Targeting of the influenza A virus polymerase PB1-PB2 interface indicates strain-specific assembly differences

Assembly of the heterotrimeric influenza virus polymerase complex from the individual subunits PB1, PA, and PB2 is a prerequisite for viral replication. The conserved protein-protein interaction sites have been suggested as potential drug targets. To characterize the PB1-PB2 interface, we fused the PB1-binding domain of PB2 to green fluorescent protein (PB2(1-37)-GFP) and determined its competitive inhibitory effect on the polymerase activity of influenza A virus strains. Coexpression of PB2(1-37)-GFP in a polymerase reconstitution system led to substantial inhibition of the polymerase of A/WSN/33 (H1N1). Surprisingly, polymerases of other strains, including A/SC35M (H7N7), A/Puerto Rico/8/34 (H1N1), A/Hamburg/4/2009 (H1N1), and A/Thailand/1(KAN-1)/2004 (H5N1), showed various degrees of resistance. Individual exchange of polymerase subunits and the nucleoprotein between the sensitive WSN polymerase and the insensitive SC35M polymerase mapped the resistance to both PB1 and PA of SC35M polymerase. While PB2(1-37)-GFP bound equally well to the PB1 subunits of both virus strains, PB1-PA dimers of SC35M polymerase showed impaired binding compared to PB1-PA dimers of WSN polymerase. The use of PA(SC35M/WSN) chimeras revealed that the reduced affinity of the SC35M PB1-PA dimer was mediated by the N-terminal 277 amino acids of PA. Based on these observations, we speculate that the PB1-PA dimer formation of resistant polymerases shields the PB2(1-37) binding site, whereas sensitive polymerases allow this interaction, suggesting different assembly strategies of the trimeric polymerase complex between different influenza A virus strains.     

DNA Polymerase in Virions of a Reptilian Type C Virus

A study was made of the DNA polymerase of reptilian type C virus isolated from Russell's viper spleen cells. Simultaneous detection experiments demonstrated the presence of 70S RNA and RNA-dependent DNA polymerase activity in reptilian type C virions. The endogenous activity was dependent on the addition of all four deoxynucleotide triphosphates and demonstrated an absolute requirement for a divalent cation. The reptilian viral DNA polymerase elutes from phosphocellulose at 0.22 M salt. In this respect, it is similar to the avian (avian myeloblastosis virus; AMV) viral enzyme but is different from the mammalian (Rauscher leukemia virus; RLV) viral enzyme which elutes at 0.4 M salt. The molecular weight of the viper DNA polymerase as estimated from glycerol gradient centrifugation is 109,000. It is a smaller enzyme than the AMV DNA polymerase (180,000 daltons) and somewhat larger than the RLV enzyme (70,000 daltons). A comparison of other properties of the type C reptilian DNA polymerase with the enzyme found in other type C oncogenic viruses is made.     

Biochemical characterization of avian influenza viral polymerase containing PA or PB2 subunit from human influenza A virus

Adaptive mutations in viral polymerase, which is composed of PB1, PB2, and PA, of avian influenza viruses are major genetic determinants of the host range. In this study, to elucidate the molecular mechanism of mammalian adaptation of avian viral polymerase, we performed cell-based vRNP reconstitution assays and biochemical analyses using purified recombinant viral polymerase complexes. We found that avian viral polymerase from A/duck/Pennsylvania/10,218/84 (DkPen) enhances the viral polymerase activity in mammalian cells by replacing the PA or PB2 gene with that from human influenza virus A/WSN/33 (WSN). Chimeric constructs between DkPen PA and WSN PA showed that the N-terminal endonuclease domain of WSN PA was essential for the mammalian adaptation of DkPen viral polymerase. We also found that the cap-snatching activity of purified DkPen viral polymerase was more than 5 times weaker than that of WSN in vitro in a PB2 Glu627-dependent manner. However, the cap-snatching activity of DkPen viral polymerase was hardly increased by replacing DkPen PA to WSN PA. These results suggest that the activity of viral genome replication may be enhanced in the DkPen reassortant containing WSN PA.     

PA residues in the 2009 H1N1 pandemic influenza virus enhance avian influenza virus polymerase activity in mammalian cells

The 2009 pandemic influenza virus (pH1N1) is a swine-origin reassortant containing human, avian, and swine influenza genes. We have previously shown that the polymerase complex of the pH1N1 strain A/California/04/2009 (Cal) is highly active in mammalian 293T cells, despite the avian origin of both its PA and PB2. In this study, we analyzed the polymerase residues that are responsible for high pH1N1 polymerase activity in the mammalian host. Characterization of polymerase complexes containing various combinations of Cal and avian influenza virus A/chicken/Nanchang/3-120/01 (H3N2) (Nan) by reporter gene assay indicates that Cal PA, but not PB2, is a major contributing factor to high Cal polymerase activity in 293T cells. In particular, Cal PA significantly activates the otherwise inactive Nan polymerase at 37 and 39°C but not at the lower temperature of 34°C. Further analysis using site-directed mutagenesis showed that the Cal PA residues 85I, 186S, and 336M contribute to enhanced activity of the Cal polymerase. Recombinant A/WSN/33 (H1N1) (WSN) viruses containing Nan NP and polymerase (PA, PB1, PB2) genes with individual mutations in PA at residues 85, 186, and 336 produced higher levels of viral protein than the virus containing wild-type (WT) Nan PA. Interestingly, compared to the WT, the virus containing the 85I mutation grew faster in human A549 cells and the 336M mutation most significantly enhanced pathogenicity in a mouse model, among the three PA mutations tested. Our results suggest that multiple mutations in PA, which were rarely present in previous influenza isolates, are involved in mammalian adaptation and pathogenicity of the 2009 pH1N1.     

Serological Analysis of the Deoxyribonucleic Acid Polymerase of Avian Oncornaviruses II. Comparison of Avian Deoxyribonucleic Acid Polymerases

Monospecific antiserum prepared against the isolated deoxyribonucleic acid (DNA) polymerase of avian myeloblastosis virus (AMV) neutralized the endogenous ribonucleic acid-instructed DNA polymerase activity of detergent-disrupted virus. The viral polymerase was serologically unrelated to the seven major structural polypeptides of AMV. Furthermore, the viral enzyme was distinguished from normal cellular DNA polymerases by serological criteria; thus, antiserum against the viral enzyme neutralized its homologous antigen but not normal cellular DNA polymerases. Neutralization by antibody of viral DNA polymerase activity was observed with all avian leukemia-sarcoma viruses tested, irrespective of viral antigenic subtype. The DNA polymerase activity of avian reticuloendotheliosis virus, and of a variety of mammalian oncornaviruses, was not neutralized by antisera against the AMV polymerase. Immunological analysis of the RSValpha(O) mutant, which is deficient in DNA polymerase activity, shows this mutant to lack demonstrable polymerase antigen. Viral polymerase was identified by immunofluorescence as a cytoplasmic constituent in virus-producing chicken cells; polymerase antigen was not detected in uninfected (gs(-)) chicken cells.     

Full Factorial Analysis of Mammalian and Avian Influenza Polymerase Subunits Suggests a Role of an Efficient Polymerase for Virus Adaptation

Amongst all the internal gene segments (PB2. PB1, PA, NP, M and NS), the avian PB1 segment is the only one which was reassorted into the human H2N2 and H3N2 pandemic strains. This suggests that the reassortment of polymerase subunit genes between mammalian and avian influenza viruses might play roles for interspecies transmission. To test this hypothesis, we tested the compatibility between PB2, PB1, PA and NP derived from a H5N1 virus and a mammalian H1N1 virus. All 16 possible combinations of avian-mammalian chimeric viral ribonucleoproteins (vRNPs) were characterized. We showed that recombinant vRNPs with a mammalian PB2 and an avian PB1 had the strongest polymerase activities in human cells at all studied temperature. In addition, viruses with this specific PB2-PB1 combination could grow efficiently in cell cultures, especially at a high incubation temperature. These viruses were potent inducers of proinflammatory cytokines and chemokines in primary human macrophages and pneumocytes. Viruses with this specific PB2-PB1 combination were also found to be more capable to generate adaptive mutations under a new selection pressure. These results suggested that the viral polymerase activity might be relevant for the genesis of influenza viruses of human health concern.     

Mutations in polymerase genes enhanced the virulence of 2009 pandemic H1N1 influenza virus in mice

Influenza A virus can infect a wide variety of animal species with illness ranging from mild to severe, and is a continual cause for concern. Genetic mutations that occur either naturally or during viral adaptation in a poorly susceptible host are key mechanisms underlying the evolution and virulence of influenza A virus. Here, the variants containing PA-A36T or PB2-H357N observed in the mouse-adapted descendants of 2009 pandemic H1N1 virus (pH1N1), A/Sichuan/1/2009 (SC), were characterized. Both mutations enhanced polymerase activity in mammalian cells. These effects were confirmed using recombinant SC virus containing polymerase genes with wild type (WT) or mutant PA or PB2. The PA-A36T mutant showed enhanced growth property compared to the WT in both human A549 cells and porcine PK15 cells in vitro, without significant effect on viral propagation in murine LA-4 cells and pathogenicity in mice; however, it did enhance the lung virus titer. PB2-H357N variant demonstrated growth ability comparable to the WT in A549 cells, but replicated well in PK15, LA-4 cells and in mice with an enhanced pathogenic phenotype. Despite such mutations are rare in nature, they could be observed in avian H5 and H7 subtype viruses which were currently recognized to pose potential threat to human. Our findings indicated that pH1N1 may adapt well in mammals when acquiring these mutations. Therefore, future molecular epidemiological surveillance should include scrutiny of both markers because of their potential impact on pathogenesis.     

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

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.     

Additional Evidence That the Polymerase Subunits Contribute to the Viral Replication and the Virulence of H5N1 Avian Influenza Virus Isolates in Mice

Genetically similar H5N1 viruses circulating in the avian reservoir exhibit different levels of pathogenicity in mice. In this study, we characterized two highly pathogenic H5N1 avian isolates—A/Hunan/316/2005 (HN05), which is highly pathogenic in mice, and A/Hubei/489/2004 (HB04), which is nonpathogenic. In mammalian cells, HN05 replicates more efficiently than HB04, although both viruses have similar growth kinetics in avian cells. We used reverse genetics to generate recombinant H5N1 strains containing genes from HN05 and HB04 and examined their virulence. HN05 genes encoding the polymerase complex determine pathogenicity and viral replication ability both in vitro and in vivo. The PB2 subunit plays an important role in enhancing viral replication, and the PB1 and PA subunits contribute mainly to pathogenicity in mice. These results can be used to elucidate host-range expansion and the molecular basis of the high virulence of H5N1 viruses in mammalian species.     

构建基于杆状病毒的BV-T7杂合表达体系及利用其在哺乳动物和禽类细胞中表达eGFP基因

【目的】研究重组杆状病毒BV-T7杂合表达体系能否有效转导禽类细胞并在禽类细胞中表达外源基因(eGFP),从而构建能在禽类细胞中高效稳定表达外源基因的重组杆状病毒表达系统。【方法】本研究利用Bac-to-Bac杆状病毒表达系统,结合T7表达系统,通过对eGFP表达水平的调控来把握噬菌体T7 RNA聚合酶(T7 RNAP)的功能。利用两支重组杆状病毒,pFastBac-CMV-T7 RNAP重组杆状病毒为哺乳动物细胞启动子CMV调控的噬菌体T7 RNA聚合酶的cDNA;pFB-T7pro-IRES-GFP-T7ter重组杆状病毒为T7启动子控制的eGFP报告基因。将两支重组杆状病毒共同侵染哺乳动物OL(oligodendrocyte)细胞、鸡胚成纤维细胞和鸡胚骨骼肌细胞。【结果】两支重组杆状病毒利用T7启动子和T7 RNAP,在OL细胞、鸡胚成纤维细胞和鸡胚骨骼肌细胞中成功表达eGFP报告基因,而且未引起细胞病变,但在鸡胚原代细胞中eGFP的表达相对弱于在OL细胞中的表达。在OL细胞中重组杆状病毒对细胞的转导效率为59.5%,在鸡胚成纤维细胞和鸡胚骨骼肌细胞中转导效率分别为23.2%和33.1%。【结论】本研究构建的基于杆状病毒、T7RNA聚合酶、T7启动子(BV-T7)杂合表达体系能够在哺乳类细胞及禽类细胞中表达T7 RNAP,并利用T7RNAP继续高效而稳定地表达外源基因。这为难于体外操作的RNA病毒提供了有效的研究方法,并对新型基因工程疫苗的研制提供了一个高效而稳定的表达载体系统。     

Two active forms of RD-114 virus DNA polymerase in infected cells.

Two forms of DNA polymerase are present in RD-114-infected human, dog, and mink cells, but are not detectable in uninfected cells. The two enzymes are indistinguishable catalytically and immunologically, but differ with respect to molecular weight and elution position from (dT)12-18-cellulose and phosphocellulose. The large enzyme (equivalent 95,000 daltons) is found in the infected cells, but not the virions produced by these cells. The virions contain only the smaller enzyme (equivalent 70,000 daltons). The larger form may represent a mammalian viral equivalent to the beta subunit of avian RNA tumor virus DNA polymerase.     

H9N2 virus-derived M1 protein promotes H5N6 virus release in mammalian cells: Mechanism of avian influenza virus inter-species infection in humans

H5N6 highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4 not only exhibits unprecedented intercontinental spread in poultry, but can also cause serious infection in humans, posing a public health threat. Phylogenetic analyses show that 40% (8/20) of H5N6 viruses that infected humans carried H9N2 virus-derived internal genes. However, the precise contribution of H9N2 virus-derived internal genes to H5N6 virus infection in humans is unclear. Here, we report on the functional contribution of the H9N2 virus-derived matrix protein 1 (M1) to enhanced H5N6 virus replication capacity in mammalian cells. Unlike H5N1 virus-derived M1 protein, H9N2 virus-derived M1 protein showed high binding affinity for H5N6 hemagglutinin (HA) protein and increased viral progeny particle release in different mammalian cell lines. Human host factor, G protein subunit beta 1 (GNB1), exhibited strong binding to H9N2 virus-derived M1 protein to facilitate M1 transport to budding sites at the cell membrane. GNB1 knockdown inhibited the interaction between H9N2 virus-derived M1 and HA protein, and reduced influenza virus-like particles (VLPs) release. Our findings indicate that H9N2 virus-derived M1 protein promotes avian H5N6 influenza virus release from mammalian, in particular human cells, which could be a major viral factor for H5N6 virus cross-species infection.