Gut microbiota-derived butyrate promotes coronavirus TGEV infection through impairing RIG-I-triggered local type I interferon responses via class I HDAC inhibition.

Gut microbiota-derived metabolites are important for the replication and pathogenesis of many viruses. However, the roles of bacterial metabolites in swine enteric coronavirus (SECoV) infection remain poorly understood. Recent studies show that SECoVs infection in vivo significantly alters the composition of short-chain fatty acids (SCFAs)-producing gut microbiota. This prompted us to investigate whether and how SCFAs impact SECoV infection. Employing alphacoronavirus transmissible gastroenteritis virus (TGEV), a major cause of diarrhea in piglets, as a model, we found that SCFAs, particularly butyrate, enhanced TGEV infection both in porcine intestinal epithelial cells and swine testicular (ST) cells at the late stage of viral infection. This effect depended on the inhibited productions of virus-induced type I interferon (IFN) and downstream antiviral IFN-stimulated genes (ISGs) by butyrate. Mechanistically, butyrate suppressed the expression of retinoic acid-inducible gene I (RIG-I), a key viral RNA sensor, and downstream mitochondrial antiviral-signaling (MAVS) aggregation, thereby impairing type I IFN responses and increasing TGEV replication. Using pharmacological and genetic approaches, we showed that butyrate inhibited RIG-I-induced type I IFN signaling by suppressing class I histone deacetylase (HDAC). In summary, we identified a novel mechanism where butyrate enhances TGEV infection by suppressing RIG-I-mediated type I IFN responses. Our findings highlight that gut microbiota-derived metabolites like butyrate can be exploited by SECoV to dampen innate antiviral immunity and establish infection in the intestine.IMPORTANCESwine enteric coronaviruses (SECoVs) infection in vivo alters the composition of short-chain fatty acids (SCFAs)-producing gut microbiota, but whether microbiota-derived SCFAs impact coronavirus gastrointestinal infection is largely unknown. Here, we demonstrated that SCFAs, particularly butyrate, substantially increased alphacoronavirus TGEV infection at the late stage of infection, without affecting viral attachment or internalization. Furthermore, enhancement of TGEV by butyrate depended on impeding virus-induced type I interferon (IFN) responses. Mechanistically, butyrate suppressed the cytoplasmic viral RNA sensor RIG-I expression and downstream type I IFN signaling activation by inhibiting class I HDAC, thereby promoting TGEV infection. Our work reveals novel functions of gut microbiota-derived SCFAs in enhancing enteric coronavirus infection by impairing RIG-I-dependent type I IFN responses. This implies that bacterial metabolites could be therapeutic targets against SECoV infection by modulating antiviral immunity in the intestine.

Long-Term Expanding Porcine Airway Organoids Provide Insights into the Pathogenesis and Innate Immunity of Porcine Respiratory Coronavirus Infection.

Respiratory coronaviruses cause serious health threats to humans and animals. Porcine respiratory coronavirus (PRCoV), a natural transmissible gastroenteritis virus (TGEV) mutant with partial spike deletion, causes mild respiratory disease and is an interesting animal respiratory coronavirus model for human respiratory coronaviruses. However, the absence of robust ex vivo models of porcine airway epithelium hinders an understanding of the pathogenesis of PRCoV infection. Here, we generated long-term porcine airway organoids (AOs) derived from basal epithelial cells, which recapitulate the in vivo airway complicated epithelial cellularity. Both 3D and 2D AOs are permissive for PRCoV infection. Unlike TGEV, which established successful infection in both AOs and intestinal organoids, PRCoV was strongly amplified only in AOs, not intestinal organoids. Furthermore, PRCoV infection in AOs mounted vigorous early type I and III interferon (IFN) responses and upregulated the expression of overzealous inflammatory genes, including pattern recognition receptors (PRRs) and proinflammatory cytokines. Collectively, these data demonstrate that stem-derived porcine AOs can serve as a promising disease model for PRCoV infection and provide a valuable tool to study porcine respiratory infection. IMPORTANCE Porcine respiratory CoV (PRCoV), a natural mutant of TGEV, shows striking pathogenetic similarities to human respiratory CoV infection and provides an interesting animal model for human respiratory CoVs, including SARS-CoV-2. The lack of an in vitro model recapitulating the complicated cellularity and structure of the porcine respiratory tract is a major roadblock for the study of PRCoV infection. Here, we developed long-term 3D airway organoids (AOs) and further established 2D AO monolayer cultures. The resultant 3D and 2D AOs are permissive for PRCoV infection. Notably, PRCoV mediated pronounced IFN and inflammatory responses in AOs, which recapitulated the inflammatory responses associated with PRCoV in vivo infection. Therefore, porcine AOs can be utilized to characterize the pathogenesis of PRCoV and, more broadly, can serve as a universal platform for porcine respiratory infection.

Coronavirus Porcine Deltacoronavirus Upregulates MHC Class I Expression through RIG-I/IRF1-Mediated NLRC5 Induction.

Major histocompatibility complex class I (MHC-I) and MHC-II molecules, mainly being responsible for the processing and presentation of intracellular or extracellular antigen, respectively, are critical for antiviral immunity. Here, we reported that porcine deltacoronavirus (PDCoV) with the zoonotic potential and potential spillover from pigs to humans, upregulated the expressions of porcine MHC-I (swine leukocyte antigen class I, SLA-I) molecules and SLA-I antigen presentation associated genes instead of porcine MHC-II (SLA-II) molecules both in primary porcine enteroids and swine testicular (ST) cells at the late stage of infection, and this finding was verified in vivo. Moreover, the induction of SLA-I molecules by PDCoV infection was mediated through enhancing the expression of NOD-like receptor (NLR) family caspase recruitment domain-containing 5 (NLRC5). Mechanistic studies demonstrated that PDCoV infection robustly elevated retinoic acid-inducible gene I (RIG-I) expression, and further initiated the downstream type I interferon beta (IFN-ß) production, which led to the upregulation of NLRC5 and SLA-I genes. Likewise, interferon regulatory factor 1 (IRF1) elicited by PDCoV infection directly activated the promoter activity of NLRC5, resulting in an increased expression of NLRC5 and SLA-I upregulation. Taken together, our findings advance our understanding of how PDCoV manipulates MHC molecules, and knowledge that could help inform the development of therapies and vaccines against PDCoV. IMPORTANCE MHC-I molecules play a crucial role in antiviral immunity by presenting intracellular antigens to CD8+T lymphocytes and eliminating virus-infected cells by natural killer cells' "missing-self recognition." However, the manipulation of MHC molecules by coronaviruses remains poorly understood. Here, we demonstrated that PDCoV, a zoonotic potential coronavirus efficiently infecting cells from broad species, greatly increased the expressions of porcine MHC-I (SLA-I) molecules and MHC-I antigen presentation associated genes but not porcine MHC-II (SLA-II) molecules both in vitro and in vivo. Mechanistically, the upregulation of MHC-I molecules by PDCoV infection required the master transactivator of MHC-I, NLRC5, which was mediated not only by RIG-I-initiated type I IFN signaling pathway but also by IRF1 induced by PDCoV as it could activate NLRC5 promoter activity. These results provide significant insights into the modification of the MHC class I pathway and may provide a potential therapeutic intervention for PDCoV.

Different Mechanisms Are Utilized by Coronavirus Transmissible Gastroenteritis Virus To Regulate Interferon Lambda 1 and Interferon Lambda 3 Production.

Type III interferons (IFN-λ) are shown to be preferentially produced by epithelial cells, which provide front-line protection at barrier surfaces. Transmissible gastroenteritis virus (TGEV), belonging to the genus Alphacoronavirus of the family Coronaviridae, can cause severe intestinal injuries in porcine, resulting in enormous economic losses for the swine industry, worldwide. Here, we demonstrated that although IFN-λ1 had a higher basal expression, TGEV infection induced more intense IFN-λ3 production in vitro and in vivo than did IFN-λ1. We explored the underlying mechanism of IFN-λ induction by TGEV and found a distinct regulation mechanism of IFN-λ1 and IFN-λ3. The classical RIG-I-like receptor (RLR) pathway is involved in IFN-λ3 but not IFN-λ1 production. Except for the signaling pathways mediated by RIG-I and MDA5, TGEV nsp1 induces IFN-λ1 and IFN-λ3 by activating NF-κB via the unfolded protein responses (UPR) PERK-eIF2α pathway. Furthermore, functional domain analysis indicated that the induction of IFN-λ by the TGEV nsp1 protein was located at amino acids 85 to 102 and was dependent on the phosphorylation of eIF2α and the nuclear translocation of NF-κB. Moreover, the recombinant TGEV with the altered amino acid motif of nsp1 85-102 was constructed, and the nsp1 (85-102sg) mutant virus significantly reduced the production of IFN-λ, compared with the wild strain. Compared to the antiviral activities of IFN-λ1, the administration of IFN-λ3 showed greater antiviral activity against TGEV infections in IPEC-J2 cells. In summary, our data point to the significant role of IFN-λ in the host innate antiviral responses to coronavirus infections within mucosal organs and in the distinct mechanisms of IFN-λ1 and IFN-λ3 regulation. IMPORTANCE Coronaviruses cause infectious diseases in various mammals and birds and exhibit an epithelial cell tropism in enteric and respiratory tracts. It is critical to explore how coronavirus infections modulate IFN-λ, a key innate cytokine against mucosal viral infection. Our results uncovered the different processes of IFN-λ1 and IFN-λ3 production that are involved in the classical RLR pathway and determined that TGEV nsp1 induces IFN-λ1 and IFN-λ3 production by activating NF-κB via the PERK-eIF2α pathway in UPR. These studies highlight the unique regulation of antiviral defense in the intestine during TGEV infection. We also demonstrated that IFN-λ3 induced greater antiviral activity against TGEV replication than did IFN-λ1 in IPEC-J2 cells, which is helpful in finding a novel strategy for the treatment of coronavirus infections.

A Bispecific Antibody Targeting RBD and S2 Potently Neutralizes SARS-CoV-2 Omicron and Other Variants of Concern.

Emerging severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) variants, especially the Omicron variant, have impaired the efficacy of existing vaccines and most therapeutic antibodies, highlighting the need for additional antibody-based tools that can efficiently neutralize emerging SARS-CoV-2 variants. The use of a "single" agent to simultaneously target multiple distinct epitopes on the spike is desirable in overcoming the neutralizing escape of SARS-CoV-2 variants. Herein, we generated a human-derived IgG-like bispecific antibody (bsAb), Bi-Nab35B5-47D10, which successfully retained parental specificity and simultaneously bound to the two distinct epitopes on receptor-binding domain (RBD) and S2. Bi-Nab35B5-47D10 showed improved spike binding breadth among wild-type (WT) SARS-CoV-2, variants of concern (VOCs), and variants being monitored (VBMs) compared with its parental monoclonal antibodies (MAbs). Furthermore, pseudotyped virus neutralization demonstrated that Bi-Nab35B5-47D10 can efficiently neutralize VBMs, including Alpha (B.1.1.7), Beta (B.1.351), and Kappa (B.1.617.1), as well as VOCs, including Delta (B.1.617.2), Omicron BA.1, and Omicron BA.2. Crucially, Bi-Nab35B5-47D10 substantially improved neutralizing activity against Omicron BA.1 (IC50 = 0.15 nM) and Omicron BA.2 (IC50 = 0.67 nM) compared with its parental MAbs. Therefore, Bi-Nab35B5-47D10 represents a potential effective countermeasure against SARS-CoV-2 Omicron and other variants of concern. IMPORTANCE The new, highly contagious SARS-CoV-2 Omicron variant caused substantial breakthrough infections and has become the dominant strain in countries across the world. Omicron variants usually bear high mutations in the spike protein and exhibit considerable escape of most potent neutralization monoclonal antibodies and reduced efficacy of current COVID-19 vaccines. The development of neutralizing antibodies with potent efficacy against the Omicron variant is still an urgent priority. Here, we generated a bsAb, Bi-Nab35B5-47D10, which simultaneously targets SARS-CoV-2 RBD and S2 and improves the neutralizing potency and breadth against SARS-CoV-2 WT and the tested variants compared with their parental antibodies. Notably, Bi-Nab35B5-47D10 has more potent neutralizing activity against the VOC Omicron pseudotyped virus. Therefore, Bi-Nab35B5-47D10 is a feasible and potentially effective strategy by which to treat and prevent COVID-19.

Vaccine induction of antibodies against a structurally heterogeneous site of immune pressure within HIV-1 envelope protein variable regions 1 and 2.

The RV144 HIV-1 trial of the canary pox vector (ALVAC-HIV) plus the gp120 AIDSVAX B/E vaccine demonstrated an estimated efficacy of 31%, which correlated directly with antibodies to HIV-1 envelope variable regions 1 and 2 (V1-V2). Genetic analysis of trial viruses revealed increased vaccine efficacy against viruses matching the vaccine strain at V2 residue 169. Here, we isolated four V2 monoclonal antibodies from RV144 vaccinees that recognize residue 169, neutralize laboratory-adapted HIV-1, and mediate killing of field-isolate HIV-1-infected CD4(+) T cells. Crystal structures of two of the V2 antibodies demonstrated that residue 169 can exist within divergent helical and loop conformations, which contrasted dramatically with the ß strand conformation previously observed with a broadly neutralizing antibody PG9. Thus, RV144 vaccine-induced immune pressure appears to target a region that may be both sequence variable and structurally polymorphic. Variation may signal sites of HIV-1 envelope vulnerability, providing vaccine designers with new options.

Aminopeptidase N Expression, Not Interferon Responses, Determines the Intestinal Segmental Tropism of Porcine Deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is an economically important enteropathogen of swine with worldwide distribution. PDCoV primarily infects the small intestine instead of the large intestine in vivo However, the underlying mechanism of PDCoV tropism to different intestinal segments remains poorly understood as a result of the lack of a suitable in vitro intestinal model that recapitulates the cellular diversity and complex functions of the gastrointestinal tract. Here, we established the PDCoV infection model of crypt-derived enteroids from different intestinal segments. Enteroids were susceptible to PDCoV, and multiple types of different functional intestinal epithelia were infected by PDCoV in vitro and in vivo We further found that PDCoV favorably infected the jejunum and ileum and restrictedly replicated in the duodenum and colon. Mechanistically, enteroids from different intestinal regions displayed a distinct gene expression profile, and the differential expression of primary viral receptor host aminopeptidase N (APN) instead of the interferon (IFN) responses determined the susceptibility of different intestinal segments to PDCoV, although PDCoV substantially elicited antiviral genes production in enteroids after infection. Additional studies showed that PDCoV infection significantly induced the expression of type I and III IFNs at the late stage of infection, and exogenous IFN inhibited PDCoV replication in enteroids. Hence, our results provide critical inputs to further dissect the molecular mechanisms of PDCoV-host interactions and pathogenesis.IMPORTANCE The zoonotic potential of the PDCoV, a coronavirus efficiently infecting cells from a broad range species, including porcine, chicken, and human, emphasizes the urgent need to further study the cell and tissue tropism of PDCoV in its natural host. Herein, we generated crypt stem cell-derived enteroids from porcine different intestinal regions, which well recapitulated the events in vivo of PDCoV infection that PDCoV targeted multiple types of intestinal epithelia and preferably infected the jejunum and ileum over the duodenum and colon. Mechanistically, we demonstrated that the expression of APN receptor rather than the IFN responses determined the susceptibility of different regions of the intestines to PDCoV infection, though PDCoV infection markedly elicited the IFN responses. Our findings provide important insights into how the distinct gene expression profiles of the intestinal segments determine the cell and tissue tropism of PDCoV.

Basic Amino Acid Substitution at Residue 367 of the Envelope Protein of Tembusu Virus Plays a Critical Role in Pathogenesis.

Tembusu virus (TMUV) is a flavivirus responsible for panzootic outbreaks of severe egg-drop and fatal encephalitis of domestic waterfowl in China. Although TMUV can be attenuated by in vitro passaging, experimental evidence supporting the role of specific genetic changes in virulence attenuation is currently lacking. Here, we performed site-directed mutagenesis on five envelope (E) protein amino acid residues in accordance with the attenuated TMUV generated in our recent study. Our results showed that the Thr-to-Lys mutation of residue 367 in E protein (E367) plays a predominant role in viral cell adaptation and virulence attenuation in ducks compared with mutations in other residues. We further demonstrated that the positively charged basic amino acid substitution at E367 enhanced the viral binding affinity for glycosaminoglycans (GAGs) and reduced viremia levels and the efficiency of replication in major target organs in subcutaneously inoculated ducks. Interestingly, the T367K mutation increased viral neutralization sensitivity to the early immune sera. Together, our findings provide the first evidence that a basic amino acid substitution at E367 strongly impacts the in vitro and in vivo infection of TMUV.IMPORTANCE Outbreaks of Tembusu virus (TMUV) infection have caused huge economic losses in the production of domestic waterfowl since the virus was first recognized in China in 2010. To control TMUV infection, a live-attenuated vaccine candidate of TMUV was developed in our previous study, but the mechanisms of virulence attenuation are not fully understood. Here, we found that the Thr-to-Lys substitution at E367 is a crucial determinant of TMUV virulence attenuation in ducks. We demonstrated that the T367K mutation attenuates TMUV through reducing viral replication in the blood, brain, heart (ducklings), and ovaries. These data provide new insights into understanding the pathogenesis of TMUV and the rational development of novel TMUV vaccines.

The Epigenetic Regulator EZH2 Instructs CD4 T Cell Response to Acute Viral Infection via Coupling of Cell Expansion and Metabolic Fitness.

The protection of a majority of viral vaccines is mediated by CD4 T cell-dependent humoral immunity. The methyltransferase enhancer of zeste homolog 2 (EZH2) dictates the differentiation of naive CD4 T cells into distinct effector T helper subsets at the onset of acute viral infection. However, whether and how EZH2 manipulates differentiated virus-specific CD4 T cell expansion remain to be elucidated. Here, we found that EZH2 is integral for virus-specific CD4 T cell expansion in a mouse model of acute viral infection. By a mechanism that involves fine-tuning the mechanistic target of rapamycin (mTOR) signaling, EZH2 participates in integrating metabolic pathways to support cell expansion. The genetic ablation of EZH2 leads to impaired cellular metabolism and, consequently, poor CD4 T cell response to acute viral infection. Thus, we identified EZH2 as a novel regulator in virus-specific CD4 T cell expansion during acute viral infection.IMPORTANCE The CD4 T cell response is critical in curtailing viral infection or eliciting efficacious viral vaccination. Highly efficient expansion of virus-specific CD4 T cells culminates in a qualified CD4 T cell response. Here, we found that the epigenetic regulator EZH2 is a prerequisite for the virus-specific CD4 T cell response, with a mechanism coupling cell expansion and metabolism. Thus, our study provides valuable insights for strategies targeting EZH2 to improve the efficacy of CD4 T cell-based viral vaccines and to help treat diseases associated with aberrant CD4 T cell responses.

Comparative Transcriptomic and Proteomic Analyses Prove that IFN-λ1 is a More Potent Inducer of ISGs than IFN-α against Porcine Epidemic Diarrhea Virus in Porcine Intestinal Epithelial Cells.

Type III interferon (IFN-λ) is currently considered to be largely nonredundant to type I interferon (IFN-α) in antivirus infection, especially in epithelial cells. Previous studies reported that, compared with IFN-α, IFN-λ exhibited stronger induction of interferon-stimulated genes (ISGs) at the transcriptional level in intestinal epithelial cells and stronger inhibition of porcine epidemic diarrhea virus (PEDV). In this study, the different mechanisms of ISG upregulation induced by IFN-α and IFN-λ1 were compared at the mRNA and protein levels in the porcine intestinal epithelial cell model (IPEC-J2). It was proved that IFN-λ1 consistently exhibited stronger stimulation effects at both levels. At the mRNA level, 132 genes were significantly upregulated upon IFN-λ1 stimulation, while 42 genes upon IFN-α stimulation. At the protein level, 47 proteins were significantly upregulated upon IFN-λ1 stimulation, but only 8 proteins were upregulated upon IFN-α stimulation. The shared upregulated genes/proteins by IFN-λ1 in both transcriptional and translational omics, especially the regulation factors of ISG15, were involved in the JAK-STAT signaling pathway. Compared to IFN-α, IFN-λ1 could induce more consistent upregulation of the key ISGs (ISG15, USP18, OASL, and RSAD2) at 3-24 h postinduction as measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) validation. It was further confirmed through functional analysis that ISG15 and RSAD2 could inhibit PEDV infection in dose-dependent manners. This study provided solid evidence that IFN-λ1 could induce a more unique and higher ISG expression level, which exhibited anti-PEDV effects on porcine intestinal epithelial cells.

Porcine Intestinal Enteroids: a New Model for Studying Enteric Coronavirus Porcine Epidemic Diarrhea Virus Infection and the Host Innate Response.

Porcine epidemic diarrhea virus (PEDV), a member of the group of alphacoronaviruses, is the pathogen of a highly contagious gastrointestinal swine disease. The elucidation of the events associated with the intestinal epithelial response to PEDV infection has been limited by the absence of good in vitro porcine intestinal models that recapitulate the multicellular complexity of the gastrointestinal tract. Here, we generated swine enteroids from the intestinal crypt stem cells of the duodenum, jejunum, or ileum and found that the generated enteroids are able to satisfactorily recapitulate the complicated intestinal epithelium in vivo and are susceptible to infection by PEDV. PEDV infected multiple types of cells, including enterocytes, stem cells, and goblet cells, and exhibited segmental infection discrepancies compared with ileal enteroids and colonoids, and this finding was verified in vivo Moreover, the clinical isolate PEDV-JMS propagated better in ileal enteroids than the cell-adapted isolate PEDV-CV777, and PEDV infection suppressed interferon (IFN) production early during the infection course. IFN lambda elicited a potent antiviral response and inhibited PEDV in enteroids more efficiently than IFN alpha (IFN-α). Therefore, swine enteroids provide a novel in vitro model for exploring the pathogenesis of PEDV and for the in vitro study of the interplay between a host and a variety of swine enteric viruses.IMPORTANCE PEDV is a highly contagious enteric coronavirus that causes significant economic losses, and the lack of a good in vitro model system is a major roadblock to an in-depth understanding of PEDV pathogenesis. Here, we generated a porcine intestinal enteroid model for PEDV infection. Utilizing porcine intestinal enteroids, we demonstrated that PEDV infects multiple lineages of the intestinal epithelium and preferably infects ileal enteroids over colonoids and that enteroids prefer to respond to IFN lambda 1 over IFN-α. These events recapitulate the events that occur in vivo This study constitutes the first use of a primary intestinal enteroid model to investigate the susceptibility of porcine enteroids to PEDV and to determine the antiviral response following infection. Our study provides important insights into the events associated with PEDV infection of the porcine intestine and provides a valuable in vitro model for studying not only PEDV but also other swine enteric viruses.

The PERK Arm of the Unfolded Protein Response Negatively Regulates Transmissible Gastroenteritis Virus Replication by Suppressing Protein Translation and Promoting Type I Interferon Production.

Coronavirus replication is closely associated with the endoplasmic reticulum (ER), the primary cellular organelle for protein synthesis, folding, and modification. ER stress is a common consequence in coronavirus-infected cells. However, how the virus-induced ER stress influences coronavirus replication and pathogenesis remains controversial. Here, we demonstrated that infection with the alphacoronavirus transmissible gastroenteritis virus (TGEV) induced ER stress and triggered the unfolded protein response (UPR) in vitro and in vivo, and ER stress negatively regulated TGEV replication in vitro Although TGEV infection activated all three UPR pathways (activating transcription factor 6 [ATF6], inositol-requiring enzyme 1 [IRE1], and protein kinase R-like ER kinase [PERK]), the virus-triggered UPR suppressed TGEV replication in both swine testicular (ST) and IPEC-J2 cells primarily through activation of the PERK-eukaryotic initiation factor 2α (eIF2α) axis, as shown by functional studies with overexpression, small interfering RNA (siRNA), or specific chemical inhibitors. Moreover, we demonstrated that PERK-eIF2α axis-mediated inhibition of TGEV replication occurs through phosphorylated eIF2α-induced overall attenuation of protein translation. In addition to direct inhibition of viral production, the PERK-eIF2α pathway activated NF-κB and then facilitated type I IFN production, resulting in TGEV suppression. Taken together, our results suggest that the TGEV-triggered PERK-eIF2α pathway negatively regulates TGEV replication and represents a vital aspect of host innate responses to invading pathogens.IMPORTANCE The induction of ER stress is a common outcome in cells infected with coronaviruses. The UPR initiated by ER stress is actively involved in viral replication and modulates the host innate responses to the invading viruses, but these underlying mechanisms remain incompletely understood. We show here that infection with the alphacoronavirus TGEV elicited ER stress in vitro and in vivo, and the UPR PERK-eIF2α branch was predominantly responsible for the suppression of TGEV replication by ER stress. Furthermore, the PERK-eIF2α axis inhibited TGEV replication through direct inhibition of viral proteins due to global translation inhibition and type I IFN induction. These findings highlight a critical role of the UPR PERK-eIF2α pathway in modulating host innate immunity and coronavirus replication.

The Coronavirus Transmissible Gastroenteritis Virus Evades the Type I Interferon Response through IRE1α-Mediated Manipulation of the MicroRNA miR-30a-5p/SOCS1/3 Axis.

In host innate immunity, type I interferons (IFN-I) are major antiviral molecules, and coronaviruses have evolved diverse strategies to counter the IFN-I response during infection. Transmissible gastroenteritis virus (TGEV), a member of the Alphacoronavirus family, induces endoplasmic reticulum (ER) stress and significant IFN-I production after infection. However, how TGEV evades the IFN-I antiviral response despite the marked induction of endogenous IFN-I has remained unclear. Inositol-requiring enzyme 1 α (IRE1α), a highly conserved ER stress sensor with both kinase and RNase activities, is involved in the IFN response. In this study, IRE1α facilitated TGEV replication via downmodulating the host microRNA (miR) miR-30a-5p abundance. miR-30a-5p normally enhances IFN-I antiviral activity by directly targeting the negative regulators of Janus family kinase (JAK)-signal transducer and activator of transcription (STAT), the suppressor of cytokine signaling protein 1 (SOCS1), and SOCS3. Furthermore, TGEV infection increased SOCS1 and SOCS3 expression, which dampened the IFN-I antiviral response and facilitated TGEV replication. Importantly, compared with mock infection, TGEV infection in vivo resulted in decreased miR-30a-5p levels and significantly elevated SOCS1 and SOCS3 expression in the piglet ileum. Taken together, our data reveal a new strategy used by TGEV to escape the IFN-I response by engaging the IRE1α-miR-30a-5p/SOCS1/3 axis, thus improving our understanding of how TGEV escapes host innate immune defenses.IMPORTANCE Type I interferons (IFN-I) play essential roles in restricting viral infections. Coronavirus infection induces ER stress and the interferon response, which reflects different adaptive cellular processes. An understanding of how coronavirus-elicited ER stress is actively involved in viral replication and manipulates the host IFN-I response has remained elusive. Here, TGEV inhibited host miR-30a-5p via the ER stress sensor IRE1α, which led to the increased expression of negative regulators of JAK-STAT signaling cascades, namely, SOCS1 and SOCS3. Increased SOCS1 or SOCS3 expression impaired the IFN-I antiviral response, promoting TGEV replication. These findings enhance our understanding of the strategies used by coronaviruses to antagonize IFN-I innate immunity via IRE1α-mediated manipulation of the miR-30a-5p/SOCS axis, highlighting the crucial role of IRE1α in innate antiviral resistance and the potential of IRE1α as a novel target against coronavirus infection.

HIV-1-Specific IgA Monoclonal Antibodies from an HIV-1 Vaccinee Mediate Galactosylceramide Blocking and Phagocytosis.

Vaccine-elicited humoral immune responses comprise an array of antibody forms and specificities, with only a fraction contributing to protective host immunity. Elucidation of antibody effector functions responsible for protective immunity against human immunodeficiency virus type 1 (HIV-1) acquisition is a major goal for the HIV-1 vaccine field. Immunoglobulin A (IgA) is an important part of the host defense against pathogens; however, little is known about the role of vaccine-elicited IgA and its capacity to mediate antiviral functions. To identify the antiviral functions of HIV-1-specific IgA elicited by vaccination, we cloned HIV-1 envelope-specific IgA monoclonal antibodies (MAbs) by memory B cell cultures from peripheral blood mononuclear cells from an RV144 vaccinee and produced two IgA clonal cell lines (HG129 and HG130) producing native, nonrecombinant IgA MAbs. The HG129 and HG130 MAbs mediated phagocytosis by monocytes, and HG129 blocked HIV-1 Env glycoprotein binding to galactosylceramide, an alternative HIV-1 receptor. These findings elucidate potential antiviral functions of vaccine-elicited HIV-1 envelope-specific IgA that may act to block HIV-1 acquisition at the portal of entry by preventing HIV-1 binding to galactosylceramide and mediating antibody Fc receptor-mediated virion phagocytosis. Furthermore, these findings highlight the complex and diverse interactions of vaccine-elicited IgA with pathogens that depend on IgA fine specificity and form (e.g., multimeric or monomeric) in the systemic circulation and mucosal compartments.IMPORTANCE Host-pathogen interactions in vivo involve numerous immune mechanisms that can lead to pathogen clearance. Understanding the nature of antiviral immune mechanisms can inform the design of efficacious HIV-1 vaccine strategies. Evidence suggests that both neutralizing and nonneutralizing antibodies can mediate some protection against HIV in animal models. Although numerous studies have characterized the functional properties of HIV-1-specific IgG, more studies are needed on the functional attributes of HIV-1-specific IgA, specifically for vaccine-elicited IgA. Characterization of the functional properties of HIV-1 Env-specific IgA monoclonal antibodies from human vaccine clinical trials are critical toward understanding the capacity of the host immune response to block HIV-1 acquisition.

Interferon gamma inhibits transmissible gastroenteritis virus infection mediated by an IRF1 signaling pathway.

Interferon gamma (IFN-γ) is best known for its ability to regulate host immune responses; however, its direct antiviral activity is less well studied. Transmissible gastroenteritis virus (TGEV) is an economically important swine enteric coronavirus and causes acute diarrhea in piglets. At present, little is known about the function of IFN-γ in the control of TGEV infection. In this study, we demonstrated that IFN-γ inhibited TGEV infection directly in ST cells and intestine epithelial IPEC-J2 cells and that the anti-TGEV activity of IFN-γ was independent of IFN-α/ß. Moreover, IFN-γ suppressed TGEV infection in ST cells more efficiently than did IFN-α, and the combination of IFN-γ and IFN-α displayed a synergistic effect against TGEV. Mechanistically, using overexpression and functional knockdown experiments, we demonstrated that porcine interferon regulatory factor 1 (poIRF1) elicited by IFN-γ primarily mediated IFN-γ signaling cascades and the inhibition of TGEV infection by IFN-γ. Importantly, we found that TGEV elevated the expression of poIRF1 and IFN-γ in infected small intestines and peripheral blood mononuclear cells. Thus, IFN-γ plays a crucial role in curtailing enteric coronavirus infection and may serve as an effective prophylactic and/or therapeutic agent against TGEV infection.

Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses.

Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine-induced antibodies and therapeutic antibodies will enable a better understanding of their capacity to prevent and/or control HIV-1 infection in vivo.

Expression and characterization of a recombinant porcinized antibody against the E2 protein of classical swine fever virus.

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious and economically important disease of pigs. The envelope glycoprotein E2 of CSFV is the major antigen that induces neutralizing antibodies and confers protection against CSFV infections. Previously, we developed a murine monoclonal antibody (MAb), HQ06, against the E2 protein of CSFV. To produce the antibody conveniently and stably, the genes coding for the variable regions of the heavy and light chains of HQ06 and constant region genes from the swine antibody were fused and cloned into lentiviral expression vectors to express a recombinant porcinized MAb (rHQ06Sw) in mammalian cells. rHQ06Sw was able to react with the E2 protein or the CSFV virions specifically in different assays. Notably, rHQ06Sw could neutralize CSFV infection in a dose-dependent manner. Taken together, the functional porcinized MAb rHQ06Sw was generated, which can be used to develop novel diagnostic assays or to investigate the structure and functions of the E2 protein.

MicroRNA-30a-5p Inhibits the Growth of Renal Cell Carcinoma by Modulating GRP78 Expression.

BACKGROUND/AIMS: MiR-30a-5p, a member of the microRNA-30 family (miR-30), is known to function as a tumor suppressor in several different cancers. However, the expression levels, biological function, and underlying mechanisms of miR-30a-5p in renal cell carcinoma (RCC) remain unclear. Glucose-regulated protein78 (GRP78) is a common cancer biomarker and promotes the growth and survival of cancer cells. The expression of GRP78 has been reported to be modulated by miR-30a in neurons. In this study, the expression profile of miR-30a-5p in clear cell renal cell carcinoma (ccRCC) and its effect on ccRCC through regulating GRP78 expression was investigated. METHODS: MiR-30a-5p expression was analyzed using bioinformatic software on open microarray datasets from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), and confirmed by quantitative RT-PCR (qRT-PCR) in ccRCC cell lines. Cell proliferation was investigated using CCK-8 and cell count assays. Western blotting, immunohistochemistry, luciferase reporter assays, and flow cytometry were employed to investigate the mechanisms of the effect of miR-30a-5p on ccRCC Results: MiR-30a-5p was down-regulated in ccRCC and related to the clinicopathological factors and prognosis of ccRCC. MiR-30a-5p was found to both suppress the growth of ccRCC cells and promote apoptosis of ccRCC cells in vitro. GRP78 was the direct target gene of miR-30a-5p, and the GRP78 expression was inversely correlated with the expression of miR-30a-5p in vivo and in vitro. The functional studies of GRP78 overexpression or knockdown demonstrated that GRP78 promoted proliferation and anti-apoptosis of ccRCC cells, and the oncogenic activity of GRP78 resulting in by miR-30a-5p overexpression. CONCLUSION: MiR-30a-5p is a bona fide negative regulator of GRP78 expression, and the anti-tumor activity of miR-30a-5p in ccRCC is due at least in part to down-regulating GRP78 expression and modulating the unfolded protein response (UPR) pathway. Thus, miR-30-GRP78 interaction provides a novel therapeutic candidate target in ccRCC treatment.

Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques.

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.