Chi-Jene Chen

Anti-Influenza Drug Discovery: Structure-Activity Relationship and Mechanistic Insight into Novel Angelicin Derivatives

By using a cell-based high throughput screening campaign, a novel angelicin derivative 6a was identified to inhibit influenza A (H1N1) virus induced cytopathic effect in Madin-Darby canine kidney cell culture in low micromolar range. Detailed structure-activity relationship studies of 6a revealed that the angelicin scaffold is essential for activity in pharmacophore B, while meta-substituted phenyl/2-thiophene rings are optimal in pharmacophore A and C. The optimized lead 4-methyl-9-phenyl-8-(thiophene-2-carbonyl)-furo[2,3-h]chromen-2-one (8g, IC(50) = 70 nM) showed 64-fold enhanced activity compared to the high throughput screening (HTS) hit 6a. Also, 8g was found effective in case of influenza A (H3N2) and influenza B virus strains similar to approved anti-influenza drug zanamivir (4). Preliminary mechanistic studies suggest that these compounds act as anti-influenza agents by inhibiting ribonucleoprotein (RNP) complex associated activity and have the potential to be developed further, which could form the basis for developing additional defense against influenza pandemics.

Differential Localization and Function of PB1-F2 Derived from Different Strains of Influenza A Virus

ABSTRACT PB1-F2 is a viral protein that is encoded by the PB1 gene of influenza A virus by alternative translation. It varies in length and sequence context among different strains. The present study examines the functions of PB1-F2 proteins derived from various human and avian viruses. While H1N1 PB1-F2 was found to target mitochondria and enhance apoptosis, H5N1 PB1-F2, surprisingly, did not localize specifically to mitochondria and displayed no ability to enhance apoptosis. Introducing Leu into positions 69 (Q69L) and 75 (H75L) in the C terminus of H5N1 PB1-F2 drove 40.7% of the protein to localize to mitochondria compared with the level of mitochondrial localization of wild-type H5N1 PB1-F2, suggesting that a Leu-rich sequence in the C terminus is important for targeting of mitochondria. However, H5N1 PB1-F2 contributes to viral RNP activity, which is responsible for viral RNA replication. Lastly, although the swine-origin influenza virus (S-OIV) contained a truncated form of PB1-F2 (12 amino acids [aa]), potential mutation in the future may enable it to contain a full-length product. Therefore, the functions of this putative S-OIV PB1-F2 (87 aa) were also investigated. Although this PB1-F2 from the mutated S-OIV shares only 54% amino acid sequence identity with that of seasonal H1N1 virus, it also increased viral RNP activity. The plaque size and growth curve of the viruses with and without S-OIV PB1-F2 differed greatly. The PB1-F2 protein has various lengths, amino acid sequences, cellular localizations, and functions in different strains, which result in strain-specific pathogenicity. Such genetic and functional diversities make it flexible and adaptable in maintaining the optimal replication efficiency and virulence for various strains of influenza A virus.

Regulation Mechanisms of Viral IRES-Driven Translation

Internal ribosome entry sites (IRESs) can be found in the mRNA of many viruses as well as in cellular genes involved in the stress response, cell cycle, and apoptosis. IRES-mediated translation can occur when dominant cap-dependent translation is inhibited, and viruses can take advantage of this to subvert host translation machinery. In this review, we focus on the four major types of IRES identified in RNA viruses, and outline their distinct structural properties and requirements of translational factors. We further discuss auxiliary host factors known as IRES trans-acting factors (ITAFs), which are involved in the modulation of optimal IRES activity. Currently known strategies employed by viruses to harness ITAFs and regulate IRES activity are also highlighted.

Mechanism of action of the suppression of influenza virus replication by Ko-Ken Tang through inhibition of the phosphatidylinositol 3-kinase/Akt signaling pathway and viral RNP nuclear export

AIMS OF THE STUDY Ko-Ken Tang (KKT, aka kakkon-to), a conventional Chinese herbal medicine, has been used for the treatment of the common cold, fever and influenza virus infection. However, the underlying mechanism of its activity against influenza virus infection remains elusive. In this study, the antiviral effect and its underlying mechanism was evaluated, including the investigation of anti-influenza virus activity of KKT on MDCK cells and corresponding mechanism related to phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and its consecutive viral RNP nuclear export. MATERIALS AND METHODS The antiviral activity of non-toxic concentration of KKT was examined against various strains of influenza virus and enterovirus 71 by neutralization assay. PI3K/Akt signaling activated by influenza virus was inspected in A549 cells by western blot. Inhibition of influenza polymerase activity by KKT was measured with plasmid-based reverse genetics using primer extension assay and luciferase reporter assay. Inhibition of viral vRNP nuclear export was demonstrated by laser confocal microscopy and interspecies heterokaryon assay. RESULTS KKT inhibits influenza virus replication but not entry, and it exhibits a broad spectrum inhibitory activity against human influenza A viruses and enterovirus 71. KKT does not inhibit viral polymerase activity but directly blocks the virus-induced phosphatidylinositol 3-kinase/Akt signaling pathway, which in turns causes retention of viral nucleoprotein in the nucleus, thereby interfering with virus propagation. The inhibition by KKT of the nuclear export of viral protein was further confirmed by heterokaryon assay. CONCLUSIONS The results obtained in this study give scientific support to KKT for the treatment of influenza virus infection. KKT could be of potential use in the management of seasonal pandemic influenza virus infection in addition to other clinically available drugs.

Caspase-1 Deficient Mice Are More Susceptible to Influenza A Virus Infection With PA Variation

BACKGROUND Reassortment within polymerase genes causes changes in the pathogenicity of influenza A viruses. We previously reported that the 2009 pH1N1 PA enhanced the pathogenicity of seasonal H1N1. We examined the effects of the PA gene from the HPAI H5N1 following its introduction into currently circulating seasonal influenza viruses. METHODS To evaluate the role of H5N1 PA in altering the virulence of seasonal influenza viruses, we generated a recombinant seasonal H3N2 (3446) that expressed the H5N1 PA protein (VPA) and evaluated the RNP activity, growth kinetics, and pathogenicity of the reassortant virus in mice. RESULTS Compared with the wild-type 3446 virus, the substitution of the H5N1 PA gene into the 3446 virus (VPA/3446) resulted in increased RNP activity and an increased replication rate in A549 cells. The recombinant VPA/3446 virus also caused more severe pneumonia in Casp 1(-/-) mice than in IL1β(-/-) and wild-type B6 mice. CONCLUSIONS Although the PA from H5N1 is incidentally compatible with a seasonal H3N2 backbone, the H5N1 PA affected the virulence of seasonal H3N2, particularly in inflammasome-related innate immunity deficient mice. These findings highlight the importance of monitoring PA reassortment in seasonal flu, and confirm the role of the Caspase-1 gene in influenza pathogenesis.

Computational analysis and mapping of novel open reading frames in influenza A viruses.

The influenza A virus contains 8 segmented genomic RNAs and was considered to encode 10 viral proteins until investigators identified the 11th viral protein, PB1-F2, which uses an alternative reading frame of the PB1 gene. The recently identified PB1-N40, PA-N155 and PA-N182 influenza A proteins have shown the potential for using a leaking ribosomal scanning mechanism to generate novel open reading frames (ORFs). These novel ORFs provide examples of the manner in which the influenza A virus expands its coding capacity by using overlapping reading frames. In this study, we performed a computational search, based on a ribosome scanning mechanism, on all influenza A coding sequences to identify possible forward-reading ORFs that could be translated into novel viral proteins. We specified that the translated products had a prevalence ≥5% to eliminate sporadic ORFs. A total of 1,982 ORFs were thus identified and presented in terms of their locations, lengths and Kozak sequence strengths. We further provided an abridged list of ORFs by requiring every candidate an upstream start codon (within the upstream third of the primary transcript), a strong Kozak consensus sequence and high prevalence (≥95% and ≥50% for in-frame and alternative-frame ORFs, respectively). The PB1-F2, PB1-N40, PA-N155 and PA-N182 proteins all fulfilled our filtering criteria. Subject to these three stringent settings, we additionally named 16 novel ORFs for all influenza A genomes except for HA and NA, for which 43 HA and 11 NA ORFs from their respective subtypes were also recognized.

Altered Pathogenicity for Seasonal Influenza Virus by Single Reassortment of the RNP Genes Derived From the 2009 Pandemic Influenza Virus

BACKGROUND The 2009 influenza A pandemic virus (H1N1(pdm)) may reassort with old seasonal influenza A virus (H1N1₁₄₁) in humans and potentially change their pathogenicity. METHODS AND RESULTS This study focuses on the reassortment of ribonucleoproteins (RNPs) among H1N1(pdm) and seasonal influenza A viruses. A single RNP gene reassortment altered reporter gene expression levels driven by polymerase complex in transfection system. The growth rates of recombinant viruses with different RNP recombinations were changed in A549 cells. Mice were infected with recombinant viruses containing single RNP gene reassortment, and pathogenicity was examined. The results demonstrated that the median lethal dose (LD₅₀) of the PB2₁₄₁/PB1₁₄₁/PA(pdm)/NP₁₄₁ recombinant virus was lower than that of the seasonal H1N1 virus. Viral titers of this reassorted virus in the lung and spleen were significantly higher than that in seasonal H1N1 virus-challenged mice. CONCLUSIONS Although the changes of RNP activity did not exactly reflect to mice virulence, we consistently observed that the PA gene of H1N1(pdm) results in increased polymerase activity, better replication in mice, and lower LD₅₀. Our findings suggest that monitoring of gene reassortment for the 2009 pandemic influenza and seasonal human viruses is also important, which would help to constrain the potential emergence of a more virulent influenza A variant.

Cellular hnRNP A2/B1 interacts with the NP of influenza A virus and impacts viral replication

The viral ribonucleoprotein (vRNP) of influenza A virus is formed by virion RNA (vRNA), viral polymerase complex, and nucleoprotein (NP). The NP plays an important role in facilitating the replication and stabilization of viral RNA. To explore host factors that may be involved in the regulation of viral replication through interactions with NP, we conducted an immunoprecipitation experiment followed by mass spectrometry to identify NP-associated cellular proteins. Here, we demonstrate that NP can interact and colocalize with heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 in mammalian cells and that the interaction may occur via direct binding to the glycine-rich domain (GRD) of hnRNP A2/B1. In addition, two residues in the tail loop of NP, F412 and R422, are required for the interaction of hnRNP A2/B1. Because the knockdown of hnRNP A2/B1 expression reduces viral RNP activity, hnRNP A2/B1 may act as a positive regulator in viral RNA synthesis of influenza A virus. More importantly, the findings in this research demonstrate that host proteins can regulate the replication of influenza A virus by interacting with NP.

Interactome Analysis of NS1 Protein Encoded by Influenza A H7N9 Virus Reveals an Inhibitory Role of NS1 in Host mRNA Maturation

Influenza A virus infections can result in severe respiratory diseases. The H7N9 subtype of avian influenza A virus has been transmitted to humans and caused severe disease and death. Nonstructural protein 1 (NS1) of influenza A virus is a virulence determinant during viral infection. To elucidate the functions of the NS1 encoded by influenza A H7N9 virus (H7N9 NS1), interaction partners of H7N9 NS1 in human cells were identified with immunoprecipitation followed by SDS-PAGE coupled with liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). We identified 36 cellular proteins as the interacting partners of the H7N9 NS1, and they are involved in RNA processing, mRNA splicing via spliceosome, and the mRNA surveillance pathway. Two of the interacting partners, cleavage and polyadenylation specificity factor subunit 2 (CPSF2) and CPSF7, were confirmed to interact with H7N9 NS1 using coimmunoprecipitation and immunoblotting based on the previous finding that the two proteins are involved in pre-mRNA polyadenylation machinery. Furthermore, we illustrate that overexpression of H7N9 NS1, as well as infection by the influenza A H7N9 virus, interfered with pre-mRNA polyadenylation in host cells. This study comprehensively profiled the interactome of H7N9 NS1 in host cells, and the results demonstrate a novel endotype for H7N9 NS1 in inhibiting host mRNA maturation.

Evolutionarily conserved residues at an oligomerization interface of the influenza A virus neuraminidase are essential for viral survival.

Neuraminidase (NA) is a homotetramer viral surface glycoprotein that is essential for virus release during influenza virus infections. Previous studies have not explored why influenza NA forms a tetramer when the bacterial monomer NA already exhibits excellent NA enzymatic activity levels. In this study, we focused on 28 highly conserved residues among all NA subtypes, identifying 21 of 28 positions as crucial residues for viral survival by using reverse genetics. Maintaining NA enzymatic activity levels is critical and numerous conserved residues were located at the oligomerization interface; however, these mutations did not affect NA enzymatic activity levels or NA cellular localization, but rather affected the stability of NA oligomerization, suggesting that the oligomerization of NA is essential for viral viability. An increased understanding of the biological functions of NA, in particular NA oligomerization, could facilitate an alternative design for antivirals to combat influenza virus infections.