The packaging signal of influenza viral RNA molecules

Abstract

The packaging signal present in influenza viral RNA molecules is shown not to constitute a separate structural element, but to reside within the 59-bulged promoter structure, as caused by the central unpaired residue A10 in its 59 branch. Upon insertion of two uridine residues in the 39 branch opposite A10, the minus-strand viral RNA (vRNA) promoter is converted into a 39-bulged structure, whereas the plus-strand cRNA promoter instead adopts the 59-bulged conformation. In this promoter variant it is exclusively the cRNA that is found packaged in the progeny virions. Upon insertion of only a single uridine nucleotide opposite 59A10, the two debulged structures of the vRNA and cRNA promoters are rendered identical, and both vRNA and cRNA molecules are packaged indiscriminately, in a 1:1 ratio, but at lower rates. We propose that the binding interactions of viral polymerase with either of the two differently bulged vRNA and cRNA promoter structures result in two different conformations of the enzyme protein. Only the 59 bulged RNA-associated polymerase conformation appears to be recognized for nuclear export, which depends on nuclear matrix protein M1 and nonstructural protein NS2. And the respective wild-type vRNP-or insertion mutant cRNP complex is observed to enter the cytoplasm and hence is included in the viral encapsidation process, which takes place at the plasma membrane.

Intergenic subset organization within a set of geographically-defined viral sequences from the 2009 H1N1 influenza A pandemic

“…The centrality of the M1 node in the network may reflect the important role that M1 plays [14], in concert with other influenza components [15] in the packaging and cellular exteriorization of the virus. Moreover, these biological functions of the M1 gene, necessary for viral production, are known to be susceptible to synonymous mutation [15,16].…”

Genome packaging in influenza A virus

“…1a), the UTRs of each segment consist of 12-13 nt of highly conserved sequence, forming a panhandle structure required for polymerase binding (Neumann et al, 2004), as well as a variable amount (typically around 25 nt) of segment-specific sequence. Studies showed that the UTRs of segments 4, 5 or 8 were sufficient, in the presence of a helper virus, to direct replication of the segment, to package it into virions and, in some experiments, to maintain it for several passages, indicating that the UTRs contained the minimal determinants of segment packaging (Luytjes et al, 1989;Neumann et al, 2004;Neumann & Hobom, 1995;Tchatalbachev et al, 2001). This approach was then used to produce a virus with nine distinct segments (Enami et al, 1991).…”

“…The partial complementarity of the 59-and 39-termini means that a similar but not identical panhandle structure is present in the plus-sense cRNA replicative intermediates from which progeny vRNAs are transcribed (Neumann et al, 2004). In the context of a synthetic vRNA lacking an influenza gene and therefore a specific packaging signal, the unpaired A10 residue in the 59-arm of vRNA is apparently crucial for differentiating vRNA from cRNA, operating to prevent packaging of the latter by not supporting its nuclear export (Tchatalbachev et al, 2001). …”

“…Although the evidence for a specific packaging method is overwhelming, the mechanism is clearly not perfect. The existence of nine segment viruses (Enami et al, 1991;Laver & Downie, 1976;Nakajima & Sugiura, 1977;Scholtissek et al, 1978) and the low levels of 'background' packaging of reporter vRNAs lacking specific packaging signals seen by several laboratories and for all segments (Bancroft & Parslow, 2002;Dos Santos Afonso et al, 2005;Fujii et al, 2003;Luytjes et al, 1989;Muramoto et al, 2006;Neumann et al, 1994;Tchatalbachev et al, 2001) suggest that there is a measurable degree of imprecision in the mechanism. We are also presented with the apparent paradox, that in the laboratory, small mutations (even a single nucleotide) can significantly disrupt packaging of a particular segment to the point where the virus replicates noticeably more poorly (e.g.…”

Rescue of influenza B virus from eight plasmids

“…The non coding regions contain cis-acting signals for the regulation of transcription and replication of viral RNA (35,36). Type-specific differences of the NCRs between members of the Orthomyxovirus family were analyzed in the last decade mostly by using in vitro transcription assays or in vivo reconstitution of vRNA-like templates encoding reporter genes such as chloramphenicol acetyltransferase (37)(38)(39)(40). Those studies can now be extended to characterize sequence elements that are type specific and those that modulate gene expression and can be exchanged between types in the context of infectious viruses.…”