Fangchinoline Inhibits African Swine Fever Virus Replication by Suppressing the AKT/mTOR/NF-κB Signaling Pathway in Porcine Alveolar Macrophages.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is one of the most important infectious diseases that cause high morbidity and mortality in pigs and substantial economic losses to the pork industry of affected countries due to the lack of effective vaccines. The need to develop alternative robust antiviral countermeasures, especially anti-ASFV agents, is of the utmost urgency. This study shows that fangchinoline (FAN), a bisbenzylisoquinoline alkaloid found in the roots of Stephania tetrandra of the family Menispermaceae, significantly inhibits ASFV replication in porcine alveolar macrophages (PAMs) at micromolar concentrations (IC50 = 1.66 µM). Mechanistically, the infection of ASFV triggers the AKT/mTOR/NF-κB signaling pathway. FAN significantly inhibits ASFV-induced activation of such pathways, thereby suppressing viral replication. Such a mechanism was confirmed using an AKT inhibitor MK2206 as it inhibited AKT phosphorylation and ASFV replication in PAMs. Altogether, the results suggest that the AKT/mTOR pathway could potentially serve as a treatment strategy for combating ASFV infection and that FAN could potentially emerge as an effective novel antiviral agent against ASFV infections and deserves further in vivo antiviral evaluations.

The MGF300-2R Protein of African Swine Fever Virus Promotes IKKß Ubiquitination by Recruiting the E3 Ubiquitin Ligase TRIM21.

African swine fever (ASF) is an acute, hemorrhagic, highly contagious disease in pigs caused by African swine fever virus (ASFV). Our previous study identified that the ASFV MGF300-2R protein functions as a virulence factor and found that MGF300-2R degrades IKKß via selective autophagy. However, the E3 ubiquitin ligase responsible for IKKß ubiquitination during autophagic degradation still remains unknown. In order to solve this problem, we first pulled down 328 proteins interacting with MGF300-2R through immunoprecipitation-mass spectrometry. Next, we analyzed and confirmed the interaction between the E3 ubiquitin ligase TRIM21 and MGF300-2R and demonstrated the catalytic role of TRIM21 in IKKß ubiquitination. Finally, we indicated that the degradation of IKKß by MGF300-2R was dependent on TRIM21. In summary, our results indicate TRIM21 is the E3 ubiquitin ligase involved in the degradation of IKKß by MGF300-2R, thereby augmenting our understanding of the functions of MGF300-2R and offering insights into the rational design of live attenuated vaccines and antiviral strategies against ASF.

The proteomic analysis uncovers the cellular responses to the African swine fever virus membrane proteins p54, p17, and pB117L.

African swine fever virus (ASFV) infection causes African swine fever (ASF), a highly contagious and fatal disease that poses severe threat to swine production. To gain insights into the host responses to ASFV, we generated recombinant adenovirus Ad5 expressing viral membrane proteins p54, p17, and pB117L individually and infected an alveolar cell line, 3D4/21, with these recombinant viruses. Then, the cell lysates were analyzed using label-free quantification proteomic analysis method. A total of 2158 differentially expressed proteins (DEPs) were identified, of which 817, 466, and 875 proteins were from Ad5-p54-, Ad5-p17-, Ad5-pB117L-infected 3D4/21 cells, respectively. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed distinct yet interconnecting patterns of protein interaction networks. Specifically, the Ad5-p54 virus infection enriched the DEPs primarily involved in the metabolic pathways, endocytosis, adherens junction, and SNARE interactions in vesicular transport. The Ad5-p17 virus infection enriched the DEPs in endocytosis, ubiquitin-mediated proteolysis, N-Glycan biosynthesis, and apoptosis, while the Ad5-pB117L virus infection enriched the DEPs in metabolic pathways, endocytosis, oxidative phosphorylation, and focal adhesion. In summary, these results provide a comprehensive proteinomics analysis of the cellular responses to three ASFV membrane proteins, thus facilitating our understanding of ASFV pathogenesis.

Function investigation of p11.5 in ASFV infection.

Virus replication relies on complex interactions between viral proteins. In the case of African swine fever virus (ASFV), only a few such interactions have been identified so far. In this study, we demonstrate that ASFV protein p72 interacts with p11.5 using co-immunoprecipitation and liquid chromatography-mass spectrometry (LC-MS). It was found that protein p72 interacts specifically with p11.5 â€‹at sites amino acids (aa) 1-216 of p72 and aa 1-68 of p11.5. To assess the importance of p11.5 in ASFV infection, we developed a recombinant virus (ASFVGZΔA137R) by deleting the A137R gene from the ASFVGZ genome. Compared with ASFVGZ, the infectious progeny virus titers of ASFVGZΔA137R were reduced by approximately 1.0 logs. In addition, we demonstrated that the growth defect was partially attributable to a higher genome copies-to-infectious virus titer ratios produced in ASFVGZΔA137R-infected MA104 â€‹cells than in those infected with ASFVGZ. This finding suggests that MA104 â€‹cells infected with ASFVGZΔA137R may generate larger quantities of noninfectious particles. Importantly, we found that p11.5 did not affect virus-cell binding or endocytosis. Collectively, we show for the first time the interaction between ASFV p72 and p11.5. Our results effectively provide the relevant information of the p11.5 protein. These results extend our understanding of complex interactions between viral proteins, paving the way for further studies of the potential mechanisms and pathogenesis of ASFV infection.

African swine fever virus MGF505-6R attenuates type I interferon production by targeting STING for degradation.

African swine fever (ASF) is an acute hemorrhagic and devastating infectious disease affecting domestic pigs and wild boars. It is caused by the African swine fever virus (ASFV), which is characterized by genetic diversity and sophisticated immune evasion strategies. To facilitate infection, ASFV encodes multiple proteins to antagonize host innate immune responses, thereby contributing to viral virulence and pathogenicity. The molecular mechanisms employed by ASFV-encoded proteins to modulate host antiviral responses have not been comprehensively elucidated. In this study, it was observed that the ASFV MGF505-6R protein, a member of the multigene family 505 (MGF505), effectively suppressed the activation of the interferon-beta (IFN-ß) promoter, leading to reduced mRNA levels of antiviral genes. Additional evidence has revealed that MGF505-6R antagonizes the cGAS-STING signaling pathway by interacting with the stimulator of interferon genes (STING) for degradation in the autophagy-lysosomal pathway. The domain mapping revealed that the N-terminal region (1-260aa) of MGF505-6R is the primary domain responsible for interacting with STING, while the CTT domain of STING is crucial for its interaction with MGF505-6R. Furthermore, MGF505-6R also inhibits the activation of STING by reducing the K63-linked polyubiquitination of STING, leading to the disruption of STING oligomerization and TANK binding kinase 1 (TBK1) recruitment, thereby impairing the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3). Collectively, our study elucidates a novel strategy developed by ASFV MGF505-6R to counteract host innate immune responses. This discovery may offer valuable insights for further exploration of ASFV immune evasion mechanisms and antiviral strategies.

African swine fever virus infection regulates pyroptosis by cleaving gasdermin A via active caspase-3 and caspase-4.

African swine fever, caused by the African swine fever virus (ASFV), is a viral hemorrhagic disease that affects domestic pigs and wild boars. ASFV infection causes extensive tissue damage, and the associated mechanism is poorly understood. Pyroptosis is characterized by the activation of inflammatory caspases and pore formation in the cellular plasma membrane, resulting in the release of inflammatory cytokines and cell damage. How ASFV infection regulates pyroptosis remains unclear. Here, using siRNA assay and overexpression methods, we report that ASFV infection regulated pyroptosis by cleaving the pyroptosis execution protein gasdermin A (GSDMA). ASFV infection activated caspase-3 and caspase-4, which specifically cleaved GSDMA at D75-P76 and D241-V242 to produce GSDMA into five fragments, including GSDMA-N1-75, GSDMA-N1-241, and GSDMA-N76-241 fragments at the N-terminal end of GSDMA. Only GSDMA-N1-241, which was produced in the late stage of ASFV infection, triggered pyroptosis and inhibited ASFV replication. The fragments, GSDMA-N1-75 and GSDMA-N76-241, lose the ability to induce pyroptosis. Overall ASFV infection differentially regulates pyroptosis by GSDMA in the indicated phase, which may be conducive to its own replication. Our findings reveal a novel molecular mechanism for the regulation of pyroptosis.

Infection of Human Macrophage-Like Cells by African Swine Fever Virus.

BACKGROUND: The African swine fever (ASF) virus (ASFV) and ASF-like viral sequences were identified in human samples and sewage as well as in different water environments. Pigs regularly experience infections by the ASFV. The considerable stability of the virus in the environment suggests that there is ongoing and long-term contact between humans and the ASFV. However, humans exhibit resistance to the ASFV, and the decisive factor in developing infection in the body is most likely the reaction of target macrophages to the virus. Therefore, this study aimed to characterize the responses of human macrophages to the virus and explore the distinct features of the viral replication cycle within human macrophages. METHODS: The ASFV Armenia/07 strain was used in all experiments. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the ASFV gene expression; flow cytometry analysis was performed to evaluate the effects of the inactive and active ASFV (inASFV and aASFV) treatments on the phenotype of THP-1-derived macrophages (Mφ0) and inflammatory markers. Moreover, other methods such as cell viability and apoptosis assays, staining techniques, phagocytosis assay, lysosome-associated membrane protein (LAMP-1) cytometry, and cytokine detection were used during experiments. RESULTS: Our findings showed that the virus initiated replication by entering human macrophages. Subsequently, the virus shed its capsid and initiated the transcription of numerous viral genes, and at least some of these genes executed their functions. In THP-1-derived macrophages (Mφ0), the ASFV implemented several functions to suppress cell activity, although the timing of their implementation was slower compared with virus-sensitive porcine alveolar macrophages (PAMs). Additionally, the virus could not complete the entire replication cycle in human Mφ0, as indicated by the absence of viral factories and a decrease in infectious titers of the virus with each subsequent passage. Overall, the infection of Mφ0 with the ASFV caused significant alterations in their phenotype and functions, such as increased TLR2, TLR3, CD80, CD36, CD163, CXCR2, and surface LAMP-1 expression. Increased production of the tumor necrosis factor (TNF) and interleukin (IL)-10 and decreased production of interferon (IFN)-α were also observed. Taken together, the virus enters human THP-1-derived macrophages, starts transcription, and causes immunological responses by target cells but cannot complete the replicative cycle. CONCLUSION: These findings suggest that there may be molecular limitations within human macrophages that at least partially restrict the complete replication of the ASFV. Understanding the factors that hinder viral replication in Mφ0 can provide valuable insights into the host-virus interactions and the mechanisms underlying the resistance of human macrophages to the ASFV.

Deoxycholic acid inhibits ASFV replication by inhibiting MAPK signaling pathway.

African swine fever (ASF) is an acute, febrile, highly contagious infection of pigs caused by the African swine fever virus (ASFV). The purpose of this study is to understand the molecular mechanism of ASFV infection and evaluate the effect of DCA on MAPK pathway, so as to provide scientific basis for the development of new antiviral drugs. The transcriptome analysis found that ASFV infection up-regulated the IL-17 and MAPK signaling pathways to facilitate viral replication. Metabolome analysis showed that DCA levels were up-regulated after ASFV infection, and that exogenous DCA could inhibit activation of the MAPK pathway by ASFV infection and thus inhibit viral replication. Dual-luciferase reporter assays were used to screen the genes of ASFV and revealed that I73R could significantly up-regulate the transcription level of AP-1 transcription factor in the MAPK pathway. Confocal microscopy demonstrated that I73R could promote AP-1 entry into the nucleus, and that DCA could inhibit the I73R-mediated nuclear entry of AP-1, inhibiting MAPK pathway, and I73R interacts with AP-1. These results indicated that DCA can inhibit ASFV-mediated activation of the MAPK pathway, thus inhibiting ASFV replication. This study provides a theoretical basis for research on ASF pathogenesis and for antiviral drug development.

African swine fever virus pH240R enhances viral replication via inhibition of the type I IFN signaling pathway.

African swine fever (ASF) is an acute, hemorrhagic, and severe infectious disease caused by ASF virus (ASFV) infection. At present, there are still no safe and effective drugs and vaccines to prevent ASF. Mining the important proteins encoded by ASFV that affect the virulence and replication of ASFV is the key to developing effective vaccines and drugs. In this study, ASFV pH240R, a capsid protein of ASFV, was found to inhibit the type I interferon (IFN) signaling pathway. Mechanistically, pH240R interacted with IFNAR1 and IFNAR2 to disrupt the interaction of IFNAR1-TYK2 and IFNAR2-JAK1. Additionally, pH240R inhibited the phosphorylation of IFNAR1, TYK2, and JAK1 induced by IFN-α, resulting in the suppression of the nuclear import of STAT1 and STAT2 and the expression of IFN-stimulated genes (ISGs). Consistent with these results, H240R-deficient ASFV (ASFV-∆H240R) infection induced more ISGs in porcine alveolar macrophages compared with its parental ASFV HLJ/18. We also found that pH240R enhanced viral replication via inhibition of ISGs expression. Taken together, our results clarify that pH240R enhances ASFV replication by inhibiting the JAK-STAT signaling pathway, which highlights the possibility of pH240R as a potential drug target.IMPORTANCEThe innate immune response is the host's first line of defense against pathogen infection, which has been reported to affect the replication and virulence of African swine fever virus (ASFV) isolates. Identification of ASFV-encoded proteins that affect the virulence and replication of ASFV is the key step in developing more effective vaccines and drugs. In this study, we found that pH240R interacted with IFNAR1 and IFNAR2 by disrupting the interaction of IFNAR1-TYK2 and IFNAR2-JAK1, resulting in the suppression of the expression of interferon (IFN)-stimulated genes (ISGs). Consistent with these results, H240R-deficient ASFV (ASFV-∆H240R) infection induces more ISGs' expression compared with its parental ASFV HLJ/18. We also found that pH240R enhanced viral replication via inhibition of ISGs' expression. Taken together, our findings showed that pH240R enhances ASFV replication by inhibiting the IFN-JAK-STAT axis, which highlights the possibility of pH240R as a potential drug target.

African Swine Fever Virus Host-Pathogen Interactions.

African swine fever virus is a complex double-stranded DNA virus that exhibits tropism for cells of the mononuclear phagocytic system. Virus replication is a multi-step process that involves the nucleus of the host cell as well the formation of large perinuclear sites where progeny virions are assembled prior to transport to, and budding through, the plasma membrane. Like many viruses, African swine fever virus reorganises the cellular architecture to facilitate its replication and has evolved multiple mechanisms to avoid the potential deleterious effects of host cell stress response pathways. However, how viral proteins and virus-induced structures trigger cellular stress pathways and manipulate the subsequent responses is still relatively poorly understood. African swine fever virus alters nuclear substructures, modulates autophagy, apoptosis and the endoplasmic reticulum stress response pathways. The viral genome encodes for at least 150 genes, of which approximately 70 are incorporated into the virion. Many of the non-structural genes have not been fully characterised and likely play a role in host range and modifying immune responses. As the field moves towards approaches that take a broader view of the effect of expression of individual African swine fever genes, we summarise how the different steps in virus replication interact with the host cell and the current state of knowledge on how it modulates the resulting stress responses.

Interactome between ASFV and host immune pathway proteins.

IMPORTANCE: African swine fever (ASF), caused by African swine fever virus (ASFV), has become a major crisis for the pork industry in recent years. The mechanism for ASFV pathology and the clinical symptoms difference of ASF between domestic pigs and reservoir hosts remain to be elucidated. We deciphered the comprehensive protein-protein interaction (PPI) network between ASFV and host immune pathways. The intensive PPI network contained both ASFV-host immune pathway PPI and ASFV-ASFV PPI information, providing a comprehensive ASFV-host interaction landscape. Furthermore, the ASFV-host PPI difference between domestic pigs and warthogs was explored, which will be instructive for exploring essential candidates involved in ASFV pathology. Moreover, we screened the inhibitory effect of ASFV proteins in the PPI with cGAS-STING pathway on IFN-I and NF-κB, further providing possible functions of ASFV-host PPI network in innate immune regulation.

African swine fever virus protein p17 promotes mitophagy by facilitating the interaction of SQSTM1 with TOMM70.

Viruses have developed different strategies to hijack mitophagy to facilitate their replication. However, whether and how African swine fever virus (ASFV) regulates mitophagy are largely unknown. Here, we found that the ASFV-encoded p17 induced mitophagy. Coimmunoprecipitation/mass spectrometry assays identified translocase of outer mitochondrial membrane 70 (TOMM70) as the protein that interacted with p17. The binding of TOMM70 to p17 promoted the binding of the mitophagy receptor SQSTM1 to TOMM70, led to engulfment of mitochondria by autophagosomes, and consequently decreased the number of mitochondria. Consistently, the levels of TOMM70 and TOMM20 decreased substantially after p17 expression or ASFV infection. Furthermore, p17-mediated mitophagy resulted in the degradation of mitochondrial antiviral signalling proteins and inhibited the production of IFN-α, IL-6 and TNFα. Overall, our findings suggest that ASFV p17 regulates innate immunity by inducing mitophagy via the interaction of SQSTM1 with TOMM70.

African swine fever virus pS273R antagonizes stress granule formation by cleaving the nucleating protein G3BP1 to facilitate viral replication.

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs are regulated by different stimulators including viral infection, which is involved in the antiviral activity of host cells to limit viral propagation. To survive, several viruses have been reported to execute various strategies, such as modulating SG formation, to create optimal surroundings for viral replication. African swine fever virus (ASFV) is one of the most notorious pathogens in the global pig industry. However, the interplay between ASFV infection and SG formation remains largely unknown. In this study, we found that ASFV infection inhibited SG formation. Through SG inhibitory screening, we found that several ASFV-encoded proteins are involved in inhibition of SG formation. Among them, an ASFV S273R protein (pS273R), the only cysteine protease encoded by the ASFV genome, significantly affected SG formation. ASFV pS273R interacted with G3BP1 (Ras-GTPase-activating protein [SH3 domain] binding protein 1), a vital nucleating protein of SG formation. Furthermore, we found that ASFV pS273R cleaved G3BP1 at the G140-F141 to produce two fragments (G3BP1-N1-140 and G3BP1-C141-456). Interestingly, both the pS273R-cleaved fragments of G3BP1 lost the ability to induce SG formation and antiviral activity. Taken together, our finding reveals that the proteolytic cleavage of G3BP1 by ASFV pS273R is a novel mechanism by which ASFV counteracts host stress and innate antiviral responses.

African Swine Fever Virus MGF110-7L Induces Host Cell Translation Suppression and Stress Granule Formation by Activating the PERK/PKR-eIF2α Pathway.

African swine fever (ASF) is a highly contagious and often lethal disease of pigs caused by ASF virus (ASFV) and recognized as the biggest killer in global swine industry. Despite exhibiting incredible self-sufficiency, ASFV remains unconditionally dependent on the host translation machinery for its mRNA translation. However, less is yet known regarding how ASFV-encoded proteins regulate host translation machinery in infected cells. Here, we examined how ASFV interacts with the eukaryotic initiation factor 2α (eIF2α) signaling axis, which directs host translation control and adaptation to cellular stress. We found that ASFV MGF110-7L, a previously uncharacterized member of the multigene family 110, remarkably enhanced the phosphorylation level of eIF2α. In porcine alveolar macrophage 3D4/21 and porcine kidney-15 cells, MGF110-7L triggered eIF2α signaling and the integrated stress response, resulting in the suppression of host translation and the formation of stress granules (SGs). Mechanistically, MGF110-7L-induced phosphorylation of eIF2α was mediated via protein kinase R (PKR) and PKR-like endoplasmic reticulum (ER) kinase (PERK), and this process was essential for host translation repression and SG formation. Notably, our subsequent analyses confirmed that MGF110-7L was overwhelmingly retained in the ER and caused a specific reorganization of the secretory pathway. Further proteomic analyses and biochemical experiments revealed that MGF110-7L could trigger ER stress and activate the unfolded protein response, thus contributing to eIF2α phosphorylation and translation reprogramming. Overall, our study both identifies a novel mechanism by which ASFV MGF110-7L subverts the host protein synthesis machinery and provides further insights into the translation regulation that occurs during ASFV infection. IMPORTANCE African swine fever (ASF) has become a socioeconomic burden and a threat to food security and biodiversity, but no commercial vaccines or antivirals are available currently. Understanding the viral strategies to subvert the host translation machinery during ASF virus (ASFV) infection could potentially lead to new vaccines and antiviral therapies. In this study, we dissected how ASFV MGF110-7L interacts with the eIF2α signaling axis controlling translational reprogramming, and we addressed the role of MGF110-7L in induction of cellular stress responses, eIF2α phosphorylation, translation suppression, and stress granule formation. These results define several molecular interfaces by which ASFV MGF110-7L subverts host cell translation, which may guide research on antiviral strategies and dissection of ASFV pathogenesis.

Functional Analysis and Proteomics Profiling of Extracellular Vesicles From Swine Plasma Infected by African Swine Fever Virus.

African swine fever (ASF) has brought excellent barriers to swine production in China and the world. Studies have shown that extracellular vesicles mediate the RNA and protein spread of pathogenic microorganisms and RNA and proteins. After infection by pathogenic microorganisms causes significant differences in the proteins contained within extracellular vesicles. Based on the above studies, the extracellular vesicles were extracted from ASF virus (ASFV)-infected swine plasma. And qPCR, western blot, and confocal experiment were carried out. The research shows that extracted extracellular vesicles significantly promote the replication of ASFV in susceptible and non-susceptible cells Proteomics analysis of the extracellular vesicle proteins revealed that ASFV infection could cause significant differences in the protein profile. This study demonstrates that extracellular vesicles play a critical role in ASFV replication and transmission and cause significant differences in the protein profile encapsulated in extracellular vesicles.

Role of African Swine Fever Virus Proteins EP153R and EP402R in Reducing Viral Persistence in Blood and Virulence in Pigs Infected with BeninΔDP148R.

The limited knowledge on the role of many of the approximately 170 proteins encoded by African swine fever virus restricts progress toward vaccine development. Previously, the DP148R gene was deleted from the genome of genotype I virulent Benin 97/1 isolate. This virus, BeninΔDP148R, induced transient moderate clinical signs after immunization and high levels of protection against challenge. However, the BeninΔDP148R virus and genome persisted in blood over a prolonged period. In the current study, deletion of either EP402R or EP153R genes individually or in combination from BeninΔDP148R genome was shown not to reduce virus replication in macrophages in vitro. However, deletion of EP402R dramatically reduced the period of infectious virus persistence in blood in immunized pigs from 28 to 14 days and virus genome from 59 to 14 days while maintaining high levels of protection against challenge. The additional deletion of EP153R (BeninΔDP148RΔEP153RΔEP402R) further attenuated the virus, and no viremia or clinical signs were observed postimmunization. This was associated with decreased protection and detection of moderate levels of challenge virus in blood. Interestingly, the deletion of EP153R alone from BeninΔDP148R did not result in further virus attenuation and did not reduce the period of virus persistence in blood. These results show that EP402R and EP153R have a synergistic role in reducing clinical signs and levels of virus in blood. IMPORTANCE African swine fever virus (ASFV) causes a disease of domestic pigs and wild boar which results in death of almost all infected animals. The disease has a high economic impact, and no vaccine is available. We investigated the role of two ASFV proteins, called EP402R and EP153R, in determining the levels and length of time virus persists in blood from infected pigs. EP402R causes ASFV particles and infected cells to bind to red blood cells. Deletion of the EP402R gene dramatically reduced virus persistence in blood but did not reduce the level of virus. Deletion of the EP153R gene alone did not reduce the period or level of virus persistence in blood. However, deleting both EP153R and EP402R resulted in undetectable levels of virus in blood and no clinical signs showing that the proteins act synergistically. Importantly, the infected pigs were protected following infection with the wild-type virus that kills pigs.

Deletion of the H240R Gene of African Swine Fever Virus Decreases Infectious Progeny Virus Production Due to Aberrant Virion Morphogenesis and Enhances Inflammatory Cytokine Expression in Porcine Macrophages.

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus that causes African swine fever, a lethal hemorrhagic disease that currently threatens the pig industry. Recent studies have identified the viral structural proteins of infectious ASFV particles. However, the functional roles of several ASFV structural proteins remain largely unknown. Here, we characterized the function of the ASFV structural protein H240R (pH240R) in virus morphogenesis. pH240R was identified as a capsid protein by using immunoelectron microscopy and interacted with the major capsid protein p72 by pulldown assays. Using a recombinant ASFV, ASFV-ΔH240R, with the H240R gene deleted from the wild-type ASFV (ASFV-WT) genome, we revealed that the infectious progeny virus titers were reduced by approximately 2.0 logs compared with those of ASFV-WT. Furthermore, we demonstrated that the growth defect was due to the generation of noninfectious particles with a higher particle-to-infectious titer ratio in ASFV-ΔH240R-infected primary porcine alveolar macrophages (PAMs) than in those infected with ASFV-WT. Importantly, we found that pH240R did not affect virus-cell binding, endocytosis, or egress but did affect ASFV assembly; noninfectious virions containing large aberrant tubular and bilobulate structures comprised nearly 98% of all virions observed in ASFV-ΔH240R-infected PAMs by electron microscopy. Notably, we demonstrated that ASFV-ΔH240R infection induced high-level expression of inflammatory cytokines in PAMs. Collectively, we show for the first time that pH240R is essential for ASFV icosahedral capsid formation and infectious particle production. Also, these results highlight the importance of pH240R in ASFV morphogenesis and provide a novel target for the development of ASF vaccines and antivirals. IMPORTANCE African swine fever is a lethal hemorrhagic disease of global concern that is caused by African swine fever virus (ASFV). Despite extensive research, there exist relevant gaps in knowledge of the fundamental biology of the viral life cycle. In this study, we identified pH240R as a capsid protein that interacts with the major capsid protein p72. Furthermore, we showed that pH240R was required for the efficient production of infectious progeny virions as indicated by the H240R-deleted ASFV mutant (ASFV-ΔH240R). More specifically, pH240R directs the morphogenesis of ASFV toward the icosahedral capsid in the process of assembly. In addition, ASFV-ΔH240R infection induced high-level expression of inflammatory cytokines in primary porcine alveolar macrophages. Our results elucidate the role of pH240R in the process of ASFV assembly, which may instruct future research on effective vaccines or antiviral strategies.

African Swine Fever Virus Regulates Host Energy and Amino Acid Metabolism To Promote Viral Replication.

African swine fever is one of the most serious viral diseases caused by African swine fever virus (ASFV). The metabolic changes induced by ASFV infection remain unknown. Here, porcine alveolar macrophages (PAMs) infected with ASFV was analyzed by ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) in combination with multivariate statistical analysis. A total of 90 metabolites were significantly changed after ASFV infection, and most of them were amino acids and tricarboxylic acid (TCA) cycle intermediates. ASFV infection induced an increase in most of amino acids in the host during the early stages of infection, and amino acids decreased in the late stages of infection. ASFV infection did not significantly affect the glycolysis pathway, whereas it induced increases in citrate, succinate, α-ketoglutarate, and oxaloacetate levels in the TCA cycle, suggesting that ASFV infection promoted the TCA cycle. The activities of aspartate aminotransferase and glutamate production were significantly elevated in ASFV-infected cells and pigs, resulting in reversible transition between TCA cycle and amino acid synthesis. Aspartate, glutamate, and TCA cycle were essential for ASFV replication. In addition, ASFV infection induced an increase in lactate level using lactate dehydrogenase, which led to low expression of beta interferon (IFN-ß) and increased ASFV replication. Our data, for the first time, indicate that ASFV infection controls IFN-ß production through RIG-I-mediated signaling pathways. These data identified a novel mechanism evolved by ASFV to inhibit host innate immune responses and provide insights for development of new preventive or therapeutic strategies targeting the altered metabolic pathways. IMPORTANCE In order to promote viral replication, viruses often cause severe immunosuppression and seize organelles to synthesize a large number of metabolites required for self-replication. African swine fever virus (ASFV) has developed many strategies to evade host innate immune responses. However, the impact of ASFV infection on host cellular metabolism remains unknown. Here, for the first time, we analyzed the metabolomic profiles of ASFV-infected PAMs. ASFV infection increased host TCA cycle and amino acid metabolism. Aspartate, glutamate, and TCA cycle promoted ASFV replication. ASFV infection also induced the increase of lactate production to inhibit innate immune responses for self-replication. This study identified novel immune evasion mechanisms utilized by ASFV and provided insights into ASFV-host interactions, which is critical for guiding the design of new prevention strategies against ASFV targeting the altered metabolic pathways.

The A179L Gene of African Swine Fever Virus Suppresses Virus-Induced Apoptosis but Enhances Necroptosis.

A179L, a non-structural protein of African swine fever virus (ASFV), is capable of suppressing apoptosis by binding the BH3 domain of the pro-apoptotic Bcl-2 family proteins via a conserved ligand binding groove. Our present study aims to determine if A179L affects necroptosis, the second form of programmed cell death induced by DNA and RNA viruses. Here we report that A179L enhanced TNF-α or TSZ (TNF-α, Smac, and Z-Vad)-induced receptor-interacting protein kinase (RIPK1), RIPK3, and mixed lineage kinase domain like peudokinase (MLKL) phosphorylation in L929 cells, a murine fibrosarcoma cell line. Sytox green staining revealed that A179L significantly increased the number of necroptotic cells in TSZ-treated L929 cells. Using human herpes simplex virus 1 (HSV-1) to model DNA virus-induced cell death, we found that A179L blocked the HSV-1-induced cleavage of poly (ADP-ribose) polymerase (PARP), caspase 8, and caspase 3 and decreased the number of apoptotic cells in HSV-1-infected IPEC-DQ cells, a porcine intestinal epithelial cell line. In contrast, A179L transfection of IPEC-DQ cells enhanced HSV-1-induced RIPK1, RIPK3, and MLKL phosphorylation and increased the number of necroptotic cells. Consistently, A179L also suppressed apoptosis but enhanced the necroptosis induced by two RNA viruses, Sendai virus (SeV) and influenza virus (IAV). Our study uncovers a previously unrecognized role of A179L in regulating cell death and suggests that A179L re-directs its anti-apoptotic activity to necroptosis.

African Swine Fever Virus A528R Inhibits TLR8 Mediated NF-κB Activity by Targeting p65 Activation and Nuclear Translocation.

African swine fever (ASF) is mainly an acute hemorrhagic disease which is highly contagious and lethal to domestic pigs and wild boars. The global pig industry has suffered significant economic losses due to the lack of an effective vaccine and treatment. The African swine fever virus (ASFV) has a large genome of 170-190 kb, encoding more than 150 proteins. During infection, ASFV evades host innate immunity via multiple viral proteins. A528R is a very important member of the polygene family of ASFV, which was shown to inhibit IFN-ß production by targeting NF-κB, but its mechanism is not clear. This study has shown that A528R can suppress the TLR8-NF-κB signaling pathway, including the inhibition of downstream promoter activity, NF-κB p65 phosphorylation and nuclear translocation, and the antiviral and antibacterial activity. Further, we found the cellular co-localization and interaction between A528R and p65, and ANK repeat domains of A528R and RHD of p65 are involved in their interaction and the inhibition of p65 activity. Therefore, we conclude that A528R inhibits TLR8-NF-κB signaling by targeting p65 activation and nuclear translocation.