Journal of Virology

Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease in pigs. The low-density lipoprotein receptor (LDLR) is a transcriptional target of the sterol-regulatory element-binding proteins (SREBPs) and participates in the uptake of LDL-derived cholesterol. However, the involvement of LDLR in PRV infection has not been well characterized. We observed an increased expression level of LDLR mRNA in PRV-infected 3D4/21, PK-15, HeLa, RAW264.7, and L929 cells. The LDLR protein level was also upregulated by PRV infection in PK-15 cells and in murine lung and brain. The treatment of cells with the SREBP inhibitor, fatostatin, or with SREBP2-specific small interfering RNA prevented the PRV-induced upregulation of LDLR expression as well as viral protein expression and progeny virus production. This suggested that PRV activated SREBPs to induce LDLR expression. Furthermore, interference in LDLR expression affected PRV proliferation, while LDLR overexpression promoted it. This indicated that LDLR was involved in PRV infection. The study also demonstrated that LDLR participated in PRV invasions. The overexpression of LDLR or inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which binds to LDLR and targets it for lysosomal degradation, significantly enhanced PRV attachment and entry. Mechanistically, LDLR interacted with PRV on the plasma membrane, and pretreatment of cells with LDLR antibodies was able to neutralize viral entry. An in vivo study indicated that the treatment of mice with the PCSK9 inhibitor SBC-115076 promoted PRV proliferation. The data from the study indicate that PRV hijacks LDLR for viral entry through the activation of SREBPs. IMPORTANCE Pseudorabies virus (PRV) is a herpesvirus that primarily manifests as fever, pruritus, and encephalomyelitis in various domestic and wild animals. Owing to its lifelong latent infection characteristics, PRV outbreaks have led to significant financial setbacks in the global pig industry. There is evidence that PRV variant strains can infect humans, thereby crossing the species barrier. Therefore, gaining deeper insights into PRV pathogenesis and developing updated strategies to contain its spread are critical. This study posits that the low-density lipoprotein receptor (LDLR) could be a co-receptor for PRV infection. Hence, strategies targeting LDLR may provide a promising avenue for the development of effective PRV vaccines and therapeutic interventions.

The ubiquitin-proteasome system is one of the most important protein stability regulation systems. It can precisely regulate host immune responses by targeting signaling proteins. TRAF6 is a crucial E3 ubiquitin ligase in host antiviral signaling pathway. Here, we discovered that EF-hand domain-containing protein D2 (EFHD2) collaborated with the E3 ubiquitin ligase Smurf1 to potentiate the degradation of TRAF6, hence facilitating RNA virus Siniperca chuatsi rhabdovirus infection. The mechanism analysis revealed that EFHD2 interacted with Smurf1 and enhanced its protein stability by impairing K48-linked polyubiquitination of Smurf1, thereby promoting Smurf1-catalyzed degradation of TRAF6. This study initially demonstrated a novel mechanism by which viruses utilize host EFHD2 to achieve immune escape and provided a new perspective on the exploration of mammalian innate immunity. IMPORTANCE Viruses induce host cells to activate several antiviral signaling pathways. TNF receptor-associated factor 6 (TRAF6) plays an essential role in these pathways. Numerous studies have been done on the mechanisms of TRAF6-mediated resistance to viral invasion. However, little is known about the strategies that viruses employ to antagonize TRAF6-mediated antiviral signaling pathway. Here, we discovered that EFHD2 functions as a host factor to promote viral replication. Mechanistically, EFHD2 potentiates Smurf1 to catalyze the ubiquitin-proteasomal degradation of TRAF6 by promoting the deubiquitination and stability of Smurf1, which in turn inhibits the production of proinflammatory cytokines and interferons. Our study also provides a new perspective on mammalian resistance to viral invasion.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with high case mortality rates, which is caused by Dabie bandavirus (DBV), a novel pathogen also termed as SFTS virus (SFTSV). Currently, no specific therapeutic drugs or vaccines are available for SFTS. Myxovirus resistance protein A (MxA) has been shown to inhibit multiple viral pathogens; however, the role of MxA in DBV infection is unknown. Here, we demonstrated that DBV stimulates MxA expression which, in turn, restricts DBV infection. Mechanistic target analysis revealed that MxA specifically interacts with the viral nucleocapsid protein (NP) in a manner independent of RNA. Minigenome reporter assay showed that in agreement with its targeting of NP, MxA inhibits DBV ribonucleoprotein (RNP) activity. In detail, MxA interacts with the NP N-terminal and disrupts the interaction of NP with the viral RNA-dependent RNA polymerase (RdRp) but not NP multimerization, the critical activities of NP for RNP formation and function. Furthermore, MxA N-terminal domain was identified as the functional domain inhibiting DBV infection, and, consistently, then was shown to interact with NP and obstruct the NP-RdRp interaction. Additionally, threonine 103 within the N-terminal domain is important for MxA inhibition to DBV, and its mutation (T103A) attenuates MxA binding to NP and obstruction of the NP-RdRp interaction. This study uncovers MxA inhibition of DBV with a series of functional and mechanistical analyses, providing insights into the virus−host interactions and probably helping inform the development of antiviral agents in the future. IMPORTANCE DBV/SFTSV is an emerging high-pathogenic virus. Since its first identification in China in 2009, cases of DBV infection have been reported in many other countries, posing a significant threat to public health. Uncovering the mechanisms of DBV-host interactions is necessary to understand the viral pathogenesis and host response and may advance the development of antiviral therapeutics. Here, we found that host factor MxA whose expression is induced by DBV restricts the virus infection. Mechanistically, MxA specifically interacts with the viral NP and blocks the NP-RdRp interaction, inhibiting the viral RNP activity. Further studies identified the key domain and amino acid residue required for MxA inhibition to DBV. Consistently, they were then shown to be important for MxA targeting of NP and obstruction of the NP-RdRp association. These findings unravel the restrictive role of MxA in DBV infection and the underlying mechanism, expanding our knowledge of the virus-host interactions.

Both influenza A virus genome transcription (vRNA→mRNA) and replication (vRNA→cRNA→vRNA), catalyzed by the influenza RNA polymerase (FluPol), are dynamically regulated across the virus life cycle. It has been reported that the last amino acid I 121 of the viral NS2 protein plays a critical role in promoting viral genome replication in influenza mini-replicon systems. Here, we performed a 20 natural amino acid substitution screening at residue NS2-I 121 in the context of virus infection. We found that the hydrophobicity of the residue 121 is essential for virus survival. Interestingly, through serial passage of the rescued mutant viruses, we further identified adaptive mutations PA-K19E and PB1-S713N on FluPol which could effectively compensate for the replication-promoting defect caused by NS2-I 121 mutation in the both mini-replicon and virus infection systems. Structural analysis of different functional states of FluPol indicates that PA-K19E and PB1-S713N could stabilize the replicase conformation of FluPol. By using a cell-based NanoBiT complementary reporter assay, we further demonstrate that both wild-type NS2 and PA-K19E/PB1-S713N could enhance FluPol dimerization, which is necessary for genome replication. These results reveal the critical role NS2 plays in promoting viral genome replication by coordinating with FluPol. IMPORTANCE The intrinsic mechanisms of influenza RNA polymerase (FluPol) in catalyzing viral genome transcription and replication have been largely resolved. However, the mechanisms of how transcription and replication are dynamically regulated remain elusive. We recently reported that the last amino acid of the viral NS2 protein plays a critical role in promoting viral genome replication in an influenza mini-replicon system. Here, we conducted a 20 amino acid substitution screening at the last residue 121 in virus rescue and serial passage. Our results demonstrate that the replication-promoting function of NS2 is important for virus survival and efficient multiplication. We further show evidence that NS2 and NS2-I 121 adaptive mutations PA-K19E/PB1-S713N regulate virus genome replication by promoting FluPol dimerization. This work highlights the coordination between NS2 and FluPol in fulfilling efficient genome replication. It further advances our understanding of the regulation of viral RNA synthesis for influenza A virus.

Circular RNAs (CircRNAs) have been shown to play a critical role in regulating viral infection and replication. We performed RNA sequencing to identify differentially expressed circRNAs in A549 human lung adenocarcinoma cells with and without Zika virus (ZIKV) infection. Notably, hsa_circ_0007321 was significantly downregulated after ZIKV infection. We then explored the role and mechanism of hsa_circ_0007321 in the regulation of ZIKV replication. Hsa_circ_0007321, derived from exons 2, 3, 4, and 5 of the DIS3L2 (DIS3 like 3'−5' exoribonuclease 2) gene, was mainly enriched in cytoplasm. We found that the knockdown of hsa_circ_0007321 significantly increased microRNA-492 levels to decrease NFKBID (NFKB inhibitor delta) expression, leading to the activation of the nuclear factor-κB (NF-κB) signaling pathway, inflammatory cytokine production, and subsequently, inhibition of ZIKV replication. These findings illustrate that hsa_circ_0007321 acts as a competitive endogenous RNA that regulates ZIKV replication through the miR-492/NFKBID NF-κB signaling pathway. IMPORTANCE Over the past decade, increasing evidence has shown that circular RNAs (circRNAs) play important regulatory roles in viral infection and host antiviral responses. However, reports on the role of circRNAs in Zika virus (ZIKV) infection are limited. In this study, we identified 45 differentially expressed circRNAs in ZIKV-infected A549 cells by RNA sequencing. We clarified that a downregulated circRNA, hsa_circ_0007321, regulates ZIKV replication through targeting of miR-492 and the downstream gene NFKBID. NFKBID is a negative regulator of nuclear factor-κB (NF-κB), and we found that inhibition of the NF-κB pathway promotes ZIKV replication. Therefore, this finding that hsa_circ_0007321 exerts its regulatory role on ZIKV replication through the miR-492/NFKBID/NF-κB signaling pathway has implications for the development of strategies to suppress ZIKV and possibly other viral infections.

The human immunodeficiency virus (HIV) persists in HIV viral reservoirs despite antiretroviral therapy (ART). Hepatitis C virus (HCV) coinfection is associated with increased HIV reservoir size and residual HIV transcription during ART. Herein, we investigated the impact of direct acting antivirals (DAA)-mediated HCV cure on the size/transcriptional activity of the HIV reservoirs and investigated predictors of HIV reservoirs decline in HCV+/HIV+ coinfected individuals. HCV+/HIV+ ( n = 20) and HCV+/HIV− ( n = 14) participants were examined prior to DAA treatment (baseline), at the end of treatment (EOT), and at 12–24 weeks after EOT (follow-up). In HCV+/HIV+ individuals, DAA-mediated HCV cure significantly reduced integrated HIV DNA levels, mainly in participants infected with HCV prior to HIV. Integrated HIV DNA, unspliced (US), and multiply spliced (MS) HIV RNA levels were quantified in sorted CD4+ T-cells. Despite the transient elevation of US HIV RNA at EOT, changes in US and MS HIV RNA were not statistically significant. Plasma inflammation markers were measured and DAA also reduced plasma sCD163 and sCD14. Changes in immunological/virological parameters were analyzed using multivariate random forest analysis where pre-ART peak HIV viremia and HCV viral load predicted integrated HIV DNA and US RNA changes. In conclusion, we demonstrate the beneficial impact of DAA on HIV reservoirs and immune activation, with a fraction of HIV reservoirs being DAA-sensitive in people infected with HCV before HIV. Furthermore, we identify HIV/HCV viremia as the top predictors of DAA-mediated changes in the HIV reservoirs. These findings support the need for early ART and DAA treatment in HIV/HCV coinfections. IMPORTANCE Antiretroviral therapy (ART) for human immunodeficiency virus (HIV) can control virus replication and prolong the life of people living with HIV (PLWH). However, the virus remains dormant within immune cells in what is called the HIV reservoir. Furthermore, 2.3 million PLWH are also coinfected with hepatitis C virus (HCV) and are at risk of developing chronic liver disease and cancer. HCV treatment with direct acting antivirals (DAA) can completely cure the infection in more than 95% of treated individuals and improve their long-term health outcomes. In this study, we investigated how HCV treatment and cure affect the HIV reservoir. We demonstrate the beneficial impact of DAA treatment as it reduces the HIV reservoirs in particular in people infected with HCV before HIV. These results support the need for early ART and DAA treatment in HIV/HCV coinfections.

Relative humidity (RH) is an environmental variable that affects mosquito physiology and can impact pathogen transmission. Low RH can induce dehydration in mosquitoes, leading to alterations in physiological and behavioral responses such as blood-feeding and host-seeking behavior. We evaluated the effects of a temporal drop in RH (RH shock) on mortality and Mayaro virus vector competence in Ae. aegypti. While dehydration induced by humidity shock did not impact virus infection, we detected a significant effect of dehydration on mosquito mortality and blood-feeding frequency, which could significantly impact transmission dynamics.

Viruses are constantly evolving to promote propagation in the host. Here, we show that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes host RAD51 for replication. Silencing of RAD51 impaired SARS-CoV-2 propagation. Viral RNA colocalized with RAD51 in the cytoplasm of SARS-CoV-2-infected cells, suggesting that both viral RNA and RAD51 may form a replication complex. We, therefore, evaluated RAD51 inhibitors as possible therapeutic agents against SARS-CoV-2. Indeed, RAD51 inhibitors exerted antiviral activities against not only Wuhan but also variants of SARS-CoV-2. Molecular docking model shows that RAD51 inhibitors impede SARS-CoV-2 propagation by interfering with dimerization of RAD51. These data suggest that RAD51 may represent a novel host-based drug target for coronavirus disease 2019 treatment.

Enterovirus D68 (EV-D68) is an emerging human pathogen associated with respiratory diseases and/or acute flaccid myelitis. Neutralizing antigenic sites of EV-D68 have not yet been comprehensively studied. In this study, we generated multiple neutralizing monoclonal antibodies (MAbs) directed against EV-D68 prototype or clinical strains. All these antibodies can inhibit EV-D68 attachment. The antibody epitopes were identified by selection and sequence analysis of prototype or clinical strain-derived neutralization-resistant mutants. The epitopes were then grouped into four distinct neutralizing antigenic sites (I to IV) by cross-neutralization analysis of the mutants with the MAbs and by spatial considerations. Site I, including residues 81, 85, and 87 of VP1 protein, is located in the VP1 BC loop, near the fivefold axis, and at the north rim of the canyon (the receptor binding site). Site II, involving residues 137, 139, and 142 of VP2, is situated in the VP2 EF loop and at the south rim of the canyon. Site III is composed of VP1 C-terminal residues 285 and 293 and resides on the south side of the canyon of neighboring asymmetric unit. Site IV contains residue 70 (βB strand) of VP2 from an asymmetric unit and residues 74 and 79 (BC loop) of VP3 from an adjacent unit and is located around the threefold axis. The four antigenic sites show various degrees of sequence variation. The identification of the four neutralizing antigenic sites on EV-D68 capsid provides a better understanding of the recognition of EV-D68 by neutralizing antibodies and viral evolution and immune escape. IMPORTANCE Enterovirus D68 (EV-D68) is an emerging respiratory pathogen associated with acute flaccid myelitis. Currently, no approved vaccines or antiviral drugs are available. Here, we report four functionally independent neutralizing antigenic sites (I to IV) by analyses of neutralizing monoclonal antibody (MAb)-resistant mutants. Site I is located in the VP1 BC loop near the fivefold axis. Site II resides in the VP2 EF loop, and site III is situated in VP1 C-terminus; both sites are located at the south rim of the canyon. Site IV is composed of residue in VP2 βB strand and residues in the VP3 BC loop and resides around the threefold axis. The developed MAbs targeting the antigenic sites can inhibit viral binding to cells. These findings advance the understanding of the recognition of EV-D68 by neutralizing antibodies and viral evolution and immune escape and also have important implications for the development of novel EV-D68 vaccines.

Most protease-targeted antiviral development evaluates the ability of small molecules to inhibit the cleavage of artificial substrates. However, before they can cleave any other substrates, viral proteases need to cleave themselves out of the viral polyprotein in which they have been translated. This can occur either intra- or inter-molecularly. Whether this process occurs intra- or inter-molecularly has implications for the potential for precursors to accumulate and for the effectiveness of antiviral drugs. We argue that evaluating candidate antivirals for their ability to block these cleavages is vital to drug development because the buildup of uncleaved precursors can be inhibitory to the virus and potentially suppress the selection of drug-resistant variants.

Porcine epidemic diarrhea virus (PEDV) infection in pigs is characterized by vomiting, dehydration, and diarrhea. Structural proteins of PEDV play crucial roles in viral entry, release, assembly, budding, and host immune regulation. Similar to other viruses, PEDV relies heavily on the host cellular machinery for productive infection. However, the host factors involved in PEDV infection remain unidentified. Thus, this study aims to map the PEDV structural proteins interacting with host factors in Vero cells. Results revealed karyopherin α 2 (KPNA2) as a potential host factor to suppress PEDV replication. KPNA2 overexpression in target cells significantly inhibited PEDV infection, whereas KPNA2 silencing by small interfering RNA promoted PEDV infection. Mechanistically, KPNA2 interacted with PEDV E, which led to the degradation of PEDV E protein. These results indicate the probable involvement of KPNA2 in the host antiviral response against PEDV. This study provides novel KPNA2-mediated viral restriction mechanisms in which KPNA2 overexpression suppresses PEDV replication by targeting and degrading the viral E protein by autophagy. KPNA2 may serve as a target in developing strategies to control PEDV infection. IMPORTANCE Porcine epidemic diarrhea, characterized by vomiting, dehydration, and diarrhea, is an acute and highly contagious enteric disease caused by porcine epidemic diarrhea virus (PEDV) in neonatal piglets. This disease has caused large economic losses to the porcine industry worldwide. Thus, identifying the host factors involved in PEDV infection is important to develop novel strategies to control PEDV transmission. This study shows that PEDV infection upregulates karyopherin α 2 (KPNA2) expression in Vero and intestinal epithelial (IEC) cells. KPNA2 binds to and degrades the PEDV E protein via autophagy to suppress PEDV replication. These results suggest that KPNA2 plays an antiviral role against PEDV. Specifically, knockdown of endogenous KPNA2 enhances PEDV replication, whereas its overexpression inhibits PEDV replication. Our data provide novel KPNA2-mediated viral restriction mechanisms in which KPNA2 suppresses PEDV replication by targeting and degrading the viral E protein through autophagy. These mechanisms can be targeted in future studies to develop novel strategies to control PEDV infection.

The pandemic of the coronavirus disease 2019 (COVID-19) has created the largest global health crisis in almost a century. Low frequencies of functional SARS-CoV-2-specific CD4 ⁺ and CD8 ⁺ T cells in the lungs of COVID-19 patients have been associated with severe cases of COVID-19. Low levels of T cell-attracting CXCL9, CXCL10, and CXCL11 chemokines in infected lungs may not be sufficient for the migration of CD4 ⁺ and CD8 ⁺ T cells from circulation into infected lungs. We hypothesize that a coronavirus vaccine strategy that boosts the frequencies of functional SARS-CoV-2-specific CD4 ⁺ and CD8 ⁺ T cells in the lungs would lead to better protection from COVID-19-like symptoms. In the present study, we designed and pre-clinically tested the safety, immunogenicity, and protective efficacy of a novel multi-epitope/CXCL11 prime/pull mucosal coronavirus vaccine. This prime/pull vaccine strategy consists of intranasal delivery of a lung-tropic adeno-associated virus type 9 vector that incorporates highly conserved human CD4 ⁺ and CD8 ⁺ cell epitopes of SARS-CoV-2 ( prime ) followed by recruitment of the primed T cells into the lungs using the T cell-attracting chemokine, CXCL-11 ( pull ). We demonstrated that the immunization of HLA-DR*0101/HLA-A*0201/hACE2 triple transgenic mice with this multi-epitope/CXCL11 prime/pull coronavirus mucosal vaccine: (i) increased the frequencies of functional CD4 ⁺ and CD8 ⁺ T EM , T CM , and T RM cells in the lungs and (ii) reduced COVID-19-like symptoms, lowered virus replication, and prevented deaths following challenge with SARS-CoV-2. These findings demonstrate that bolstering the number of functional lung-resident memory CD4 ⁺ and CD8 ⁺ T cells improved protection against SARS-CoV-2 infection, COVID-19-like symptoms, and death. IMPORTANCE Although the current rate of SARS-CoV-2 infections has decreased significantly, COVID-19 still ranks very high as a cause of death worldwide. As of October 2023, the weekly mortality rate is still at 600 deaths in the United States alone, which surpasses even the worst mortality rates recorded for influenza. Thus, the long-term outlook of COVID-19 is still a serious concern outlining the need for the next-generation vaccine. This study found that a prime/pull coronavirus vaccine strategy increased the frequency of functional SARS-CoV-2-specific CD4 ⁺ and CD8 ⁺ memory T cells in the lungs of SARS-CoV-2-infected triple transgenic HLA-DR*0101/HLA-A*0201/hACE2 mouse model, thereby resulting in low viral titer and reduced COVID-19-like symptoms.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes zoonotic disease. Dromedary camels are the source of zoonotic infection. We performed sequence analysis of clade B MERS-CoV after excluding potential bias arising multiple sequences from a single zoonotic transmission chain and identified a substitution of amino acid leucine to phenylalanine in the codon 232 position of the non-structural protein 6 (nsp6) (nsp6 L232F) that occurs preferentially in human clade B MERS-CoV, with a rate of 16.9% (20/118) in human sequences vs a 0.6% (1/160) in camel sequences. Using a human clade B MERS-CoV strain GD01 as backbone, we generated a pair of isogenic recombinant MERS-CoV with nsp6 232L and 232F residues, respectively, and showed that the nsp6 L232F substitution confers higher replication competence in ex vivo culture of human nasal and bronchial tissues and in lungs of mice experimentally infected in vivo . Mechanistically, the nsp6 L232F substitution was found to associate with higher exocytic virus egress, while innate immune responses, autophagic restriction, and zippering activity of the endoplasmic reticulum remained unaffected. Our study suggests an adaptive mutation that occurs in clade B MERS-CoV associated with inter-species transmission to humans that may facilitate higher viral replication in the human respiratory tract. This highlights the importance of MERS-CoV as a zoonotic threat and the need for continued virus surveillance in camels and humans. IMPORTANCE Viral host adaptation plays an important role in inter-species transmission of coronaviruses and influenza viruses. Multiple human-adaptive mutations have been identified in influenza viruses but not so far in MERS-CoV that circulates widely in dromedary camels in the Arabian Peninsula leading to zoonotic transmission. Here, we analyzed clade B MERS-CoV sequences and identified an amino acid substitution L232F in nsp6 that repeatedly occurs in human MERS-CoV. Using a loss-of-function reverse genetics approach, we found the nsp6 L232F conferred increased viral replication competence in vitro , in cultures of the upper human respiratory tract ex vivo, and in lungs of mice infected in vivo . Our results showed that nsp6 L232F may be an adaptive mutation associated with zoonotic transmission of MERS-CoV. This study highlighted the capacity of MERS-CoV to adapt to transmission to humans and also the need for continued surveillance of MERS-CoV in camels.

Classical swine fever virus nonstructural protein NS5A is essential for viral genome replication, protein translation, virus assembly, and autophagy. In the present paper, the interaction of cellular PPP2R1A, PP2Ac, and DAPK3 with NS5A has been confirmed. These interactions mediate the dissociation of PP2A from Beclin 1 and its association with DAPK3. Downregulation of DAPK3 or PPP2R1A inhibits CSFV replication. NS5A induced phosphorylation of Ser88 (PK-15 cells) or Ser90 (HEK293T cells) on Beclin 1. Knockdown of Beclin 1 inhibited NS5A-induced autophagy, indicating that NS5A induces Beclin 1-dependent autophagy. Downregulation of DAPK3, PPP2R1A, or PP2Ac by siRNA reduced Beclin 1 phosphorylation and autophagy mediated by NS5A, showing a critical role of PP2A and DAPK3 in Beclin 1 phosphorylation and autophagy triggered by NS5A. In vitro analysis of Beclin 1 phosphorylation revealed PP2A as being essential for DAPK3-mediated phosphorylation of Beclin 1, indicating that PP2A may dephosphorylate DAPK3 to activate its protein kinase activity, with activated DAPK3 phosphorylated Beclin 1, and then triggering autophagy. These data show for the first time that DAPK3 can be activated through dephosphorylation by PP2A. These findings reveal a novel mechanism whereby CSFV NS5A protein activates autophagy via PP2A-DAPK3-Beclin 1 axis to favor viral replication. IMPORTANCE Autophagy is a conserved degradation process that maintains cellular homeostasis and regulates native and adaptive immunity. Viruses have evolved diverse strategies to inhibit or activate autophagy for their benefit. The paper reveals that CSFV NS5A mediates the dissociation of PP2A from Beclin 1 and the association of PP2A with DAPK3 by interaction with PPP2R1A and DAPK3, PP2A dephosphorylates DAPK3 to activate its protein kinase activity, and activated DAPK3 phosphorylates Beclin 1 to trigger autophagy, indicating that NS5A activates autophagy via the PP2A-DAPK3-Beclin 1 axis. These data highlight a novel mechanism by which CSFV activates autophagy to favor its replication, thereby contributing to the development of antiviral strategies.

Viruses interact with receptors on the cell surface to initiate and coordinate infection. The distribution of receptors on host cells can be a key determinant of viral tropism and host infection. Unravelling the complex nature of virus-receptor interactions is, therefore, of fundamental importance to understanding viral pathogenesis. Noroviruses are non-enveloped, icosahedral, positive-sense RNA viruses of global importance to human health, with no approved vaccine or antiviral agent available. Here, we use murine norovirus as a model to study the molecular mechanisms of virus-receptor interactions. We show that variation at a single amino acid residue in the major viral capsid protein, VP1 301, has a key impact on the interaction between virus and receptor. This variation did not affect virion replication or virus growth kinetics, but a specific amino acid was rapidly selected through evolution experiments and significantly improved cellular attachment when infecting cells in suspension. However, modulating plasma membrane mobility counteracted this phenotype, suggesting a role for membrane fluidity in norovirus cellular attachment. When the infectivity of a panel of recombinant viruses with single amino acid substitutions at this residue was compared in vivo , there were differences in the tissue distribution of viruses in a murine host, suggesting a role for VP1 301 in dissemination in vivo . Overall, these results highlight how capsid evolution can influence infectivity and dissemination in the host. IMPORTANCE All viruses initiate infection by utilizing receptors to attach to target host cells. These virus-receptor interactions can therefore dictate viral replication and pathogenesis. Understanding the nature of virus-receptor interactions could also be important for the development of novel therapies. Noroviruses are non-enveloped icosahedral viruses of medical importance. They are a common cause of acute gastroenteritis with no approved vaccine or therapy and are a tractable model for studying fundamental virus biology. In this study, we utilized the murine norovirus model system to show that variation in a single amino acid of the major capsid protein alone can affect viral infectivity through improved attachment to suspension cells. Modulating plasma membrane mobility reduced infectivity, suggesting an importance of membrane mobility for receptor recruitment and/or receptor conformation. Furthermore, different substitutions at this site altered viral tissue distribution in a murine model, illustrating how in-host capsid evolution could influence viral infectivity and/or immune evasion.

The mechanism by which lipid metabolism regulates innate immunity is unknown. Here, we report that the key enzyme in cholesterol synthesis, 3β-hydroxysteroid-Δ24 reductase (DHCR24), is inhibited by viral infection. DHCR24 deficiency significantly promotes interferon production and interferon-stimulated gene expression. Inhibition of DHCR24 enzyme activity or the addition of the precursor 24-dehydrocholesterol (24-DHC) can augment innate immunity. Mechanistically, DHCR24 interacts with MAVS or STING, and DHCR24 impairs K27-linked ubiquitination of MAVS mediated by TRIM21 and K27-linked ubiquitination of STING mediated by AMFR, blocking the activation of MAVS and STING, respectively. Collectively, DHCR24 plays a negative role in regulating innate immune responses that may be targeted to improve immunity. IMPORTANCE The precise regulation of the innate immune response is essential for the maintenance of homeostasis. MAVS and STING play key roles in immune signaling pathways activated by RNA and DNA viruses, respectively. Here, we showed that DHCR24 impaired the antiviral response by targeting MAVS and STING. Notably, DHCR24 interacts with MAVS and STING and inhibits TRIM21-triggered K27-linked ubiquitination of MAVS and AMFR-triggered K27-linked ubiquitination of STING, restraining the activation of MAVS and STING, respectively. Together, this study elucidates how one cholesterol key enzyme orchestrates two antiviral signal transduction pathways.

Human metapneumovirus (hMPV) is a major respiratory pathogen worldwide. Here, we report the crystal structure of a neutralizing antibody M8C10 isolated from human B cells that targets the viral fusion protein (hMPV-F) trimerization interface. This is a novel prefusion-specific epitope that is highly conserved across hMPV strains and other related viruses. The interactions between M8C10 and hMPV-F are largely driven by residues on the light chain with most hypermutated light chain residues on or near the interface. This highlights the immune recognition of a buried trimerization interface during the in vivo affinity maturation process. In addition, in vivo cotton rat challenge studies were performed that show M8C10 provides strong lung protection against hMPV A viral challenge. The hMPV-F:M8C10 Fab structure reported reveals a class of hMPV antibodies in the human immune repertoire with a novel neutralization mechanism, targeting the trimerization interface of hMPV-F antigen. IMPORTANCE Human metapneumovirus (hMPV) is a common pathogen causing lower respiratory tract infections worldwide and can develop severe symptoms in high-risk populations such as infants, the elderly, and immunocompromised patients. There are no approved hMPV vaccines or neutralizing antibodies available for therapeutic or prophylactic use. The trimeric hMPV fusion F protein is the major target of neutralizing antibodies in human sera. Understanding the immune recognition of antibodies to hMPV-F antigen will provide critical insights into developing efficacious hMPV monoclonal antibodies and vaccines.

Porcine circovirus type 3 (PCV3) is a virus that causes a wide range of illnesses, including porcine dermatitis nephropathy syndrome, reproductive failure, and multisystemic inflammation. Nucleolin is predominantly localized in the nucleus and is important in viral replication. Mass spectrometry has revealed that the PCV3 capsid (Cap) protein interacts with nucleolin. However, the effects of nucleolin on PCV3 replication remain unclear. This study confirmed that PCV3 Cap interacts with nucleolin and that the Cap nuclear location signal domain and nucleolin amino acids 1–110 are essential for the Cap-nucleolin interaction. Further examinations indicated that lysine residue 102 of nucleolin is conjugated to K48-linked polyubiquitination chains by the E3 ligase RNF34, which is transported by PCV3 Cap from the cytoplasm to the nucleolus, resulting in nucleolin degradation. PCV3 replication was increased by small interfering RNA-mediated nucleolin knockdown and was decreased by nucleolin overexpression. Inhibition of PCV3 replication by nucleolin overexpression was associated with nucleolin-promoted release of interferon β. These findings suggest that nucleolin functions as an antiviral protein to combat PCV3 infection by activating innate immunity. The findings provide important insights concerning the prevention and treatment of PCV3 infection. IMPORTANCE Porcine circovirus type 3 (PCV3) is an emerging pathogen that causes multisystem disease in pigs and poses a severe threat to the swine industry. However, the mechanisms of how PCV3 uses host proteins to regulate its own life cycle are not well understood. In this study, we found that PCV3 capsid protein interacts with nucleolin and degrades it. Degradation of nucleolin by the PCV3 capsid protein requires recruitment of the enzyme RNF34, which is transported to the nucleolus from the cytoplasm in the presence of the PCV3 capsid protein. Nucleolin also decreases PCV3 replication by promoting the release of interferon β. These findings clarify the mechanism by which nucleolin modulates PCV3 replication in cells, thereby facilitating to provide an important strategy for preventing and controlling PCV3 infection.

The central nervous system (CNS) is a major human immunodeficiency virus type-1 (HIV-1) reservoir. Microglia are the primary target cell of HIV-1 infection in the CNS. Current models have not allowed the precise molecular pathways of acute and chronic CNS microglial infection to be tested with in vivo genetic methods. Here, we describe a novel-humanized mouse model utilizing human induced pluripotent stem cell (iPSC)-derived microglia to xenograft into murine hosts. These mice are additionally engrafted with human peripheral blood mononuclear cells that serve as a medium to establish a peripheral infection that then spreads to the CNS microglia xenograft, modeling a trans-blood-brain barrier route of acute CNS HIV-1 infection with human target cells. The approach is compatible with iPSC genetic engineering, including inserting targeted transgenic reporter cassettes to track the xenografted human cells, enabling the testing of novel treatment and viral tracking strategies in a comparatively simple and cost-effective in vivo model for neuroHIV. IMPORTANCE Our mouse model is a powerful tool for investigating the genetic mechanisms governing central nervous system (CNS) human immunodeficiency virus type-1 (HIV-1) infection and latency in the CNS at a single-cell level. A major advantage of our model is that it uses induced pluripotent stem cell-derived microglia, which enables human genetics, including gene function and therapeutic gene manipulation, to be explored in vivo , which is more challenging to study with current hematopoietic stem cell-based models for neuroHIV. Our transgenic tracing of xenografted human cells will provide a quantitative medium to develop new molecular and epigenetic strategies for reducing the HIV-1 latent reservoir and to test the impact of therapeutic inflammation-targeting drug interventions on CNS HIV-1 latency.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has accumulated more than 700 million infection cases and 6.9 million deaths. New variants have affected antibody interaction with the surface spike protein. We defined the domain specificities and measured hexa-proline stabilized spike protein (HexaPro) binding kinetics of a large panel of antibodies sourced by the Coronavirus Immunotherapeutics Consortium. Epitope binning analysis of antibodies competing for HexaPro binding separated the fine specificities of the majority of antibodies to four regions: top, outer, mesa/valley, or cryptic site of receptor binding domain (RBD). Most of the top-RBD-specific antibodies showed >3-fold loss of binding and authentic-virus neutralization activity for the B.1.351 variant. Remarkably, among RBD mesa/valley-specific or cryptic-site-specific antibodies, 55% showed >3-fold stronger affinities, and at least 60% maintained neutralization activity for the B.1.351 variant. These data also highlighted the diversity of SARS-CoV-2-specific antibodies that retain high spike affinities and antiviral functions across variants. IMPORTANCE Multiple SARS-CoV-2 variants of concern have emerged and caused a significant number of infections and deaths worldwide. These variants of concern contain mutations that might significantly affect antigen-targeting by antibodies. It is therefore important to further understand how antibody binding and neutralization are affected by the mutations in SARS-CoV-2 variants. We highlighted how antibody epitope specificity can influence antibody binding to SARS-CoV-2 spike protein variants and neutralization of SARS-CoV-2 variants. We showed that weakened spike binding and neutralization of Beta (B.1.351) and Omicron (BA.1) variants compared to wildtype are not universal among the panel of antibodies and identified antibodies of a specific binding footprint exhibiting consistent enhancement of spike binding and retained neutralization to Beta variant. These data and analysis can inform how antigen-targeting by antibodies might evolve during a pandemic and prepare for potential future sarbecovirus outbreaks.

Marek’s disease (MD) is a poultry disease characterized by severe immunosuppression and the development of T cell lymphomas caused by Marek’s disease virus (MDV). Our study focuses on the role of the viral LORF9 gene in MD pathogenesis and vaccine development. We found that a LORF9 gene deletion mutant (Md5BAC Δ LORF9 ) showed significantly reduced pathogenicity compared with the parental strain. The LORF9 gene is essential for the early cytolytic infection in B cells. Interestingly, Md5BAC Δ LORF9 provided adequate immunological protection against very virulent MDV challenge and induced a distinct host immune response compared with the CVI988/Rispens vaccine strain. Transcriptome analysis revealed that the deletion of LORF9 led to a significantly different expression of innate immunity-associated genes. Overall, our study provides insights into the role of LORF9 in MD pathogenesis and highlights its importance in MDV gene deletion vaccine development. IMPORTANCE Marek’s disease virus (MDV) is a highly infectious and oncogenic virus that can induce severe T cell lymphomas in chickens. MDV encodes more than 100 genes, most of which have unknown functions. This work indicated that the LORF9 gene is necessary for MDV early cytolytic replication in B lymphocyte. In addition, we have found that the LORF9 deletion mutant has a comparative immunological protective effect with CVI988/Rispens vaccine strain against very virulent MDV challenge. This is a significant discovery that LORF9 can be exploited as a possible target for the development of an MDV gene deletion vaccine.

Vaccinia virus (VACV) infection induces prominent changes in host cell metabolism. Little is known about the global metabolic reprogramming that takes place in whole tissue during viral infection. Here, we performed a longitudinal metabolomics study in VACV-infected mouse skin to investigate metabolic changes in the tissue during infection. We assessed metabolites in homogenized skin of the ear pinnae over time in the presence or absence of antigen-specific T cells using untargeted mass spectrometry. VACV infection induced several significant metabolic changes in the tissue, including in the levels of nucleic acid metabolites (reflecting the impact of viral replication on the skin metabolome). Furthermore, monocyte- and antiviral T cell-produced metabolites, such as itaconic acid and glutamine, were significantly increased following infection, highlighting the immune response’s contribution to the global skin metabolome. Additional RNA-Seq of infected skin tissue recapitulated metabolic changes identified by metabolomics analysis. Overall, our study reveals the metabolic balance of viral replication and the antiviral immune response in the skin, elucidating metabolic pathways that could contribute to cutaneous poxvirus control in vivo . IMPORTANCE Human poxvirus infections have caused significant public health burdens both historically and recently during the unprecedented global Mpox virus outbreak. Although vaccinia virus (VACV) infection of mice is a commonly used model to explore the anti-poxvirus immune response, little is known about the metabolic changes that occur in vivo during infection. We hypothesized that the metabolome of VACV-infected skin would reflect the increased energetic requirements of both virus-infected cells and immune cells recruited to sites of infection. Therefore, we profiled whole VACV-infected skin using untargeted mass spectrometry to define the metabolome during infection, complementing these experiments with flow cytometry and transcriptomics. We identified specific metabolites, including nucleotides, itaconic acid, and glutamine, that were differentially expressed during VACV infection. Together, this study offers insight into both virus-specific and immune-mediated metabolic pathways that could contribute to the clearance of cutaneous poxvirus infection.

Porcine epidemic diarrhea virus (PEDV) is a pig coronavirus that causes severe diarrhea and high mortality in piglets, threatening the global pig industry. Clinically effective medicines against this virus are still not available. To find an optional natural feed compound, viral titers were detected, and the intestinal contents of specific pathogen-free pigs more effectively blocked PEDV invasion than those of the other two breeds investigated. Based on proteomic and metabolic analyses of the intestinal content, 10 metabolites were selected for further investigation, and linoleic acid (LA) was the most promising according to the selectivity index. By detecting viral gene expression, LA inhibited viral replication and release from Vero-E6 cells, mainly by influencing the PI3K pathway and, in particular, inhibiting AKT phosphorylation. In addition, LA bound to viral NSP5 and attenuated NSP5-activated AKT phosphorylation. In the in vivo pig experiment, oral administration of the higher dose of LA protected two-thirds of pigs from death, both of which completely recovered from the infection, while the lower dose of LA protected one-third of pigs from death but with the induction of severe diarrhea. In conclusion, LA can be used as a candidate medicine for the clinical prevention and treatment of PEDV, and its mechanism of inhibiting the PI3K signaling pathway provides some data support for the subsequent exploration of antiviral drugs for coronavirus infections. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) is a pig coronavirus that causes severe diarrhea and high mortality in piglets, but as no effective drugs are available, this virus threatens the pig industry. Here, we found that the intestinal contents of specific pathogen-free pigs effectively blocked PEDV invasion. Through proteomic and metabolic analyses of the intestinal contents, we screened 10 metabolites to investigate their function and found that linoleic acid (LA) significantly inhibited PEDV replication. Further investigations revealed that LA inhibited viral replication and release mainly by binding with PEDV NSP5 to regulate the PI3K pathway and, in particular, inhibiting AKT phosphorylation. In vivo experiments illustrated that orally administered LA protected pigs from PEDV challenge and severe diarrhea. These findings provide strong support for exploring antiviral drugs for coronavirus treatment.

The traditional view that betacoronaviruses exit occurs via the constitutive secretory route has recently been questioned by studies suggesting that this process involves lysosomal exocytosis. Here, we demonstrated that porcine hemagglutinating encephalomyelitis virus (PHEV), a neurotropic betacoronavirus, traffics to lysosomes prior to engaging in Arl8b-dependent lysosomal exocytosis. Notably, PHEV induces active or passive lysosomal acidification and activates lysosomal degradative enzymes. PHEV release depends on vacuolar H ⁺ -ATPase-mediated lysosomal acidification. In addition, PHEV transmission and PHEV-induced brain damage in the central nervous system can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Taken together, lysosome plays a critical role in PHEV egress in vitro and in vivo . IMPORTANCE Betacoronaviruses, including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and mouse hepatitis virus (MHV), exploit the lysosomal exocytosis pathway for egress. However, whether all betacoronaviruses members use the same pathway to exit cells remains unknown. Here, we demonstrated that porcine hemagglutinating encephalomyelitis virus (PHEV) egress occurs by Arl8b-dependent lysosomal exocytosis, a cellular egress mechanism shared by SARS-CoV-2 and MHV. Notably, PHEV acidifies lysosomes and activates lysosomal degradative enzymes, while SARS-CoV-2 and MHV deacidify lysosomes and limit the activation of lysosomal degradative enzymes. In addition, PHEV release depends on V-ATPase-mediated lysosomal pH. Furthermore, this is the first study to evaluate βCoV using lysosome for spreading through the body, and we have found that lysosome played a critical role in PHEV neural transmission and brain damage caused by virus infection in the central nervous system. Taken together, different betacoronaviruses could disrupt lysosomal function differently to exit cells.

This satellite symposium was focused on the molecular arms race between bacteria and their predators, the bacteriophages: who’s the friend and who’s the foe? This Gem recounts highlights of the talks and presents food for thought and additional reflections on the current state of the field.