Selective incorporation of influenza virus RNA segments into virions

Abstract

The genome of influenza A virus is comprised of eight viral RNA (vRNA) segments. Although the products of all eight vRNA segments must be present for viral replication, little is known about the mechanism(s) responsible for incorporation of these segments into virions. Two models have been proposed for the generation of infectious virions containing eight vRNA segments. The randomincorporation model assumes a common structural feature in all the vRNAs, enabling any combination of vRNAs to be incorporated randomly into virions. The selective-incorporation model predicts the presence of specific structures in each vRNA segment, leading to the incorporation of a set of eight vRNA segments into virions. Here we demonstrate that eight different vRNA segments must be present for efficient virion formation and that sequences within the coding region of (and thus unique to) the neuraminidase vRNA possess a signal that drives incorporation of this segment into virions. These findings indicate a unique contribution from individual vRNA segments and thus suggest a selective (rather than random) mechanism of vRNA recruitment into virions. The neuraminidase vRNA incorporation signal and others yet to be identified should provide attractive targets for the attenuation of influenza viruses in vaccine production and the design of new antiviral drugs.T he influenza A virus genome consists of eight segments of single-stranded RNA with negative polarity (complementary to mRNA). Each viral RNA (vRNA) segment resides within a complex [the viral ribonucleoprotein complex (vRNP)] of nucleoprotein and three polymerase subunits designated PA, PB1, and PB2. After transport of M1 and NS2 proteins into the nucleus, vRNPs formed in this compartment are exported to the cytoplasm, where presumably they interact with viral membraneassociated proteins including hemagglutinin (HA), neuraminidase (NA), and M1 to ensure the correct assembly of virions and their release from the host cell. Although all eight vRNPs must be present for efficient viral replication, little is known about the mechanism(s) responsible for incorporation of vRNA segments into virions and their stable maintenance during repeated cycles of replication.Two models have been proposed for the generation of infectious virions containing eight vRNA segments. The randomincorporation model assumes a common structural feature in all the vRNPs, enabling them to be incorporated randomly into virions. Support for this model comes from the observation that an influenza A virion can possess more than eight vRNPs (1). The selective-incorporation model predicts the presence of specific structures in each vRNA segment, leading to their individual incorporation into virions. This hypothesis was suggested by data showing that the presence of excess amounts of internally deleted segments encoding polymerase proteins resulted in a corresponding reduction of full-length segments in virions (2, 3).Air and coworkers (4-6) produced an influenza A virus with a large internal deletion in the NA vRNA s...

Rewiring the RNAs of influenza virus to prevent reassortment

“…Surprisingly, except for the 12 conserved nucleotides at the 3Ј end and the 13 nucleotides at the 5Ј end of the vRNA, which are required for both polymerase recognition and binding (1), no other conserved motif has been identified in the remainder of the packaging sequences. In addition, the minimum lengths required for efficient packaging, either at the 3Ј or 5Ј ends of the vRNA, differ from one segment to another (3)(4)(5)(6)(7)(8)(9)(10)(11). Although much is known about the role of these packaging sequences in influenza RNA packaging, their mechanism has yet to be fully elucidated.…”

“…A previous study showed that soon after budding, the influenza A virus ribonucleoprotein (RNP) complexes inside the virions form a specific architecture: a central segment surrounded by the seven other vRNPs (2), which suggests that influenza viral RNA (vRNA) packaging is not a random process. Moreover, segment-specific RNA packaging sequences have been identified on each segment of the influenza A/WSN/33 virus (3)(4)(5)(6)(7)(8)(9)(10)(11). A common feature of these packaging signals is that both the 3Ј and 5Ј noncoding regions (NCRs), as well as coding sequences at the two ends of each open reading frame (ORF) are important [(3-11) and Fig.…”

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

“…Using reverse genetics, cispackaging signals of the human H1N1 WSN and PR8 strains were found to reside at both ends of each vRNA, including the UTRs, along with up to 80 bases of adjacent coding sequences (21)(22)(23)(24)(25)(26)(27)(28). In this study, we generated reassortant viruses in vitro from avian H5N2 and human H3N2 viruses to identify incompatibilities between the two parental viruses arising at the vRNA level.…”

Development of high-yield influenza B virus vaccine viruses

“…The use of a universal backbone for both influenza A and B viruses would simplify the vaccine production process. Reassortants between influenza A and B viruses do not occur naturally, likely because of type-specific viral packaging signals located in the 5′ and 3′ terminal regions of influenza vRNA segments (14,15). We, therefore, generated vRNA segments in which the ectodomains of the influenza A PR8 virus HA and NA proteins were replaced with influenza B virus counterparts (SI Appendix, Fig.…”