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Influenza A virus nucleoprotein selectively decreases neuraminidase gene-segment packaging while enhancing viral fitness and transmissibility.

ABSTRACT: The influenza A virus (IAV) genome is divided into eight distinct RNA segments believed to be copackaged into virions with nearly perfect efficiency. Here, we describe a mutation in IAV nucleoprotein (NP) that enhances replication and transmission in guinea pigs while selectively reducing neuraminidase (NA) gene segment packaging into virions. We show that incomplete IAV particles lacking gene segments contribute to the propagation of the viral population through multiplicity reactivation under conditions of widespread coinfection, which we demonstrate commonly occurs in the upper respiratory tract of guinea pigs. NP also dramatically altered the functional balance of the viral glycoproteins on particles by selectively decreasing NA expression. Our findings reveal novel functions for NP in selective control of IAV gene packaging and balancing glycoprotein expression and suggest a role for incomplete gene packaging during host adaptation and transmission.

SUBMITTER: Brooke CB

PROVIDER: S-EPMC4250133 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Influenza A virus nucleoprotein selectively decreases neuraminidase gene-segment packaging while enhancing viral fitness and transmissibility.

Brooke Christopher B CB Ince William L WL Wei Jiajie J Bennink Jack R JR Yewdell Jonathan W JW

Proceedings of the National Academy of Sciences of the United States of America 20141110 47

The influenza A virus (IAV) genome is divided into eight distinct RNA segments believed to be copackaged into virions with nearly perfect efficiency. Here, we describe a mutation in IAV nucleoprotein (NP) that enhances replication and transmission in guinea pigs while selectively reducing neuraminidase (NA) gene segment packaging into virions. We show that incomplete IAV particles lacking gene segments contribute to the propagation of the viral population through multiplicity reactivation under ...[more]

PMID: 25385602

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Project description:The genome of the influenza A virus is an eight-segmented negative-strand RNA (vRNA). Progeny vRNAs replicated in the nucleus selectively assemble into a single set of eight different segments, probably in the cytoplasm, and are packaged into progeny virions at the cell membrane. In these processes, a region of approximately 100 nucleotides at both ends of each segment is thought to function as a selective assembly/packaging signal; however, the details of the mechanism, such as the required sequences, are still unknown. In this study, we focused on the 5'-terminus of the sixth neuraminidase gene segment vRNA (Seg.6) to identify the essential sequence for selective packaging. The 5'-terminal region of the A/Puerto Rico/8/34 strain Seg.6 was divided into seven regions of 15 nucleotides each from A to G, and mutations were introduced into each region by complementary base substitutions or synonymous codon substitutions. Mutant viruses were generated and compared for infectious titers, and the relative ratios of the eight segments packaged into virions were measured. We also ascertained whether mutant vRNA was eliminated by competitive packaging with wild-type vRNA. Mutations in the A-C regions reduced infectious titers and eliminated mutant vRNAs by competition with wild-type vRNA. Even under non-competitive conditions, the packaging efficiency of the A or B region mutant Seg.6 was reduced. Next, we designed an artificial vRNA with a 50-nucleotide duplication at the 5'-terminal region. Using this, a virus library was created by randomly replacing each region, which became an untranslated region (UTR), with complementary bases. After selecting proliferative viruses from the library, nine wild-type nucleotides in the A and B regions were identified as essential bases, and we found that these bases were highly conserved in Seg.6 vRNAs encoding the N1 subtype neuraminidase. From these results, we conclude that the identified bases function as the 5'-terminal packaging signal for the N1 subtype Seg.6 vRNA.

Project description:The increased detection of H3 C-IVA (1990.4.a) clade influenza A viruses (IAVs) in US swine in 2019 was associated with a reassortment event to acquire an H1N1pdm09 lineage nucleoprotein (pdmNP) gene, replacing a TRIG lineage NP (trigNP). We hypothesized that acquiring the pdmNP conferred a selective advantage over prior circulating H3 viruses with a trigNP. To investigate the role of NP reassortment in transmission, we identified two contemporary 1990.4.a representative strains (NC/19 and MN/18) with different evolutionary origins of the NP gene. A reverse genetics system was used to generate wild-type (wt) strains and swap the pdm and TRIG lineage NP genes, generating four viruses: wtNC/19-pdmNP, NC/19-trigNP, wtMN/18-trigNP, and MN/18-pdmNP. The pathogenicity and transmission of the four viruses were compared in pigs. All four viruses infected 10 primary pigs and transmitted to five indirect contact pigs per group. Pigs infected via contact with MN/18-pdmNP shed virus 2 days earlier than pigs infected with wtMN/18-trigNP. The inverse did not occur for wtNC/19-pdmNP and NC/19-trigNP. This suggests that pdmNP reassortment resulted in a combination of genes that improved transmission efficiency when paired with the 1990.4.a hemagglutinin (HA). This is likely a multigenic trait, as replacing the trigNP gene did not diminish the transmission of a wild-type IAV in swine. This study demonstrates how reassortment and evolutionary change of internal genes can result in more transmissible viruses that influence HA clade detection frequency. Thus, rapidly identifying novel reassortants paired with dominant hemagglutinin/neuraminidase may improve the prediction of strains to include in vaccines.IMPORTANCEInfluenza A viruses (IAVs) are composed of eight non-continuous gene segments that can reassort during coinfection of a host, creating new combinations. Some gene combinations may convey a selective advantage and be paired together preferentially. A reassortment event was detected in swine in the United States that involved the exchange of two lineages of nucleoprotein (NP) genes (trigNP to pdmNP) that became a predominant genotype detected in surveillance. Using a transmission study, we demonstrated that exchanging the trigNP for a pdmNP caused the virus to shed from the nose at higher levels and transmit to other pigs more rapidly. Replacing a pdmNP with a trigNP did not hinder transmission, suggesting that transmission efficiency depends on interactions between multiple genes. This demonstrates how reassortment alters IAV transmission and that reassortment events can provide an explanation for why genetically related viruses with different internal gene combinations experience rapid fluxes in detection frequency.

Dataset Information

Influenza A virus nucleoprotein selectively decreases neuraminidase gene-segment packaging while enhancing viral fitness and transmissibility.

Publications

Influenza A virus nucleoprotein selectively decreases neuraminidase gene-segment packaging while enhancing viral fitness and transmissibility.

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