 ◽

2020 ◽

Author(s):

Sho Miyamoto ◽

Yukiko Muramoto ◽

Keiko Shindo ◽

Yoko Fujita ◽

Takeshi Morikawa ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Influenza Viruses ◽

Packaging Signal ◽

Genome Packaging ◽

Coding Regions ◽

Signal Region ◽

Rna Interaction ◽

Negative Sense

AbstractThe genome of the influenza A virus is composed of eight single-stranded negative-sense RNA segments (vRNAs). The eight different vRNAs are selectively packaged into progeny virions. This process likely involves specific interactions among vRNAs via segment-specific packaging signals located in the 3’ and 5’ terminal coding regions of vRNAs. To identify vRNA(s) that interact with hemagglutinin (HA) vRNA during genome packaging, we generated a mutant virus, HA 5m2, which possessed five silent mutations in the 5’ packaging signal region of HA vRNA. The HA 5m2 virus had a specific defect in HA vRNA incorporation, which reduced the viral replication efficiency. After serial passaging in cells, the virus acquired additional mutations in the 5’ terminal packaging signal regions of both HA and PB2 vRNAs. These mutations contributed to recovery of viral growth and packaging efficiency of HA vRNA. A direct RNA-RNA interaction between the 5’ ends of HA and PB2 vRNAs was confirmed in vitro. Our results indicate that direct interactions of HA vRNA with PB2 vRNA via their packaging signal regions are important for selective genome packaging and enhance our knowledge on the emergence of pandemic influenza viruses through genetic reassortment.

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Identification and targeting of a pan-genotypic influenza A virus RNA structure that mediates packaging and disease.

10.1101/2021.08.21.457170 ◽

2021 ◽

Author(s):

Rachel J. Hagey ◽

Menashe Elazar ◽

Siqi Tian ◽

Edward A. Pham ◽

Wipapat Kladwang ◽

...

Keyword(s):

Influenza A Virus ◽

Rna Structure ◽

Influenza A ◽

Locked Nucleic Acid ◽

Antiviral Resistance ◽

Packaging Signal ◽

Oseltamivir Carboxylate ◽

Virus Rna ◽

Different Strains

Currently approved anti-influenza drugs target viral proteins, are subtype limited, and are challenged by rising antiviral resistance. To overcome these limitations, we sought to identify a conserved essential RNA secondary structure within the genomic RNA predicted to have greater constraints on mutation in response to therapeutics targeting this structure. Here, we identified and genetically validated an RNA stemloop structure we termed PSL2, which serves as a packaging signal for genome segment PB2 and is highly conserved across influenza A virus (IAV) isolates. RNA structural modeling rationalized known packaging-defective mutations and allowed for predictive mutagenesis tests. Disrupting and compensating mutations of PSL2's structure give striking attenuation and restoration, respectively, of in vitro virus packaging and mortality in mice. Antisense Locked Nucleic Acid oligonucleotides (LNAs) designed against PSL2 dramatically inhibit IAV in vitro against viruses of different strains and subtypes, possess a high barrier to the development of antiviral resistance, and are equally effective against oseltamivir carboxylate-resistant virus. A single dose of LNA administered 3 days after, or 14 days before, a lethal IAV inoculum provides 100% survival. Moreover, such treatment led to the development of strong immunity to rechallenge with a ten-fold lethal inoculum. Together, these results have exciting implications for the development of a versatile novel class of antiviral therapeutics capable of prophylaxis, post-exposure treatment, and 'just-in-time' universal vaccination against all IAV strains, including drug-resistant pandemics.

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Detection and Characterization of Influenza A Virus PA-PB2 Interaction through a Bimolecular Fluorescence Complementation Assay

Journal of Virology ◽

10.1128/jvi.02300-08 ◽

2009 ◽

Vol 83 (8) ◽

pp. 3944-3955 ◽

Cited By ~ 55

Author(s):

Joseph N. Hemerka ◽

Dan Wang ◽

Yuejin Weng ◽

Wuxun Lu ◽

Radhey S. Kaushik ◽

...

Keyword(s):

Influenza A Virus ◽

Protein Interactions ◽

Influenza A ◽

Specific Interaction ◽

Polymerase Activity ◽

Bimolecular Fluorescence Complementation ◽

Polymerase Complex ◽

Binary Complexes ◽

Fluorescence Complementation ◽

Bimolecular Fluorescence

ABSTRACT The influenza virus polymerase complex, consisting of the PA, PB1, and PB2 subunits, is required for the transcription and replication of the influenza A viral genome. Previous studies have shown that PB1 serves as a core subunit to incorporate PA and PB2 into the polymerase complex by directly interacting with PA and PB2. Despite numerous attempts, largely involving biochemical approaches, a specific interaction between PA and PB2 subunits has yet to be detected. In the current study, we developed and utilized bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in the assembly of the influenza A virus polymerase complex. Proof-of-concept experiments demonstrated that BiFC can specifically detect PA-PB1 interactions in living cells. Strikingly, BiFC demonstrated an interaction between PA and PB2 that has not been reported previously. Deletion-based BiFC experiments indicated that the N-terminal 100 amino acid residues of PA are responsible for the PA-PB2 interaction observed in BiFC. Furthermore, a detailed analysis of subcellular localization patterns and temporal nuclear import of PA-PB2 binary complexes suggested that PA and PB2 subunits interacted in the cytoplasm initially and were subsequently transported as a dimer into the nucleus. Taken together, results of our studies reveal a previously unknown PA-PB2 interaction and provide a framework for further investigation of the biological relevance of the PA-PB2 interaction in the polymerase activity and viral replication of influenza A virus.

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Conserved secondary structures predicted within the 5′ packaging signal region of influenza A virus PB2 segment

Meta Gene ◽

10.1016/j.mgene.2017.11.006 ◽

2018 ◽

Vol 15 ◽

pp. 75-79 ◽

Cited By ~ 2

Author(s):

Yuki Kobayashi ◽

Oliver G. Pybus ◽

Takuya Itou ◽

Yoshiyuki Suzuki

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Secondary Structures ◽

Packaging Signal ◽

Signal Region

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Identification of the 5′-Terminal Packaging Signal of the H1N1 Influenza A Virus Neuraminidase Segment at Single-Nucleotide Resolution

Frontiers in Microbiology ◽

10.3389/fmicb.2021.709010 ◽

2021 ◽

Vol 12 ◽

Author(s):

Erika Seshimo ◽

Fumitaka Momose ◽

Yuko Morikawa

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Synonymous Codon ◽

Gene Segment ◽

H1n1 Influenza ◽

Terminal Region ◽

Selective Assembly ◽

Wild Type ◽

Packaging Signal ◽

Nucleotide Resolution

The genome of the influenza A virus is an eight-segmented negative-strand RNA (vRNA). Progeny vRNAs replicated in the nucleus selectively assemble into a single set of eight different segments, probably in the cytoplasm, and are packaged into progeny virions at the cell membrane. In these processes, a region of approximately 100 nucleotides at both ends of each segment is thought to function as a selective assembly/packaging signal; however, the details of the mechanism, such as the required sequences, are still unknown. In this study, we focused on the 5′-terminus of the sixth neuraminidase gene segment vRNA (Seg.6) to identify the essential sequence for selective packaging. The 5′-terminal region of the A/Puerto Rico/8/34 strain Seg.6 was divided into seven regions of 15 nucleotides each from A to G, and mutations were introduced into each region by complementary base substitutions or synonymous codon substitutions. Mutant viruses were generated and compared for infectious titers, and the relative ratios of the eight segments packaged into virions were measured. We also ascertained whether mutant vRNA was eliminated by competitive packaging with wild-type vRNA. Mutations in the A–C regions reduced infectious titers and eliminated mutant vRNAs by competition with wild-type vRNA. Even under non-competitive conditions, the packaging efficiency of the A or B region mutant Seg.6 was reduced. Next, we designed an artificial vRNA with a 50-nucleotide duplication at the 5′-terminal region. Using this, a virus library was created by randomly replacing each region, which became an untranslated region (UTR), with complementary bases. After selecting proliferative viruses from the library, nine wild-type nucleotides in the A and B regions were identified as essential bases, and we found that these bases were highly conserved in Seg.6 vRNAs encoding the N1 subtype neuraminidase. From these results, we conclude that the identified bases function as the 5′-terminal packaging signal for the N1 subtype Seg.6 vRNA.

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Packaging signal of influenza A virus

Virology Journal ◽

10.1186/s12985-021-01504-4 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Xiuli Li ◽

Min Gu ◽

Qinmei Zheng ◽

Ruyi Gao ◽

Xiufan Liu

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Packaging Signal ◽

Genome Packaging ◽

Genetic Reassortment ◽

A Genome ◽

Virus Genotypes ◽

Virus Packaging ◽

Negative Sense ◽

Packaging Signals

AbstractInfluenza A virus (IAV) contains a genome with eight single-stranded, negative-sense RNA segments that encode 17 proteins. During its assembly, all eight separate viral RNA (vRNA) segments are incorporated into virions in a selective manner. Evidence suggested that the highly selective genome packaging mechanism relies on RNA-RNA or protein-RNA interactions. The specific structures of each vRNA that contribute to mediating the packaging of the vRNA into virions have been described and identified as packaging signals. Abundant research indicated that sequences required for genome incorporation are not series and are varied among virus genotypes. The packaging signals play important roles in determining the virus replication, genome incorporation and genetic reassortment of influenza A virus. In this review, we discuss recent studies on influenza A virus packaging signals to provide an overview of their characteristics and functions.

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