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The non-classical major-histocompatibility-complex II protein SLA-DM is crucial for African swine fever virus replication

ABSTRACT: African swine fever virus (ASFV) is a lethal animal pathogen which enters its host cells through endocytosis. So far, host factors specifically required for ASFV replication have been barely identified. In this study a genome-wide CRISPR/Cas9 knockout screen in porcine cells indicated that the genes RFXANK, RFXAP, SLA-DMA, SLA-DMB, and CIITA are important for productive ASFV infection. The proteins encoded by these genes belong to the major-histocompatibility-complex II (MHC II), or swine-leucocyte-antigen-complex II (SLA II). RFXAP and CIITA are MHC II-specific transcription factors, whereas SLA-DMA/B are subunits of the non-classical MHC II molecule SLA-DM. Targeted knockout of either of these genes led to severe replication defects of different ASFV isolates, reflected by substantially reduced plating efficiency, cell-to-cell spread, and progeny virus titers. For the characterization of the knockouts on a proteome level the protein contents of the knockout cell lines were analyzed by mass spectrometry.

INSTRUMENT(S): maXis

ORGANISM(S): Sus Scrofa Domesticus (domestic Pig)

SUBMITTER: Axel Karger

LAB HEAD: Axel Karger

PROVIDER: PXD034242 | Pride | 2023-08-21

REPOSITORIES: Pride

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			Action	DRS
	10_RFXAP1_Slot1-37_1_1855.RAW.zip	Raw
	10_RFXAP1_Slot1-37_1_1855.pep.xml	Pepxml
	10_RFXAP1_Slot1-37_1_1855_calibrated.mgf	Mgf
	11_RFXAP2_Slot1-38_1_1856.RAW.zip	Raw
	11_RFXAP2_Slot1-38_1_1856.pep.xml	Pepxml

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Project description:African swine fever virus (ASFV) is an infectious disease with a mortality rate of nearly 100% in pigs; however, no safe commercial vaccines or antiviral drugs are currently available, which seriously threatens the global pig industry. Therefore, effective biologicals against ASFV are urgently needed. Here, we screened 138 Bacillus subtilis strains, four of which evidently inhibited ASFV replication in vitro. Pigs fed with biologics of different types from the four B. subtilis strains showed reduced pathological changes and viral loads in tissues, with a survival rate of up to 100%. The antiviral activity of B. subtilis was attributed to small-molecule metabolites, rather than to secretory proteins. A total of 169 small molecules were obtained from the metabolites of B. subtilis using LC-MS/MS, arctiin and genistein, which showed the highest inhibition efficiency, suppressing ASFV proliferation at the mid-stage of infection. These molecules acted as competitive inhibitors by complexing the ATP-binding domain of viral topoisomerase II, as demonstrated using molecular docking, biolayer interferometry binding and a competitive decatenation assay, thereby disrupting the catalytic activity of the enzyme and inhibiting ASFV replication. Furthermore, pigs administered arctiin and genistein orally showed decreased mortality and tissue damage. Collectively, these results suggest that the four B. subtilis strains screened may be preventative biologicals against ASFV infection. Our findings pave the way for ASFV prevention and control strategies in the pig industry to curb the economic losses caused by the disease. IMPORTANCE: African swine fever virus (ASFV) is a highly fatal swine disease that severely affects the pig industry. Although ASFV has been prevalent for more than 100 years, effective vaccines or antiviral strategies are still lacking. In this study, we identified four Bacillus subtilis strains that inhibited ASFV proliferation in vitro. Pigs fed with liquid biologicals or powders derived from four B. subtilis strains mixed with pellet feed showed reduced morbidity and mortality when challenged with ASFV. Further analysis showed that the antiviral activity of B. subtilis was based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II. Our findings offer a promising new strategy for the prevention and control of ASFV that may significantly alleviate the economic losses in the pig industry.

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The non-classical major-histocompatibility-complex II protein SLA-DM is crucial for African swine fever virus replication

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