Influenza Virus PB1 and Neuraminidase Gene Segments Can Cosegregate during Vaccine Reassortment Driven by Interactions in the PB1 Coding Region

Abstract

Egg-grown influenza vaccine yields are maximized by infection with a seed virus produced by "classical reassortment" of a seasonal isolate with a highly egg-adapted strain. Seed viruses are selected based on a high-growth phenotype and the presence of the seasonal hemagglutinin (HA) and neuraminidase (NA) surface antigens. Retrospective analysis of H3N2 vaccine seed viruses indicated that, unlike other internal proteins that were predominantly derived from the high-growth parent A/Puerto Rico/8/34 (PR8), the polymerase subunit PB1 could be derived from either parent depending on the seasonal strain. We have recently shown that A/Udorn/307/72 (Udorn) models a seasonal isolate that yields reassortants bearing the seasonal PB1 gene. This is despite the fact that the reverse genetics-derived virus that includes Udorn PB1 with Udorn HA and NA on a PR8 background has inferior growth compared to the corresponding virus with PR8 PB1. Here we use competitive plasmid transfections to investigate the mechanisms driving selection of a less fit virus and show that the Udorn PB1 gene segment cosegregates with the Udorn NA gene segment. Analysis of chimeric PB1 genes revealed that the coselection of NA and PB1 segments was not directed through the previously identified packaging sequences but through interactions involving the internal coding region of the PB1 gene. This study identifies associations between viral genes that can direct selection in classical reassortment for vaccine production and which may also be of relevance to the gene constellations observed in past antigenic shift events where creation of a pandemic virus has involved reassortment. IMPORTANCEInfluenza vaccine must be produced and administered in a timely manner in order to provide protection during the winter season, and poor-growing vaccine seed viruses can compromise this process. To maximize vaccine yields, manufacturers create hybrid influenza viruses with gene segments encoding the surface antigens from a seasonal virus isolate, important for immunity, and others from a virus with high growth properties. This involves coinfection of cells with both parent viruses and selection of dominant progeny bearing the seasonal antigens. We show that this method of creating hybrid viruses does not necessarily select for the best yielding virus because preferential pairing of gene segments when progeny viruses are produced determines the genetic makeup of the hybrids. This not only has implications for how hybrid viruses are selected for vaccine production but also sheds light on what drives and limits hybrid gene combinations that arise in nature, leading to pandemics.

In vitro vRNA–vRNA interactions in the H1N1 influenza A virus genome

“…Similarly, the PB1–NA complex was detected for H1N1 WSN, requiring MgCl 2 , suggesting the necessary requirement of Mg 2+ for detecting vRNA–vRNA interactions. These results are consistent with those from previous studies showing that NA vRNA is copackaged with PB1 vRNA into progeny virions in both H1N1 PR8 and H3N2 Udorn viruses . By contrast, HA and M vRNAs of the H1N1 WSN virus did not form a complex even when the concentration of MgCl 2 was increased (Figure b), supporting a specific interaction between H1N1 WSN PB1 and NA vRNAs.…”

Influenza NA and PB1 Gene Segments Interact during the Formation of Viral Progeny: Localization of the Binding Region within the PB1 Gene

“…The resulting viral progeny in the transfection supernatants were then analysed by RT-PCR from plaque-picks to determine the source of the competing PB1 gene. Initially we competed the wildtype PR8 and Udorn PB1 genes to re-affirm data we have published previously [24] showing that the Udorn PB1 gene is incorporated at a significantly higher frequency than PR8 PB1 ( p < 0.0001) in the presence of the Udorn NA gene on this background (Figure 3A).…”

“…When given the choice of PB1 genes, packaged progeny viruses predominantly contained the Udorn PB1. Similar experiments linked the preferential co-segregation of Udorn PB1 to the Udorn NA and not HA segment [24]. Therefore it was postulated that incorporation of H3N2 PB1 into vaccine seeds was likely due to this same co-selection mechanism with potential pairing with the H3N2 NA segment.…”

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

“…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.…”

“…These results (18)(19)(20)(21) support the hypothesis that there is cooperation between homologous PB1 and HA/NA. Indeed, it was shown recently that the selection of the high-growth 3 ϩ 5 reassortants of A/PR/8/1934 could be explained by favorable interactions between the homologous vRNA segments encoding PB1 and NA during the packaging of vRNAs into virus particles (22).…”