African swine fever virus-induced polypeptides in porcine alveolar macrophages and in Vero cells: two-dimensional gel analysis

High-resolution two-dimensional electrophoresis followed by computer analysis has been used to study quantitatively the patterns of protein synthesis produced in porcine alveolar macrophages and in Vero cells infected with African swine fever virus (ASFV). Initially, a protein database for each cell type was constructed. The porcine alveolar macrophage database includes 995 polypeptides (818 acidic, isoelectric focusing (IEF) and 177 basic, nonequilibrium pH gradient electrophoresis (NEPHGE)) whereas the Vero database contains 1,398 polypeptides (1,127 acidic, IEF and 271 basic, NEPHGE). Taking these databases as reference, ASFV highly virulent strain E70 induces 57 acid and 43 basic polypeptides in porcine alveolar macrophages, which account for most of the information content of the virus DNA. The kinetics of synthesis of the virus-induced polypeptides showed the existence of three classes of proteins: one whose synthesis starts early after infection, continues for a period and then switches off; another whose synthesis also starts early but continues for prolonged periods; and a third which requires DNA replication. The attenuated, cell adapted, strain BA71V induces 92 acidic and 37 basic proteins in Vero cells. Significant differences were observed when comparing the patterns of polypeptides induced by the two viral strains. In both cell systems studied, ASFV infection produces a general shutoff of protein synthesis that affects up to 65% of the cellular proteins. Interestingly, 28 proteins of porcine alveolar macrophages and 48 proteins of Vero cells are stimulated at least two times by ASFV infection.     

African swine fever virus protein p54 interacts with the microtubular motor complex through direct binding to light-chain dynein

Dynein is a minus-end-directed microtubule-associated motor protein involved in cargo transport in the cytoplasm. African swine fever virus (ASFV), a large DNA virus, hijacks the microtubule motor complex cellular transport machinery during virus infection of the cell through direct binding of virus protein p54 to the light chain of cytoplasmic dynein (LC8). Interaction of p54 and LC8 occurs both in vitro and in cells, and the two proteins colocalize at the microtubular organizing center during viral infection. p50/dynamitin, a dominant-negative inhibitor of dynein-dynactin function, impeded ASFV infection, suggesting an essential role for dynein during virus infection. A 13-amino-acid domain of p54 was sufficient for binding to LC8, an SQT motif within this domain being critical for this binding. Direct binding of a viral structural protein to LC8, a small molecule of the dynein motor complex, could constitute a molecular mechanism for microtubule-mediated virus transport.     

A Virally Encoded Chaperone Specialized for Folding of the Major Capsid Protein of African Swine Fever Virus

It is generally believed that cellular chaperones facilitate the folding of virus capsid proteins, or that capsid proteins fold spontaneously. Here we show that p73, the major capsid protein of African swine fever virus (ASFV) failed to fold and aggregated when expressed alone in cells. This demonstrated that cellular chaperones were unable to aid the folding of p73 and suggested that ASFV may encode a chaperone. An 80-kDa protein encoded by ASFV, termed the capsid-associated protein (CAP) 80, bound to the newly synthesized capsid protein in infected cells. The 80-kDa protein was released following conformational maturation of p73 and dissociated before capsid assembly. Coexpression of the 80-kDa protein with p73 prevented aggregation and allowed the capsid protein to fold with kinetics identical to those seen in infected cells. CAP80 is, therefore, a virally encoded chaperone that facilitates capsid protein folding by masking domains exposed by the newly synthesized capsid protein, which are susceptible to aggregation, but cannot be accommodated by host chaperones. It is likely that these domains are ultimately buried when newly synthesized capsid proteins are added to the growing capsid shell.     

Characterization of a ubiquitinated protein which is externally located in African swine fever virions.

An antiserum was raised against the African swine fever virus (ASFV)-encoded ubiquitin-conjugating enzyme (UBCv1) and used to demonstrate by Western blotting (immunoblotting) and immunofluorescence that the enzyme is present in purified extracellular virions, is expressed both early and late after infection of cells with ASFV, and is cytoplasmically located. Antiubiquitin serum was used to identify novel ubiquitin conjugates present during ASFV infections. This antiserum stained virus factories late after infection, suggesting that virion proteins may be ubiquitinated. This possibility was confirmed by Western blotting, which identified three major antiubiquitin-immunoreactive proteins with molecular masses of 5, 18, and 58 kDa in purified extracellular virions. The 18-kDa protein was solubilized from virions at relatively low concentrations of the detergent n-octyl-beta-D-glucopyranoside, indicating that it is externally located and is possibly in the virus capsid. The 18-kDa protein was purified, and N-terminal amino acid sequencing confirmed that the protein was ubiquitinated and was ASFV encoded. The ASFV gene encoding this protein (PIG1) was sequenced, and the encoded protein expressed in an Escherichia coli expression vector. Recombinant PIG1 was ubiquitinated in the presence of E. coli expressed UBCv1 in vitro. These results suggest that PIG1 may be a substrate for UBCv1. The predicted molecular masses of the PIG1 protein and recombinant ubiquitinated protein were larger than the 18-kDa molecular mass of the ubiquitinated protein present in virions. Therefore, during viral replication, a precursor protein may undergo limited proteolysis to generate the ubiquitinated 18-kDa protein.     

Mechanism of collapse of endoplasmic reticulum cisternae during African swine fever virus infection

Infection of cells with African swine fever virus (ASFV) can lead to the formation of zipper-like stacks of structural proteins attached to collapsed endoplasmic reticulum (ER) cisternae. We show that the collapse of ER cisternae observed during ASFV infection is dependent on the viral envelope protein, J13Lp. Expression of J13Lp alone in cells is sufficient to induce collapsed ER cisternae. Collapse was dependent on a cysteine residue in the N-terminal domain of J13Lp exposed to the ER lumen. Luminal collapse was also dependent on the expression of J13Lp within stacks of ER where antiparallel interactions between the cytoplasmic domains of J13Lp orientated N-terminal domains across ER cisternae. Cisternal collapse was then driven by disulphide bonds between N-terminal domains arranged in antiparallel arrays across the ER lumen. This provides a novel mechanism for biogenesis of modified stacks of ER present in cells infected with ASFV, and may also be relevant to cellular processes.     

Regulation of Host Translational Machinery by African Swine Fever Virus

African swine fever virus (ASFV), like other complex DNA viruses, deploys a variety of strategies to evade the host''s defence systems, such as inflammatory and immune responses and cell death. Here, we analyse the modifications in the translational machinery induced by ASFV. During ASFV infection, eIF4G and eIF4E are phosphorylated (Ser1108 and Ser209, respectively), whereas 4E-BP1 is hyperphosphorylated at early times post infection and hypophosphorylated after 18 h. Indeed, a potent increase in eIF4F assembly is observed in ASFV-infected cells, which is prevented by rapamycin treatment. Phosphorylation of eIF4E, eIF4GI and 4E-BP1 is important to enhance viral protein production, but is not essential for ASFV infection as observed in rapamycin- or {"type":"entrez-protein","attrs":{"text":"CGP57380","term_id":"877393391","term_text":"CGP57380"}}CGP57380-treated cells. Nevertheless, eIF4F components are indispensable for ASFV protein synthesis and virus spread, since eIF4E or eIF4G depletion in COS-7 or Vero cells strongly prevents accumulation of viral proteins and decreases virus titre. In addition, eIF4F is not only activated but also redistributed within the viral factories at early times of infection, while eIF4G and eIF4E are surrounding these areas at late times. In fact, other components of translational machinery such as eIF2α, eIF3b, eIF4E, eEF2 and ribosomal P protein are enriched in areas surrounding ASFV factories. Notably, the mitochondrial network is polarized in ASFV-infected cells co-localizing with ribosomes. Thus, translation and ATP synthesis seem to be coupled and compartmentalized at the periphery of viral factories. At later times after ASFV infection, polyadenylated mRNAs disappear from the cytoplasm of Vero cells, except within the viral factories. The distribution of these pools of mRNAs is similar to the localization of viral late mRNAs. Therefore, degradation of cellular polyadenylated mRNAs and recruitment of the translation machinery to viral factories may contribute to the inhibition of host protein synthesis, facilitating ASFV protein production in infected cells.     

New insights into the role of endosomal proteins for African swine fever virus infection

African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection.     

非洲猪瘟病毒内囊膜蛋白p17与宿主互作蛋白的初步鉴定

非洲猪瘟病毒(African swine fever virus,ASFV)的感染导致猪的死亡率高达100%,给养猪业造成毁灭性灾难。因此,开展针对ASFV感染复制的研究有着重大的意义。目前发现ASFV有超过150个开放阅读框,其中D117L基因编码的内囊膜蛋白p17参与病毒二十面体结构的形成,但是对p17调控宿主细胞功能的机制知之甚少。研究通过免疫沉淀技术联合蛋白质谱分析,初步筛选出与ASFV p17潜在的宿主互作蛋白。通过进一步免疫共沉淀技术和激光共聚焦实验确认了p17与线粒体外膜蛋白TOMM70(translocase of outer mitochondrial membrane 70)、热休克蛋白HSPA8(heat shock 70 kDa protein 8)的互作。该研究为进一步探索p17在ASFV感染过程中的功能提供了重要信息。     

CD2v Interacts with Adaptor Protein AP-1 during African Swine Fever Infection

African swine fever virus (ASFV) CD2v protein is believed to be involved in virulence enhancement, viral hemadsorption, and pathogenesis, although the molecular mechanisms of the function of this viral protein are still not fully understood. Here we describe that CD2v localized around viral factories during ASFV infection, suggesting a role in the generation and/or dynamics of these viral structures and hence in disturbing cellular traffic. We show that CD2v targeted the regulatory trans-Golgi network (TGN) protein complex AP-1, a key element in cellular traffic. This interaction was disrupted by brefeldin A even though the location of CD2v around the viral factory remained unchanged. CD2v-AP-1 binding was independent of CD2v glycosylation and occurred on the carboxy-terminal part of CD2v, where a canonical di-Leu motif previously reported to mediate AP-1 binding in eukaryotic cells, was identified. This motif was shown to be functionally interchangeable with the di-Leu motif present in HIV-Nef protein in an AP-1 binding assay. However, we demonstrated that it was not involved either in CD2v cellular distribution or in CD2v-AP-1 binding. Taken together, these findings shed light on CD2v function during ASFV infection by identifying AP-1 as a cellular factor targeted by CD2v and hence elucidate the cellular pathways used by the virus to enhance infectivity.     

Two African swine fever virus proteins derived from a common precursor exhibit different nucleocytoplasmic transport activities

African swine fever virus (ASFV), a large icosahedral deoxyvirus, is the causative agent of an economically relevant hemorrhagic disease that affects domestic pigs. The major purpose of the present study was to investigate the nuclear transport activities of the ASFV p37 and p14 proteins, which result from the proteolytic processing of a common precursor. Experiments were performed by using yeast-based nucleocytoplasmic transport assays and by analysis of the subcellular localization of different green fluorescent and Myc fusion proteins in mammalian cells. The results obtained both in yeast and mammalian cells clearly demonstrated that ASFV p14 protein is imported into the nucleus but not exported to the cytoplasm. The ability of p37 protein to be exported from the nucleus to the cytoplasm of both yeast and mammalian cells was also demonstrated, and the results clearly indicate that p37 nuclear export is dependent on the interaction of the protein with the CRM-1 receptor. In addition, p37 was shown to exhibit nuclear import activity in mammalian cells. The p37 protein nuclear import and export abilities described here constitute the first report of a nucleocytoplasmic shuttling protein encoded by the ASFV genome. Overall, the overlapping results obtained for green fluorescent protein fusions and Myc-tagged proteins undoubtedly demonstrate that ASFV p37 and p14 proteins exhibit nucleocytoplasmic transport activities. These findings are significant for understanding the role these proteins play in the replication cycle of ASFV.     

African Swine Fever Virus Protein E199L Promotes Cell Autophagy through the Interaction of PYCR2

African swine fever virus(ASFV), as a member of the large DNA viruses, may regulate autophagy and apoptosis by inhibiting programmed cell death. However, the function of ASFV proteins has not been fully elucidated, especially the role of autophagy in ASFV infection. One of three Pyrroline-5-carboxylate reductases(PYCR), is primarily involved in conversion of glutamate to proline. Previous studies have shown that depletion of PYCR2 was related to the induction of autophagy. In the present study, we found for the first time that ASFV E199 L protein induced a complete autophagy process in Vero and HEK-293 T cells. Through co-immunoprecipitation coupled with mass spectrometry(CoIP-MS)analysis, we firstly identified that E199 L interact with PYCR2 in vitro. Importantly, our work provides evidence that E199 L down-regulated the expression of PYCR2, resulting in autophagy activation. Overall, our results demonstrate that ASFV E199 L protein induces complete autophagy through interaction with PYCR2 and down-regulate the expression level of PYCR2, which provide a valuable reference for the role of autophagy during ASFV infection and contribute to the functional clues of PYCR2.     

Porcine Immunoglobulin Fc Fused P30/P54 Protein of African Swine Fever Virus Displaying on Surface of S. cerevisiae Elicit Strong Antibody Production in Swine

African swine fever virus(ASFV) infects domestic pigs and European wild boars with strong, hemorrhagic and high mortality. The primary cellular targets of ASFV is the porcine macrophages. Up to now, no commercial vaccine or effective treatment available to control the disease. In this study, three recombinant Saccharomyces cerevisiae(S. cerevisiae) strains expressing fused ASFV proteins-porcine Ig heavy chains were constructed and the immunogenicity of the S. cerevisiae-vectored cocktail ASFV feeding vaccine was further evaluated. To be specific, the P30-Fcc and P54-Fca fusion proteins displaying on surface of S. cerevisiae cells were produced by fusing the Fc fragment of porcine immunoglobulin Ig G1 or IgA1 with p30 or p54 gene of ASFV respectively. The recombinant P30-Fcc and P54-Fca fusion proteins expressed by S. cerevisiae were verified by Western blotting, flow cytometry and immunofluorescence assay.Porcine immunoglobulin Fc fragment fused P30/P54 proteins elicited P30/P54-specific antibody production and induced higher mucosal immunity in swine. The absorption and phagocytosis of recombinant S. cerevisiae strains in IPEC-J2 cells or porcine alveolar macrophage(PAM) cells were significantly enhanced, too. Here, we introduce a kind of cheap and safe oral S. cerevisiae-vectored vaccine, which could activate the specific mucosal immunity for controlling ASFV infection.     

The MyD116 African swine fever virus homologue interacts with the catalytic subunit of protein phosphatase 1 and activates its phosphatase activity

The DP71L protein of African swine fever virus (ASFV) shares sequence similarity with the herpes simplex virus ICP34.5 protein over a C-terminal domain. We showed that the catalytic subunit of protein phosphatase 1 (PP1) interacts specifically with the ASFV DP71L protein in a yeast two-hybrid screen. The chimeric full-length DP71L protein, from ASFV strain Badajoz 71 (BA71V), fused to glutathione S-transferase (DP71L-GST) was expressed in Escherichia coli and shown to bind specifically to the PP1-alpha catalytic subunit expressed as a histidine fusion protein (6xHis-PP1alpha) in E. coli. The functional effects of this interaction were investigated by measuring the levels of PP1 and PP2A in ASFV-infected Vero cells. This showed that infection with wild-type ASFV strain BA71V activated PP1 between two- and threefold over that of mock-infected cells. This activation did not occur in cells infected with the BA71V isolate in which the DP71L gene had been deleted, suggesting that expression of DP71L leads to PP1 activation. In contrast, no effect was observed on the activity of PP2A following ASFV infection. We showed that infection of cells with wild-type BA71V virus resulted in decreased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha). ICP34.5 recruits PP1 to dephosphorylate the alpha subunit of eukaryotic translational initiation factor 2 (also known as eIF-2alpha); possibly the ASFV DP71L protein has a similar function.     

Aggresomes resemble sites specialized for virus assembly

The large cytoplasmic DNA viruses such as poxviruses, iridoviruses, and African swine fever virus (ASFV) assemble in discrete perinuclear foci called viral factories. Factories exclude host proteins, suggesting that they are novel subcellular structures induced by viruses. Novel perinuclear structures, called aggresomes are also formed by cells in response to misfolded protein (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. Cell Biol. 143:1883--1898; García-Mata, R., Z. Beb?k, E.J. Sorscher, and E.S. Sztul. 1999. J. Cell Biol. 146:1239--1254). In this study, we have investigated whether aggresomes and viral factories are related structures. Aggresomes were compared with viral factories produced by ASFV. Aggresomes and viral factories were located close to the microtubule organizing center and required an intact microtubular network for assembly. Both structures caused rearrangement of intermediate filaments and the collapse of vimentin into characteristic cages, and both recruited mitochondria and cellular chaperones. Given that ASFV factories resemble aggresomes, it is possible that a cellular response originally designed to reduce the toxicity of misfolded proteins is exploited by cytoplasmic DNA viruses to concentrate structural proteins at virus assembly sites.     

African swine fever virus protein p30 interaction with heterogeneous nuclear ribonucleoprotein K (hnRNP-K) during infection

Heterogeneous nuclear ribonucleoprotein K (hnRNP-K) was identified as interacting cellular protein with the abundant immediate early protein p30 from African swine fever virus (ASFV) in a macrophage cDNA library screening. The interacting regions of hnRNP-K with p30 were established within residues 35-197, which represent KH1 and KH2 domains responsible for RNA binding. Colocalization of hnRNP-K and p30 was observed mainly in the nucleus, but not in the cytoplasm of infected cells and infection modified hnRNP-K subcellular distribution and decreased the incorporation of 5-fluorouridine into nascent RNA. Since similar effects were observed in cells transiently expressing p30, this interaction provides new insights into p30 function and could represent a possible additional mechanism by which ASFV downregulates host cell mRNA translation.     

Vimentin rearrangement during African swine fever virus infection involves retrograde transport along microtubules and phosphorylation of vimentin by calcium calmodulin kinase II

African swine fever virus (ASFV) infection leads to rearrangement of vimentin into a cage surrounding virus factories. Vimentin rearrangement in cells generally involves phosphorylation of N-terminal domains of vimentin by cellular kinases to facilitate disassembly and transport of vimentin filaments on microtubules. Here, we demonstrate that the first stage in vimentin rearrangement during ASFV infection involves a microtubule-dependent concentration of vimentin into an "aster" within virus assembly sites located close to the microtubule organizing center. The aster may play a structural role early during the formation of the factory. Conversion of the aster into a cage required ASFV DNA replication. Interestingly, viral DNA replication also resulted in the activation of calcium calmodulin-dependent protein kinase II (CaM kinase II) and phosphorylation of the N-terminal domain of vimentin on serine 82. Immunostaining showed that vimentin within the cage was phosphorylated on serine 82. Significantly, both viral DNA replication and Ser 82 phosphorylation were blocked by KN93, an inhibitor of CaM kinase II, suggesting a link between CaM kinase II activation, DNA replication, and late gene expression. Phosphorylation of vimentin on serine 82 may be necessary for cage formation or may simply be a consequence of activation of CaM kinase II by ASFV. The vimentin cage may serve a cytoprotective function and prevent movement of viral components into the cytoplasm and at the same time concentrate late structural proteins at sites of virus assembly.     

African swine fever virus causes microtubule-dependent dispersal of the trans-golgi network and slows delivery of membrane protein to the plasma membrane

Viral interference with secretory cargo is a common mechanism for pathogen immune evasion. Selective down regulation of critical immune system molecules such as major histocompatibility complex (MHC) proteins enables pathogens to mask themselves from their host. African swine fever virus (ASFV) disrupts the trans-Golgi network (TGN) by altering the localization of TGN46, an organelle marker for the distal secretory pathway. Reorganization of membrane transport components may provide a mechanism whereby ASFV can disrupt the correct secretion and/or cell surface expression of host proteins. In the study reported here, we used the tsO45 temperature-sensitive mutant of the G protein of vesicular stomatitis virus to show that ASFV significantly reduces the rate at which the protein is delivered to the plasma membrane. This is linked to a general reorganization of the secretory pathway during infection and a specific, microtubule-dependent disruption of structural components of the TGN. Golgin p230 and TGN46 are separated into distinct vesicles, whereupon TGN46 is depleted. These data suggest that disruption of the TGN by ASFV can slow membrane traffic during viral infection. This may be functionally important because infection of macrophages with virulent isolates of ASFV increased the expression of MHC class I genes, but there was no parallel increase in MHC class I molecule delivery to the plasma membrane.     

Generation of filamentous instead of icosahedral particles by repression of African swine fever virus structural protein pB438L

The mechanisms involved in the construction of the icosahedral capsid of the African swine fever virus (ASFV) particle are not well understood at present. Capsid formation requires protein p72, the major capsid component, but other viral proteins are likely to play also a role in this process. We have examined the function of the ASFV structural protein pB438L, encoded by gene B438L, in virus morphogenesis. We show that protein pB438L associates with membranes during the infection, behaving as an integral membrane protein. Using a recombinant ASFV that inducibly expresses protein pB438L, we have determined that this structural protein is essential for the formation of infectious virus particles. In the absence of the protein, the virus assembly sites contain, instead of icosahedral particles, large aberrant tubular structures of viral origin as well as bilobulate forms that present morphological similarities with the tubules. The filamentous particles, which possess an aberrant core shell domain and an inner envelope, are covered by a capsid-like layer that, although containing the major capsid protein p72, does not acquire icosahedral morphology. This capsid, however, is to some extent functional, as the filamentous particles can move from the virus assembly sites to the plasma membrane and exit the cell by budding. The finding that, in the absence of protein pB438L, the viral particles formed have a tubular structure in which the icosahedral symmetry is lost supports a role for this protein in the construction or stabilization of the icosahedral vertices of the virus particle.