Abstract
The genome of influenza A viruses (IAV) consists of eight single-stranded negative sense viral RNAs (vRNAs) encapsidated into viral ribonucleoproteins (vRNPs). It is now well established that genome packaging (i.e., the incorporation of a set of eight distinct vRNPs into budding viral particles), follows a specific pathway guided by segment-specific cis-acting packaging signals on each vRNA. However, the precise nature and function of the packaging signals, and the mechanisms underlying the assembly of vRNPs into sub-bundles in the cytoplasm and their selective packaging at the viral budding site, remain largely unknown. Here, we review the diverse and complementary methods currently being used to elucidate these aspects of the viral cycle. They range from conventional and competitive reverse genetics, single molecule imaging of vRNPs by fluorescence in situ hybridization (FISH) and high-resolution electron microscopy and tomography of budding viral particles, to solely in vitro approaches to investigate vRNA-vRNA interactions at the molecular level.
Highlights
Influenza A viruses (IAVs) are responsible for yearly flu epidemics that cause three to five million cases of severe illness, claim 250,000 to 500,000 lives annually and greatly impact the global economy.Increased morbidity and mortality can result from occasional pandemics, the latest one being in2009
RNA polymerase I (PolI) promoter sequences and either a PolI terminator or a ribozyme sequence that generate the correct 51 and 31 ends, respectively. These plasmids were transfected into human embryonic kidney cells expressing the simian virus 40 (SV40) large T antigen (HEK293T) along with expression plasmids under the control of the RNA polymerase II (PolII) promoter for, at minima, the synthesis of the viral PB1, PB2, polymerase acidic (PA) and NP proteins, which results in the reconstitution of viral ribonucleoproteins (vRNPs) and allows the initiation of a viral replication cycle [11,12]
We review the diverse experimental approaches available to map the cis-acting packaging signals on individual viral RNA segments (vRNAs) and to understand how they function to promote the assembly and packaging of a set of eight distinct vRNAs into viral particles
Summary
Influenza A viruses (IAVs) are responsible for yearly flu epidemics that cause three to five million cases of severe illness, claim 250,000 to 500,000 lives annually and greatly impact the global economy. IAVs are members of the Orthomyxoviridae family Their genome consists of eight single-stranded negative sense viral RNA segments (vRNAs), varying in length from 2341 to 890 nucleotides (nt), numbered from 1 to 8 or named after the main protein they encode. Despite their difference in length, all vRNAs share the same genetic organization: the functional open reading frame (ORF), in antisense orientation, is flanked by two non-coding regions (NCRs) that differ in length and in sequence between vRNAs, except for the 12- and 13-nt long sequences at the 31 and 51 end, respectively, that are highly conserved between vRNAs and between species (Figure 1a).
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