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.     

稳定表达非洲猪瘟病毒P54蛋白的Vero细胞系的建立

旨为建立稳定表达非洲猪瘟病毒(ASFV)P54蛋白的Vero细胞系,将ASFV-P54基因与绿色荧光基因Azami Green的融合基因片段,将其克隆至慢病毒载体pLV-puro中构建重组慢病毒质粒pLV-ASFV-P54-AG,将该质粒与慢病毒包装质粒pH1和pH2共转染HEK-293V细胞,包装表达ASFV-P54蛋白的慢病毒。将重组慢病毒在聚凝胺(Polybrene)的介导下感染Vero细胞,筛选出一株稳定表达ASFV-P54蛋白的Vero细胞系,命名为Vero-AG-ASFV-P54。间接免疫荧光试验表明,该细胞系能够与P54多克隆抗体反应;经波兰国家兽医研究所进一步验证,结果显示,该细胞系与ASFV抗体阳性血清也能发生反应,并且与阴性血清无反应。结果表明,Vero-AG-ASFV-P54细胞系能够稳定高效的表达具有生物活性的ASFV-P54蛋白。     

Deletion of a CD2-Like Gene, 8-DR, from African Swine Fever Virus Affects Viral Infection in Domestic Swine

An African swine fever virus (ASFV) gene with similarity to the T-lymphocyte surface antigen CD2 has been found in the pathogenic African isolate Malawi Lil-20/1 (open reading frame [ORF] 8-DR) and a cell culture-adapted European virus, BA71V (ORF EP402R) and has been shown to be responsible for the hemadsorption phenomenon observed for ASFV-infected cells. The structural and functional similarities of the ASFV gene product to CD2, a cellular protein involved in cell-cell adhesion and T-cell-mediated immune responses, suggested a possible role for this gene in tissue tropism and/or immune evasion in the swine host. In this study, we constructed an ASFV 8-DR gene deletion mutant (Δ8-DR) and its revertant (8-DR.R) from the Malawi Lil-20/1 isolate to examine gene function in vivo. In vitro, Δ8-DR, 8-DR.R, and the parental virus exhibited indistinguishable growth characteristics on primary porcine macrophage cell cultures. In vivo, 8-DR had no obvious effect on viral virulence in domestic pigs; disease onset, disease course, and mortality were similar for the mutant Δ8-DR, its revertant 8-DR.R, and the parental virus. Altered viral infection was, however, observed for pigs infected with Δ8-DR. A delay in spread to and/or replication of Δ8-DR in the draining lymph node, a delay in generalization of infection, and a 100- to 1,000-fold reduction in virus titers in lymphoid tissue and bone marrow were observed. Onset of viremia for Δ8-DR-infected animals was significantly delayed (by 2 to 5 days), and mean viremia titers were reduced approximately 10,000-fold at 5 days postinfection and 30- to 100-fold at later times; moreover, unlike in 8-DR.R-infected animals, the viremia was no longer predominantly erythrocyte associated but rather was equally distributed among erythrocyte, leukocyte, and plasma fractions. Mitogen-dependent lymphocyte proliferation of swine peripheral blood mononuclear cells in vitro was reduced by 90 to 95% following infection with 8-DR.R but remained unaltered following infection with Δ8-DR, suggesting that 8-DR has immunosuppressive activity in vitro. Together, these results suggest an immunosuppressive role for 8-DR in the swine host which facilitates early events in viral infection. This may be of most significance for ASFV infection of its highly adapted natural host, the warthog.     

African Swine Fever Virus Protein p17 Is Essential for the Progression of Viral Membrane Precursors toward Icosahedral Intermediates

The first morphological evidence of African swine fever virus (ASFV) assembly is the appearance of precursor viral membranes, thought to derive from the endoplasmic reticulum, within the assembly sites. We have shown previously that protein p54, a viral structural integral membrane protein, is essential for the generation of the viral precursor membranes. In this report, we study the role of protein p17, an abundant transmembrane protein localized at the viral internal envelope, in these processes. Using an inducible virus for this protein, we show that p17 is essential for virus viability and that its repression blocks the proteolytic processing of polyproteins pp220 and pp62. Electron microscopy analyses demonstrate that when the infection occurs under restrictive conditions, viral morphogenesis is blocked at an early stage, immediately posterior to the formation of the viral precursor membranes, indicating that protein p17 is required to allow their progression toward icosahedral particles. Thus, the absence of this protein leads to an accumulation of these precursors and to the delocalization of the major components of the capsid and core shell domains. The study of ultrathin serial sections from cells infected with BA71V or the inducible virus under permissive conditions revealed the presence of large helicoidal structures from which immature particles are produced, suggesting that these helicoidal structures represent a previously undetected viral intermediate.African swine fever virus (ASFV) (61, 72) is the only known DNA-containing arbovirus and the sole member of the Asfarviridae family (24). Infection by this virus of its natural hosts, the wild swine warthogs and bushpigs and the argasid ticks of the genus Ornithodoros, results in a mild disease, often asymptomatic, with low viremia titers, that in many cases develops into a persistent infection (3, 43, 71). In contrast, infection of domestic pigs leads to a lethal hemorrhagic fever for which the only available methods of disease control are the quarantine of the affected area and the elimination of the infected animals (51).The ASFV genome is a lineal molecule of double-stranded DNA of 170 to 190 kbp in length with convalently closed ends and terminal inverted repeats. The genome encodes more than 150 open reading frames, half of which lack any known or predictable function (16, 75).The virus particle, with an overall icosahedral shape and an average diameter of 200 nm (11), is organized in several concentric layers (6, 11, 15) containing more than 50 structural proteins (29). Intracellular particles are formed by an inner viral core, which contains the central nucleoid surrounded by a thick protein coat, referred to as core shell. This core is enwrapped by an inner lipid envelope (7, 34) on top of which the icosahedral capsid is assembled (26, 27, 31). Extracellular virions possess an additional membrane acquired during the budding from the plasma membrane (11). Both forms of the virus, intracellular and extracellular, are infective (8).The assembly of ASFV particles occurs in the cytoplasm of the infected cell, in viral factories located close to the cell nucleus (6, 13, 49). ASFV factories possess several characteristics similar to those of the cellular aggresomes (35), which are accumulations of aggregates of cellular proteins that form perinuclear inclusions (44).Current models propose that ASFV assembly begins with the modification of endoplasmic reticulum (ER) membranes, which are subsequently recruited to the viral factories and transformed into viral precursor membranes. These ER-derived viral membranes represent the precursors of the inner viral envelope and are the first morphological evidence of viral assembly (7, 60). ASFV viral membrane precursors evolve into icosahedral intermediates and icosahedral particles by the progressive assembly of the outer capsid layer at the convex face of the precursor membranes (5, 26, 27, 31) through an ATP- and calcium-dependent process (19). At the same time, the core shell is formed underneath the concave face of the viral envelope, and the viral DNA and nucleoproteins are packaged and condensed to form the innermost electron-dense nucleoid (6, 9, 12, 69). However, the assembly of the capsid and the internal envelope appears to be largely independent of the components of the core of the particle, since the absence of the viral polyprotein pp220 during assembly produces empty virus-like particles that do not contain the core (9).Comparative genome analysis suggests that ASFV shares a common origin with the members of the proposed nucleocytoplasmic large DNA viruses (NCLDVs) (40, 41). The reconstructed phylogeny of NCLDVs as well as the similitude in the structures and organizations of the genomes indicates that ASFV is more closely related to poxviruses than to other members of the NCLDVs. A consensus about the origin and nature of the envelope of the immature form of vaccinia virus (VV), the prototypical poxvirus, seems to be emerging (10, 17, 20, 54). VV assembly starts with the appearance of crescent-shaped structures within specialized regions of the cytoplasm also known as viral factories (21, 23). The crescent membranes originate from preexisting membranes derived from some specialized compartment of the ER (32, 37, 52, 53, 67), and an operative pathway from the ER to the crescent membrane has recently been described (38, 39). VV crescents apparently grow in length while maintaining the same curvature until they become closed circles, spheres in three dimensions, called immature virions (IV) (22). The uniform curvature is produced by a honeycomb lattice of protein D13L (36, 70), which attaches rapidly to the membranes so that nascent viral membranes always appear to be coated over their entirety. The D13L protein is evolutionarily related to the capsid proteins of the other members of the NCLDV group, including ASFV, but lacks the C-terminal jelly roll motif (40). This structural difference is probably related to the fact that poxviruses are the only member of this group without an icosahedral capsid; instead, the spherical D13L coat acts as a scaffold during the IV stage but is discarded in subsequent steps of morphogenesis (10, 28, 46, 66). Thus, although crescents in VV and precursors of the inner envelope in ASFV are the first morphogenetic stages discernible in the viral factories of these viruses, they seem to be different in nature. Crescents are covered by the D13L protein and are more akin to the icosahedral intermediates of ASFV assembly, whereas ASFV viral membrane precursors are more similar to the naked membranes seen when VV morphogenesis is arrested by rifampin treatment (33, 47, 48, 50) or when the expression of the D13L and A17L proteins are repressed during infection with lethal conditional VV viruses (45, 55, 56, 68, 74, 76).Although available evidence strongly supports the reticular origin of the ASFV inner envelope (7, 60), the mechanism of acquisition remains unknown, and the number of membranes present in the inner envelope is controversial. The traditional view of the inner envelope as formed by two tightly opposed membranes derived from ER collapsed cisternae (7, 59, 60) has recently been challenged by the careful examination of the width of the internal membrane of viral particles and the single outer mitochondrial membrane, carried out using chemical fixation, cryosectioning, and high-pressure freezing (34). The results suggest that the inner envelope of ASFV is a single lipid bilayer, which raises the question of how such a structure can be generated and stabilized in the precursors of the ASFV internal envelope. In the case of VV, the coat of the D13L protein has been suggested to play a key role in the stabilization of the single membrane structure of the crescent (10, 17, 36), but the ASFV capsid protein p72 is not a component of the viral membrane precursors. The identification and functional characterization of the proteins involved in the generation of these structures are essential for the understanding of the mechanisms involved in these early stages of viral assembly. For this reason, we are focusing our interest on the study of abundant structural membrane proteins that reside at the inner envelope of the viral particle. We have shown previously that one of these proteins, p54, is essential for the recruitment of ER membranes to the viral factory (59). Repression of protein p54 expression has a profound impact on virus production and leads to an early arrest in virion morphogenesis, resulting in the virtual absence of membranes in the viral factory.Protein p17, encoded by the late gene D117L in the BA71V strain, is an abundant structural protein (60, 65). Its sequence, which is highly conserved among ASFV isolates (16), does not show any significant similarity with the sequences present in the databases. Protein p17 is an integral membrane protein (18) that is predicted to insert in membranes with a Singer type I topology and has been localized in the envelope precursors as well as in both intracellular and extracellular mature particles (60), suggesting that it resides at the internal envelope, the only membranous structure of the intracellular particles.In this work, we analyze the role of protein p17 in viral assembly by means of an IPTG (isopropyl-β-d-thiogalactopyranoside)-dependent lethal conditional virus. The data presented indicate that protein p17 is essential for viral morphogenesis. The repression of this protein appears to block assembly at the level of viral precursor membranes, resulting in their accumulation at the viral factory.From the electron microscopy analysis of serial sections of viral factories at very early times during morphogenesis, we present experimental evidence that suggests that, during assembly, viral precursor membranes and core material organize into large helicoidal intermediates from which icosahedral particles emerge. The possible role of these structures during ASFV morphogenesis is discussed.     

Comprehensive Phylogenetic Reconstructions of African Swine Fever Virus: Proposal for a New Classification and Molecular Dating of the Virus

African swine fever (ASF) is a highly lethal disease of domestic pigs caused by the only known DNA arbovirus. It was first described in Kenya in 1921 and since then many isolates have been collected worldwide. However, although several phylogenetic studies have been carried out to understand the relationships between the isolates, no molecular dating analyses have been achieved so far. In this paper, comprehensive phylogenetic reconstructions were made using newly generated, publicly available sequences of hundreds of ASFV isolates from the past 70 years. Analyses focused on B646L, CP204L, and E183L genes from 356, 251, and 123 isolates, respectively. Phylogenetic analyses were achieved using maximum likelihood and Bayesian coalescence methods. A new lineage-based nomenclature is proposed to designate 35 different clusters. In addition, dating of ASFV origin was carried out from the molecular data sets. To avoid bias, diversity due to positive selection or recombination events was neutralized. The molecular clock analyses revealed that ASFV strains currently circulating have evolved over 300 years, with a time to the most recent common ancestor (TMRCA) in the early 18^th century.     

Resistance Evolution against Host-directed Antiviral Agents: Buffalopox Virus Switches to Use p38-ϒ under Long-term Selective Pressure of an Inhibitor Targeting p38-α

Host-dependency factors have increasingly been targeted to minimize antiviral drug resistance. In this study, we have demonstrated that inhibition of p38 mitogen-activated protein kinase (a cellular protein) suppresses buffalopox virus (BPXV) protein synthesis by targeting p38-MNK1-eIF4E signaling pathway. In order to provide insights into the evolution of drug resistance, we selected resistant mutants by long-term sequential passages (P; n = 60) in the presence of p38 inhibitor (SB239063). The P60-SB239063 virus exhibited significant resistance to SB239063 as compared to the P60-Control virus. To provide mechanistic insights on the acquisition of resistance by BPXV-P60-SB239063, we generated p38-α and p38-ϒ (isoforms of p38) knockout Vero cells by CRISPR/Cas9-mediated genome editing. It was demonstrated that unlike the wild type (WT) virus which is dependent on p38-α isoform, the resistant virus (BPXV-P60-SB239063) switches over to use p38-ϒ so as to efficiently replicate in the target cells. This is a rare evidence wherein a virus was shown to bypass the dependency on a critical cellular factor under selective pressure of a drug.     

Functionality and Cell Anchorage Dependence of the African Swine Fever Virus Gene A179L,a Viral bcl-2 Homolog,in Insect Cells

The African swine fever virus gene A179L has been shown to be a functional member of the ced9/bcl-2 family of apoptosis inhibitors in mammalian cell lines. In this work we have expressed the A179L gene product (p21) under the control of the baculovirus polyhedrin promoter using a baculovirus system. Expression of the A179L gene neither altered the baculovirus replication phenotype nor delayed the shutoff of cellular protein synthesis, but it extended the survival of the infected insect cells to very late times postinfection. The increase in cell survival rates correlated with a marked apoptosis reduction after baculovirus infection. Interestingly, prevention of apoptosis was observed when recombinant baculovirus infections were carried out in monolayer cell cultures but not when cells were infected in suspension, suggesting a cell anchorage dependence for p21 function in insect cells. Cell survival was enhanced under optimal conditions of cell attachment and cell-to-cell contact as provided by extracellular matrix components or poly-d-lysine. Since it was observed that cytoskeleton organization varied depending on culture conditions of insect cells (grown in monolayer versus grown in suspension), these results suggested that A179L might regulate apoptosis in insect cells only when the cytoskeletal support of intracellular signaling is maintained upon cell adhesion. Thus, cell shape and cytoskeleton status might allow variations in intracellular transduction of signals related to cell survival in virus-infected cells.African swine fever virus (ASFV), the causative agent for an important disease of swine, is a large double-stranded DNA virus that replicates not only in members of the Suidae family but also in soft ticks of the Ornithodoros genus (24, 31, 41). ASFV infects a variety of cells of the mononuclear phagocytic system and produces a characteristic apoptotic cell death in infected swine macrophages and bystander uninfected lymphocytes (33–35). Virus-induced apoptosis in target cells is produced late during infection, suggesting the existence of viral genes that prevent early apoptosis to support the productive infection. An ASFV gene, 5HL (A179L in Ba71V virus), with sequence similarity to the ced9/bcl-2 gene family has been described (28). This gene encodes a protein of approximately 21 kDa (p21) that is synthesized in infected cells at both early and late times postinfection (28). The similarity of this gene to bcl-2 has pointed to its possible role in apoptosis inhibition during ASFV infection. We have recently demonstrated that expression of the A179L gene by a recombinant vaccinia virus inhibits apoptosis mediated by the interferon-induced double-stranded RNA-activated protein kinase in HeLa and BSC-40 cells (5). Also, the A179L product is able to suppress apoptotic cell death in the FL5.12 mouse prolymphocytic cell line and in the K562 human myeloid leukemia cell line (1, 37). Thus, the ability of the A179L gene to suppress apoptosis in mammalian cells has been clearly shown. However, the function in insect cells of the bcl-2 gene, the prototype of a death-regulator gene family, remains controversial (3, 8, 11), and the function of A179L during ASFV infection of nonmammalian cells is still unknown. Other ASFV genes potentially involved in the regulation of intracellular apoptosis pathways are A238L and 23NL, which have sequence similarity to the IκB factor (32) and the ICP34.5 gene of herpes simplex virus I (14, 40), respectively. The existence of an ASFV gene (A224L) with similarity to the iap family of apoptosis inhibitors suggested that A179L and A224L could function as host range genes (9). Nevertheless, there are no current data supporting the function of A224L as an apoptosis inhibitor, and studies conducted to analyze its role during ASFV infection demonstrated that this gene is dispensable for ASFV growth in swine macrophages (29).Therefore, the study of the apoptosis regulatory functions of the A179L gene in different cell lines and under different culture conditions is important to an understanding of the role of this gene during ASFV infection in its different hosts. To examine these functions, we have expressed the A179L gene in Spodoptera frugiperda (Sf9) cells using a baculovirus system. We demonstrated that this gene is functional and prevents virus-induced apoptosis in these cells only when intracellular signaling upon cell adhesion is maintained.

Expression of the A179L gene product in Sf9 cells.

The construction of recombinant baculovirus expressing the A179L gene product was carried out as previously described (21, 30). Briefly, DNA amplification of the A179L gene from the ASFV isolate E70 was carried out by PCR with ampliTaq DNA polymerase (Perkin-Elmer Cetus) with the primers (i) 5′-AAATATAGGGATCCGCTATGGAGGG (5′ primer) and (ii) 5′-CCGCGTGGATCCTATATCAAATTGC (3′ primer). Both primers contain the recognition sequence for the BamHI restriction enzyme. The PCR product was digested with BamHI and cloned into the BamHI site of the baculovirus transfer vector pBacPAK8 (Clontech) under the control of the polyhedrin promoter. The cloned gene was sequenced by the dideoxynucleotide chain terminator method by using specific primers to check possible sequence changes introduced by PCR amplification of the gene. Then, Sf9 cells were cotransfected with the transfer recombinant vector (pBacPAK-A179L) and the purified, noninfectious BacPAK6 DNA (Bsu36I digested), which contains the β-galactosidase gene under the control of the polyhedrin promoter. Isolation of recombinant baculovirus was achieved by negative selection due to replacement of the β-galactosidase gene by the newly introduced recombinant gene. The selected virus was further purified after three successive plaque assays in Sf9 cells. The recombinant baculovirus expressing the A179L gene was denominated BVA179L. Another recombinant baculovirus bearing the A179L gene cloned in the opposite orientation (antisense, BVA179L-AS) was constructed by similar methods.Expression of the A179L gene by the recombinant baculovirus BVA179L was analyzed by Western blotting at 72 h postinfection (p.i.) in cells from suspension and monolayer cultures (Fig. ​(Fig.1A).1A). Cell extracts with similar numbers of infected cells from both cultures were lysed, electrophoresed in sodium dodecyl sulfate (SDS)–15% polyacrylamide gels, transferred to nitrocellulose filters (Bio-Rad), and then incubated with a polyclonal antiserum raised against the product of the ASFV gene 5HL expressed in Escherichia coli cells (28). The serum reacted with a 21-kDa polypeptide (p21) but failed to react with any protein product of this electrophoretic mobility in mock-infected cells (data not shown) or in cells infected with the recombinant baculovirus BVA179L-AS (Fig. ​(Fig.1A).1A). Open in a separate windowFIG. 1Expression of the ASFV A179L gene by using a baculovirus system. (A) Western blot analysis of infected Sf9 cell extracts. Cell lysates from Sf9 cells synchronously infected either with the recombinant baculovirus BVA179L or with BVA179L-AS reacted at 72 h p.i. with a specific antiserum raised against the A179L gene product expressed in E. coli cells. (B) Immunofluorescence of BVA179L- or BVA179L-AS-infected insect cells in suspension, analyzed at 72 h p.i. by using the same serum against A179L stained with fluorescein isothiocyanate (FITC). Green cytoplasmic fluorescence was detected only in cells infected with BVA179L. The cell nucleus was contrasted with propidium iodide (PI) (right). (C) Growth curves of BVA179L and BVA179L-AS recombinant baculoviruses in Sf9 monolayer cell cultures. Extracellular virus obtained from culture supernatants at the indicated time points was titrated in a conventional plaque assay. Squares, BVA179L-AS; circles, BVA179L.Indirect immunofluorescence of fixed and permeabilized BVA179L- or BVA179L-AS-infected Sf9 cells, using the same anti-p21 serum, revealed predominantly cytoplasmic staining for p21 (Fig. ​(Fig.1B,1B, center), which contrasted with the nuclear localization of propidium iodide, a DNA-intercalating agent (Fig. ​(Fig.1B,1B, right). This distribution of p21 was similar in cells grown in suspension or in adherent growth conditions.The effect of p21 expression on recombinant baculovirus growth was also investigated (Fig. ​(Fig.1C).1C). Cells were infected (multiplicity of infection [MOI], 2), supernatants were collected at different times postinfection for titration, and fresh medium was added to the cultures. Infection with BVA179L or BVA179L-AS yielded similar virus titers, suggesting that overexpression of the A179L gene did not alter the baculovirus replication phenotype in Sf9 cells. Maximum viral titers were reached at 48 h p.i. with both recombinant viruses (Fig. ​(Fig.1C).1C). As expected, virus yields dropped drastically after this time point, because the experiment measured virus release to the extracellular medium rather than virus accumulation. This result suggested the occurrence of a strong shutoff of protein synthesis due to baculovirus infection at late time points.In order to confirm this fact, long-term synthesis of baculovirus-induced proteins in the presence of the apoptosis inhibitor p21 was analyzed by metabolic pulse labeling of infected cells at different times postinfection. Sf9 cells (10⁵) were mock infected or infected at a MOI of 10 in 96-well culture plates. At 22, 46, 70, and 94 h p.i. the medium was replaced with Grace’s medium lacking methionine (Gibco) and maintained for 1 h prior to the addition of fresh methionine-deficient medium containing 200 μCi of [³⁵S]methionine/ml (>1,000 Ci/mmol). At the selected time points cell pellets were harvested, lysed in SDS buffer, and analyzed by autoradiography after SDS-polyacrylamide gel electrophoresis. A strong shutoff of protein synthesis was observed starting from 48 h p.i. in monolayer cultures infected with BVA179L, BVA179L-AS, or a baculovirus expressing β-galactosidase (data not shown). At 96 h p.i. no newly synthesized cellular or viral-induced proteins were detected in any infected culture.

Functionality of the A179L gene in insect cells.

To determine the possible role of the A179L gene in prevention of baculovirus-induced apoptosis, viability of cell cultures infected at MOI of 2, 10 and 100 was determined at various times postinfection by trypan blue exclusion by counting 1 × 10³ to 1.4 × 10³ cells in five independent fields each (Fig. ​(Fig.2).2). High MOI were used to assure a synchronized infection with 99% infected cells. Viral infections on monolayer cell cultures were carried out in multiwell plastic dishes (Nunc). For the infection of suspension cultures, 250-ml flasks in a rotary shaker, with constant stirring at 80 rpm, were used. Both monolayer and suspension cultures were maintained in Grace’s insect medium (Gibco-BRL) supplemented with 10% fetal bovine serum. Cells were inoculated with extracellular, budded virus, and viral titers were determined by plaque assay on 10⁶ cells seeded onto 35-mm dishes and overlaid with a mixture of 0.7% agarose (Sigma) in 10% fetal bovine serum–Grace’s medium. Infections with a control baculovirus expressing the reporter gene β-galactosidase at a MOI of 10 or higher yielded more than 95% cells expressing the reporter gene in both monolayer and suspension (data not shown). With all recombinants, all cells showed a clear cytopathic effect characteristic of baculovirus productive infection. Open in a separate windowFIG. 2Effect of A179L gene expression on the survival of baculovirus-infected Sf9 cells. Viability of insect cells grown in monolayers (A) or in suspension (B) and infected with BVA179L-AS (squares) or BVA179L (circles) at a MOI of 100 was determined by trypan blue exclusion at different times postinfection. Means and standard errors were calculated from three independent experiments. (C) A representative field of trypan blue-stained monolayer cultures of BVA179L- or BVA179L-AS-infected Sf9 cells at 144 h p.i. Original magnification, ×200.The viability levels of baculovirus-infected cells correlated with the postinfection time. Differences in cell viability were consistently found after 48 h p.i., when viral yields reached a maximum and protein shutoff was more evident. In monolayer cultures (Fig. ​(Fig.2A),2A), fewer than 20% of the BVA179L-AS-infected cells survived to infection at 144 h p.i. (Fig. ​(Fig.2A).2A). In contrast, cells infected with the baculovirus expressing the A179L gene presented only a slight decrease in viability at this time point (Fig. ​(Fig.2A).2A). A representative field of cells infected with recombinant baculoviruses stained with trypan blue at 144 h p.i. is shown in Fig. ​Fig.2C.2C. Thus, the expression of p21 at late times postinfection increased the survival of baculovirus-infected Sf9 cells cultured in monolayer.However, in suspension, viability experiments carried out with cells infected with either BVA179L or BVA179L-AS did not result in demonstrable differences in survival rates (Fig. ​(Fig.2B).2B). High proportions of dead cells (about 50%) were found at 48 h p.i. in spite of p21 expression. These discrepancies found between monolayer and suspension cultures suggested that the effect of p21 expression on Sf9 viability could be related to the lack of cell attachment to a substrate in suspended cultures.We then investigated whether the increased viability found in BVA179L-infected cells in monolayer cultures was due to apoptosis inhibition. Since both viruses expressed identical levels of baculovirus p35 at early times (11), infection-induced apoptosis should be similar in both cases, thus minimizing the differences in survival rates before 48 h p.i. Beyond this time point, when p35 is no longer functional (8, 11), differences between BVA179L and BVA179L-AS, not expressing p21, might become evident. As programmed cell death is relatively highly conserved during evolution and inhibitors of apoptosis are functionally interchangeable among distant species, it might be reasonable to suggest a function for A179L in insect cells similar to that displayed in mammalian cells. However, the bcl-2 homolog gene function in insect cells still remains controversial. Alnemri et al. (3) found that overexpression of human bcl-2 increased survival of baculovirus-infected Sf9 cells by prevention of apoptosis. Since the gene encoding p21 is a bcl-2 homolog (1, 5, 37), it seems likely that both genes act in similar apoptosis pathways. Nevertheless, it was reported that expression of bcl-2 or the adenovirus gene E1B-19K did not rescue the wild-type phenotypes of baculoviruses lacking the p35 gene (8, 11), so it was postulated that early apoptosis induction prevented by p35 expression could be mediated by bcl-2-independent mechanisms in Sf9 cells. Baculovirus infection could then trigger two different apoptosis mechanisms, an early apoptosis blocked by p35 but not by bcl-2 or its homologs, such as p21, and a late apoptosis induction in which p21/bcl-2 might be functional.The chromatin fragmentation of baculovirus-infected cells by different methods was then analyzed (Fig. ​(Fig.3A3A to C). First we carried out a comparative Hoechst 33258 staining of infected cells in monolayer at different hours postinfection with BVA179L or BVA179L-AS viruses (Fig. ​(Fig.3A).3A). Cells were methanol fixed for 10 min before incubation with 10 μg of Hoechst dye per ml in phosphate-buffered saline for 30 min at room temperature. BVA179L-AS-virus-infected cells exhibited a chromatin fragmentation pattern characteristic of apoptosis (Fig. ​(Fig.3A,3A, right) which increased in a time-dependent fashion (data not shown). In contrast, at 144 h p.i. Sf9 cells infected with the BVA179L virus presented very few figures of apoptosis (Fig. ​(Fig.3A,3A, left). This result indicated a correlation between cell viability and occurrence of chromatin fragmentation, which was confirmed by DNA laddering analysis (Fig. ​(Fig.3C)3C) in 1.6% agarose gels (5). These experiments confirmed the lack of apoptosis prevention by p21 under nonadherent cell culture growth conditions (Fig. ​(Fig.3C,3C, right). Open in a separate windowFIG. 3DNA fragmentation in infected Sf9 cells (MOI of 10). (A) Hoechst 33258 staining of BVA179L- or BVA179L-AS-infected Sf9 cells grown in plates at 144 h p.i. Original magnification, ×400. (B) ELISA quantitation of the DNA linked to histone proteins in the cytoplasmic fraction of apoptotic Sf9 cells infected in monolayer at different time points. Squares: BVA179L-AS-infected cells; circles, BVA179L-infected cells; triangles, mock-infected cells. Means and standard errors were calculated from three independent experiments. (C) Agarose gel electrophoresis of the internucleosomal DNA laddering detected in infected or mock-infected Sf9 cells in monolayer cultures at 96 and 144 h p.i. (left) or in suspension cultures at 96 h p.i. (right). M, molecular size markers.Quantitation of histone-associated DNA fragments released to the cytoplasm was carried out by a specific enzyme-linked immunosorbent assay (ELISA) (Boehringer Mannheim) (5). The results obtained after this analysis clearly indicated that expression of the ASFV gene A179L prevented the onset of apoptosis induced by baculovirus infection of cells cultured in monolayer (Fig. ​(Fig.3B).3B). In contrast, at 144 h p.i. BVA179L-AS-virus-infected cells (Fig. ​(Fig.3B)3B) yielded higher apoptosis rates than cells expressing p21.The above results strongly suggest that the antiapoptotic function of p21 is dependent on cell attachment, because in suspended cells, A179L expression was unable to prevent baculovirus-induced apoptosis. Cell attachment is important for many cell functions. In fact, most types of normal cells require extracellular matrix attachment to respond to growth factor stimulation and other signals controlling cell proliferation or survival. When detached from their matrix, some cells undergo apoptosis (7, 15, 19, 26).

Effect of cell attachment on p21 function.

In an attempt to confirm if the activity of p21 was dependent on cell attachment, we performed experiments by infecting Sf9 cells with recombinant baculoviruses under conditions that improved cell adhesion to the culture surface (Fig. ​(Fig.4A).4A). Sf9 cells were cultured on extracellular matrix components, such as rat collagen type I and human fibronectin, or on poly-d-lysine-coated glass plates (Falcon). Then, cells were infected with BVA179L or BVA179L-AS viruses at a MOI of 10. Figure ​Figure4A4A shows the apoptosis indices (AIs) of those cultures measured by anti-histone quantitative ELISA (inverse correlation). In BVA179L-infected cultures, apoptosis rate reduction was detected with all substrates that facilitated cell attachment by any pathway. Either extracellular matrix components (collagen type I and fibronectin) that support integrin-mediated cell adhesion or a substrate that mediates adhesion by nonspecific interactions (poly-d-lysine) enhanced apoptosis protection with respect to apoptosis of cells grown on uncoated nonadherent glass surfaces (AI of ≤1). Interestingly, infections performed on cells on a collagen type I matrix yielded more differences in apoptosis inhibition by p21 (AI of ≤0.5) with respect to apoptosis of cells on untreated surfaces (Fig. ​(Fig.4A).4A). This could be related to the fact that Sf9 cells grown on this type of surface also had increased cell-to-cell interactions (not shown). Open in a separate windowFIG. 4(A) Apoptosis induction of Sf9 cells (expressing p21) expressed as the ratio to that in BVA179L-AS-infected cells grown on different substrates to improve adhesiveness, measured by ELISA. The AI is given by the quotient (BVA179L-infected cells/BVA179L-AS-infected cells) of mean absorbance values at 405 nm from two replicate experiments at 96 h p.i. FN, human fibronectin; PDL, poly-d-lysine; COLI, rat tail collagen type I; UNTREATED, BVA179L-infected cells. (B) Actin cytoskeleton organization in adherent insect cells (left). Cells cultured in suspended growth conditions presented actin focalization (right). Cellular F-actin was stained with phalloidin-tetramethyl rhodamine isothiocyanate. (C) Characteristic individual infected Sf9 cells cultured in monolayer (upper) or in suspension (lower). Actin clumped in coarse fragments showed intense red staining. Original magnification, ×600 (panels B and C).Cell attachment to underlying extracellular matrix is mediated by specialized membrane proteins called integrins, which interact with determined extracellular matrix components (6, 22). Integrin-mediated cell adhesion initiates a cascade of events that allow the transduction of survival signals that can block programmed cell death (13, 25). Induction of this survival pathway includes the upregulation of antiapoptotic proteins such as bcl-2 family members (16, 27, 42, 43). Also, cell-to-cell interaction inversely correlates with apoptosis associated with bcl-2 protein expression (4); in our results, aggregation of Sf9 cells plated on collagen I matrix resulted in enhanced survival. Apoptosis inhibition by p21 was also obtained with a nonspecific adhesive, such as poly-d-lysine, a synthetic compound altering surface charge, that increases cell adhesion not mediated by integrins. Our results indicate that cell attachment alone is sufficient to allow for the antiapoptotic activity of p21 in Sf9 cells infected by baculovirus. In fact, recent findings focus on cell shape changes and cytoskeleton integrity as supportive of rescue from apoptosis (10, 38).

Cytoskeleton organization in insect cells during infection with baculovirus in presence of p21.

Since conditions of cell anchorage may modify cytoskeleton organization, we analyzed the actin cytoskeleton of insect cells cultured under adherent and suspended growth conditions (Fig. ​(Fig.4).4). Sf9 cells were grown either directly on 96-well multiwell plates or in spinner flasks. Cells from suspension cultures were removed and allowed to sediment on glass slides. In both cases, cells were fixed with 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 in phosphate-buffered saline, and stained for 30 min with 1:300 phalloidin-tetramethyl rhodamine isothiocyanate (Sigma), a marker for F-actin. Uninfected Sf9 cells attached to a surface displayed morphology different from that of cells that were grown in suspension. A profuse and fine surface microvillar network present in attached cells (Fig. ​(Fig.4B,4B, left) was lacking in suspension. In contrast, suspended cells showed marked focalization of actin staining, indicating the occurrence of cytoskeleton reorganization (Fig. ​(Fig.4B,4B, right). Cells in suspension infected with either BVA179L or BVA179L-AS baculoviruses showed irregular clumping of actin (Fig. ​(Fig.4C,4C, lower). Dense actin staining was found concentrated in coarse fragments, and such elements were found in the cultures in proportions similar to that in the nonviable cell fraction (Fig. ​(Fig.2A2A and B). However, this pattern of actin clumping was not found in attached cells infected with BVA179L (Fig. ​(Fig.4C,4C, upper left). Moreover, in monolayer, the overall intensity of actin staining decreased in a time-dependent manner in cells infected with BVA179L-AS, but it was maintained longer in infected cells expressing p21 (not shown). This result suggests that the expression of p21 in BVA179L-infected cells could contribute to the preservation of the cellular actin cytoskeleton at late postinfection times.Cell adhesion to underlying extracellular matrix is mediated by sites of tight adhesion, called focal adhesions, that develop in cells in culture (12). Focal adhesions provide a structural link between the actin cytoskeleton and the extracellular matrix and are regions of signal transduction related to gene expression, growth control, and cell survival. It was recently suggested that cell attachment to matrix or integrin binding per se is not sufficient for maintaining cell viability and that cells need to undergo some minimal degree of shape changes to survive (10, 36, 38). It was recently reported that suspended endothelial cells acquired rounded shape, presented cytoskeleton disorganization, and underwent apoptosis (36). In contrast, when cells were grown on fibronectin or vitronectin, they became flattened, showed actin microfilament organization, and retained viability (36). Interleukin 4, which is able to activate neutrophil cytoskeletal rearrangements, produces a delay of apoptosis (20). Also, epithelial cells cultured on extracellular matrix components or laminin had a more well-developed actin cytoskeleton than cells cultured on noncoated dishes, which underwent apoptosis (2). The organization of the actin cytoskeleton in Sf9 cells grown attached to a surface was quite different from that displayed by cells grown in suspension. Consequently, actin organization and cell shape changes might provide the conditions for p21 protective function.The in vivo relevance of changes in cell anchorage has been mainly focused on to date, either in the leukocyte movement out of vessels along endothelial cells in inflammation (23, 39) or in the loss of adherence of transformed cells in metastasis production (17, 18). Our findings suggest a role for cell shape and cytoskeleton status in viral diseases as well. Variations in those conditions in determined tissues or cell types in viral infections might explain differences in intracellular transduction of signals related to cell growth and survival. The cell anchorage dependence demonstrated by the ASFV bcl-2 homolog could have important consequences in the infection among the different cell compartments in vivo. Nevertheless, the physiological relevance of the biological effects of a protein overexpressed to the levels reported here should be an object of future studies. Based on these findings, it should be further analyzed if the absence of function of A179L apoptosis inhibitor in nonattached infected cells, such as circulating cells, might favor an early apoptosis induction and death of those cells. In fact, during in vivo infection with ASFV, only small percentages of infected monocytes are detected in peripheral blood (33). In contrast, tissue-fixed macrophages attached to extracellular matrix could be more prone to support the function of the bcl-2 viral homolog in vivo, leading to apoptosis inhibition in these cells, and could constitute a viral reservoir during persistent infection.In conclusion, the above-presented data demonstrate that the product of the ASFV A179L gene, p21, is functional in insect cells and prevents late apoptosis after baculovirus infection. p21 increases the viability of infected cells in the context of a strong shutoff of protein synthesis and without modifying the baculovirus infection cycle. This suggests that p21 probably inhibits, in a way similar to human bcl-2, a highly conserved component of the apoptosis execution program.     

Involvement of ZO-1 in Cadherin-based Cell Adhesion through Its Direct Binding to α Catenin and Actin Filaments

ZO-1, a 220-kD peripheral membrane protein consisting of an amino-terminal half discs large (dlg)-like domain and a carboxyl-terminal half domain, is concentrated at the cadherin-based cell adhesion sites in non-epithelial cells. We introduced cDNAs encoding the full-length ZO-1, its amino-terminal half (N-ZO-1), and carboxyl-terminal half (C-ZO-1) into mouse L fibroblasts expressing exogenous E-cadherin (EL cells). The full-length ZO-1 as well as N-ZO-1 were concentrated at cadherin-based cell–cell adhesion sites. In good agreement with these observations, N-ZO-1 was specifically coimmunoprecipitated from EL transfectants expressing N-ZO-1 (NZ-EL cells) with the E-cadherin/α, β catenin complex. In contrast, C-ZO-1 was localized along actin stress fibers. To examine the molecular basis of the behavior of these truncated ZO-1 molecules, N-ZO-1 and C-ZO-1 were produced in insect Sf9 cells by recombinant baculovirus infection, and their direct binding ability to the cadherin/catenin complex and the actin-based cytoskeleton, respectively, were examined in vitro. Recombinant N-ZO-1 bound directly to the glutathione-S-transferase fusion protein with α catenin, but not to that with β catenin or the cytoplasmic domain of E-cadherin. The dissociation constant between N-ZO-1 and α catenin was ~0.5 nM. On the other hand, recombinant C-ZO-1 was specifically cosedimented with actin filaments in vitro with a dissociation constant of ~10 nM. Finally, we compared the cadherin-based cell adhesion activity of NZ-EL cells with that of parent EL cells. Cell aggregation assay revealed no significant differences among these cells, but the cadherin-dependent intercellular motility, i.e., the cell movement in a confluent monolayer, was significantly suppressed in NZ-EL cells. We conclude that in nonepithelial cells, ZO-1 works as a cross-linker between cadherin/catenin complex and the actin-based cytoskeleton through direct interaction with α catenin and actin filaments at its amino- and carboxyl-terminal halves, respectively, and that ZO-1 is a functional component in the cadherin-based cell adhesion system.     

Receptor Inhibition of Pheromone Signaling Is Mediated by the Ste4p Gβ Subunit

The pheromone response pathway of the yeast Saccharomyces cerevisiae is initiated in MATa cells by binding of α-factor to the α-factor receptor. MATa cells in which the a-factor receptor is inappropriately expressed exhibit reduced pheromone signaling, a phenomenon termed receptor inhibition. In cells undergoing receptor inhibition, activation of the signaling pathway occurs normally at early time points but decreases after prolonged exposure to pheromone. Mutations that suppress the effects of receptor inhibition were obtained in the STE4 gene, which encodes the β-subunit of the G protein that transmits the pheromone response signal. These mutations mapped to the N terminus and second WD repeat of Ste4p in regions that are not part of its G_α binding surface. A STE4 allele containing several of these mutations, called STE4^SD13, reversed the signaling defect seen at late times in cells undergoing receptor inhibition but had no effect on the basal activity of the pathway. Moreover, the signaling properties of STE4^SD13 were indistinguishable from those of STE4 in wild-type MATa and MATα cells. These results demonstrate that the effect of the STE4^SD13 allele is specific to the receptor inhibition function of STE4. STE4^SD13 suppressed the signaling defect conferred by receptor inhibition in a MATa strain containing a deletion of GPA1, the G protein α-subunit gene; however, STE4^SD13 had no effect in a MATα strain containing a GPA1 deletion. Suppression of receptor inhibition by STE4^SD13 in a MATa strain containing a GPA1 deletion was unaffected by deletion of STE2, the α-factor receptor gene. The results presented here are consistent with a model in which an a-specific gene product other than Ste2p detects the presence of the a-factor receptor and blocks signaling by inhibiting the function of Ste4p.     

The EphA8 Receptor Regulates Integrin Activity through p110γ Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

Recent genetic studies suggest that ephrins may function in a kinase-independent Eph receptor pathway. Here we report that expression of EphA8 in either NIH 3T3 or HEK293 cells enhanced cell adhesion to fibronectin via alpha(5)beta(1)- or beta(3) integrins. Interestingly, a kinase-inactive EphA8 mutant also markedly promoted cell attachment to fibronectin in these cell lines. Using a panel of EphA8 point mutants, we have demonstrated that EphA8 kinase activity does not correlate with its ability to promote cell attachment to fibronectin. Analysis using EphA8 extracellular and intracellular domain mutants has revealed that enhanced cell adhesion is dependent on ephrin A binding to the extracellular domain and the juxtamembrane segment of the cytoplasmic domain of the receptor. EphA8-promoted adhesion was efficiently inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. Additionally, we found that EphA8 had associated PI 3-kinase activity and that the p110gamma isoform of PI 3-kinase is associated with EphA8. In vitro binding experiments revealed that the EphA8 juxtamembrane segment was sufficient for the formation of a stable complex with p110gamma. Similar results were obtained in assay using cells stripped of endogenous ephrin A ligands by treatment with preclustered ephrin A5-Fc proteins. In addition, a membrane-targeted lipid kinase-inactive p110gamma mutant was demonstrated to stably associate with EphA8 and suppress EphA8-promoted cell adhesion to fibronectin. Taken together, these results suggest the presence of a novel mechanism by which the EphA8 receptor localizes p110gamma PI 3-kinase to the plasma membrane in a tyrosine kinase-independent fashion, thereby allowing access to lipid substrates to enable the signals required for integrin-mediated cell adhesion.     

Inhibition of nonsense-mediated decay rescues p53β/γ isoform expression and activates the p53 pathway in MDM2-overexpressing and select p53-mutant cancers

Inactivation of p53 is present in almost every tumor, and hence, p53-reactivation strategies are an important aspect of cancer therapy. Common mechanisms for p53 loss in cancer include expression of p53-negative regulators such as MDM2, which mediate the degradation of wildtype p53 (p53α), and inactivating mutations in the TP53 gene. Currently, approaches to overcome p53 deficiency in these cancers are limited. Here, using non–small cell lung cancer and glioblastoma multiforme cell line models, we show that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising exons 1 to 9β or 9γ, respectively) and that lack the C-terminal MDM2-binding domain have markedly reduced susceptibility to MDM2-mediated degradation but are highly susceptible to nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53β and p53γ and restores activation of the p53 pathway. Consistent with p53 pathway activation, NMD inhibition induces tumor suppressive activities such as apoptosis, reduced cell viability, and enhanced tumor radiosensitivity, in a relatively p53-dependent manner. In addition, NMD inhibition also inhibits tumor growth in a MDM2-overexpressing xenograft tumor model. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in p53-deficient tumors bearing MDM2 overexpression or p53 mutations downstream of exon 9, subgroups that comprise approximately 6% of all cancers.