Interplay between Innate Immunity and Negative-Strand RNA Viruses: towards a Rational Model

The influenza A virus genome is comprised of eight single-stranded negative-sense viral RNA (vRNA) segments. Each of the eight vRNA segments contains segment-specific nonconserved noncoding regions (NCRs) of similar sequence and length in different influenza A virus strains. However, in the subtype-determinant segments, encoding haemagglutinin (HA) and neuraminidase (NA), the segment-specific noncoding regions are subtype-specific, varying significantly in sequence and length at both the 3´ and 5´ termini among different subtypes. The significance of these subtype-specific noncoding regions (ssNCR) in the influenza virus replication cycle is not fully understood. In this study, we show that truncations of the 3´-end H1-subtype-specific noncoding region (H1-ssNCR) resulted in recombinant viruses with decreased HA vRNA replication and attenuated growth phenotype, although the vRNA replication was not affected in single-template RNP reconstitution assays. The attenuated viruses were unstable and point mutations at nucleotide position 76 or 56 in the adjacent coding region of HA vRNA were found after serial passage. The mutations restored the HA vRNA replication and reversed the attenuated virus growth phenotype. We propose that the terminal noncoding and adjacent coding regions act synergistically to ensure optimal levels of HA vRNA replication in a multi-segment environment. These results, provide novel insights into the role of the 3´-end nonconserved noncoding regions and adjacent coding regions on template preference in multiple-segmented negative-strand RNA viruses. IMPORTANCE While most influenza A virus vRNA segments contain segment-specific nonconserved noncoding regions of similar length and sequence, these regions vary considerably both in length and sequence in the segments encoding HA and NA, the two major antigenic determinants of influenza A viruses. In this study, we investigated the function of the 3´-end H1-ssNCR and observed a synergistic effect between the 3´-end H1-ssNCR nucleotides and adjacent coding nucleotide(s) of HA segment on template preference in a multi-segment environment. The results unravel an additional level of complexity in the regulation of RNA replication in multiple-segmented negative-strand RNA viruses.

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Transcription and Replication of Nonsegmented Negative-Strand RNA Viruses

Current Topics in Microbiology and Immunology - Biology of Negative Strand RNA Viruses: The Power of Reverse Genetics ◽

10.1007/978-3-662-06099-5_3 ◽

2004 ◽

pp. 61-119 ◽

Cited By ~ 52

Author(s):

S. P. J. Whelan ◽

J. N. Barr ◽

G. W. Wertz

Keyword(s):

Rna Viruses ◽

Negative Strand ◽

Transcription And Replication

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Processing of Genome 5′ Termini as a Strategy of Negative-Strand RNA Viruses to Avoid RIG-I-Dependent Interferon Induction

PLoS ONE ◽

10.1371/journal.pone.0002032 ◽

2008 ◽

Vol 3 (4) ◽

pp. e2032 ◽

Cited By ~ 198

Author(s):

Matthias Habjan ◽

Ida Andersson ◽

Jonas Klingström ◽

Michael Schümann ◽

Arnold Martin ◽

...

Keyword(s):

Rna Viruses ◽

Negative Strand ◽

Interferon Induction

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Strand-Specific Dual RNA Sequencing of Bronchial Epithelial Cells Infected with Influenza A/H3N2 Viruses Reveals Splicing of Gene Segment 6 and Novel Host-Virus Interactions

Journal of Virology ◽

10.1128/jvi.00518-18 ◽

2018 ◽

Vol 92 (17) ◽

Cited By ~ 15

Author(s):

Giulia Fabozzi ◽

Andrew J. Oler ◽

Poching Liu ◽

Yong Chen ◽

Samuel Mindaye ◽

...

Keyword(s):

Innate Immunity ◽

Influenza Virus ◽

Epithelial Cells ◽

Rna Sequencing ◽

Influenza A ◽

Bronchial Epithelial Cells ◽

Immune Gene ◽

Rna Seq ◽

Bronchial Epithelial ◽

Negative Strand

ABSTRACT Host-influenza virus interplay at the transcript level has been extensively characterized in epithelial cells. Yet, there are no studies that simultaneously characterize human host and influenza A virus (IAV) genomes. We infected human bronchial epithelial BEAS-2B cells with two seasonal IAV/H3N2 strains, Brisbane/10/07 and Perth/16/09 (reference strains for past vaccine seasons) and the well-characterized laboratory strain Udorn/307/72. Strand-specific RNA sequencing (RNA-seq) of the infected BEAS-2B cells allowed for simultaneous analysis of host and viral transcriptomes, in addition to pathogen genomes, to reveal changes in mRNA expression and alternative splicing (AS). In general, patterns of global and immune gene expression induced by the three IAVs were mostly shared. However, AS of host transcripts and small nuclear RNAs differed between the seasonal and laboratory strains. Analysis of viral transcriptomes showed deletions of the polymerase components (defective interfering-like RNAs) within the genome. Surprisingly, we found that the neuraminidase gene undergoes AS and that the splicing event differs between seasonal and laboratory strains. Our findings reveal novel elements of the host-virus interaction and highlight the importance of RNA-seq in identifying molecular changes at the genome level that may contribute to shaping RNA-based innate immunity. IMPORTANCE The use of massively parallel RNA sequencing (RNA-seq) has revealed insights into human and pathogen genomes and their evolution. Dual RNA-seq allows simultaneous dissection of host and pathogen genomes and strand-specific RNA-seq provides information about the polarity of the RNA. This is important in the case of negative-strand RNA viruses like influenza virus, which generate positive (complementary and mRNA) and negative-strand RNAs (genome) that differ in their potential to trigger innate immunity. Here, we characterize interactions between human bronchial epithelial cells and three influenza A/H3N2 strains using strand-specific dual RNA-seq. We focused on this subtype because of its epidemiological importance in causing significant morbidity and mortality during influenza epidemics. We report novel elements that differ between seasonal and laboratory strains highlighting the complexity of the host-virus interplay at the RNA level.

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