Species difference in ANP32A underlies influenza A virus polymerase host restriction.

Influenza pandemics occur unpredictably when zoonotic influenza viruses with novel antigenicity acquire the ability to transmit amongst humans. Host range breaches are limited by incompatibilities between avian virus components and the human host. Barriers include receptor preference, virion stability and poor activity of the avian virus RNA-dependent RNA polymerase in human cells. Mutants of the heterotrimeric viral polymerase components, particularly PB2 protein, are selected during mammalian adaptation, but their mode of action is unknown. We show that a species-specific difference in host protein ANP32A accounts for the suboptimal function of avian virus polymerase in mammalian cells. Avian ANP32A possesses an additional 33 amino acids between the leucine-rich repeats and carboxy-terminal low-complexity acidic region domains. In mammalian cells, avian ANP32A rescued the suboptimal function of avian virus polymerase to levels similar to mammalian-adapted polymerase. Deletion of the avian-specific sequence from chicken ANP32A abrogated this activity, whereas its insertion into human ANP32A, or closely related ANP32B, supported avian virus polymerase function. Substitutions, such as PB2(E627K), were rapidly selected upon infection of humans with avian H5N1 or H7N9 influenza viruses, adapting the viral polymerase for the shorter mammalian ANP32A. Thus ANP32A represents an essential host partner co-opted to support influenza virus replication and is a candidate host target for novel antivirals

Identification of a PA-Binding Peptide with Inhibitory Activity against Influenza A and B Virus Replication

There is an urgent need for new drugs against influenza type A and B viruses due to incomplete protection by vaccines and the emergence of resistance to current antivirals. The influenza virus polymerase complex, consisting of the PB1, PB2 and PA subunits, represents a promising target for the development of new drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between the PB1 and PA subunits of the polymerase complex of influenza A virus using a small peptide derived from the PA-binding domain of PB1. However, this influenza A virus-derived peptide did not affect influenza B virus polymerase activity. Here we report that the PA-binding domain of the polymerase subunit PB1 of influenza A and B viruses is highly conserved and that mutual amino acid exchange shows that they cannot be functionally exchanged with each other. Based on phylogenetic analysis and a novel biochemical ELISA-based screening approach, we were able to identify an influenza A-derived peptide with a single influenza B-specific amino acid substitution which efficiently binds to PA of both virus types. This dual-binding peptide blocked the viral polymerase activity and growth of both virus types. Our findings provide proof of principle that protein-protein interaction inhibitors can be generated against influenza A and B viruses. Furthermore, this dual-binding peptide, combined with our novel screening method, is a promising platform to identify new antiviral lead compounds

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

Identification of a strong enhancer element upstream from the pregenomic RNA start site of the duck hepatitis B virus genome.

The genome of the duck hepatitis B virus (DHBV) contains an enhancer element. This sequence, of 192 bp, is located in the 3'-terminal coding region of the DNA polymerase gene (nucleotides 2159 to 2351), upstream from the pregenomic RNA start site. This enhancer potentiates a marked increased activity from the heterologous thymidine kinase promoter in an orientation-independent manner and at a proximal, as well as a distal, location. The DHBV enhancer activates transcription in a relatively cell-type-independent manner. Sequence homologies with the nuclear factor EF-C binding site are located in the DHBV enhancer. By using the HepG2 nuclear extracts and the DHBV enhancer as probes, a complex was observed in mobility shift assays

Binding of Nuclear Factors to Functional Domains of the Duck Hepatitis B Virus Enhancer

International audienceWe have analyzed the structures, relative organization, and activities of binding sites for nuclear factors in the duck hepatitis B virus (duck HBV) enhancer. DNase I footprinting analysis and mobility shift assays demonstrate that this enhancer of 192 bp contains at least three binding sites for transcription factors: one for hepatocyte-adipocyte C/EBP, a second for the liver-specific transactivator hepatocyte nuclear factor 1 HNF-1, and a third for a factor, called F3, which binds to a DNA sequence bearing some resemblance to that for the ubiquitous factor EF-C. Analysis of transcriptional activity reveals that oligonucleotides corresponding to the individual binding sites, inserted upstream from a heterologous promoter, display very weak enhancer activity, whereas the enhancer encompassing these three sites displays very high activity. Analysis of duck HBV enhancer mutants indicates that the deletion of any of these sites leads to a modification of transcriptional enhancer activity. The hepatocyte nuclear factor 1 binding site is crucial, since an internal deletion of 14 bp abolishes the activity. The C/EBP site can act as repressor, and the F3 site is required for full activity. Comparative analysis reveals that the nuclear factors are similar to those bound to the human HBV enhancer but that the organization of their binding sites in the duck HBV enhancer is different

Incorporation of the influenza A virus NA segment into virions does not require cognate non-coding sequences.

&lt;p&gt;For each influenza virus genome segment, the coding sequence is flanked by non-coding (NC) regions comprising shared, conserved sequences and specific, non-conserved sequences. The latter and adjacent parts of the coding sequence are involved in genome packaging, but the precise role of the non-conserved NC sequences is still unclear. The aim of this study is to better understand the role of the non-conserved non-coding sequences in the incorporation of the viral segments into virions. The NA-segment NC sequences were systematically replaced by those of the seven other segments. Recombinant viruses harbouring two segments with identical NC sequences were successfully rescued. Virus growth kinetics and serial passages were performed, and incorporation of the viral segments was tested by real-time RT-PCR. An initial virus growth deficiency correlated to a specific defect in NA segment incorporation. Upon serial passages, growth properties were restored. Sequencing revealed that the replacing 5&amp;#39;NC sequence length drove the type of mutations obtained. With sequences longer than the original, point mutations in the coding region with or without substitutions in the 3&amp;#39;NC region were detected. With shorter sequences, insertions were observed in the 5&amp;#39;NC region. Restoration of viral fitness was linked to restoration of the NA segment incorporation.&lt;/p&gt;</p

Chimeric NP Non Coding Regions between Type A and C Influenza Viruses Reveal Their Role in Translation Regulation

International audienceExchange of the non coding regions of the NP segment between type A and C influenza viruses was used to demonstrate the importance not only of the proximal panhandle, but also of the initial distal panhandle strength in type specificity. Both elements were found to be compulsory to rescue infectious virus by reverse genetics systems. Interestingly, in type A influenza virus infectious context, the length of the NP segment 5' NC region once transcribed into mRNA was found to impact its translation, and the level of produced NP protein consequently affected the level of viral genome replication

The Panhandle formed by influenza A and C virus NS non-coding regions determines NS segment expression.

&lt;p&gt;Exchange of the extremities of the NS segment of type A and C influenza viruses in reverse genetics systems was used to assess their putative role in type specificity. Restoration of each specific proximal panhandle was mandatory to allow the rescue of viruses with heterotypic extremities. Moreover, the transcription level of the modified segment seemed to be directly affected by the distal panhandle strength.&lt;/p&gt;</p