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

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.

Section: Resultssupporting

confidence: 81%

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

“…NP coprecipitated significantly smaller amounts of PB2 K627E from human cells than the WT (Fig. 4A and B), in agreement with previous findings (9,11,12,40). The amount of coprecipitated PB2 was directly related to polymerase activity for all of the P[F/P]AAAPP and basic face mutants.…”

Section: G T D T Q I I K L L P a A A P P Q S R M Q S S L T V N V R V supporting

confidence: 81%

“…The holoenzyme further assembles with genomic RNA and NP to form higher-order vRNPs. Defects or the intentional perturbation of any of these assembly steps can impair polymerase activity (9,11,(38)(39)(40). Polymerase activity was reconstituted with the P[P/F]AAAPP or basic face mutants, and immunoprecipitation assays were used to test whether impaired polymerase activity resulted from defects in the assembly process.…”

Section: G T D T Q I I K L L P a A A P P Q S R M Q S S L T V N V R V mentioning

confidence: 99%

Conserved Features of the PB2 627 Domain Impact Influenza Virus Polymerase Function and Replication

Kirui

Bucci

Poole

et al. 2014

Successful replication of influenza virus requires the coordinated expression of viral genes and replication of the genome by the viral polymerase, composed of the subunits PA, PB1, and PB2. Polymerase activity is regulated by both viral and host factors, yet the mechanisms of regulation and how they contribute to viral pathogenicity and tropism are poorly understood. To characterize these processes, we created a series of mutants in the 627 domain of the PB2 subunit. This domain contains a conserved "P[F/ P]AAAPP" sequence motif and the well-described amino acid 627, whose identity regulates host range. A lysine present at position 627 in most mammalian viral isolates creates a basic face on the domain surface and confers high-level activity in humans compared to the glutamic acid found at this position in avian isolates. Mutation of the basic face or the P[F/P]AAAPP motif impaired polymerase activity, assembly of replication complexes, and viral replication. Most of these residues are required for general polymerase activity, whereas PB2 K586 and R589 were preferentially required for function in human versus avian cells. Thus, these data identify residues in the 627 domain and other viral proteins that regulate polymerase activity, highlighting the importance of the surface charge and structure of this domain for virus replication and host adaptation. IMPORTANCE Influenza virus faces barriers to transmission across species as it emerges from its natural reservoir in birds to infect mammals.The viral polymerase is an important regulator of this process and undergoes discrete changes to adapt to replication in mammals. Many of these changes occur in the polymerase subunit PB2. Here we describe the systematic analysis of a key region in PB2 that controls species-specific polymerase activity. We report the importance of conserved residues that contribute to the overall charge of the protein as well as those that likely affect protein structure. These findings provide further insight into the molecular events dictating species-specific polymerase function and viral replication.

“…In a replication assay that lacks NP, activity of reconstituted viral polymerases was nonetheless reduced in mammalian cells with PB2 627E compared to those with PB2 627K (45). In addition, enhanced coimmunoprecipitation of NP with PB2 627K compared to that with PB2 627E has been shown by some groups to be dependent on the presence of a vRNA template (42,75). This observation has led to the suggestion that increased PB2-NP interactions with PB2 627K arise not due to an intrinsic improvement in PB2-NP binding but cells.…”

Section: Discussionmentioning

confidence: 93%

“…due to increases in the number active vRNP complexes within the infected cell (75). We have not established herein whether NP-PB2 binding is affected by PB2 E627K or Q591K in the dk/AB/76 virus background.…”

Section: Discussionmentioning

confidence: 94%

Mutations to PB2 and NP Proteins of an Avian Influenza Virus Combine To Confer Efficient Growth in Primary Human Respiratory Cells

Danzy

Studdard

Manicassamy

et al. 2014

Influenza pandemics occur when influenza A viruses (IAV) adapted to other host species enter humans and spread through the population. Pandemics are relatively rare due to host restriction of IAV: strains adapted to nonhuman species do not readily infect, replicate in, or transmit among humans. IAV can overcome host restriction through reassortment or adaptive evolution, and these are mechanisms by which pandemic strains arise in nature. To identify mutations that facilitate growth of avian IAV in humans, we have adapted influenza A/duck/Alberta/35/1976 (H1N1) (dk/AB/76) virus to a high-growth phenotype in differentiated human tracheo-bronchial epithelial (HTBE) cells. Following 10 serial passages of three independent lineages, the bulk populations showed similar growth in HTBE cells to that of a human seasonal virus. The coding changes present in six clonal isolates were determined. The majority of changes were located in the polymerase complex and nucleoprotein (NP), and all isolates carried mutations in the PB2 627 domain and regions of NP thought to interact with PB2. Using reverse genetics, the impact on growth and polymerase activity of individual and paired mutations in PB2 and NP was evaluated. The results indicate that coupling of the mammalian-adaptive mutation PB2 E627K or Q591K to selected mutations in NP further augments the growth of the corresponding viruses. In addition, minimal combinations of three (PB2 Q236H, E627K, and NP N309K) or two (PB2 Q591K and NP S50G) mutations were sufficient to recapitulate the efficient growth in HTBE cells of dk/AB/76 viruses isolated after 10 passages in this substrate. IMPORTANCEInfluenza A viruses adapted to birds do not typically grow well in humans. However, as has been seen recently with H5N1 and H7N9 subtype viruses, productive and virulent infection of humans with avian influenza viruses can occur. The ability of avian influenza viruses to adapt to new host species is a consequence of their high mutation rate that supports their zoonotic potential. Understanding of the adaptation of avian viruses to mammals strengthens public health efforts aimed at controlling influenza. In particular, it is critical to know how readily and through mutation to which functional components avian influenza viruses gain the ability to grow efficiently in humans. Our data show that as few as three mutations, in the PB2 and NP proteins, support robust growth of a low-pathogenic, H1N1 duck isolate in primary human respiratory cells.