cRNA Promoter Research Articles

Influenza virus RNA encapsidation: discrimination for vRNA versus cRNA molecules

The packaging signal of influenza viral RNA is shown to reside within the 5′ bulged promoter structure, caused by the central unpaired residue A10 in its 5′ branch. Upon insertion of two uridine residues in the 3′ branch opposite A10, the minus-strand vRNA promoter is converted into a 3′ bulged structure, while the plus-strand cRNA promoter adopts the 5′ bulged conformation. The cRNA is packaged exclusively in the progeny virions of this promoter variant. Upon insertion of a single uridine nucleotide opposite A10, the two de-bulged structures of the vRNA and cRNA promoters are rendered identical, and both vRNA and cRNA molecules are packaged indiscriminately, in a 1:1 ratio. We propose that interaction of viral polymerase with the two differently bulged vRNA and cRNA promoter structures results in either of two conformations of the protein, which in turn are recognized in a two-step procedure by matrix protein M1. While both, viral nucleoprotein particles (vRNPs) and cRNPs, are exported from the nucleus in association with M1, attachment to only the 5′ bulged vRNPs is maintained during encapsidation at the plasma membrane.

Functional analysis of the influenza A virus cRNA promoter and construction of an ambisense transcription system.

While influenza A viral RNA is known to act as a template for the synthesis of both viral mRNA and complementary cRNA, the latter has been observed so far only to function as an intermediate in replication and give rise to progeny vRNA molecules. Here it is shown that the cRNA promoter is also capable of initiating viral mRNA synthesis, similar to vRNA-promoted transcription adhering to the cap-snatching mode of primer recruitment. Detection of cRNA promoted transcription required an inversion of the reporter gene coding sequence plus relocation of the viral polyadenylation signal. Construction of cRNA promoter variants through RNA polymerase I reverse genetics allowed us to determine the RNA polymerase-associated, base-paired conformation in a reporter gene read-out system. It again turned out to adhere to the "corkscrew" model, similar, but slightly different in its binding interactions from the corresponding vRNA conformation. The observation of two transcription reactions, initiated in either direction from influenza vRNA and cRNA template molecules, allowed us to construct bicistronic, ambisense RNA molecules for simultaneous expression of two proteins from a single segment of viral RNA.

The packaging signal of influenza viral RNA molecules

The packaging signal present in influenza viral RNA molecules is shown not to constitute a separate structural element, but to reside within the 5'-bulged promoter structure, as caused by the central unpaired residue A10 in its 5' branch. Upon insertion of two uridine residues in the 3' branch opposite A10, the minus-strand viral RNA (vRNA) promoter is converted into a 3'-bulged structure, whereas the plus-strand cRNA promoter instead adopts the 5'-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 5'A10, 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 5' 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.

An endonuclease switching mechanism in the virion RNA and cRNA promoters of Thogoto orthomyxovirus.

An in vitro assay was developed to investigate endonuclease activity of Thogoto virus, a tick-borne orthomyxovirus. Endonuclease activity relied on an interaction between the 3' and 5' termini of virion RNA (vRNA) and not those of cRNA. Evidence was obtained that cap structures are cleaved directly from cap donors and that cleavage does not occur after pyrimidines. A 5' hook structure, present in the vRNA promoter but not the cRNA promoter, was introduced into cRNA promoter mutants. These mutants stimulated endonuclease activity, although at levels slightly lower than that of vRNA. The ability of the cRNA promoter to stimulate endonuclease activity when mutated to contain a 5' hook structure indicates that this structure constitutes a switching mechanism for endonuclease activity between the vRNA and cRNA promoters.

The Thogoto orthomyxovirus cRNA promoter functions as a panhandle but does not stimulate cap snatching in vitro.

The cRNA promoter of Thogoto virus, a tick-borne orthomyxovirus, was investigated using an in vitro polymerase assay based on purified viral cores and synthetic oligoribonucleotides corresponding to the 3' and 5' ends of cRNA. In vitro polymerase activity relied on an interaction between the 3' and 5' ends of cRNA and was ApG primer-dependent. Mutational analysis of the promoter showed that interstrand base-pairing of residues 11 and 12 of the 3' promoter arm with residues 10 and 11 of the 5' promoter arm, respectively, was essential for polymerase activity. These data provide the first clear evidence for a cRNA panhandle in an orthomyxovirus. No evidence was obtained for the presence of a 5' or 3' hook structure in the cRNA promoter, and transcription could not be primed with rabbit globin mRNA or synthetic cap analogues. This demonstrates that cap snatching activity relies on the presence of the vRNA terminal sequences.

Mutational analysis of the promoter required for influenza virus virion RNA synthesis.

An in vitro RNA synthesis system was established in which the influenza virus virion (minus-sense) RNA was made from the synthetic plus-sense RNA (cRNA) template by the purified viral polymerase complex. The cRNA promoter was studied by mutational analysis using the in vitro system, and on the basis of these experiments, the first 11 nucleotides of the 3' noncoding sequence were found to contain the minimum promoter required for virion RNA synthesis. The addition of extra nucleotides at the 3' end decreased the promoter activity of the templates, indicating that the viral polymerase does not recognize an internal promoter efficiently. The wild-type and mutated RNA templates were also tested in vivo by using the ribonucleoprotein transfection system. In contrast to the in vitro system, it was found that the majority of mutations at the 3'-terminal sequence significantly decreased or abolished chloramphenicol acetyltransferase (CAT) expression. These results suggest that the cRNA promoter overlaps other essential cis elements required for chloramphenicol acetyltransferase expression in vivo.