Weinan Zheng

Assessment of the Internal Genes of Influenza A (H7N9) Virus Contributing to High Pathogenicity in Mice

ABSTRACT The recently identified H7N9 influenza A virus has caused severe economic losses and worldwide public concern. Genetic analysis indicates that its six internal genes all originated from H9N2 viruses. However, the H7N9 virus is more highly pathogenic in humans than H9N2, which suggests that the internal genes of H7N9 have mutated. To analyze which H7N9 virus internal genes contribute to its high pathogenicity, a series of reassortants was generated by reverse genetics, with each virus containing a single internal gene of the typical A/Anhui/1/2013 (H7N9) (AH-H7N9) virus in the genetic background of the A/chicken/Shandong/lx1023/2007 (H9N2) virus. The replication ability, polymerase activity, and pathogenicity of these viruses were then evaluated in vitro and in vivo. These recombinants displayed high genetic compatibility, and the H7N9-derived PB2, M, and NP genes were identified as the virulence genes for the reassortants in mice. Further investigation confirmed that the PB2 K627 residue is critical for the high pathogenicity of the H7N9 virus and the reassortant containing the H7N9-derived PB2 segment (H9N2-AH/PB2). Notably, the H7N9-derived PB2 gene displayed greater compatibility with the H9N2 genome than that of H7N9, endowing the H9N2-AH/PB2 reassortant with greater viability and virulence than the parental H7N9 virus. In addition, the H7N9 virus, with the exception of the H9N2 reassortants, could effectively replicate in human A549 cells. Our results indicate that PB2, M, and NP are the key virulence genes, together with the surface hemagglutinin (HA) and neuraminidase (NA) proteins, contributing to the high infectivity of the H7N9 virus in humans. IMPORTANCE To date, the novel H7N9 influenza A virus has caused 437 human infections, with approximately 30% mortality. Previous work has primarily focused on the two viral surface proteins, HA and NA, but the contribution of the six internal genes to the high pathogenicity of H7N9 has not been systematically studied. Here, the H9N2 virus was used as a genetic backbone to evaluate the virulence genes of H7N9 virus in vitro and in vivo. Our data indicate that the PB2, M, and NP genes play important roles in viral infection in mice and, together with HA and NA, contribute to the high infectivity of the H7N9 virus in humans.

Phosphorylation Controls the Nuclear-Cytoplasmic Shuttling of Influenza A Virus Nucleoprotein

ABSTRACT The nucleoprotein (NP) is a major component of the viral ribonucleoprotein (vRNP) complex. During the replication of influenza virus, the vRNP complex undergoes nuclear-cytoplasmic shuttling, during which NP serves as one of the determinants. To date, many phosphorylation sites on NP have been identified, but the biological functions of many of these phosphorylation sites remain unknown. In the present study, the functions of the phosphorylation sites S9, Y10, and Y296 were characterized. These residues are highly conserved, and their phosphorylation was essential for virus growth in cell culture and in a mouse model by regulating the activity of the viral polymerase and the nuclear-cytoplasmic shuttling of NP. The phosphorylation and dephosphorylation of S9 and Y10 controlled nuclear import of NP by affecting the binding affinity between NP and different isoforms of importin-α. In addition, the phosphorylation of Y296 caused nuclear retention of NP by reducing the interaction between NP and CRM1. Furthermore, tyrosine phosphorylation of NP during the early stage of virus infection was ablated when Y296 was mutated to F. However, at later stages of infection, it was weakened by the Y10F mutation. Taken together, the present data indicate that the phosphorylation and dephosphorylation of NP control the shuttling of NP between the nucleus and the cytoplasm during virus replication. IMPORTANCE It is well known that phosphorylation regulates the functions of viral proteins and the life cycle of influenza A virus. As NP is the most abundant protein in the vRNP complex of influenza A virus, several phosphorylation sites on this protein have been identified. However, the functions of these phosphorylation sites were unknown. The present study demonstrates that the phosphorylation status of these sites on NP can mediate its nuclear-cytoplasmic shuttling, which drives the trafficking of vRNP complexes in infected cells. The present data suggest that the phosphorylated residues of NP are multistep controllers of the virus life cycle and new targets for the design of anti-influenza drugs.

Cyclophilin A-regulated ubiquitination is critical for RIG-I-mediated antiviral immune responses

RIG-I is a key cytosolic pattern recognition receptor that interacts with MAVS to induce type I interferons (IFNs) against RNA virus infection. In this study, we found that cyclophilin A (CypA), a peptidyl-prolyl cis/trans isomerase, functioned as a critical positive regulator of RIG-I-mediated antiviral immune responses. Deficiency of CypA impaired RIG-I-mediated type I IFN production and promoted viral replication in human cells and mice. Upon Sendai virus infection, CypA increased the interaction between RIG-I and its E3 ubiquitin ligase TRIM25, leading to enhanced TRIM25-mediated K63-linked ubiquitination of RIG-I that facilitated recruitment of RIG-I to MAVS. In addition, CypA and TRIM25 competitively interacted with MAVS, thereby inhibiting TRIM25-induced K48-linked ubiquitination of MAVS. Taken together, our findings reveal an essential role of CypA in boosting RIG-I-mediated antiviral immune responses by controlling the ubiquitination of RIG-I and MAVS. DOI: http://dx.doi.org/10.7554/eLife.24425.001

Robust Lys63-Linked Ubiquitination of RIG-I Promotes Cytokine Eruption in Early Influenza B Virus Infection

ABSTRACT Influenza A and B virus infections both cause a host innate immunity response. Here, we report that the robust production of type I and III interferons (IFNs), IFN-stimulated genes, and proinflammatory factors can be induced by influenza B virus rather than influenza A virus infection in alveolar epithelial (A549) cells during early infection. This response is mainly dependent on the retinoic acid-inducible gene I (RIG-I)-mediated signaling pathway. Infection by influenza B virus promotes intense Lys63-linked ubiquitination of RIG-I, resulting in cytokine eruption. It is known that the influenza A virus NS1 protein (NS1-A) interacts with RIG-I and TRIM25 to suppress the activation of RIG-I-mediated signaling. However, the present results indicate that the influenza B virus NS1 protein (NS1-B) is unable to interact with RIG-I but engages in the formation of a RIG-I/TRIM25/NS1-B ternary complex. Furthermore, we demonstrate that the N-terminal RNA-binding domain (RBD) of NS1-B is responsible for interaction with TRIM25 and that this interaction blocks the inhibitory effect of the NS1-B C-terminal effector domain (TED) on RIG-I ubiquitination. Our findings reveal a novel mechanism for the host cytokine response to influenza B virus infection through regulatory interplay between host and viral proteins. IMPORTANCE Influenza B virus generally causes local mild epidemics but is occasionally lethal to individuals. Existing studies describe the broad characteristics of influenza B virus epidemiology and pathology. However, to develop better prevention and treatments for the disease, determining the concrete molecular mechanisms of pathogenesis becomes pivotal to understand how the host reacts to the challenge of influenza B virus. Thus, we aimed to characterize the host innate immune response to influenza B virus infection. Here, we show that vigorous Lys63-linked ubiquitination of RIG-I and cytokine eruption dependent on RIG-I-mediated signal transduction are induced by virus infection. Additionally, TRIM25 positively regulates RIG-I-mediated signaling by ablating the inhibitory function of NS1-B on RIG-I ubiquitination.

Threonine 80 phosphorylation of non-structural protein 1 regulates the replication of influenza A virus by reducing the binding affinity with RIG-I.

Influenza A virus evades host antiviral defense through hijacking innate immunity by its non‐structural protein 1 (NS1). By using mass spectrometry, threonine 80 (T80) was identified as a novel phosphorylated residue in the NS1 of the influenza virus A/WSN/1933(H1N1). By generating recombinant influenza viruses encoding NS1 T80 mutants, the roles of this phosphorylation site were characterized during viral replication. The T80E (phosphomimetic) mutant attenuated virus replication, whereas the T80A (non‐phosphorylatable) mutant did not. Similar phenotypes were observed for these mutants in a mouse model experiment. In further study, the T80E mutant decreased the binding capacity between NS1 and viral nucleoprotein (NP), leading to impaired viral ribonucleoprotein (vRNP)‐mediated viral transcription. The T80E mutant was also unable to inhibit interferon (IFN) production by reducing the binding affinity between NS1 and retinoic acid‐induced gene 1 protein (RIG‐I), causing attenuation of virus replication. Taken together, the present study reveals that T80 phosphorylation of NS1 reduced influenza virus replication through controlling RIG‐I‐mediated IFN production and vRNP activity.

Nucleocytoplasmic Shuttling of Influenza A Virus Proteins

Influenza viruses transcribe and replicate their genomes in the nuclei of infected host cells. The viral ribonucleoprotein (vRNP) complex of influenza virus is the essential genetic unit of the virus. The viral proteins play important roles in multiple processes, including virus structural maintenance, mediating nucleocytoplasmic shuttling of the vRNP complex, virus particle assembly, and budding. Nucleocytoplasmic shuttling of viral proteins occurs throughout the entire virus life cycle. This review mainly focuses on matrix protein (M1), nucleoprotein (NP), nonstructural protein (NS1), and nuclear export protein (NEP), summarizing the mechanisms of their nucleocytoplasmic shuttling and the regulation of virus replication through their phosphorylation to further understand the regulation of nucleocytoplasmic shuttling in host adaptation of the viruses.

Cyclophilin A protects mice against infection by influenza A virus

Our previous studies indicate that Cyclophilin A (CypA) impairs the replication of influenza A virus in vitro. To further evaluate the antiviral functions of CypA and explore its mechanism, transgenic mice with overexpression of CypA by two specific promoters with SPC (CypA-SPC) or CMV (CypA-CMV) were developed. After challenge with the A/WSN/33(H1N1) influenza virus, CypA-SPC and CypA-CMV transgenic mice displayed nearly 2.5- and 3.8-fold stronger disease resistance to virus infection, respectively, compared to wild-type animals. Virus replication, pathological lesions and inflammatory cytokines were substantially reduced in both lines of transgenic mice. In addition, after infection there was an upregulation of genes associated with cell migration, immune function, and organ development; and a downregulation of genes associated with the positive regulation of immune cells and apoptosis in the peritoneal macrophages of CypA-overexpressing transgenic mice (CypA+). These results indicate that CypA is a key modulator of influenza virus resistance in mice, and that CypA+ mice constitutes an important model to study the roles of CypA in the regulation of immune responses and infections.

Differential nucleocytoplasmic shuttling of the nucleoprotein of influenza a viruses and association with host tropism.

The nucleoprotein (NP) of influenza A virus plays a crucial role in virus replication, infectivity, and host adaptation. As a major component of the viral ribonucleoprotein complexes (vRNP), NP initiates vRNP shuttling between the nucleus and cytoplasm in the host cell. However, the characteristics of the nucleocytoplasmic shuttling of NP from H1N1 influenza A virus still remain unclear. In the present study, the subcellular localization and the related key residues of the H1N1 influenza virus NP were identified and evaluated. The NP of influenza virus A/WSN/33 (H1N1; WSN) displayed a more obvious nuclear accumulation than A/Anhui/1/2013 (H7N9; AH) and A/chicken/Shandong/lx1023/2007 (H9N2; SD). NP residue K4, located in NLS1, and residue F253, located in NES3, from WSN NP are not conserved in H7N9 and H9N2, which instead encode Q4 and I253, respectively. Crucially, these residues are involved in the regulation of NP nucleocytoplasmic shuttling through interactions with CRM1 and importin‐α. Moreover, residues at position 253 also play important roles in the replication of the virus, resulting in an increase in vRNP polymerase activity and an alteration of the cell tropism and pathogenicity in mice. The present data revealed a pivotal role of the Q4 and I253 residues of NP from H7N9 in enhancing the cytoplasmic accumulation of NP and vRNP activity compared to the K4 and F253 residues in WSN‐NP. In addition, an F253I substitution in the NP of WSN altered the survival ratio of infected mice and the growth curve in infected avian‐origin cells (DF‐1). The current data indicate that the F253I mutation results in attenuated pathogenicity of the virus in mice and altered cell tropism. The present study demonstrated the dissimilarity in subcellular NP transport processes between H1N1 virus WSN and other influenza A virus strains, as well as uncovered the mechanism responsible for this difference.

Phosphorylation and dephosphorylation of threonine 188 in nucleoprotein is crucial for the replication of influenza A virus

Nucleoprotein (NP) is a major component of the viral ribonucleoprotein (vRNP) complex that is responsible for viral replication, transcription and packaging of influenza A virus. Phosphorylation of NP plays an important role during viral infection. In the present study, we identified threonine 188 (T188) as a novel phosphorylated residue in the NP of influenza A virus by using mass spectrometry. T188 is located within nuclear export signal 2 (NES2) which is chromosome region maintenance 1 (CRM1)-independent. We observed that the phosphorylation and dephosphorylation of residue T188 regulated viral replication by controlling NES2-dependent NP nuclear export and the polymerase activity of the vRNP complex. Our findings provide further insights for understanding the replication of influenza A virus.

CDC25B promotes influenza A virus replication by regulating the phosphorylation of nucleoprotein

Cell division cycle 25 B (CDC25B) is a member of the CDC25 phosphatase family. It can dephosphorylate cyclin-dependent kinases and regulate the cell division cycle. Moreover, siRNA knockdown of CDC25B impairs influenza A virus (IAV) replication. Here, to further understand the regulatory mechanism of CDC25B for IAV replication, a CDC25B-knockout (KO) 293T cell line was constructed using CRISPR/Cas9. The present data indicated that the replication of IAV was decreased in CDC25B-KO cells. Additionally, CDC25B deficiency damaged viral polymerase activity, nucleoprotein (NP) self-oligomerization, and NP nuclear export. Most importantly, we found that the NP phosphorylation levels were significantly increased in CDC25B-KO cells. These findings indicate that CDC25B facilitates the dephosphorylation of NP, which is vital for regulating NP functions and the life cycle of IAV.