One influenza virus particle packages eight unique viral RNAs as shown by FISH analysis

Chou, Yi-ying; Vafabakhsh, Reza; Doğanay, Sultan; Gao, Qinshan; Ha, Taekjip; Palese, Peter

doi:10.1073/pnas.1206069109

Cited by 156 publications

(159 citation statements)

References 35 publications

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“…The observation that IAV can selectively reduce the relative packaging frequency of individual gene segments undermines the dogma that IAV genome segments are copackaged in equimolar ratios with high efficiency (9)(10)(11). Instead, packaging efficiency is clearly variable and can be modulated rapidly (via a singlenucleotide substitution) in response to selective pressure.…”

Section: Discussionmentioning

confidence: 99%

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

Brooke

Ince

Wei

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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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.influenza virus | genome segmentation | genome packaging | nucleoprotein | host adaptation S easonal influenza A virus (IAV) remains a major public health threat, causing tens of thousands of deaths each year in the United States alone (1). Morbidity and mortality rates can increase dramatically when a zoonotic strain adapts to circulate in humans, triggering a pandemic. The continuing toll exerted by IAV stems from its remarkable adaptability, which enables it to move between widely divergent host species and also evade herd immunity within each species. Defining the specific mechanisms that mediate IAV adaptation is essential to improving anti-IAV vaccines, therapeutics, and pandemic surveillance.The IAV genome consists of eight negative-sense RNA segments, each of which is required for productive infection. Genome segmentation complements the high mutation rate of IAV by facilitating reassortment, which can maximize positive intergenic epistasis (2-5) and allow selective elimination of segments with deleterious mutations (6, 7). Although reassortment is the most obvious and best-characterized benefit of segmentation, there are likely additional evolutionary advantages.Genome segmentation imposes the substantial constraint of maintaining a gene-packaging mechanism to produce fully infectious virions (8). For IAV, it is widely believed that a single copy of each segment is packaged into progeny virions with nearly perfect efficiency, resulting in an equimolar ratio of the segments at the population level (9-12). Confounding this model, we recently demonstrated that most IAV virions fail to express one or more gene products (13). This finding raises the possibility that in some circumstances incomplete influenza gene packaging is evolutionarily neutral and possibly even advantageous (14).The viral glycoproteins HA and neuraminidase (NA) are encoded by separate genome segments. HA attaches IAV to terminal sialic acid residues on the host cell surface, enabling viral entry. By hydrolyzing sialic acids, NA detaches budding virions and neutralizes HA-inhibiting glyco...

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Section: Discussionmentioning

confidence: 99%

“…For IAV, it is widely believed that a single copy of each segment is packaged into progeny virions with nearly perfect efficiency, resulting in an equimolar ratio of the segments at the population level (9)(10)(11)(12). Confounding this model, we recently demonstrated that most IAV virions fail to express one or more gene products (13).…”

mentioning

confidence: 92%

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

Brooke

Ince

Wei

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…We then excited the fluorophores attached to DNA molecules inside individual virus heads and the number of photobleaching steps was counted (Fig. S3) (27). Fig.…”

Section: Resultsmentioning

confidence: 99%

Single-molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4

Vafabakhsh

Kondabagil

Earnest

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Viral DNA packaging motors are among the most powerful molecular motors known. A variety of structural, biochemical, and singlemolecule biophysical approaches have been used to understand their mechanochemistry. However, packaging initiation has been difficult to analyze because of its transient and highly dynamic nature. Here, we developed a single-molecule fluorescence assay that allowed visualization of packaging initiation and reinitiation in real time and quantification of motor assembly and initiation kinetics. We observed that a single bacteriophage T4 packaging machine can package multiple DNA molecules in bursts of activity separated by long pauses, suggesting that it switches between active and quiescent states. Multiple initiation pathways were discovered including, unexpectedly, direct DNA binding to the capsid portal followed by recruitment of motor subunits. Rapid succession of ATP hydrolysis was essential for efficient initiation. These observations have implications for the evolution of icosahedral viruses and regulation of virus assembly.virus packaging | single-molecule fluorescence imaging | molecular motors A s part of a virus life cycle, genetic information needs to be incorporated into the newly produced virus particles. Tailed bacteriophages, which probably form the largest biomass of the planet (1), and many eukaryotic viruses such as herpes viruses use powerful ATPase motors to achieve this (2). These motors generate forces as high as 80-100 pN and translocate DNA into a preformed prohead until a DNA condensate of near crystalline density fills the interior (3).The viral packaging motors share a common architecture with the ASCE (additional strand, conserved E) superfamily of multimeric ring ATPases that perform diverse functions such as chromosome segregation (helicases), protein remodeling (chaperones and proteasomes), and cargo transport (dyneins) (4). Although much has been learned about the mechanochemistry of these motors, little is known about how a functional motor is assembled and its activity is initiated. The packaging motors have the difficult task of precisely inserting the end of a viral genome into the capsid at the time of initiation.In a general virus assembly pathway shared by dsDNA viruses, assembly starts at a unique fivefold vertex of the prohead called the portal vertex, which is formed from 12 molecules of the portal protein (5). A protein shell assembles around a protein scaffold and later becomes an empty prohead after the scaffold leaves, or is degraded (6). In most dsDNA bacteriophages as well as herpes viruses a complex of two proteins, known as small and large "terminase" proteins, recognize a specific sequence of DNA in the concatemeric viral genome (e.g., cos site in phage λ and pac site in phage P22) and make a cut to create a dsDNA end (7,8). The small terminase is responsible for binding to the cos or pac site, whereas the large terminase makes the cut. However, phage phi29 and adenoviruses do not require DNA cutting because the genome is a unit-length m...

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“…However, an increasing number of reports provide convincing evidence to support the selective packaging model (28,50,51). The selective packaging model claims that each of eight vRNA segments possesses a segment-specific packaging signal sequence, allowing the selective incorporation of eight vRNA segments into a fully functional virion.…”

Section: Discussionmentioning

confidence: 99%

An Eight-Segment Swine Influenza Virus Harboring H1 and H3 Hemagglutinins Is Attenuated and Protective against H1N1 and H3N2 Subtypes in Pigs

Mašić

Pyo

Babiuk

et al. 2013

J Virol

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Swine influenza virus (SIV) infections continue to cause production losses in the agricultural industry in addition to being a human public health concern. The primary method of controlling SIV is through vaccination. The killed SIV vaccines currently in use must be closely matched to the challenge virus, and their protective efficacy is limited. Live attenuated influenza vaccines (LAIV) provide strong, long-lived cell-mediated and humoral immunity against different influenza virus subtypes with no need for antigen matching. Here we report the generation of a new potential LAIV, an eight-segment SIV harboring two different SIV hemagglutinins (HAs), H1 and H3, in the genetic background of H1N1 SIV. This mutant SIV was generated by fusing the H3 HA ectodomain from A/Swine/Texas/4199-2/98 (H3N2) to the cytoplasmic tail, transmembrane domain, and stalk region of neuraminidase (NA) from A/Swine/Saskatchewan/18789/02 (H1N1) SIV. While this H1-H3 chimeric SIV, when propagated in vitro in the presence of exogenous neuraminidase, showed kinetics and growth properties similar to those of the parental wild-type virus, in vivo it was highly attenuated in pigs, demonstrating a great potential for serving as a dual LAIV. Furthermore, vaccination with the H1-H3 virus elicited robust immune responses, which conferred complete protection against infections with both H1 and H3 SIV subtypes in pigs.

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One influenza virus particle packages eight unique viral RNAs as shown by FISH analysis

Cited by 156 publications

References 35 publications

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

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

Single-molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4

An Eight-Segment Swine Influenza Virus Harboring H1 and H3 Hemagglutinins Is Attenuated and Protective against H1N1 and H3N2 Subtypes in Pigs

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