AMBRA1 promotes dsRNA- and virus-induced apoptosis through interacting with and stabilizing MAVS.

Apoptosis is an important cellular response to viral infection. In this study, we identified activating molecule in Beclin1-regulated autophagy protein 1 (AMBRA1) as a positive regulator of apoptosis triggered by double-stranded (ds)RNA. Depletion of AMBRA1 by gene editing significantly reduced dsRNA-induced apoptosis, which was largely restored by trans-complementation of AMBRA1. Mechanistically, AMBRA1 interacts with mitochondrial antiviral-signaling protein (MAVS), a key mitochondrial adaptor in the apoptosis pathway induced by dsRNA and viral infection. Further co-immunoprecipitation analysis demonstrated that the mitochondrial localization of MAVS was essential for their interaction. The impact of AMBRA1 on dsRNA-induced apoptosis relied on the presence of MAVS and caspase-8. AMBRA1 was involved in the stabilization of MAVS through preventing its dsRNA-induced proteasomal degradation. Consistently, AMBRA1 upregulated the apoptosis induced by Semliki Forest virus infection. Taken together, our work illustrated a role for AMBRA1 in virus-induced apoptosis through interacting with and stabilizing MAVS.

Inositol-Requiring Enzyme 1α Promotes Zika Virus Infection through Regulation of Stearoyl Coenzyme A Desaturase 1-Mediated Lipid Metabolism.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus which has become a global epidemic threat due to its rapid spread and association with serious consequences of infection, including neonatal microcephaly. Inositol-requiring enzyme 1α (IRE1α) is an endoplasmic reticulum (ER)-related transmembrane protein that mediates unfolded protein response (UPR) pathway and has been indicated to play an important role in flavivirus replication. However, the mechanism of how IRE1α affects ZIKV replication remains unknown. In this study, we explored the role of IRE1α in ZIKV infection in vitro and in vivo by using CRISPR/Cas9-based gene knockout and RNA interference-based gene knockdown techniques. Both knockout and knockdown of IRE1α dramatically reduced ZIKV replication levels, including viral RNA levels, protein expression, and titers in different human cell lines. Trans-complementation with IRE1α restored viral replication levels decreased by IRE1α depletion. Furthermore, the proviral effect of IRE1α was dependent on its kinase and RNase activities. Importantly, we found that IRE1α promoted the replication of ZIKV through upregulating the accumulation of monounsaturated fatty acid (MUFA) rate-limiting enzyme stearoyl coenzyme A (stearoyl-CoA) desaturase 1 (SCD1), which further affected the production of oleic acid (OA) and lipid droplet. Finally, our data demonstrated that in the brain tissues of ZIKV-infected mice, the replication levels of ZIKV and virus-related lesions were significantly suppressed by both the kinase and RNase inhibitors of IRE1α. Taken together, our results identified IRE1α as a ZIKV dependency factor which promotes viral replication through affecting SCD1-mediated lipid metabolism, potentially providing a novel molecular target for the development of anti-ZIKV agents.IMPORTANCE Zika virus (ZIKV) has been linked to serious neurologic disorders and causes widespread concern in the field of global public health. Inositol requiring enzyme 1α (IRE1α) is an ER-related transmembrane protein that mediates unfolded protein response (UPR) pathway. Here, we revealed that IRE1α is a proviral factor for ZIKV replication both in culture cells and mice model, which relies on its kinase and RNase activities. Importantly, we further provided evidence that upon ZIKV infection, IRE1α is activated and splices XBP1 mRNA which enhances the expression of monounsaturated fatty acids rate-limiting enzyme stearoyl coenzyme A (stearoyl-CoA) desaturase 1 (SCD1) and subsequent lipid droplet production. Our data uncover a novel mechanism of IRE1α proviral effect by modulating lipid metabolism, providing the first evidence of a close relationship between IRE1α-mediated UPR, lipid metabolism, and ZIKV replication and indicating IRE1α inhibitors as potentially effective anti-ZIKV agents.

Double-stranded RNA deaminase ADAR1 promotes the Zika virus replication by inhibiting the activation of protein kinase PKR.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged as a threat to global health. The family of adenosine deaminases acting on dsRNA (ADARs) are human host factors important for the genetic diversity and evolution of ZIKV. Here, we further investigated the role of ADAR1 in ZIKV replication by utilizing CRISPR/Cas9-based gene editing and RNAi-based gene knockdown techniques. Both ADAR1 knockout and knockdown significantly reduced ZIKV RNA synthesis, protein levels, and viral titers in several human cell lines. Trans-complementation with the full-length ADAR1 form p150 or the shorter form p110 lacking the Zα domain restored viral replication levels suppressed by the ADAR1 knockout. Moreover, we observed that the nuclear p110 form was redistributed to the cytoplasm in response to ZIKV infection. ADAR1 was not involved in viral entry but promoted viral protein translation by impairing ZIKV-induced activation of protein kinase regulated by dsRNA (PKR). Of note, the RNA-editing activity of ADAR1 was not required to promote ZIKV replication. We also found that the proviral role of ADAR1 was partially mediated through its ability to suppress IFN production and PKR activation. Our work identifies ADAR1 as a proviral factor involved in ZIKV replication, suggesting that ADAR1 could be a potential antiviral target.

RNA Helicase A Is an Important Host Factor Involved in Dengue Virus Replication.

Dengue virus (DENV) utilizes host factors throughout its life cycle. In this study, we identified RNA helicase A (RHA), a member of the DEAD/H helicase family, as an important host factor of DENV. In response to DENV2 infection, nuclear RHA protein was partially redistributed into the cytoplasm. The short interfering RNA-mediated knockdown of RHA significantly reduced the amounts of infectious viral particles in various cells. The RHA knockdown reduced the multistep viral growth of DENV2 and Japanese encephalitis virus but not Zika virus. Further study showed that the absence of RHA resulted in a reduction of both viral RNA and protein levels, and the data obtained from the reporter replicon assay indicated that RHA does not directly promote viral protein synthesis. RHA bound to the DENV RNA and associated with three nonstructural proteins, including NS1, NS2B3, and NS4B. Further study showed that different domains of RHA mediated its interaction with these viral proteins. The expression of RHA or RHA-K417R mutant protein lacking ATPase/helicase activity in RHA-knockdown cells successfully restored DENV2 replication levels, suggesting that the helicase activity of RHA is dispensable for its proviral effect. Overall, our work reveals that RHA is an important factor of DENV and might serve as a target for antiviral agents.IMPORTANCE Dengue, caused by dengue virus, is a rapidly spreading disease, and currently there are no treatments available. Host factors involved in the viral replication of dengue virus are potential antiviral therapeutic targets. Although RHA has been shown to promote the multiplication of several viruses, such as HIV and adenovirus, its role in the flavivirus family, including dengue virus, Japanese encephalitis virus, and emerging Zika virus, remains elusive. The current study revealed that RHA relocalized into the cytoplasm upon DENV infection and associated with viral RNA and nonstructural proteins, implying that RHA was actively engaged in the viral life cycle. We further provide evidence that RHA promoted the viral yields of DENV2 independent of its helicase activity. These findings demonstrated that RHA is a new host factor required for DENV replication and might serve as a target for antiviral drugs.

Melanoma differentiation-associated gene 5 is involved in the induction of stress granules and autophagy by protonophore CCCP.

The eukaryotic cell has evolved a variety of stress responses against external stimuli, such as innate immunity, the formation of stress granules (SGs), and autophagy. We previously demonstrated that the innate immune adaptor IFN-ß promoter stimulator 1 (IPS-1) plays an essential role in the formation of dsRNA-induced SGs, indicating a connection between SG formation and innate immunity. In this study, it was further demonstrated that melanoma differentiation-associated gene 5 (MDA5), an innate immune sensor, is involved in SG formation induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial protonophore. MDA5 knockdown had no significant impact on the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) triggered by CCCP, and MDA5 itself was not recruited to SGs, suggesting that the regulation of MDA5 in the SG response occurs downstream of eIF2α. Furthermore, the depletion of MDA5 or G3BP1 led to reduced autophagy in CCCP-stimulated cells, implying that the regulatory effect of MDA5 with respect to autophagy depends on its role in SG formation. This study uncovered an unexpected role of the innate immune protein MDA5 in SG formation and autophagy triggered by the protonophore CCCP, further supporting a correlation between different stress responses.

Characterization of the enterovirus 71 VP1 protein as a vaccine candidate.

Enterovirus 71 (EV71) is an important agent responsible for hand-foot-and-mouth disease (HFMD), which can cause severe neurological complications and death in children. However, there is no specific treatment for EV71 infection, and a safe and effective vaccine is needed urgently. In this study, an effective and economical method for the production of EV71-VP1 protein was developed, and the VP1 protein was evaluated in humoral and cellular immune responses as an EV71 vaccine. The results revealed that the VP1 protein induced high titers of cross-neutralizing antibodies for different EV71 subtypes, and elicited significant splenocyte proliferation. The high levels of IFN-r and IL-10 showed the VP1 protein induced a mixed Th1 and Th2 immune response. Vaccinated female mice could confer protection in their neonatal offspring. Compared with the inactivated EV71, the VP1 protein elicited similar humoral and cellular responses, but the engineered protein is safer, less expensive and can be produced more efficiently. Therefore, EV71-VP1 protein can induce effective immunologic protection against EV71 and is an ideal candidate against EV71 infection.

Conessine inhibits enveloped viruses replication through up-regulating cholesterol level.

Dengue virus (DENV) is one of the most prevalent arthropod-borne diseases. It may cause dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), while no effective vaccines and drugs are available. Our study demonstrated that conessine exhibits broad antiviral activity against several enveloped viruses, including DENV, vesicular stomatitis virus, and herpes simplex virus. In addition, conessine has no direct destructive effect on the integrity or infectivity of virions. Both pre-treatment and post-treatment with conessine significantly reduce DENV replication. Pre-treatment with conessine disrupts the endocytosis of enveloped viruses, while post-treatment disturbs DENV RNA replication or translation at an early stage. Through screening differentially expressed genes by transcriptome sequencing, we found that conessine may affect cholesterol biosynthesis, metabolism or homeostasis. Finally, we confirmed that conessine inhibits virus replication through up-regulating cholesterol levels. Our work suggests that conessine could be developed as a prophylactic and therapeutic treatment for infectious diseases caused by enveloped viruses.

Rottlerin plays an antiviral role at early and late steps of Zika virus infection.

Infection of Zika virus (ZIKV) may cause microcephaly and other neurological disorders, while no vaccines and drugs are available. Our study revealed that rottlerin confers a broad antiviral activity against several enveloped viruses, including ZIKV, vesicular stomatitis virus, and herpes simplex virus, but not against two naked viruses (enterovirus 71 and encephalomyocarditis virus). Rottlerin does not have a direct virucidal effect on the virions, and its antiviral effect is independent of its regulation on PKCδ or ATP. Both pretreatment and post-treatment of rottlerin effectively reduce the viral replication of ZIKV. The pretreatment of rottlerin disturbs the endocytosis of enveloped viruses, while the post-treatment of rottlerin acts at a late stage through disturbing the maturation of ZIKV. Importantly, administration of rottlerin in neonatal mice significantly decreased the ZIKV replication in vivo, and alleviated the neurological symptoms caused by ZIKV. Our work suggests that rottlerin exerts an antiviral activity at two distinct steps of viral infection, and can be potentially developed as a prophylactic and therapeutic agent.

ANKS4B Restricts Replication of Zika Virus by Downregulating the Autophagy.

Infection of Zika virus (ZIKV) has become a severe threaten to global health while no specific drug is available. In this study, we explored the relationship between ZIKV and a cellular protein, ankyrin repeat and sterile motif domain containing 4b (ANKS4B). Our data revealed that the expression of ANKS4B in cultured cells and in neonatal mice was downregulated by ZIKV infection. The reduction of ANKS4B upon ZIKV infection was caused by decrease of two hepatocyte nuclear factors HNF1α and HNF4α. Through CRISPR/Cas9 gene editing system, we generated two ANKS4B knockout (KO) cell clones in A549 and Huh7 cells respectively. In the ANKS4B-KO cells, the viral replication levels including viral RNA, protein, and titer were significantly enhanced, which was reversed by trans-complementation of ANKS4B. ANKS4B did not affect the viral entry step, but impaired the autophagy induced by ZIKV infection. Furthermore, our data showed that inhibition of autophagy led to similar replication levels of ZIKV in ANKS4B-sufficient and ANKS4B-deficient cells, suggesting the antiviral effect of ANKS4B relied on its modulation on the autophagy. Therefore, our work identified ANKS4B as a new restriction factor of ZIKV.

Role of heparan sulfate in the Zika virus entry, replication, and cell death.

Zika virus (ZIKV) is an emerging arbovirus and its infection associates with neurologic diseases. Whether heparan sulfate (HS), an attachment factor for many viruses, plays a role in the ZIKV infection remains controversial. Our study generated several HS biosynthesis-deficient cell clones by disrupting SLC35B2, B3GAT3, or B4GALT7 gene using the CRISPR/Cas9 system. The HS deficiency did not affect the viral attachment and internalization of ZIKV, but reduced the attachment of Dengue virus (DENV) 2. The early RNA and protein levels of ZIKV and DENV2 were impaired in the HS deficient cells, while the viral yields were not accordingly reduced. Our data further showed that HS promoted the cell death induced by virus infection, and inhibition of cell death significantly increased the viral replication of ZIKV and DENV2. Collectively, our study described an unexpected role of HS in the viral attachment, replication and cell death induced by ZIKV.

Hydroxycarboxylic Acid Receptor 2 Is a Zika Virus Restriction Factor That Can Be Induced by Zika Virus Infection Through the IRE1-XBP1 Pathway.

Zika virus (ZIKV) is an emerging arthropod-borne virus and belongs to the Flaviviridae family. The infection of ZIKV has become the global health crisis because of its rapid spread and association with severe neurological disorders, including congenital microcephaly and Guillain-Barre Syndrome. To identify host factors contributing to ZIKV pathogenesis, transcriptomic landscape in ZIKV-infected cells was examined with mRNA microarray analysis and we observed that the expression of hydroxycarboxylic acid receptor 2 (HCAR2) could be significantly induced by ZIKV infection. By utilizing two IRE1 inhibitors and XBP1-specific shRNAs, we revealed that the up-regulation of HCAR2 expression induced by ZIKV was dependent on the IRE1-XBP1 pathway. Through the CRISPR/Cas9 system, we generated HCAR2-deficient cell clones in two cell types (human lung carcinoma epithelial A549 cell and human hepatoma Huh7.5 cell). We found that the depletion of HCAR2 significantly increased the replication level of ZIKV, including RNA levels, protein expression levels, and viral titers. In addition, our data demonstrated that the antiviral effect of HCAR2 was not involved in viral entry process and was not dependent on its antilipolytic effect on nicotinic acid/HCAR2-mediated signaling pathway. Taken together, our results indicated that HCAR2 could function as a restriction factor in control of ZIKV replication, potentially providing a novel molecular target for anti-ZIKV therapeutics.

Characterization of the enterovirus 71 P1 polyprotein expressed in Pichia pastor as a candidate vaccine.

Human enterovirus 71 (EV71) plays an important role in hand, foot, and mouth disease (HFMD), which recently caused the death of hundreds of children in the Asia-Pacific region. However, there are no specific treatments available for EV71 infections; thus, a safe and effective vaccine is needed urgently. In this study, we developed an effective and economical method for producing EV71 polyprotein (P1 protein) in Pichia pastoris. Furthermore, we evaluated the potential of P1 protein as a candidate vaccine against EV71 virus. The data revealed that P1 protein induced persistent high cross-neutralization antibodies for different EV71 subtypes, and elicited significant splenocyte proliferation. The high levels of interleukin-10(IL-10) and interferon-gamma (IFN-γ) showed that P1 protein induced Th1 and Th2 immune responses. Interestingly, vaccinating female mice with the P1 protein conferred cross-protection against different EV71 subtypes to their neonatal offspring.Compared with heat-inactivated EV71, the P1 protein elicited improved humoral and cellular immune responses and showed good cross-protection with different EV71 subtypes. Therefore, the EV71-P1 protein produced by P. pastoris is a promising candidate vaccine against EV71.

Expression and purification of enterovirus type 71 polyprotein P1 using Pichia pastoris system.

Enterovirus type 71(EV71) causes severe hand-foot-and-mouth disease (HFMD) resulting in hundreds of deaths of children every year; However, currently, there is no effective treatment for EV71. In this study, the EV71 poly-protein (EV71-P1 protein) gene was processed and cloned into the eukaryotic expression vector pPIC9k and then expressed in Pichia pastoris strain GS115. The EV71 P1 protein with a molecular weight of 100 kD was produced and secreted into the medium. The soluble EV71 P1 protein was purified by column chromatography with a recovery efficiency of 70%. The result of the immunological analysis showed that the EV71 P1 protein had excellent immunogenicity and could stimulate the production of EV71-VP1 IgG antibody in injected rabbits. We suggest that EV71-P1 protein is an ideal candidate for an EV71 vaccine to prevent EV71 infection.