Avian Influenza A Virus Polymerase Association with Nucleoprotein, but Not Polymerase Assembly, Is Impaired in Human Cells during the Course of Infection

Abstract

Strong determinants of the host range of influenza A viruses have been identified on the polymerase complex formed by the PB1, PB2, and PA subunits and on the nucleoprotein (NP). In the present study, molecular mechanisms that may involve these four core proteins and contribute to the restriction of avian influenza virus multiplication in human cells have been investigated. The efficiencies with which the polymerase complexes of a human and an avian influenza virus isolate assemble and interact with the viral NP and cellular RNA polymerase II proteins were compared in mammalian and in avian infected cells. To this end, recombinant influenza viruses expressing either human or avian-derived core proteins with a PB2 protein fused to the One-Strep purification tag at the N or C terminus were generated. Copurification experiments performed on infected cell extracts indicate that the avian-derived polymerase is assembled and interacts physically with the cellular RNA polymerase II at least as efficiently as does the human-derived polymerase in human as well as in avian cells. Restricted growth of the avian isolate in human cells correlates with low levels of the core proteins in infected cell extracts and with poor association of the NP with the polymerase compared to what is observed for the human isolate. The NP-polymerase association is restored by a Glu-to-Lys substitution at residue 627 of PB2. Overall, our data point to viral and cellular factors regulating the NP-polymerase interaction as key determinants of influenza A virus host range. Recombinant viruses expressing a tagged polymerase should prove useful for further studies of the molecular interactions between viral polymerase and host factors during the infection cycle.

Adaptive strategies of the influenza virus polymerase for replication in humans

“…3). This recapitulates the restriction of avian viruses in human cells and is in agreement with other studies of viruses containing an avian polymerase (12,14). Virus with the SR polymorphism replicated 9-to 70-fold higher than WT S009 in A549 cells at the indicated time points.…”

“…Discrete mutations within the polymerase have also been associated with an extended host range. A notable example is the species-specific preference for lysine at PB2 position 627 resulting in high levels of viral replication and/or polymerase activity in animal models and human cells (5,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Other changes within PB2, such as the D701N mutation, are associated with increased host range, polymerase activity, and pathogenicity in mammalian systems and humans (33)(34)(35).…”

“…PB2 derived from human viral isolates almost exclusively possesses a lysine at position 627 (K627), whereas glutamic acid (E627) at this position predominates in avian viruses (6). PB2 K627 correlates with enhanced polymerase activity, virus replication, transmission, and in certain cases, pathogenicity and mortality in mammals (5,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). PB2 K627 also correlates with moderately enhanced influenza virus replication in pigs, consistent with pigs serving as an intermediary viral reservoir between birds and humans (17).…”

Virulence determinants of pandemic influenza viruses

“…In addition, H5N1 viruses with a PB2 E627K mutation have increased growth in the nasal passages of mice compared with the wild-type virus (81). The cold sensitivity of avian virus polymerases with PB2 627E may be the result of a disrupted interaction between PB2 and NP (86), the presence of a cellular restriction factor (87), or, conversely, the lack of a compatible host cofactor for polymerase activity (88). The amino acid at PB2 627 also affects the transmissibility of influenza viruses.…”

Reassortment and Mutation of the Avian Influenza Virus Polymerase PA Subunit Overcome Species Barriers

“…The origin of the polymerase genes can alter substantially the activity of the influenza virus polymerase in a host-specific fashion (1). Viruses with avian polymerases generally have reduced transmissibility in animal models, replicate at lower levels with reduced pathogenicity in mice and ferrets, and display low levels of polymerase activity in human cells (23,27,35,38,44,49). To overcome this natural restriction, avian viruses acquire mutations that lead to enhanced activity in mammalian hosts.…”