Critical role of segment-specific packaging signals in genetic reassortment of influenza A viruses

McCrae

Journal of General Virology

et al. 2016

The process by which eukaryotic viruses with segmented genomes select a complete set of genome segments for packaging into progeny virus particles is not understood. In this study a model based on the association of genome segments through specific RNA-RNA interactions driven by base pairing was formalized and tested in the Orbivirus genus of the Reoviridae family. A strategy combining screening of the genomic sequences for inter-segment complementarity with direct functional testing of inter-segment RNA-RNA interactions using reverse genetics is described in the type species of the Orbivirus genus, Bluetongue virus (BTV). Two examples, involving four of the ten BTV genomic segments, of specific intersegment interaction motifs whose maintenance is essential for the generation of infectious virus, were identified. Equivalent inter-segment complementarities were found between the identified regions of the orthologous genome segments of all orbiviruses, including phylogenetically distant species. Specific interaction of the participating RNA segments was confirmed in vitro using electrophoretic mobility shift assays, with the interactions inhibited using oligonucleotides complementary to the interaction motif of one of the interacting partners, and also through mutagenesis of the motifs. In each example, the base pairing rather than the absolute sequence was critical to the formation of a functional inter-segment interaction, with mutations only being tolerated in rescued virus if compensating changes were made in the interacting partner to restore uninterrupted base pairing. The absolute sequence of the complementarity motifs varied between species, indicating that this newly identified phenomenon may contribute to the observed lack of reassortment between Orbivirus species.

Section: Discussionmentioning

confidence: 95%

Inter-segment complementarity in orbiviruses: a driver for co-ordinated genome packaging in the Reoviridae?

McCrae

Journal of General Virology

et al. 2016

“…The reverse genetics plasmids used to generate PR8-sciIAV (31) and the influenza A virus ⌬HA/GFP plasmid pPolI HA(45)GFP (80) have been previously described (28). The reverse genetics plasmids used to rescue B/Brisbane/60/2008 were generated by extracting RNA (kindly provided by the CDC) using an RNeasy minikit (Qiagen).…”

Section: Methodsmentioning

confidence: 99%

“…Evaluation of vRNA incorporation into virions. Rescue transfections where sciIAV or sciIBV was each rescued with the ⌬HA/GFP construct pPolI influenza A virus HA(45)GFP (80) or pPolI influenza B virus HA(228)GFP(108) were performed as previously described (26). Briefly, 293T cells (10 6 cells, 6-well plate format) were cotransfected with seven ambisense rescue plasmids (for PB2, PB1, PA, NP, NA, M, and NS) for PR8 (31) or B/Bris (37), together with pCAGGS HA protein expression plasmids to facilitate initial rescue (26), and ⌬HA/GFP vRNA expression plasmids, using Lipofectamine 2000 (1:1 ratio).…”

Section: Methodsmentioning

confidence: 99%

Influenza A and B Virus Intertypic Reassortment through Compatible Viral Packaging Signals

Baker

Nogales

Finch

et al. 2014

J Virol

104

Influenza A and B viruses cocirculate in humans and together cause disease and seasonal epidemics. These two types of influenza viruses are evolutionarily divergent, and exchange of genetic segments inside coinfected cells occurs frequently within types but never between influenza A and B viruses. Possible mechanisms inhibiting the intertypic reassortment of genetic segments could be due to incompatible protein functions of segment homologs, a lack of processing of heterotypic segments by influenza virus RNA-dependent RNA polymerase, an inhibitory effect of viral proteins on heterotypic virus function, or an inability to specifically incorporate heterotypic segments into budding virions. Here, we demonstrate that the full-length hemagglutinin (HA) of prototype influenza B viruses can complement the function of multiple influenza A viruses. We show that viral noncoding regions were sufficient to drive gene expression for either type A or B influenza virus with its cognate or heterotypic polymerase. The native influenza B virus HA segment could not be incorporated into influenza A virus virions. However, by adding the influenza A virus packaging signals to full-length influenza B virus glycoproteins, we rescued influenza A viruses that possessed HA, NA, or both HA and NA of influenza B virus. Furthermore, we show that, similar to single-cycle infectious influenza A virus, influenza B virus cannot incorporate heterotypic transgenes due to packaging signal incompatibilities. Altogether, these results demonstrate that the lack of influenza A and B virus reassortants can be attributed at least in part to incompatibilities in the virus-specific packaging signals required for effective segment incorporation into nascent virions. IMPORTANCEReassortment of influenza A or B viruses provides an evolutionary strategy leading to unique genotypes, which can spawn influenza A viruses with pandemic potential. However, the mechanism preventing intertypic reassortment or gene exchange between influenza A and B viruses is not well understood. Nucleotides comprising the coding termini of each influenza A virus gene segment are required for specific segment incorporation during budding. Whether influenza B virus shares a similar selective packaging strategy or if packaging signals prevent intertypic reassortment remains unknown. Here, we provide evidence suggesting a similar mechanism of influenza B virus genome packaging. Furthermore, by appending influenza A virus packaging signals onto influenza B virus segments, we rescued recombinant influenza A/B viruses that could reassort in vitro with another influenza A virus. These findings suggest that the divergent evolution of packaging signals aids with the speciation of influenza A and B viruses and is in part responsible for the lack of intertypic viral reassortment.

“…Importantly, packaging signals and patterns of intersegment interactions in the network can differ from one virus strain to another, and these differences can affect the outcome of the reassortment between two viruses (for reviews, see references 23, 39, and 40). Thus, strain-specific interactions between gene segments might be responsible for the coselection of two or more homologous segments during the emergence of reassortant pandemic viruses (22,38). In our experiments, the combination of homologous avian HA and PB1 segments did not significantly enhance fitness of the viruses compared to fitness with single-gene PB1 reassortants.…”

Section: Discussionmentioning

confidence: 52%

“…To corroborate these findings, we studied competition of the PB1 segments during the process of virus preparation from reverse genetics plasmids (37,38). Viruses were generated using nine plasmids, namely, seven non-PB1 plasmids required for the preparation of either the PB1 pair or the HAϩPB1 pair plus the 1:1 mixture of PB1 Cal and PB1 HK plasmids (Fig.…”

Section: Resultsmentioning

confidence: 95%

The Avian-Origin PB1 Gene Segment Facilitated Replication and Transmissibility of the H3N2/1968 Pandemic Influenza Virus

Wendel

Rubbenstroth

Doedt

et al. 2015

J Virol

IMPORTANCEDespite the high impact of influenza pandemics on human health, some mechanisms underlying the emergence of pandemic influenza viruses still are poorly understood. Thus, it was unclear why both H2N2/1957 and H3N2/1968 reassortant pandemic viruses contained, in addition to the avian HA, the PB1 gene segment of the avian parent. Here, we addressed this long-standing question by modeling the emergence of the H3N2/1968 virus from its putative human and avian precursors. We show that the avian PB1 segment increased activity of the viral polymerase and facilitated viral replication. Our results suggest that in addition to the acquisition of antigenically novel HA (i.e., antigenic shift), enhanced viral polymerase activity is required for the emergence of pandemic influenza viruses from their seasonal human precursors. Influenza A viruses are single-stranded negative-sense RNA viruses. Their genome consists of 8 RNA segments, each of which encodes 1 to 3 proteins (1). Pandemic influenza has been occurring at irregular intervals for at least 500 years, causing significant, occasionally catastrophic mortality in the human population (2, 3). Genetic data available for the last four pandemic viruses (H1N1/1918, H2N2/1957, H3N2/1968, and H1N1/2009) indicate that they all contained an antigenically novel hemagglutinin (HA) which was derived from animal influenza A viruses (reviewed in references 2-5). The origin of the other seven gene segments of the pandemic viruses varied (3,4,6). The source of the non-HA gene segments of the H1N1/1918 virus is not clear due to the lack of sequence data for coexistent avian and mammalian influenza A viruses. The precursor of the H1N1/2009 virus emerged and circulated in pigs; this virus was transmitted and adapted to humans as a whole. Two other pandemic viruses emerged via gene mixing (reassortment) between animal viruses and contemporary human viruses and contained either 5 gene segments (H2N2/1957) or 6 gene segments (H3N2/1968) of the human virus parent.Avian influenza viruses do not replicate efficiently in humans. It is believed that exchange via reassortment of nonadapted gene segments from an avian virus by segments from a human-adapted parent might allow the emerging pandemic virus to overcome host range restriction (2,7,8). Surprisingly, both 1957 and 1968 pandemic viruses contained the avian PB1 segment (gene segment 2) (9). This segment encodes three proteins, polymerase basic protein 1 (PB1), PB1-F2, and PB1-N40, which are translated from the start codons 1, 4, and 5, respectively (1, 10). PB1 is the core subunit of the trimeric viral RNA-dependent RNA polymerase complex (PB1, PB2, and PA), which is responsible for transcription and replication of the viral genome (1, 11). PB1-F2 and PB1-N40 are not essential for virus replication, but they can affect replication efficiency and contribute to pathogenicity (10, 12).