Antisense Oligonucleotides Targeting Influenza A Segment 8 Genomic RNA Inhibit Viral Replication

ABSTRACT Influenza A virus expresses three viral polymerase (P) subunits—PB1, PB2, and PA—all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain α, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain α. The role of the minimal PB1 domain α in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

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Antisense oligonucleotides directed against the viral RNA polymerase gene enhance survival of mice infected with influenza A

Nature Biotechnology ◽

10.1038/9893 ◽

1999 ◽

Vol 17 (6) ◽

pp. 583-587 ◽

Cited By ~ 41

Author(s):

Tadashi Mizuta ◽

Masatoshi Fujiwara ◽

Toshifumi Hatta ◽

Takayuki Abe ◽

Naoko Miyano-Kurosaki ◽

...

Keyword(s):

Rna Polymerase ◽

Antisense Oligonucleotides ◽

Influenza A ◽

Viral Rna ◽

Polymerase Gene ◽

Rna Polymerase Gene

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Influenza A viruses balance ER stress with host protein synthesis shutoff

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2024681118 ◽

2021 ◽

Vol 118 (36) ◽

pp. e2024681118

Author(s):

Beryl Mazel-Sanchez ◽

Justyna Iwaszkiewicz ◽

Joao P. P. Bonifacio ◽

Filo Silva ◽

Chengyue Niu ◽

...

Keyword(s):

Viral Replication ◽

Er Stress ◽

Host Cell ◽

Messenger Rna ◽

Influenza A ◽

Nonstructural Protein ◽

Host Protein ◽

Stress Induction ◽

Nonstructural Protein 1

Excessive production of viral glycoproteins during infections poses a tremendous stress potential on the endoplasmic reticulum (ER) protein folding machinery of the host cell. The host cell balances this by providing more ER resident chaperones and reducing translation. For viruses, this unfolded protein response (UPR) offers the potential to fold more glycoproteins. We postulated that viruses could have developed means to limit the inevitable ER stress to a beneficial level for viral replication. Using a relevant human pathogen, influenza A virus (IAV), we first established the determinant for ER stress and UPR induction during infection. In contrast to a panel of previous reports, we identified neuraminidase to be the determinant for ER stress induction, and not hemagglutinin. IAV relieves ER stress by expression of its nonstructural protein 1 (NS1). NS1 interferes with the host messenger RNA processing factor CPSF30 and suppresses ER stress response factors, such as XBP1. In vivo viral replication is increased when NS1 antagonizes ER stress induction. Our results reveal how IAV optimizes glycoprotein expression by balancing folding capacity.

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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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Replication protein of tobacco mosaic virus cotranslationally binds the 5' untranslated region of genomic RNA to enable viral replication

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1321660111 ◽

2014 ◽

Vol 111 (16) ◽

pp. E1620-E1628 ◽

Cited By ~ 32

Author(s):

K. Kawamura-Nagaya ◽

K. Ishibashi ◽

Y.-P. Huang ◽

S. Miyashita ◽

M. Ishikawa

Keyword(s):

Viral Replication ◽

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Untranslated Region ◽

Replication Protein ◽

Genomic Rna

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Tissue Tropisms of Avian Influenza A Viruses Affect Their Spillovers from Wild Birds to Pigs

Journal of Virology ◽

10.1128/jvi.00847-20 ◽

2020 ◽

Vol 94 (24) ◽

Cited By ~ 1

Author(s):

Xiaojian Zhang ◽

Fred L. Cunningham ◽

Lei Li ◽

Katie Hanson-Dorr ◽

Liyuan Liu ◽

...

Keyword(s):

Viral Replication ◽

Receptor Binding ◽

Influenza A ◽

Phenotypic Trait ◽

Influenza A Viruses ◽

Replication Efficiency ◽

Binding Preference ◽

Upper Respiratory ◽

Tissue Tropisms ◽

Viral Replication Efficiency

ABSTRACT Wild aquatic birds maintain a large, genetically diverse pool of influenza A viruses (IAVs), which can be transmitted to lower mammals and, ultimately, humans. Through phenotypic analyses of viral replication efficiency, only a small set of avian IAVs were found to replicate well in epithelial cells of the swine upper respiratory tract, and these viruses were shown to infect and cause virus shedding in pigs. Such a phenotypic trait of the viral replication efficiency appears to emerge randomly and is distributed among IAVs across multiple avian species and geographic and temporal orders. It is not determined by receptor binding preference but is determined by other markers across genomic segments, such as those in the ribonucleoprotein complex. This study demonstrates that phenotypic variants of viral replication efficiency exist among avian IAVs but that only a few of these may result in viral shedding in pigs upon infection, providing opportunities for these viruses to become adapted to pigs, thus posing a higher potential risk for creating novel variants or detrimental reassortants within pig populations. IMPORTANCE Swine serve as a mixing vessel for generating pandemic strains of human influenza virus. All hemagglutinin subtypes of IAVs can infect swine; however, only sporadic cases of infection with avian IAVs are reported in domestic swine. The molecular mechanisms affecting the ability of avian IAVs to infect swine are still not fully understood. From the findings of phenotypic analyses, this study suggests that the tissue tropisms (i.e., in swine upper respiratory tracts) of avian IAVs affect their spillovers from wild birds to pigs. It was found that this phenotype is determined not by receptor binding preference but is determined by other markers across genomic segments, such as those in the ribonucleoprotein complex. In addition, our results show that such a phenotypic trait was sporadically and randomly distributed among IAVs across multiple avian species and geographic and temporal orders. This study suggests an efficient way for assessment of the risk posed by avian IAVs, such as in evaluating their potentials to be transmitted from birds to pigs.

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