Release of HIV-1 particles from macrophages is promoted by an anchored cytoskeleton and driven by mechanical constraints.

A feature of HIV-1 replication in macrophages is that viral assembly occurs at the limiting membrane of a compartment often named the virus-containing compartment (VCC). Assembled virions accumulate in the lumen of the VCC, from where they can be released into the extracellular medium via mechanisms that remain poorly described. Here, we show that the actin cytoskeleton contributes to this process by performing experiments combining pharmacological and mechanical perturbations with imaging and biochemical analysis. We found that jasplakinolide inhibited HIV-1 release from macrophages and led to scattering of the compartment. Concomitantly, both the integrin CD18 (ß2-integrin) and the phosphorylated form of PYK2 (also known as PTK2B) were displaced away from the VCC. Inhibition of PYK2 activity promoted retention of viral particles in VCCs that lost their connections to the surface. Finally, in infected macrophages undergoing frustrated phagocytosis, VCCs rapidly trafficked to the basal membrane and released their viral content, in a manner dependent on their association with the actin cytoskeleton. These results highlight that the trafficking of VCCs and virus release are intimately linked to a reorganization of the macrophage actin cytoskeleton that can be modulated by external physical cues.

The Zika virus infection remodels the expression of the synaptotagmin-9 secretory protein.

The exact mechanisms involved in flaviviruses virions' release and the specific secretion of viral proteins, such as the Non Structural protein-1 (NS1), are still unclear. While these processes might involve vesicular transport to the cell membrane, NS1 from some flaviviruses was shown to participate in viral assembly and release. Here, we assessed the effect of the Zika virus (ZIKV) NS1 expression on the cellular proteome to identify trafficking-related targets that may be altered in the presence of the viral protein. We detected an increase in the synaptotagmin-9 (SYT9) secretory protein, which participates in the intracellular transport of protein-laden vesicles. We confirmed the effect of NS1 on SYT9 levels by transfection models while also detecting a significant subcellular redistribution of SYT9. We found that ZIKV prM-Env proteins, required for the viral particle release, also increased SYT9 levels and changed its localization. Finally, we demonstrated that ZIKV cellular infection raises SYT9 levels and promotes changes in its subcellular localization, together with a co-distribution with both Env and NS1. Altogether, the data suggest SYT9's implication in the vesicular transport of viral proteins or virions during ZIKV infection, showing for the first time the association of synaptotagmins with the flavivirus' life cycle.

Epstein-Barr Virus BBLF1 Mediates Secretory Vesicle Transport to Facilitate Mature Virion Release.

Enveloped viruses undergo a complex multistep process of assembly, maturation, and release into the extracellular space utilizing host secretory machinery. Several studies of the herpesvirus subfamily have shown that secretory vesicles derived from the trans-Golgi network (TGN) or endosomes transport virions into the extracellular space. However, the regulatory mechanism underlying the release of Epstein-Barr virus, a human oncovirus, remains unclear. We demonstrate that disruption of BBLF1, a tegument component, suppressed viral release and resulted in the accumulation of viral particles on the inner side of the vesicular membrane. Organelle separation revealed the accumulation of infectious viruses in fractions containing vesicles derived from the TGN and late endosomes. Deficiency of an acidic amino acid cluster in BBLF1 reduced viral secretion. Moreover, truncational deletion of the C-terminal region of BBLF1 increased infectious virus production. These findings suggest that BBLF1 regulates the viral release pathway and reveal a new aspect of tegument protein function. IMPORTANCE Several viruses have been linked to the development of cancer in humans. Epstein-Barr virus (EBV), the first identified human oncovirus, causes a wide range of cancers. Accumulating literature has demonstrated the role of viral reactivation in tumorigenesis. Elucidating the functions of viral lytic genes induced by reactivation, and the mechanisms of lytic infection, is essential to understanding pathogenesis. Progeny viral particles synthesized during lytic infection are released outside the cell after the assembly, maturation, and release steps, leading to further infection. Through functional analysis using BBLF1-knockout viruses, we demonstrated that BBLF1 promotes viral release. The acidic amino acid cluster in BBLF1 was also important for viral release. Conversely, mutants lacking the C terminus exhibited more efficient virus production, suggesting that BBLF1 is involved in the fine-tuning of progeny release during the EBV life cycle.

Fish ELOVL7a is involved in virus replication via lipid metabolic reprogramming.

The elongation of very long chain fatty acids (ELOVL) proteins are key rate-limiting enzymes that catalyze fatty acid synthesis to form long chain fatty acids. ELOVLs also play regulatory roles in the lipid metabolic reprogramming induced by mammalian viruses. However, little is known about the roles of fish ELOVLs during virus infection. Here, a homolog of ELOVL7 was cloned from Epinephelus coioides (EcELOVL7a), and its roles in red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV) infection were investigated. The transcription level of EcELOVL7a was significantly increased upon RGNNV and SGIV infection or other pathogen-associated molecular patterns stimulation in grouper spleen (GS) cells. Subcellular localization analysis showed that EcELOVL7a encoded an endoplasmic reticulum (ER) related protein. Overexpression of EcELOVL7a promoted the viral production and virus release during SGIV and RGNNV infection. Furthermore, the lipidome profiling showed that EcELOVL7a overexpression reprogrammed cellular lipid components in vitro, evidenced by the increase of glycerophospholipids, sphingolipids and glycerides components. In addition, VLCFAs including FFA (20:2), FFA (20:4), FFA (22:4), FFA (22:5) and FFA (24:0), were enriched in EcELOVL7a overexpressed cells. Consistently, EcELOVL7a overexpression upregulated the transcription level of the key lipid metabolic enzymes, including fatty acid synthase (FASN), phospholipase A 2α (PLA 2α), and cyclooxygenases -2 (COX-2), LPIN1, and diacylglycerol acyltransferase 1α (DGAT1α). Together, our results firstly provided the evidence that fish ELOVL7a played an essential role in SGIV and RGNNV replication by reprogramming lipid metabolism.

Nonlytic Quasi-Enveloped Hepatovirus Release Is Facilitated by pX Protein Interaction with the E3 Ubiquitin Ligase ITCH.

Hepatoviruses are atypical hepatotropic picornaviruses that are released from infected cells without lysis in small membranous vesicles. These exosome-like, quasi-enveloped virions (eHAV) are infectious and the only form of hepatitis A virus (HAV) found circulating in blood during acute infection. eHAV is released through multivesicular endosomes in a process dependent on endosomal sorting complexes required for transport (ESCRT). Capsid protein interactions with the ESCRT-associated Bro1 domain proteins, ALG-2-interacting protein X (ALIX) and His domain-containing protein tyrosine phosphatase (HD-PTP), which are both recruited to the pX domain of 1D (VP1pX), are critical for this process. Previous proteomics studies suggest pX also binds the HECT domain, NEDD4 family E3 ubiquitin ligase, ITCH. Here, we confirm this interaction and show ITCH binds directly to the carboxy-terminal half of pX from both human and bat hepatoviruses independently of ALIX. A small chemical compound (compound 5) designed to disrupt interactions between WW domains of NEDD4 ligases and substrate molecules blocked ITCH binding to pX and demonstrated substantial antiviral activity against HAV. CRISPR deletion or small interfering RNA (siRNA) knockdown of ITCH expression inhibited the release of a self-assembling nanocage protein fused to pX and also impaired the release of eHAV from infected cells. The release could be rescued by overexpression of wild-type ITCH, but not a catalytically inactive ITCH mutant. Despite this, we found no evidence that ITCH ubiquitylates pX or that eHAV release is strongly dependent upon Lys residues in pX. These data indicate ITCH plays an important role in the ESCRT-dependent release of quasi-enveloped hepatovirus, although the substrate molecule targeted for ubiquitylation remains to be determined. IMPORTANCE Mechanisms underlying the cellular release of quasi-enveloped hepatoviruses are only partially understood, yet play a crucial role in the pathogenesis of this common agent of viral hepatitis. Multiple NEDD4 family E3 ubiquitin ligases, including ITCH, have been reported to promote the budding of conventional enveloped viruses but are not known to function in the release of HAV or other picornaviruses from infected cells. Here, we show that the unique C-terminal pX extension of the VP1 capsid protein of HAV interacts directly with ITCH and that ITCH promotes eHAV release in a manner analogous to its role in budding of some conventional enveloped viruses. The catalytic activity of ITCH is required for efficient eHAV release and may potentially function to ubiquitylate the viral capsid or activate ESCRT components.

Host ESCRT factors are recruited during chikungunya virus infection and are required for the intracellular viral replication cycle.

Chikungunya fever is a re-emerging zoonotic disease caused by chikungunya virus (CHIKV), a member of the Alphavirus genus in the Togaviridae family. Only a few studies have reported on the host factors required for intracellular CHIKV trafficking. Here, we conducted an imaging-based siRNA screen to identify human host factors for intracellular trafficking that are involved in CHIKV infection, examined their interactions with CHIKV proteins, and investigated the contributions of these proteins to CHIKV infection. The results of the siRNA screen revealed that host endosomal sorting complexes required for transport (ESCRT) proteins are recruited during CHIKV infection. Co-immunoprecipitation analyses revealed that both structural and nonstructural CHIKV proteins interact with hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), a component of the ESCRT-0 complex. We also observed that HGS co-localizes with the E2 protein of CHIKV and with dsRNA, a marker of the replicated CHIKV genome. Results from gene knockdown analyses indicated that, along with other ESCRT factors, HGS facilitates both genome replication and post-translational steps during CHIKV infection. Moreover, we show that ESCRT factors are also required for infections with other alphaviruses. We conclude that during CHIKV infection, several ESCRT factors are recruited via HGS and are involved in viral genome replication and post-translational processing of viral proteins.

HIV-1 Vpu Downregulates Tim-3 from the Surface of Infected CD4+ T Cells.

Along with other immune checkpoints, T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is expressed on exhausted CD4+ and CD8+ T cells and is upregulated on the surface of these cells upon infection by human immunodeficiency virus type 1 (HIV-1). Recent reports have suggested an antiviral role for Tim-3. However, the molecular determinants of HIV-1 which modulate cell surface Tim-3 levels have yet to be determined. Here, we demonstrate that HIV-1 Vpu downregulates Tim-3 from the surface of infected primary CD4+ T cells, thus attenuating HIV-1-induced upregulation of Tim-3. We also provide evidence that the transmembrane domain of Vpu is required for Tim-3 downregulation. Using immunofluorescence microscopy, we determined that Vpu is in close proximity to Tim-3 and alters its subcellular localization by directing it to Rab 5-positive (Rab 5+) vesicles and targeting it for sequestration within the trans- Golgi network (TGN). Intriguingly, Tim-3 knockdown and Tim-3 blockade increased HIV-1 replication in primary CD4+ T cells, thereby suggesting that Tim-3 expression might represent a natural immune mechanism limiting viral spread.IMPORTANCE HIV infection modulates the surface expression of Tim-3, but the molecular determinants remain poorly understood. Here, we show that HIV-1 Vpu downregulates Tim-3 from the surface of infected primary CD4+ T cells through its transmembrane domain and alters its subcellular localization. Tim-3 blockade increases HIV-1 replication, suggesting a potential negative role of this protein in viral spread that is counteracted by Vpu.

New Isolates of Pandoraviruses: Contribution to the Study of Replication Cycle Steps.

Giant viruses are complex members of the virosphere, exhibiting outstanding structural and genomic features. Among these viruses, the pandoraviruses are some of the most intriguing members, exhibiting giant particles and genomes presenting at up to 2.5 Mb, with many genes having no known function. In this work, we analyzed, by virological and microscopic methods, the replication cycle steps of three new pandoravirus isolates from samples collected in different regions of Brazil. Our data indicate that all analyzed pandoravirus isolates can deeply modify the Acanthamoeba cytoplasmic environment, recruiting mitochondria and membranes into and around the electron-lucent viral factories. We also observed that the viral factories start forming before the complete degradation of the cellular nucleus. Various patterns of pandoravirus particle morphogenesis were observed, and the assembly of the particles seemed to be started either by the apex or by the opposite side. On the basis of the counting of viral particles during the infection time course, we observed that pandoravirus particles could undergo exocytosis after their morphogenesis in a process that involved intense recruitment of membranes that wrapped the just-formed particles. The treatment of infected cells with brefeldin affected particle exocytosis in two of the three analyzed strains, indicating biological variability among isolates. Despite such particle exocytosis, the lysis of host cells also contributed to viral release. This work reinforces knowledge of and reveals important steps in the replication cycle of pandoraviruses.IMPORTANCE The emerging Pandoraviridae family is composed of some of the most complex viruses known to date. Only a few pandoravirus isolates have been described until now, and many aspects of their life cycle remain to be elucidated. A comprehensive description of the replication cycle is pivotal to a better understanding of the biology of the virus. For this report, we describe new pandoraviruses and used different methods to better characterize the steps of the replication cycle of this new group of viruses. Our results provide new information about the diversity and biology of these giant viruses.

Contribution of the Cytoplasmic Determinants of Vpu to the Expansion of Virus-Containing Compartments in HIV-1-Infected Macrophages.

HIV-1 infection of macrophages leads to the sequestration of newly formed viruses in intracellular plasma membrane-connected structures termed virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The cellular restriction factor bone marrow stromal cell antigen 2 (BST2), which prevents HIV-1 dissemination by tethering budding viral particles at the plasma membrane, can be found in VCCs. The HIV-1 accessory protein Vpu counteracts the restriction factor BST2 by downregulating its expression and removing it from viral budding sites. Numerous studies described these Vpu countermeasures in CD4+ T cells or model cell lines, but the interplay between Vpu and BST2 in VCC formation and HIV-1 production in macrophages is less explored. Here, we show that Vpu expression in HIV-1-infected macrophages enhances viral release. This effect is related to Vpu's ability to circumvent BST2 antiviral activity. We show that in absence of Vpu, BST2 is enriched in VCCs and colocalizes with capsid p24, whereas Vpu expression significantly reduces the presence of BST2 in these compartments. Furthermore, our data reveal that BST2 is dispensable for the formation of VCCs and that Vpu expression impacts the volume of these compartments. This Vpu activity partly depends on BST2 expression and requires the integrity of the Vpu transmembrane domain, the dileucine-like motif E59XXXLV64 and phosphoserines 52 and 56 of Vpu. Altogether, these results highlight that Vpu controls the volume of VCCs and promotes HIV-1 release from infected macrophages.IMPORTANCE HIV-1 infection of macrophages leads to the sequestration of newly formed viruses in virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The restriction factor BST2, which prevents HIV-1 dissemination by tethering budding viral particles, can be found in VCCs. The HIV-1 Vpu protein counteracts BST2. This study explores the interplay between Vpu and BST2 in the viral protein functions on HIV-1 release and viral particle sequestration in VCCs in macrophages. The results show that Vpu controls the volume of VCCs and favors viral particle release. These Vpu functions partly depend on Vpu's ability to antagonize BST2. This study highlights that the transmembrane domain of Vpu and two motifs of the Vpu cytoplasmic domain are required for these functions. These motifs were notably involved in the control of the volume of VCCs by Vpu but were dispensable for the prevention of the specific accumulation of BST2 in these structures.

The protective role of microRNA-21 against coxsackievirus B3 infection through targeting the MAP2K3/P38 MAPK signaling pathway.

BACKGROUND: The P38 mitogen-activated protein kinase (MAPK) pathway plays an essential role in CVB3-induced diseases. We previously demonstrated microRNA-21 has potential inhibitory effect on the MAP2K3 which locates upstream of P38 MAPK and was upregulated in mouse hearts upon CVB3 infection. However, the effect and underlying mechanism of miRNA-21 on CVB3 infection remain unclear. METHODS: We detected continuous changes of cellular miRNA-21 and P38 MAPK proteins expression profiling post CVB3 infection in vitro within 12 h. P38 MAPK signaling was inhibited by the specific inhibitor, small interfering RNA and miRNA-21 mimic in vitro, CVB3 replication, cell apoptosis rate and proliferation were detected. Viral load in the mice heart, cardiomyocyte apoptosis rate and histological of the heart were also detected in the mice model of viral myocarditis pretreated with miRNA-21-lentivirus. RESULTS: We observed significant upregulation of miRNA-21 expression followed by suppression of the MAP2K3/P38 MAPK signaling in CVB3-infected Hela cells. The inactivation of the MAP2K3/P38 MAPK signaling by P38 MAPK specific inhibitor, small interfering RNA against MAP2K3, or miRNA-21 overexpression significantly inhibited viral progeny release from CVB3-infected cells. Mechanistically, when compared with control miRNA, miRNA-21 showed no effect on capsid protein VP1 expression and viral load within host cells, while significantly reversing CVB3-induced caspase-3 activation and cell apoptosis rate, further promoting proliferation of infected cells, which indicates the inhibitory effect of miRNA-21 on CVB3 progeny release. In the in vivo study, when compared with control miRNA, miRNA-21 pretreatment remarkably inactivated the MAP2K3/P38 MAPK signaling in mice and protected them against CVB3 infection as evidenced by significantly alleviated cell apoptosis rate, reduced viral titers, necrosis in the heart as well as by remarkably prolonged survival time. CONCLUSIONS: miRNA-21 were reverse correlated with P38 MAPK activation post CVB3 infection, miRNA-21 overexpression significantly inhibited viral progeny release and decreased myocytes apoptosis rate in vitro and in vivo, suggesting that miRNA-21 may serve as a potential therapeutic agent against CVB3 infection through targeting the MAP2K3/P38 MAPK signaling.

Effect of betulinic acid and its ionic derivatives on M-MuLV replication.

Murine leukemia virus (MuLV) is a retrovirus known causing leukemia and neurological disorders in mice, and its viral life cycle and pathogenesis have been investigated extensively over the past decades. As a natural antiviral agent, betulinic acid is a pentacyclic triterpenoid that can be found in the bark of several species of plants (particularly the white birch). One of the hurdles for betulinic acid to release its antiviral potency is its poor water solubility. In this study, we synthesized more water-soluble ionic derivatives of betulinic acid, and examined their activities against Moloney MuLV (M-MuLV). The mouse fibroblast cells stably infected with M-MuLV, 43D cells, were treated with various doses of betulinic acids and its derivatives, and the viral structural protein Gag in cells and media were detected by western blots. Two ionic derivatives containing the benzalkonium cation were found to inhibit the virus production into media and decreased Gag in cells. However, a cell proliferation assay showed that the benzalkonium cation inhibited the growth of 43D cells, suggesting that our ionic derivatives limited virus production through the inhibition of metabolism in 43D cells. Interestingly, all of these betulinic acid compounds exhibited a minimum impact on the processing and release of Gag from 43D cells, which outlines the differences of viral maturation between MuLV and human immunodeficiency virus.

Heparanase Upregulation Contributes to Porcine Reproductive and Respiratory Syndrome Virus Release.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause substantial economic losses to the pig industry worldwide. Heparan sulfate (HS) is used by PRRSV for initial attachment to target cells. However, the role of HS in the late phase of PRRSV infection and the mechanism of virus release from host cells remain largely unknown. In this study, we showed that PRRSV infection caused a decrease in HS expression and upregulated heparanase, the only known enzyme capable of degrading HS. We subsequently demonstrated that the NF-κB signaling pathway and cathepsin L protease were involved in regulation of PRRSV infection-induced heparanase. In addition, we found that ablation of heparanase expression using small interfering RNA duplexes increased cell surface expression of HS and suppressed PRRSV replication and release, whereas overexpression of heparanase reduced HS surface expression and enhanced PRRSV replication and release. These data suggest that PRRSV activates NF-κB and cathepsin L to upregulate and process heparanase, and then the active heparanase cleaves HS, resulting in viral release. Our findings provide new insight into the molecular mechanism of PRRSV egress from host cells, which might help us to further understand PRRSV pathogenesis.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes great economic losses each year to the pig industry worldwide. The molecular mechanism of PRRSV release from host cells largely remains a mystery. In this study, we demonstrate that PRRSV activates NF-κB and cathepsin L to upregulate and process heparanase, and then the active heparanase is released to the extracellular space and exerts enzymatic activity to cleave heparan sulfate, resulting in viral release. Our findings provide new insight into the molecular mechanism of PRRSV egress from host cells, which might help us to further understand PRRSV pathogenesis.

c-Jun N-terminal kinase activity is required for efficient respiratory syncytial virus production.

Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly, leading to more deaths than influenza each year worldwide. With no RSV antiviral or efficacious vaccine currently available, improved understanding of the host-RSV interaction is urgently required. Here we examine the contribution to RSV infection of the host stress-regulated c-Jun N-terminal kinase (JNK), for the first time. Peak JNK1/2 phosphoactivation is observed at ∼24 h post-infection, correlating with the time of virus assembly. The release of infectious RSV virions from infected cells was significantly reduced by either JNK1/2 siRNA knockdown or treatment with the JNK-specific inhibitor, JNK-IN-VIII. High resolution microscopy confirmed RSV accumulation in the host cell cytoplasm. The results implicate JNK1/2 as a key host factor for RSV virus production, raising the possibility of agents targeting JNK activity as potential anti-RSV therapeutics.

Chlorogenic acid inhibits porcine deltacoronavirus release by targeting apoptosis.

Porcine deltacoronavirus (PDCoV), belonging to family Coronaviridae, genus Deltacoronavirus, can cause acute diarrhea in piglets, and also possesses cross-species transmission potential, leading to severe economic losses and threatening public health. However, no approved drug against PDCoV infection is available. Here, we investigated the antiviral effect of chlorogenic acid (CGA), the main active component of Lonicerae Japonicae Flos, against PDCoV infection. The results showed that CGA inhibited the replication of PDCoV significantly both in LLC-PK1 and ST cells, with a selectivity index greater than 80. CGA decreased the synthesis of PDCoV viral RNA and protein, and viral titers in a dose-dependent manner. The results of the time-of-addition assay indicated that CGA mainly affected the early stage of virus replication and viral release. Moreover, CGA significantly reduced apoptosis caused by PDCoV infection, and the application of apoptosis agonist and inhibitor revealed that apoptosis could promote progeny virus release. Further study demonstrated that CGA can inhibit virus release by directly targeting apoptosis caused by PDCoV infection. In conclusion, CGA is an effective agent against PDCoV, which provides a foundation for drug development for the treatment of PDCoV and other coronavirus infections.

Inhibition of HIV-1 release by ADAM metalloproteinase inhibitors.

HIV-1 gp120 glycan binding to C-type lectin adhesion receptor L-selectin/CD62L on CD4 T cells facilitates viral attachment and entry. Paradoxically, the adhesion receptor impedes HIV-1 budding from infected T cells and the viral release requires the shedding of CD62L. To systematically investigate CD62L-shedding mediated viral release and its potential inhibition, we screened compounds specific for serine-, cysteine-, aspartyl-, and Zn-dependent proteases for CD62L shedding inhibition and found that a subclass of Zn-metalloproteinase inhibitors, including BB-94, TAPI, prinomastat, GM6001, and GI25423X, suppressed CD62L shedding. Their inhibition of HIV-1 infections correlated with enzymatic suppression of both ADAM10 and 17 activities and expressions of these ADAMs were transiently induced during the viral infection. These metalloproteinase inhibitors are distinct from the current antiretroviral drug compounds. Using immunogold labeling of CD62L, we observed association between budding HIV-1 virions and CD62L by transmission electron microscope, and the extent of CD62L-tethering of budding virions increased when the receptor shedding is inhibited. Finally, these CD62L shedding inhibitors suppressed the release of HIV-1 virions by CD4 T cells of infected individuals and their virion release inhibitions correlated with their CD62L shedding inhibitions. Our finding reveals a new therapeutic approach targeted at HIV-1 viral release.

Evaluating anti-viral effect of Ivermectin on porcine epidemic diarrhea virus and analyzing the related genes and signaling pathway by RNA-seq in vitro.

Porcine epidemic diarrhea virus (PEDV) has catastrophic impacts on the global pig industry. However, there remains no effective drugs for PEDV infection. Ivermectin is an FDA-approved anthelmintic drug used to treat worm infections. In this study, we reported the broad-spectrum antiviral activity of Ivermectin in vitro. Ivermectin can inhibit PEDV infections of different genotypes. Avermectin derivatives can also inhibit PEDV infections. A time of addition assay showed that Ivermectin exhibited potent anti-PEDV activity when added simultaneously with or post virus infection. Furthermore, Ivermectin significantly inhibited the late stage of viral infection by affecting viral release. RNA sequencing indicates Ivermectin induces cell cycle arrest, which may be related to its ability to inhibit viral release. Interestingly, when combined with Niclosamide, Ivermectin demonstrated an enhanced anti-PEDV effect. These findings highlight Ivermectin as a novel antiviral agent with potential for the development of drugs against PEDV infection.

HDAC-Specific Inhibitors Induce the Release of Porcine Epidemic Diarrhea Virus via the COPII-Coated Vesicles.

Porcine epidemic diarrhea virus (PEDV) is an alpha-coronavirus causing acute diarrhea and high mortality in neonatal suckling piglets, resulting in huge economic losses for the global swine industry. The replication, assembly and cell egression of PEDV, an enveloped RNA virus, are mediated via altered intracellular trafficking. The underlying mechanisms of PEDV secretion are poorly understood. In this study, we found that the histone deacetylase (HDAC)-specific inhibitors, trichostatin A (TSA) and sodium butyrate (NaB), facilitate the secretion of infectious PEDV particles without interfering with its assembly. We found that PEDV N protein and its replicative intermediate dsRNA colocalize with coat protein complex II (COPII)-coated vesicles. We also showed that the colocalization of PEDV and COPII is enhanced by the HDAC-specific inhibitors. In addition, ultrastructural analysis revealed that the HDAC-specific inhibitors promote COPII-coated vesicles carrying PEDV virions and the secretion of COPII-coated vesicles. Consistently, HDAC-specific inhibitors-induced PEDV particle secretion was abolished by Sec24B knockdown, implying that the HDAC-specific inhibitors-mediated COPII-coated vesicles are required for PEDV secretion. Taken together, our findings provide initial evidence suggesting that PEDV virions can assemble in the endoplasmic reticulum (ER) and bud off from the ER in the COPII-coated vesicles. HDAC-specific inhibitors promote PEDV release by hijacking the COPII-coated vesicles.

CREB3 Plays an Important Role in HPSE-Facilitated HSV-1 Release in Human Corneal Epithelial Cells.

Herpes simplex virus type-1 (HSV-1) exploits several host factors to enhance its replication and release from infected cells. It induces the production of host enzyme heparanase (HPSE) to aid in egress. While the mechanism by which HPSE assists in viral release is well-characterized, other host factors that are recruited along with HPSE for viral release are less well understood. In this study, we identify cyclic-AMP-responsive element-binding protein3 (CREB3) as a key player in HPSE-facilitated HSV-1 egress. When CREB3 is transiently upregulated in human corneal epithelial cells, HSV-1 release from the infected cells is correspondingly enhanced. This activity is linked to HPSE expression such that HPSE-transfected corneal epithelial (HCE) cells more highly express CREB3 than wild-type cells while the cells knocked out for HPSE show very little CREB3 expression. CREB3-transfected HCE cells showed significantly higher export of HPSE upon infection than wild-type cells. Our data suggests that coat protein complex II (COPII), which mediates HPSE trafficking, is also upregulated via a CREB3-dependent pathway during HSV-1 infection. Finally, the co-transfection of CREB3 and HPSE in HCE cells shows the highest viral release compared to either treatment alone, establishing CREB3 as a key player in HPSE-facilitated HSV-1 egress.

Role of Heparanase and Syndecan-1 in HSV-1 Release from Infected Cells.

Herpes Simplex Virus 1 (HSV-1) is a neurotropic human virus that belongs to the Alphaherpesvirinae subfamily of Herpesviridae. Establishment of its productive infection and progression of disease pathologies depend largely on successful release of virions from the virus-producing cells. HSV-1 is known to exploit many host factors for its release. Recent studies have shown that heparanase (HPSE) is one such host enzyme that is recruited for this purpose. It is an endoglycosidase that cleaves heparan sulfate (HS) from the surface of infected cells. HS is a virus attachment coreceptor that is commonly found on cell surfaces as HS proteoglycans e.g., syndecan-1 (SDC-1). The current model suggests that HSV-1 during the late stage of infection upregulates HPSE, which in turn enhances viral release by removing the virus-trapping HS moieties. In addition to its role in directly enabling viral release, HPSE accelerates the shedding of HS-containing ectodomains of SDC-1, which enhances HSV-1 release via a similar mechanism by upregulating CREB3 and COPII proteins. This review outlines the role of HPSE and SDC-1 as newly assigned host factors that facilitate HSV-1 release during a lytic infection cycle.

Enhanced tumor targeting and timely viral release of mesenchymal stem cells/oncolytic virus complex due to GRP78 and inducible E1B55K expressions greatly increase the antitumor effect of systemic treatment.

Systemic delivery of oncolytic viruses has been widely regarded as an impractical option for antitumor treatment. Here, we selected two target genes as leading components, and significant therapeutic effects were obtained by simultaneously reducing the expression of transforming growth factor ß 1 (TGF-ß1) and heat shock protein 27 (HSP27) in various cancer cell types. Downregulation of HSP27 reduced the cellular levels of tumor progression-related proteins, and the simultaneous downregulation of HSP27 and TGF-ß1 increased tumor cell death beyond that observed with TGF-ß1 downregulation alone. To increase the potential for systemic administration, we generated modified mesenchymal stem cells (MSCs) to act as oncolytic adenovirus factories and carriers and assessed bioavailability in tumors after MSC injection. The MSCs were modified to express 78-kDa glucose-regulated protein (GRP78) and adenovirus early-region 1B 55 kDa (E1B55K). The tightly controlled inducible system permitted selective timing of viral release from carrier MSCs within the tumor. This approach significantly improved viral production, tumor targeting, timely viral release at the tumor site, and antitumor efficacy of the oncolytic adenovirus. These combined results demonstrate that engineered MSCs can significantly enhance the antitumor effects of oncolytic viruses without adverse safety issues, which may greatly extend the clinical applicability of oncolytic adenoviruses.