Identification of several African swine fever virus replication inhibitors by screening of a library of FDA-approved drugs.

African swine fever (ASF) caused by African swine fever virus (ASFV) is a highly infectious and lethal swine disease. Currently, there is only one novel approved vaccine and no antiviral drugs for ASFV. In the study, a high-throughput screening of an FDA-approved drug library was performed to identify several drugs against ASFV infection in primary porcine alveolar macrophages. Triapine and cytarabine hydrochloride were identified as ASFV infection inhibitors in a dose-dependent manner. The two drugs executed their antiviral activity during the replication stage of ASFV. Furthermore, molecular docking studies showed that triapine might interact with the active center Fe2+ in the small subunit of ASFV ribonucleotide reductase while cytarabine hydrochloride metabolite might interact with three residues (Arg589, Lys593, and Lys631) of ASFV DNA polymerase to block new DNA chain extension. Taken together, our results suggest that triapine and cytarabine hydrochloride displayed significant antiviral activity against ASFV in vitro.

Coreceptor AXL Facilitates African Swine Fever Virus Entry via Apoptotic Mimicry.

African swine fever (ASF) is an acute and hemorrhagic infectious disease caused by African swine fever virus (ASFV), which is listed as an animal epidemic disease that must be reported by The World Organization for Animal Health and that causes serious economic losses to China and even the whole world. Currently, the entry mechanism of ASFV is not fully understood. Especially in the early stages of virus entry, the host factors required for ASFV entry have not yet been identified and characterized. In this study, we demonstrated that ASFV externalized phosphatidylserine (PS) on the envelope functioned as viral apoptotic mimicry, which interacts with AXL, a tyrosine kinase receptor, to mediate ASFV entry into porcine alveolar macrophages (PAMs). We found that AXL was the most pronounced phosphatidylserine receptor (PSR) affecting ASFV entry in PAMs by RNA interference screening. Knockout AXL gene expression remarkably decreased ASFV internalization and replication in MA104 cells. Furthermore, the antibody against AXL extracellular domains effectively inhibited the ASFV entry. Consistent with these results, the deletion of the intracellular kinase domain of AXL and the treatment of the AXL inhibitor, R428, significantly inhibited the internalization of ASFV. Mechanistically, AXL facilitated the internalization of ASFV virions via macropinocytosis. Collectively, we provide evidence that AXL is a coreceptor for ASFV entry into PAMs, which expands our knowledge of ASFV entry and provides a theoretical basis for identifying new antiviral targets. IMPORTANCE African swine fever (ASF) is a highly contagious infectious disease caused by the ASF virus (ASFV), with a mortality rate of up to 100%. ASFV has caused huge economic losses to pig farming worldwide. Specific cellular surface receptors are considered crucial determinants of ASFV tropism. However, the host factors required for ASFV entry have not yet been identified, and the molecular mechanism of its entry remains unclear. Here, we found that ASFV utilized phosphatidylserine (PS) on the surface of virions to masquerade as apoptotic mimicry and facilitated virus entry by interacting with host factor AXL. We found that knockout of AXL remarkably decreased ASFV internalization and replication. The antibody against AXL extracellular domains and AXL inhibitor R428 significantly inhibited the internalization of ASFV via macropinocytosis. The current work deepens our understanding of ASFV entry and provides clues for the development of antiviral drugs to control ASFV infection.

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.

CD1d facilitates African swine fever virus entry into the host cells via clathrin-mediated endocytosis.

African swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease with high morbidity and mortality in domestic pigs and wild boars. The disease has become a global threat to the pig production industry and has caused enormous economic losses in many countries in recent years. However, the molecular mechanism underlying ASF virus (ASFV) entry of the host cells is not fully understood, which restricts the development of vaccines and antiviral-drugs of ASFV. In this study, we found that the host protein CD1d acts as a host factor, which mediates ASFV entry into the host cells. As the main capsid protein on the surface of ASFV virions, p72 can mediate viral entry. Using IP-MS assay, CD1d was identified as a binding partner of p72 on surface of ASFV virions. Knockdown of CD1d expression and blocking the cells with anti-pCD1d antibody, or incubating ASFV virions with soluble CD1d protein could significantly inhibit ASFV infection. CD1d is located on the membrane surface of primary porcine alveolar macrophages (PAMs) and mediates the virus entry via binding to p72. CD1d knockout or CD1d knockdown assay showed that CD1d could facilitate ASFV virions internalization via clathrin-mediated endocytosis (CME). Furthermore, CD1d interacts with EPS15 to mediate ASFV entry via clathrin-mediated endocytosis. Overall, our findings revealed that CD1d is a novel host-entry factor involved in ASFV internalization via the EPS15-clathrin endocytosis axis and a potential target for antiviral intervention.