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African swine fever virus (ASFV) encodes more than 150 proteins, most of them of unknown function. We used a high-throughput proteomic analysis to elucidate the interactome of four ASFV proteins, which potentially mediate a critical step of the infection cycle, the fusion and endosomal exit of the virions. Using affinity purification and mass spectrometry, we were able to identify potential interacting partners for those ASFV proteins P34, E199L, MGF360-15R and E248R. Representative molecular pathways for these proteins were intracellular and Golgi vesicle transport, endoplasmic reticulum organization, lipid biosynthesis, and cholesterol metabolism. Rab geranyl geranylation emerged as a significant hit, and also Rab proteins, which are crucial regulators of the endocytic pathway and interactors of both p34 and E199L. Rab proteins co-ordinate a tight regulation of the endocytic pathway that is necessary for ASFV infection. Moreover, several interactors were proteins involved in the molecular exchange at ER membrane contacts. These ASFV fusion proteins shared interacting partners, suggesting potential common functions. Membrane trafficking and lipid metabolism were important categories, as we found significant interactions with several enzymes of the lipid metabolism. These targets were confirmed using specific inhibitors with antiviral effect in cell lines and macrophages. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) is a large DNA virus that replicates predominantly in the cell cytoplasm and is the only member of the Asfarviridae family. The virus causes an acute haemorrhagic fever, African swine fever (ASF), in domestic pigs and wild boar resulting in the death of most infected animals. Apoptosis is induced at an early stage during virus entry or uncoating. However, ASFV encodes anti-apoptotic proteins which facilitate production of progeny virions. These anti-apoptotic proteins include A179L, a Bcl-2 family member; A224L, an inhibitor of apoptosis proteins (IAP) family member; EP153R a C-type lectin; and DP71L. The latter acts by inhibiting activation of the stress activated pro-apoptotic pathways pro-apoptotic pathways. The mechanisms by which these proteins act is summarised. ASF disease is characterised by massive apoptosis of uninfected lymphocytes which reduces the effectiveness of the immune response, contributing to virus pathogenesis. Mechanisms by which this apoptosis is induced are discussed. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) infection causes endosomal reorganization. Here, we show that the virus causes endosomal congregation close to the nucleus as the infection progresses, which is necessary to build a compact viral replication organelle. ASFV enters the cell by the endosomal pathway and reaches multivesicular late endosomes. Upon uncoating and fusion, the virus should exit to the cytosol to start replication. ASFV remodels endosomal traffic and redistributes endosomal membranes to the viral replication site. Virus replication also depends on endosomal membrane phosphoinositides (PtdIns) synthesized by PIKfyve. Endosomes could act as platforms providing membranes and PtdIns, necessary for ASFV replication. Our study has revealed that ASFV reorganizes endosome dynamics, in order to ensure a productive infection. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is a highly contagious viral disease of swine which causes high mortality, approaching 100%, in domestic pigs. ASF is caused by a large, double stranded DNA virus, ASF virus (ASFV), which replicates predominantly in the cytoplasm of macrophages and is the only member of the Asfarviridae family, genus Asfivirus. The natural hosts of this virus include wild suids and arthropod vectors of the Ornithodoros genus. The infection of ASFV in its reservoir hosts is usually asymptomatic and develops a persistent infection. In contrast, infection of domestic pigs leads to a lethal hemorrhagic fever for which there is no effective vaccine. Identification of ASFV genes involved in virulence and the characterization of mechanisms used by the virus to evade the immune response of the host are recognized as critical steps in the development of a vaccine. Moreover, the interplay of the viral products with host pathways, which are relevant for virus replication, provides the basic information needed for the identification of potential targets for the development of intervention strategies against this disease. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) is a double-stranded DNA virus causing a hemorrhagic fever disease with high mortality rates and severe economic losses in pigs worldwide. ASFV replicates in perinuclear sites called viral factories (VFs) that are morphologically similar to cellular aggresomes. This fact raises the possibility that both VFs and aggresomes may be the same structure. However, little is known about the process involved in the formation of these viral replication platforms. In order to expand our knowledge on the assembly of ASFV replication sites, we have analyzed the involvement of both canonical aggresome pathways in the formation of ASFV VFs: HDAC6 and BAG3. HDAC6 interacts with a component of the dynein motor complex (dynactin/p150Glued) and ubiquitinated proteins, transporting them to the microtubule-organizing center (MTOC) and leading to aggresome formation, while BAG3 is mediating the recruitment of non-ubiquitinated proteins through a similar mechanism. Tubacin-mediated HDAC6 inhibition and silencing of BAG3 pathways, individually or simultaneously, did not prevent ASFV VF formation. These findings show that HDAC6 and Bag3 are not required for VFs formation suggesting that aggresomes and VFs are not the same structures. However, alternative unexplored pathways may be involved in the formation of aggresomes. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) is an acute and persistent swine virus with a high economic burden that encodes multiple genes to evade host immune response. In this work, we have revealed that early viral protein UBCv1, the only known conjugating enzyme encoded by a virus, modulates innate immune and inflammatory signaling. Transient overexpression of UBCv1 impaired activation of NF-κB and AP-1 transcription factors induced by several agonists of these pathways. In contrast, activation of IRF3 and ISRE signaling upon stimulation with TRIFΔRIP, cGAS/STING or RIG-I-CARD remained unaltered. Experiments aimed at mapping UBCv1 inhibitory activity indicated that this viral protein acts upstream or at the level step of IKKβ. In agreement with this, UBCv1 was able to block p65 nuclear translocation upon cytokine stimulation, a key event in NF-ĸB signaling. Additionally, A549 stably transduced for UBCv1 showed a significant decrease in the levels of NF-ĸB dependent genes. Interestingly, despite the well-defined capacity of UBCv1 to conjugate ubiquitin chains, a mutant disabled for ubiquitylation activity retained similar immunomodulatory activity as the wild-type enzyme, suggesting that the two functions are segregated. Altogether these data suggest that ASFV UBCv1 manipulates the innate immune response targeting the NF-κB and AP-1 pathways and opens new questions about the multifunctionality of this enzyme. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is a hemorrhagic fever of wild and domestic pigs with a high rate of mortality. Originally endemic in Africa, this disease is currently disseminating in Europe and China, causing a large socioeconomic impact. ASF is caused by a DNA virus, African swine fever virus (ASFV). There is no vaccine available against ASFV, limiting the options for disease control. ASFV reorganizes intracellular membranes to generate viral factories (VFs) in order to amplify its genome. However, little is known about the process involved in the formation of these viral replication organelles. Membrane contact sites (MCSs) allow nonvesicular lipids and ion exchange between organelles. Lipid exchange to form VFs apparently requires a number of proteins at MCSs, such as the oxysterol-binding protein (OSBP), the acyl-coenzyme A binding domain containing 3 (ACBD3) and the phosphatidylinositol-phosphate-4-kinase III beta (PI4Kβ). Itraconazole (ITZ) is an antifungal agent that targets sterol-transport molecules such as OSBP and OSBP-related protein 4 (ORP4). 25-Hydroxycholesterol (25-HC) inhibits lipid transport by high affinity binding OSBP. In this work, we analyzed the antiviral function of ITZ and 25-HC against ASFV in Vero cell cultures using the cell-adapted Ba71V isolate. ITZ and 25-HC decreased significantly ASFV replication. Our study revealed OSBP distribution in cytoplasmic membranes in uninfected Vero cells and to the periphery of VFs in infected cells. In addition, we showed that OSBP and OSBP-related proteins, PI4Kβ and ACBD3 were recruited to VFs in the context ASFV infection. [ABSTRACT FROM AUTHOR]