Differential Effect of Nucleotide Substitutions in the 3′ Arm of the Influenza A Virus vRNA Promoter on Transcription/Replication by Avian and Human Polymerase Complexes Is Related to the Nature of PB2 Amino Acid 627

Avian-origin influenza virus polymerase activity can be dramatically increased in human cells with the PB2 E627K mutation. Previously, others have proposed that this mutation increases the stability of the viral ribonucleoprotein complex (vRNP) measured by the interaction between PB2 and NP. However, we demonstrate here that a variety of PB2 adaptive mutations, including E627K, do not enhance the stability of the vRNP but rather increase the amount of replicated RNA that results in more PB2-NP coprecipitation.

mentioning

confidence: 52%

Unstable Polymerase-Nucleoprotein Interaction Is Not Responsible for Avian Influenza Virus Polymerase Restriction in Human Cells

Cauldwell

Moncorgé

Barclay

2013

“…In fact, the template or, more specifically, promoter stability has been implicated previously in the mechanism of restriction of avian influenza virus polymerases in mammalian cells (32). In order to test the effect of promoter stability on PB2 627E polymerase activity, a range of mutations were introduced into the promoter of a full-length noncoding segment 5 template.…”

Section: Resultsmentioning

confidence: 99%

Host Restriction of Influenza Virus Polymerase Activity by PB2 627E Is Diminished on Short Viral Templates in a Nucleoprotein-Independent Manner

Paterson

Velthuis

Vreede

et al. 2014

Most avian influenza viruses do not replicate efficiently in human cells. This is partly due to the low activity of the RNA polymerase of avian influenza viruses in mammalian cells. Nevertheless, this impediment can be overcome through an E¡K adaptive mutation at residue 627 of the PB2 subunit of the polymerase. Accordingly, viral ribonucleoprotein (RNP) reconstitution assays show that a viral polymerase containing PB2 627E has impaired activity in mammalian cells compared to a viral polymerase that contains PB2 627K, characteristic of mammalian-adapted influenza viruses. In contrast, purified viral polymerases containing either PB2 627E or PB2 627K show comparable levels of activity in transcription assays that require no RNP assembly. We sought to reconcile these conflicting observations by using an NP-independent cell-based transcription/replication assay to assess viral polymerase activity. We found that PB2 627E polymerase restriction in mammalian cells is independent of NP expression but is dependent on the length of the viral RNA template. In addition, restriction of PB2 627E polymerase was overcome by mutations specific to the viral RNA template promoter sequence. Consequently, we propose that PB2 627E affects recruitment of the viral RNA promoter by the viral polymerase in mammalian cells.

“…For example, H5N1 viruses that are virulent in mice contain lysine at position 627 in the PB2 protein, whereas H5N1 viruses that are not virulent in mice, as well as other avian influenza A viruses, contain glutamic acid at this position (9). This amino acid change may represent an adaptation for efficient replication in mammals (5,40,42), although this change is not required for virulence in ferrets (48). In addition, amino acids in PB1 and PA have been implicated in virulence in ferrets and/or ducks (11,38).…”

mentioning

confidence: 99%

“…In addition, amino acids in PB1 and PA have been implicated in virulence in ferrets and/or ducks (11,38). It has been postulated that influenza virus RNA synthesis requires host factors that differ between species (5,17,25,40,42), and evidence for the participation of host factors in influenza virus RNA synthesis has been reported (12,13,21,23,24,26,39).…”

mentioning

confidence: 99%

Interaction of the Influenza A Virus Nucleocapsid Protein with the Viral RNA Polymerase Potentiates Unprimed Viral RNA Replication

Newcomb

Kuo

et al. 2009

The influenza A virus polymerase transcribes and replicates the eight virion RNA (vRNA) segments. Transcription is initiated with capped RNA primers excised from cellular pre-mRNAs by the intrinsic endonuclease of the viral polymerase. Viral RNA replication occurs in two steps: first a full-length copy of vRNA is made, termed cRNA, and then this cRNA is copied to produce vRNA. The synthesis of cRNAs and vRNAs is initiated without a primer, in contrast to the initiation of viral mRNA synthesis, and requires the viral nucleocapsid protein (NP). The mechanism of unprimed viral RNA replication is poorly understood. To elucidate this mechanism, we used purified recombinant influenza virus polymerase complexes and NP to establish an in vitro system that catalyzes the unprimed synthesis of cRNA and vRNA using 50-nucleotide-long RNA templates. The purified viral polymerase and NP are sufficient for catalyzing this RNA synthesis without a primer, suggesting that host cell factors are not required. We used this purified in vitro replication system to demonstrate that the RNA-binding activity of NP is not required for the unprimed synthesis of cRNA and vRNA. This result rules out two models that postulate that the RNA-binding activity of NP mediates the switch from capped RNA-primed transcription to unprimed viral RNA replication. Because we showed that NP lacking RNA-binding activity binds directly to the viral polymerase, it is likely that a direct interaction between NP and the viral polymerase results in a modification of the polymerase in favor of unprimed initiation.