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Selective flexible packaging pathways of the segmented genome of influenza A virus.

ABSTRACT: The genome of influenza A viruses (IAV) is encoded in eight distinct viral ribonucleoproteins (vRNPs) that consist of negative sense viral RNA (vRNA) covered by the IAV nucleoprotein. Previous studies strongly support a selective packaging model by which vRNP segments are bundling to an octameric complex, which is integrated into budding virions. However, the pathway(s) generating a complete genome bundle is not known. We here use a multiplexed FISH assay to monitor all eight vRNAs in parallel in human lung epithelial cells. Analysis of 3.9?×?105 spots of colocalizing vRNAs provides quantitative insights into segment composition of vRNP complexes and, thus, implications for bundling routes. The complexes rarely contain multiple copies of a specific segment. The data suggest a selective packaging mechanism with limited flexibility by which vRNPs assemble into a complete IAV genome. We surmise that this flexibility forms an essential basis for the development of reassortant viruses with pandemic potential.

SUBMITTER: Haralampiev I

PROVIDER: S-EPMC7455735 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Selective flexible packaging pathways of the segmented genome of influenza A virus.

Haralampiev Ivan I Prisner Simon S Nitzan Mor M Schade Matthias M Jolmes Fabian F Schreiber Max M Loidolt-Krüger Maria M Jongen Kalle K Chamiolo Jasmine J Nilson Niklaas N Winter Franziska F Friedman Nir N Seitz Oliver O Wolff Thorsten T Herrmann Andreas A

Nature communications 20200828 1

The genome of influenza A viruses (IAV) is encoded in eight distinct viral ribonucleoproteins (vRNPs) that consist of negative sense viral RNA (vRNA) covered by the IAV nucleoprotein. Previous studies strongly support a selective packaging model by which vRNP segments are bundling to an octameric complex, which is integrated into budding virions. However, the pathway(s) generating a complete genome bundle is not known. We here use a multiplexed FISH assay to monitor all eight vRNAs in parallel i ...[more]

PMID: 32859915

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Project description:The influenza A virus genome is composed of eight single-stranded negative-sense viral RNA segments (vRNAs). The eight vRNAs are selectively packaged into each progeny virion. This process likely involves specific interactions between the vRNAs via segment-specific packaging signals located in both the 3'- and 5'-terminal regions of the respective vRNAs. To assess the importance of vRNA-vRNA interactions via packaging signals for selective genome packaging, we generated mutant viruses possessing silent mutations in the packaging signal region of the hemagglutinin (HA) vRNA. A mutant virus possessing silent mutations in nucleotides (nt) 1664 to 1676 resulted in defects in HA vRNA incorporation and showed a reduction in viral growth. After serial passage, the mutant virus acquired additional mutations in the 5'-terminal packaging signal regions of both the HA and polymerase basic 2 (PB2) vRNAs. These mutations contributed to the recovery of viral growth and HA vRNA packaging efficiency. In addition, an RNA-RNA interaction between the 5' ends of HA and PB2 vRNAs was confirmed in vitro, and this interaction was disrupted following the introduction of silent mutations in the HA vRNA. Thus, our results demonstrated that RNA-RNA interactions between the packaging signal regions of HA vRNA and PB2 vRNA are important for selective genome packaging. IMPORTANCE While numerous viral genomes comprise a single genome segment, the influenza A virus possesses eight segmented genomes. Influenza A virus can benefit from having a segmented genome because the segments can reassort with other strains of the influenza virus to create new genetically distinct strains. The influenza A virus efficiently incorporates one copy of each of its eight genomic segments per viral particle. However, the mechanism by which each segment is specifically selected is poorly understood. The genome segments contain RNA signals that facilitate the incorporation of segments into virus particles. These regions may facilitate specific interactions between the genome segments, creating an eight-segment complex, which can then be packaged into individual particles. In this study, we provide evidence that RNA signals contribute to specific interactions between two of the influenza virus genome segments.

Project description:Bunyavirus genomes comprise a small (S), a medium (M), and a large (L) RNA segment of negative polarity. Although the untranslated regions have been shown to comprise signals required for transcription, replication, and encapsidation, the mechanisms that drive the packaging of at least one S, M, and L segment into a single virion to generate infectious virus are largely unknown. One of the most important members of the Bunyaviridae family that causes devastating disease in ruminants and occasionally humans is the Rift Valley fever virus (RVFV). We studied the flexibility of RVFV genome packaging by splitting the glycoprotein precursor gene, encoding the (NSm)GnGc polyprotein, into two individual genes encoding either (NSm)Gn or Gc. Using reverse genetics, six viruses with a segmented glycoprotein precursor gene were rescued, varying from a virus comprising two S-type segments in the absence of an M-type segment to a virus consisting of four segments (RVFV-4s), of which three are M-type. Despite that all virus variants were able to grow in mammalian cell lines, they were unable to spread efficiently in cells of mosquito origin. Moreover, in vivo studies demonstrated that RVFV-4s is unable to cause disseminated infection and disease in mice, even in the presence of the main virulence factor NSs, but induced a protective immune response against a lethal challenge with wild-type virus. In summary, splitting bunyavirus glycoprotein precursor genes provides new opportunities to study bunyavirus genome packaging and offers new methods to develop next-generation live-attenuated bunyavirus vaccines.Rift Valley fever virus (RVFV) causes devastating disease in ruminants and occasionally humans. Virions capable of productive infection comprise at least one copy of the small (S), medium (M), and large (L) RNA genome segments. The M segment encodes a glycoprotein precursor (GPC) protein that is cotranslationally cleaved into Gn and Gc, which are required for virus entry and fusion. We studied the flexibility of RVFV genome packaging and developed experimental live-attenuated vaccines by applying a unique strategy based on the splitting of the GnGc open reading frame. Several RVFV variants, varying from viruses comprising two S-type segments to viruses consisting of four segments (RVFV-4s), of which three are M-type, could be rescued and were shown to induce a rapid protective immune response. Altogether, the segmentation of bunyavirus GPCs provides a new method for studying bunyavirus genome packaging and facilitates the development of novel live-attenuated bunyavirus vaccines.

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Selective flexible packaging pathways of the segmented genome of influenza A virus.

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Selective flexible packaging pathways of the segmented genome of influenza A virus.

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