BAG6 inhibits influenza A virus replication by inducing viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly.

The interaction between influenza A virus (IAV) and host proteins is an important process that greatly influences viral replication and pathogenicity. PB2 protein is a subunit of viral ribonucleoprotein (vRNP) complex playing distinct roles in viral transcription and replication. BAG6 (BCL2-associated athanogene 6) as a multifunctional host protein participates in physiological and pathological processes. Here, we identify BAG6 as a new restriction factor for IAV replication through targeting PB2. For both avian and human influenza viruses, overexpression of BAG6 reduced viral protein expression and virus titers, whereas deletion of BAG6 significantly enhanced virus replication. Moreover, BAG6-knockdown mice developed more severe clinical symptoms and higher viral loads upon IAV infection. Mechanistically, BAG6 restricted IAV transcription and replication by inhibiting the activity of viral RNA-dependent RNA polymerase (RdRp). The co-immunoprecipitation assays showed BAG6 specifically interacted with the N-terminus of PB2 and competed with PB1 for RdRp complex assembly. The ubiquitination assay indicated that BAG6 promoted PB2 ubiquitination at K189 residue and targeted PB2 for K48-linked ubiquitination degradation. The antiviral effect of BAG6 necessitated its N-terminal region containing a ubiquitin-like (UBL) domain (17-92aa) and a PB2-binding domain (124-186aa), which are synergistically responsible for viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly. These findings unravel a novel antiviral mechanism via the interaction of viral PB2 and host protein BAG6 during avian or human influenza virus infection and highlight a potential application of BAG6 for antiviral drug development.

Genetic and Biological Characteristics of Duck-Origin H4N6 Avian Influenza Virus Isolated in China in 2022.

The interaction between migratory birds and domestic waterfowl facilitates viral co-infections, leading to viral reassortment and the emergence of novel viruses. In 2022, samples were collected from duck farms around Poyang Lake in Jiangxi Province, China, which is located within the East Asia-Australasia flyway. Three strains of H4N6 avian influenza virus (AIV) were isolated. Genetic and phylogenetic analyses showed that the isolated H4N6 avian influenza viruses (AIVs) belonged to new genotypes, G23 and G24. All isolated strains demonstrated dual receptor binding properties. Additionally, the isolated strains were able to replicate efficiently not only in avian cells but also in mammalian cells. Furthermore, the H4N6 AIV isolates could infect chickens, with viral replication detected in the lungs and extrapulmonary organs, and could transmit within chicken flocks through contact, with viral shedding detected only in oropharyngeal swabs from chickens in the contact group. Notably, the H4N6 AIV could infect mice without prior adaptation and replicate in the lungs with high viral titers, suggesting that it is a potential threat to humans. In conclusion, this study provides valuable insight into the characteristics of H4N6 strains currently circulating in China.

Influence of Host Sialic Acid Receptors Structure on the Host Specificity of Influenza Viruses.

Influenza viruses need to use sialic acid receptors to invade host cells, and the α-2,3 and α-2,6 sialic acids glycosidic bonds linking the terminal sialic acids are generally considered to be the most important factors influencing the cross-species transmission of the influenza viruses. The development of methods to detect the binding of influenza virus HA proteins to sialic acid receptors, as well as the development of glycobiological techniques, has led to a richer understanding of the structure of the sialylated glycan in influenza virus hosts. It was found that, in addition to the sialic acid glycosidic bond, sialic acid variants, length of the sialylated glycan, Gal-GlcNAc-linked glycosidic bond within the sialylated glycan, and sulfation/fucosylation of the GlcNAc within the sialylated glycan all affect the binding properties of influenza viruses to the sialic acid receptors, thus indirectly affecting the host specificity of influenza viruses. This paper will review the sialic acid variants, internal structural differences of sialylated glycan molecules that affect the host specificity of influenza viruses, and distribution characteristics of sialic acid receptors in influenza virus hosts, in order to provide a more reliable theoretical basis for the in-depth investigation of cross-species transmission of influenza viruses and the development of new antiviral drugs.

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.

Molecular characterization and antiapoptotic function analysis of the duck plague virus Us5 gene.

Thus far, there have been no reports on the molecular characterization and antiapoptotic function of the DPV Us5 gene. To perform molecular characterization of DPV Us5, RT-PCR and pharmacological inhibition tests were used to ascertain the kinetic class of the Us5 gene. Western blotting and an indirect immunofluorescence assay (IFA) were used to analyze the expression level and subcellular localization of Us5 in infected cells at different time points. Us5 in purified DPV virions was identified by mass spectrometry. The results of RT-PCR, Western blotting, and pharmacological inhibition tests revealed that Us5 is transcribed mainly in the late stage of viral replication. The IFA results revealed that Us5 was localized throughout DPV-infected cells but was localized only to the cytoplasm of transfected cells. Mass spectrometry and Western blot analysis showed that Us5 was a virion component. Next, to study the antiapoptotic function of DPV Us5, we found that DPV CHv without gJ could induce more apoptosis cells than DPV-CHv BAC and rescue virus. we constructed a model of apoptosis in duck embryo fibroblasts (DEFs) induced by hydrogen peroxide (H2O2). Transfected cells expressing the Us5 gene were protected from apoptosis induced by H2O2, as measured by a TUNEL assay, a caspase activation assay and Flow Cytometry assay. The TUNEL assay and Flow Cytometry assay results showed that the recombinant plasmid pCAGGS-Us5 could inhibit apoptosis induced by H2O2 in DEF cells. However, caspase-3/7 and caspase-9 protein activity upregulated by H2O2 was significantly reduced in cells expressing the recombinant plasmid pCAGGS-Us5. Overall, these results show that the DPV Us5 gene is a late gene and that the Us5 protein is a component of the virion, is localized in the cytoplasm, and can inhibit apoptosis induced by H2O2 in DEF cells.

Duck Plague Virus Promotes DEF Cell Apoptosis by Activating Caspases, Increasing Intracellular ROS Levels and Inducing Cell Cycle S-Phase Arrest.

BACKGROUND: Duck plague virus (DPV) can induce apoptosis in duck embryo fibroblasts (DEFs) and in infected ducks, but the molecular mechanism of DPV-induced apoptosis remains unknown. METHODS: We first used qRT-PCR and a Caspase-Glo assay to determine whether the caspase protein family plays an important role in DPV-induced apoptosis. Then, we used an intracellular ROS detection kit and the mitochondrial probe JC-1 to respectively detect ROS levels and mitochondrial membrane potential (MMP). Finally, flow cytometry was used to detect apoptosis and cell cycle progression. RESULTS: In this study, the mRNA levels and enzymatic activities of caspase-3, caspase-7, caspase-8, and caspase-9 were significantly increased during DPV-induced apoptosis. The caspase inhibitors Z-DEVD-FMK, Z-LEHD-FMK, and Q-VD-OphA could inhibit DPV-induced apoptosis and promote viral replication. Subsequently, a significant decrease in MMP and an increase in the intracellular ROS levels were observed. Further study showed that pretreating infected cells with NAC (a ROS scavenger) decreased the intracellular ROS levels, increased the MMP, inhibited apoptosis, and promoted viral replication. Finally, we showed that DPV infection can cause cell cycle S-phase arrest. CONCLUSIONS: This study shows that DPV causes cell cycle S-phase arrest and leads to apoptosis through caspase activation and increased intracellular ROS levels. These findings may be useful for gaining an understanding of the pathogenesis of DPV and the apoptotic pathways induced by α-herpesviruses.

Programmed cell death: the battlefield between the host and alpha-herpesviruses and a potential avenue for cancer treatment.

Programed cell death is an antiviral mechanism by which the host limits viral replication and protects uninfected cells. Many viruses encode proteins resistant to programed cell death to escape the host immune defenses, which indicates that programed cell death is more favorable for the host immune defense. Alpha-herpesviruses are pathogens that widely affect the health of humans and animals in different communities worldwide. Alpha-herpesviruses can induce apoptosis, autophagy and necroptosis through different molecular mechanisms. This review concisely illustrates the different pathways of apoptosis, autophagy, and necroptosis induced by alpha-herpesviruses. These pathways influence viral infection and replication and are a potential avenue for cancer treatment. This review will increase our understanding of the role of programed cell death in the host immune defense and provides new possibilities for cancer treatment.