Abstract
Avian influenza viruses pose a serious pandemic threat to humans. Better knowledge on cross-species adaptation is important. This study examined the replication and transcription efficiency of ribonucleoprotein complexes reconstituted by plasmid co-transfection between H5N1, H1N1pdm09 and H3N2 influenza A viruses, and to identify mutations in the RNA polymerase subunit that affect human adaptation. Viral RNA polymerase subunits PB1, PB2, PA and NP derived from influenza viruses were co-expressed with pPolI-vNP-Luc in human cells, and with its function evaluated by luciferase reporter assay. A quantitative RT-PCR was used to measure vRNA, cRNA, and mRNA levels for assessing the replication and transcription efficiency. Mutations in polymerase subunit were created to identify signature of increased human adaptability. H5N1 ribonucleoprotein complexes incorporated with PB2 derived from H1N1pdm09 and H3N2 viruses increased the polymerase activity in human cells. Furthermore, single amino acid substitutions at PB2 of H5N1 could affect polymerase activity in a temperature-dependent manner. By using a highly sensitive quantitative reverse transcription-polymerase chain reaction, an obvious enhancement in replication and transcription activities of ribonucleoproteins was observed by the introduction of lysine at residue 627 in the H5N1 PB2 subunit. Although less strongly in polymerase activity, E158G mutation appeared to alter the accumulation of H5N1 RNA levels in a temperature-dependent manner, suggesting a temperature-dependent mechanism in regulating transcription and replication exists. H5N1 viruses can adapt to humans either by acquisition of PB2 from circulating human-adapted viruses through reassortment, or by mutations at critical sites in PB2. This information may help to predict the pandemic potential of newly emerged influenza strains, and provide a scientific basis for stepping up surveillance measures and vaccine production.
Highlights
Influenza A virus contains eight single-stranded RNA segments
The negative-sense viral RNA segments act as templates for messenger RNA synthesis in transcription, and for complementary RNA synthesis which is used for replication of vRNA
At 37uC, the polymerase activity of H3N2 and H1N1pdm09 was similar to WSN H1N1, whereas that of H5N1 was significantly lower than all others (25% of WSN H1N1, P,0.001)
Summary
The negative-sense viral RNA (vRNA) segments act as templates for messenger RNA (mRNA) synthesis in transcription, and for complementary RNA (cRNA) synthesis which is used for replication of vRNA. Both transcription and replication are performed by viral RNA-dependent RNA polymerase (RdRp) inside the nucleus of infected cells [1], [2]. The RdRp complex composed of three polymerase subunits, polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2) and polymerase acid protein (PA) These proteins, in association with nucleoproteins (NP) and vRNA segments, constitute viral ribonucleoproteins (RNPs) [3]. The role of PA remains uncertain, it has been suggested to function in both transcription and replication mediated by its endonuclease [1], [9], [10], [11], and binding to vRNA promoter and cap [10]
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