Replacement of the Replication Factors of Porcine Circovirus (PCV) Type 2 with Those of PCV Type 1 Greatly Enhances Viral Replication In Vitro

Porcine circovirus type 1 (PCV1), originally isolated as a contaminant of PK-15 cells, is nonpathogenic, whereas porcine circovirus type 2 (PCV2) causes an economically important disease in pigs. To determine the factors affecting virus replication, we constructed chimeric viruses by swapping open reading frame 1 (ORF1) (rep) or the origin of replication (Ori) between PCV1 and PCV2 and compared the replication efficiencies of the chimeric viruses in PK-15 cells. The results showed that the replication factors of PCV1 and PCV2 are fully exchangeable and, most importantly, that both the Ori and rep of PCV1 enhance the virus replication efficiencies of the chimeric viruses with the PCV2 backbone.Porcine circovirus (PCV) is a single-stranded DNA virus in the family Circoviridae (34). Type 1 PCV (PCV1) was discovered in 1974 as a contaminant of porcine kidney cell line PK-15 and is nonpathogenic in pigs (31-33). Type 2 PCV (PCV2) was discovered in piglets with postweaning multisystemic wasting syndrome (PMWS) in the mid-1990s and causes porcine circovirus-associated disease (PCVAD) (1, 9, 10, 25). PCV1 and PCV2 have similar genomic organizations, with two major ambisense open reading frames (ORFs) (16). ORF1 (rep) encodes two viral replication-associated proteins, Rep and Rep′, by differential splicing (4, 6, 21, 22). The Rep and Rep′ proteins bind to specific sequences within the origin of replication (Ori) located in the intergenic region, and both are responsible for viral replication (5, 7, 8, 21, 23, 28, 29). ORF2 (cap) encodes the immunogenic capsid protein (Cap) (26). PCV1 and PCV2 share approximately 80%, 82%, and 62% nucleotide sequence identity in the Ori, rep, and cap, respectively (19).In vitro studies using a reporter gene-based assay system showed that the replication factors of PCV1 and PCV2 are functionally interchangeable (2-6, 22), although this finding has not yet been validated in a live infectious-virus system. We have previously shown that chimeras of PCV in which cap has been exchanged between PCV1 and PCV2 are infectious both in vitro and in vivo (15), and an inactivated vaccine based on the PCV1-PCV2 cap (PCV1-cap2) chimera is used in the vaccination program against PCVAD (13, 15, 18, 27).PCV1 replicates more efficiently than PCV2 in PK-15 cells (14, 15); thus, we hypothesized that the Ori or rep is directly responsible for the differences in replication efficiencies. The objectives of this study were to demonstrate that the Ori and rep are interchangeable between PCV1 and PCV2 in a live-virus system and to determine the effects of swapped heterologous replication factors on virus replication efficiency in vitro.     

The Direct Binding of Mrc1, a Checkpoint Mediator,to Mcm6, a Replication Helicase,Is Essential for the Replication Checkpoint against Methyl Methanesulfonate-Induced Stress

Mrc1 plays a role in mediating the DNA replication checkpoint. We surveyed replication elongation proteins that interact directly with Mrc1 and identified a replicative helicase, Mcm6, as a specific Mrc1-binding protein. The central portion of Mrc1, containing a conserved coiled-coil region, was found to be essential for interaction with the 168-amino-acid C-terminal region of Mcm6, and introduction of two amino acid substitutions in this C-terminal region abolished the interaction with Mrc1 in vivo. An mcm6 mutant bearing these substitutions showed a severe defect in DNA replication checkpoint activation in response to stress caused by methyl methanesulfonate. Interestingly, the mutant did not show any defect in DNA replication checkpoint activation in response to hydroxyurea treatment. The phenotype of the mcm6 mutant was suppressed when the mutant protein was physically fused with Mrc1. These results strongly suggest for the first time that an Mcm helicase acts as a checkpoint sensor for methyl methanesulfonate-induced DNA damage through direct binding to the replication checkpoint mediator Mrc1.Progression of the DNA replication machinery along chromosomes is a complex process. Replication forks pause occasionally when they encounter genomic regions that are difficult to replicate, such as highly transcribed regions, tRNA genes, and regions with specialized chromatin structure, like centromeric and heterochromatic regions (17). Replication forks also stall when treated with chemicals like methyl methanesulfonate (MMS), which causes DNA damage, or hydroxyurea (HU), which limits the cellular concentration of the deoxynucleoside triphosphate pool (17). Because de novo assembly and programming of the replisome do not occur after the onset of S phase (18), DNA replication forks must be protected from replicative stresses. The DNA replication checkpoint constitutes a surveillance mechanism for S-phase progression that safeguards replication forks from various replicative stresses (22, 38, 40), and malfunction of this checkpoint leads to chromosome instability and cancer development in higher organisms (4, 9).The Saccharomyces cerevisiae DNA replication checkpoint mediator Mrc1 is functionally conserved and is involved directly in DNA replication as a component of the replisome (1, 8, 16, 19, 29, 30). Mrc1, together with Tof1 and Csm3, is required for forming a replication pausing complex when the fork is exposed to replicative stress by HU (16). The pausing complex subsequently triggers events leading to DNA replication checkpoint activation and hence stable replicative arrest. A sensor kinase complex, Mec1-Ddc2 (ATR-ATRIP homolog of higher eukaryotes), is then recruited to the complex (14, 16). Mec1-Ddc2-mediated phosphorylation of Mrc1 activates the pausing complex, and phosphorylated Mrc1 likely recruits Rad53 (a putative homolog of CHK2 of higher eukaryotes), which is then activated via phosphorylation by Mec1-Ddc2 (1, 16, 20, 30). Activated Rad53 subsequently elicits a stress responses, i.e., stabilization of replication forks, induction of repair genes, and suppression of late-firing origins (24). It remains unclear, however, whether DNA replication checkpoint activation is induced in response to DNA damage by MMS, a reagent commonly used to study the DNA replication stress response. Several lines of evidence have suggested that MMS-induced damage is also sensed directly by the replication machinery (38, 40).Although biochemical and genetic interaction data have placed Mrc1 at the center of the replication checkpoint signal transduction cascade, its molecular function remains largely unknown. The proteins Mrc1, Tof1, and Csm3 associate with the Mcm complex (8, 27), a heterohexameric DNA helicase consisting of Mcm2 to Mcm7 proteins which unwinds the parental DNA duplex to allow replisome progression (3, 12, 18, 31, 32, 35). The Mcm complex associates with a specific set of regulatory proteins at forks to form replisome progression complexes (8). In addition to Mcm, Tof1, Csm3, and Mrc1, replisome progression complexes include factors such as Cdc45 and the GINS complex that are also required for fork progression (13, 26, 31, 32, 39). Claspin, a putative Xenopus laevis homolog of Mrc1, is also reported to associate with Cdc45, DNA polymerase ɛ (Polɛ), replication protein A, and two of the replication factor C complexes in aphidicolin-treated Xenopus egg extracts (19). Recently, Mrc1 was reported to interact directly with Polɛ (23).The aim of this study was to provide mechanistic insight into Mrc1 function in the DNA replication checkpoint. For this purpose, it was essential to identify, among all the essential proteins in the replication machinery, a specific protein that interacts with Mrc1 and to examine the role of this interaction in the DNA replication checkpoint. We found that Mrc1 interacts with Mcm6 directly and specifically. When the interaction between Mrc1 and Mcm6 was impaired, cells no longer activated the DNA replication checkpoint in response to MMS-induced replicative stress. Interestingly and unexpectedly, this interaction was not required for DNA replication checkpoint activation in response to HU-induced replicative stress. Our results provide the first mechanistic evidence that cells use separate mechanisms to transmit replicative stresses caused by MMS and HU for DNA replication checkpoint activation.     

Adeno-Associated Virus Replication Induces a DNA Damage Response Coordinated by DNA-Dependent Protein Kinase

The parvovirus adeno-associated virus (AAV) contains a small single-stranded DNA genome with inverted terminal repeats that form hairpin structures. In order to propagate, AAV relies on the cellular replication machinery together with functions supplied by coinfecting helper viruses such as adenovirus (Ad). Here, we examined the host cell response to AAV replication in the context of Ad or Ad helper proteins. We show that AAV and Ad coinfection activates a DNA damage response (DDR) that is distinct from that seen during Ad or AAV infection alone. The DDR was also triggered when AAV replicated in the presence of minimal Ad helper proteins. We detected autophosphorylation of the kinases ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and signaling to downstream targets SMC1, Chk1, Chk2, H2AX, and XRCC4 and multiple sites on RPA32. The Mre11 complex was not required for activation of the DDR to AAV infection. Additionally, we found that DNA-PKcs was the primary mediator of damage signaling in response to AAV replication. Immunofluorescence revealed that some activated damage proteins were found in a pan-nuclear pattern (phosphorylated ATM, SMC1, and H2AX), while others such as DNA-PK components (DNA-PKcs, Ku70, and Ku86) and RPA32 accumulated at AAV replication centers. Although expression of the large viral Rep proteins contributed to some damage signaling, we observed that the full response required replication of the AAV genome. Our results demonstrate that AAV replication in the presence of Ad helper functions elicits a unique damage response controlled by DNA-PK.Replication of viral genomes produces a large amount of extrachromosomal DNA that may be recognized by the cellular DNA damage machinery. This is often accompanied by activation of DNA damage response (DDR) signaling pathways and recruitment of cellular repair proteins to sites of viral replication. Viruses therefore provide good model systems to study the recognition and response to DNA damage (reviewed in reference 48). The Mre11/Rad50/Nbs1 (MRN) complex functions as a sensor of chromosomal DNA double-strand breaks (DSBs) and is involved in activation of damage signaling (reviewed in reference 41). The MRN complex also localizes to DNA DSBs and is found at viral replication compartments during infection with a number of DNA viruses (6, 40, 47, 70, 75, 77, 87, 93). The phosphatidylinositol 3-kinase-like kinases (PIKKs) ataxia telangiectasia mutated (ATM), ATM and Rad3-related kinase (ATR), and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) are involved in the signal transduction cascades activated by DNA damage (reviewed in references 43, 51, and 71). These kinases respond to distinct types of damage and regulate DSB repair during different phases of the cell cycle (5), either through nonhomologous end-joining (NHEJ) or homologous recombination pathways (reviewed in references 63, 81, and 86). The DNA-PK holoenzyme is composed of DNA-PKcs and two regulatory subunits, the Ku70 and Ku86 heterodimer. DNA-PK functions with XRCC4/DNA ligase IV to repair breaks during NHEJ, and works with Artemis to process DNA hairpin structures during VDJ recombination and during a subset of DNA DSB events (46, 50, 86). While the kinase activity of DNA-PKcs leads to phosphorylation of a large number of substrates in vitro as well as autophosphorylation of specific residues (reviewed in references 16 and 85), it is currently unclear how DNA-PKcs contributes to signaling in cells upon different types of damage.The adeno-associated virus (AAV) genome consists of a molecule of single-stranded DNA with inverted terminal repeats (ITRs) at both ends that form double-hairpin structures due to their palindromic sequences (reviewed in reference 52). The ITRs are important for replication and packaging of the viral genome and for integration into the host genome. Four viral Rep proteins (Rep78, Rep68, Rep52, and Rep40) are also required for replication and packaging of the AAV genome into virions assembled from the Cap proteins. Although the Rep and Cap genes are replaced in recombinant AAV vectors (rAAV) that retain only the ITRs flanking the gene of interest, these vectors can be replicated by providing Rep in trans (reviewed in reference 7). Productive AAV infection requires helper functions supplied by adenovirus (Ad) or other viruses such as herpes simplex virus (HSV) (reviewed in reference 27), together with components of the host cell DNA replication machinery (54, 55, 58). In the presence of helper viruses or minimal helper proteins from Ad or HSV, AAV replicates in the nucleus at centers where the viral DNA and Rep proteins accumulate (35, 76, 84, 89). Cellular and viral proteins involved in AAV replication, including replication protein A (RPA), Ad DNA-binding protein (DBP), and HSV ICP8, localize with Rep proteins at these viral centers (29, 33, 76).A number of published reports suggest associations between AAV and the cellular DNA damage machinery. For example, transduction by rAAV vectors is increased by genotoxic agents and DNA damaging treatments (1, 62, 91) although the mechanisms involved remain unclear. Additionally, the ATM kinase negatively regulates rAAV transduction (64, 92), and we have shown that the MRN complex poses a barrier to both rAAV transduction and wild-type AAV replication (11, 67). UV-inactivated AAV particles also appear to activate a DDR involving ATM and ATR kinases that perturbs cell cycle progression (39, 60, 88). It has been suggested that this response is provoked by the AAV ITRs (60) and that UV-treated particles mimic stalled replication forks in infected cells (39). In addition to AAV genome components, the viral Rep proteins have been observed to exhibit cytotoxicity and induce S-phase arrest (3, 65).The role of cellular repair proteins in AAV genome processing has also been explored by examining the molecular fate of rAAV vectors, which are converted into circular and concatemeric forms that persist episomally (18, 19, 66). Proteins shown to regulate circularization in cell culture include ATM and the MRN complex (14, 64), while in vivo experiments using mouse models have implicated ATM and DNA-PK in this process (14, 20, 72). Additionally, DNA-PKcs and Artemis have recently been shown to cleave the ITR hairpins of rAAV vectors in vivo in a tissue-dependent manner (36). Despite these studies, it is not clear how damage response factors function together and how they impact AAV transduction and replication in human cells.In this study we examined the cellular response to AAV replication in the context of Ad infection or helper proteins. We show that coinfection with AAV and Ad activates a DDR that is distinct from that seen during infection with Ad alone. The ATM and DNA-PKcs damage kinases are activated and signal to downstream substrates, but the response does not require the MRN complex and is primarily mediated by DNA-PKcs. Although expression of the large Rep proteins induced some DDR events, full signaling appeared to require AAV replication and was accompanied by accumulation of DNA-PK at viral replication compartments. Our results demonstrate that AAV replication induces a unique DNA damage signal transduction response and provides a model system for studying DNA-PK.     

A Targeted Analysis of Cellular Chaperones Reveals Contrasting Roles for Heat Shock Protein 70 in Flock House Virus RNA Replication

Cytosolic chaperones are a diverse group of ubiquitous proteins that play central roles in multiple processes within the cell, including protein translation, folding, intracellular trafficking, and quality control. These cellular proteins have also been implicated in the replication of numerous viruses, although the full extent of their involvement in viral replication is unknown. We have previously shown that the heat shock protein 40 (hsp40) chaperone encoded by the yeast YDJ1 gene facilitates RNA replication of flock house virus (FHV), a well-studied and versatile positive-sense RNA model virus. To further explore the roles of chaperones in FHV replication, we examined a panel of 30 yeast strains with single deletions of cytosolic proteins that have known or hypothesized chaperone activity. We found that the majority of cytosolic chaperone deletions had no impact on FHV RNA accumulation, with the notable exception of J-domain-containing hsp40 chaperones, where deletion of APJ1 reduced FHV RNA accumulation by 60%, while deletion of ZUO1, JJJ1, or JJJ2 markedly increased FHV RNA accumulation, by 4- to 40-fold. Further studies using cross complementation and double-deletion strains revealed that the contrasting effects of J domain proteins were reproduced by altering expression of the major cytosolic hsp70s encoded by the SSA and SSB families and were mediated in part by divergent effects on FHV RNA polymerase synthesis. These results identify hsp70 chaperones as critical regulators of FHV RNA replication and indicate that cellular chaperones can have both positive and negative regulatory effects on virus replication.The compact genomes of viruses relative to those of other infectious agents restrict their ability to encode all proteins required to complete their replication cycles. To circumvent this limitation, viruses often utilize cellular factors or processes to complete essential steps in replication. One group of cellular proteins frequently targeted by viruses are cellular chaperones, which include a diverse set of heat shock proteins (hsps) that normally facilitate cellular protein translation, folding, trafficking, and degradation (18, 64). The connection between viruses and cellular chaperones was originally identified in bacteria, where the Escherichia coli hsp40 and hsp70 homologues, encoded by dnaJ and dnaK, respectively, were identified as bacterial genes essential for bacteriophage λ DNA replication (62). Research over the past 30 years has further revealed the importance of cellular chaperones in viral replication, such that the list of virus-hsp connections is now quite extensive and includes viruses from numerous families with diverse genome structures (4, 6, 7, 16, 19, 20, 23, 25, 40, 41, 44, 51, 54, 60). These studies have demonstrated the importance of cellular chaperones in multiple steps of the viral life cycle, including entry, viral protein translation, genome replication, encapsidation, and virion release. However, the list of virus-hsp connections is likely incomplete. Further studies to explore this particular host-pathogen interaction will shed light on virus replication mechanisms and pathogenesis, and potentially highlight targets for novel antiviral agents.To study the role of cellular chaperones in the genome replication of positive-sense RNA viruses, we use flock house virus (FHV), a natural insect pathogen and well-studied member of the Nodaviridae family. The FHV life cycle shares many common features with other positive-sense RNA viruses, including the membrane-specific targeting and assembly of functional RNA replication complexes (37, 38), the exploitation of various cellular processes and host factors for viral replication (5, 23, 60), and the induction of large-scale membrane rearrangements (24, 28, 38, 39). FHV virions contain a copackaged bipartite genome consisting of RNA1 (3.1 kb) and RNA2 (1.4 kb), which encode protein A, the viral RNA-dependent RNA polymerase, and the structural capsid protein precursor, respectively (1). During active genome replication, FHV produces a subgenomic RNA3 (0.4 kb), which encodes the RNA interference inhibitor protein B2 (12, 29, 32). These viral characteristics make FHV an excellent model system to study many aspects of positive-sense RNA virus biology.In addition to the benefits of a simple genome, FHV is able to establish robust RNA replication in a wide variety of genetically tractable eukaryotic hosts, including Drosophila melanogaster (38), Caenorhabditis elegans (32), and Saccharomyces cerevisiae (46). The budding yeast S. cerevisiae has been an exceptionally useful model host to study the mechanisms of viral RNA replication complex assembly and function with FHV (31, 37, 39, 45, 53, 55, 56, 60) as well as other positive-sense RNA viruses (11). The facile genetics of S. cerevisiae, along with the vast array of well-defined cellular and molecular tools and techniques, make it an ideal eukaryotic host for the identification of cellular factors required for positive-sense RNA virus replication. Furthermore, readily available yeast libraries with deletions and regulated expression of individual proteins have led to the completion of several high-throughput screens to provide a global survey of host factors that impact virus replication (26, 42, 52). An alternative approach with these yeast libraries that reduces the inherently high false-negative rates associated with high-throughput screens is to focus on a select set of host genes associated with a particular cellular pathway, process, or location previously implicated in virus replication.We have utilized such a targeted approach and focused on examining the impact of cytosolic chaperones on FHV RNA replication. Previously, we have shown that the cellular chaperone hsp90 facilitates protein A synthesis in Drosophila cells (5, 23), and the hsp40 encoded by the yeast YDJ1 gene facilitates FHV RNA replication in yeast, in part through effects on both protein A accumulation and function (60). In this report, we further extend these observations by examining FHV RNA accumulation in a panel of yeast strains with deletions of known or hypothesized cytosolic chaperones. We demonstrate that cytosolic chaperones can have either suppressive or enhancing effects on FHV RNA accumulation. In particular, related hsp70 members encoded by the SSA and SSB yeast chaperone families have marked and dramatically divergent effects on both genomic and subgenomic RNA accumulation and viral polymerase synthesis. These results highlight the complexities of the host-pathogen interactions that influence positive-sense RNA virus replication and identify the hsp70 family of cytosolic chaperones as key regulators of FHV replication.     

Critical Role of Cyclophilin A and Its Prolyl-Peptidyl Isomerase Activity in the Structure and Function of the Hepatitis C Virus Replication Complex

Replication of hepatitis C virus (HCV) RNA occurs on intracellular membranes, and the replication complex (RC) contains viral RNA, nonstructural proteins, and cellular cofactors. We previously demonstrated that cyclophilin A (CyPA) is an essential cofactor for HCV infection and the intracellular target of cyclosporine''s anti-HCV effect. Here we investigate the mechanism by which CyPA facilitates HCV replication. Cyclosporine treatment specifically blocked the incorporation of NS5B into the RC without affecting either the total protein level or the membrane association of the protein. Other nonstructural proteins or viral RNAs in the RC were not affected. NS5B from the cyclosporine-resistant replicon was resistant to this disruption of RC incorporation. We also isolated membrane fractions from both naïve and HCV-positive cells and found that CyPA is recruited into membrane fractions in HCV-replicating cells via an interaction with RC-associated NS5B, which is sensitive to cyclosporine treatment. Finally, we introduced point mutations in the prolyl-peptidyl isomerase (PPIase) motif of CyPA and demonstrated a critical role of this motif in HCV replication in cDNA rescue experiments. We propose a model in which the incorporation of the HCV polymerase into the RC depends on its interaction with a cellular chaperone protein and in which cyclosporine inhibits HCV replication by blocking this critical interaction and the PPIase activity of CyPA. Our results provide a mechanism of action for the cyclosporine-mediated inhibition of HCV and identify a critical role of CyPA''s PPIase activity in the proper assembly and function of the HCV RC.Hepatitis C virus (HCV), of the family Flaviviridae, is an enveloped, positive-stranded RNA virus. Spread mostly by blood-borne transmission, HCV infects more than 170 million people worldwide. The viral genome is composed of a single open reading frame (ORF) plus 5′- and 3′-nontranslated regions. The ORF encodes a large polyprotein that is cleaved by cellular and viral proteases into 10 viral proteins. The structural proteins, including the capsid protein (core), two glycoproteins (E1 and E2), and a small ion channel protein (p7), reside in the N-terminal half of the polyprotein. The rest of the ORF encodes six nonstructural (NS) proteins: NS2, NS3, NS4A, NS4B, NS5A, and NS5B. NS3 through NS5B assemble into a replication complex (RC) and are necessary and sufficient for HCV RNA replication in cell culture (8, 42). NS3 is a multifunctional protein with both a serine protease and an RNA helicase activity. The protease activity is responsible for cleavage at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions (5), and the helicase activity is probably required to unwind the double-stranded RNA intermediates formed during replication (38). NS4A serves as an essential cofactor for the NS3 protease and anchors the NS3 protein to intracellular membranes (25, 36, 39). NS4B induces the formation of a “membranous web” that is probably the site of HCV replication (16). It also contains a GTP-binding motif that is required for replication (17). The web is derived from the endoplasmic reticulum (ER) compartment, although proteins of early-endosome origin have also been found to locate to the web (62). NS5A is a phosphoprotein and an integral component of the viral RC. The precise function of NS5A in replication is still unknown but appears to be regulated by phosphorylation and its interaction with several cellular proteins (19, 22, 24, 51, 52, 59, 63, 67). In addition, it may be involved in the transition from replication and particle formation (4, 45, 64). NS5B is the RNA-dependent RNA polymerase that is responsible for copying the RNA genome of the virus during replication. Several cellular cofactors interact with NS5B and modulate its activity in the context of the viral RC (22, 24, 35, 69, 71).Positive-stranded RNA viruses alter the intracellular membranes of host cells to form an RC in which RNA replication occurs. Modifications include the proliferation and reorganization of certain cellular membranes (1). HCV forms an RC associated with altered cellular membranes (16, 23), and crude RCs (CRCs) that maintain the replicase activity in vitro can be isolated by membrane sedimentation or flotation techniques (2, 3, 18, 27, 37).Cyclosporine is a widely used immunosuppressive and anti-inflammatory drug for organ transplant patients. It functions by forming an inhibitory complex with cyclophilins (CyPs) that inhibits the phosphatase activity of calcineurin, which is important for T-cell activation. In recent years, cyclosporine and its derivatives have been shown to be highly effective in suppressing HCV replication in vitro (44, 49, 53, 68) and in vivo (30). The mechanism of this inhibition is independent of its immunosuppressive function and distinct from that of interferon (IFN) (44, 53, 56, 68).We recently showed that HCV infection in vitro is inhibited when CyPA, a major intracellular target of cyclosporine, is downregulated by RNA interference, and mutations in NS5B that confer cyclosporine-resistant binding to CyPA contribute to the cyclosporine resistance of the replicons harboring these mutations (56, 71). Here we report that CyPA is recruited into the HCV RC together with NS5B in HCV replicon or in HCV-infected cells. Cyclosporine disrupts the association between RC-incorporated NS5B and CyPA and results in an exclusion of the polymerase from the viral RC. We also show that the prolyl-peptidyl isomerase (PPIase) motif of CyPA is essential for HCV replication.     

Lack of Bax Prevents Influenza A Virus-Induced Apoptosis and Causes Diminished Viral Replication

The ectopic overexpression of Bcl-2 restricts both influenza A virus-induced apoptosis and influenza A virus replication in MDCK cells, thus suggesting a role for Bcl-2 family members during infection. Here we report that influenza A virus cannot establish an apoptotic response without functional Bax, a downstream target of Bcl-2, and that both Bax and Bak are directly involved in influenza A virus replication and virus-induced cell death. Bak is substantially downregulated during influenza A virus infection in MDCK cells, and the knockout of Bak in mouse embryonic fibroblasts yields a dramatic rise in the rate of apoptotic death and a corresponding increase in levels of virus replication, suggesting that Bak suppresses both apoptosis and the replication of virus and that the virus suppresses Bak. Bax, however, is activated and translocates from the cytosol to the mitochondria; this activation is required for the efficient induction of apoptosis and virus replication. The knockout of Bax in mouse embryonic fibroblasts blocks the induction of apoptosis, restricts the infection-mediated activation of executioner caspases, and inhibits virus propagation. Bax knockout cells still die but by an alternative death pathway displaying characteristics of autophagy, similarly to our previous observation that influenza A virus infection in the presence of a pancaspase inhibitor leads to an increase in levels of autophagy. The knockout of Bax causes a retention of influenza A virus NP within the nucleus. We conclude that the cell and virus struggle to control apoptosis and autophagy, as appropriately timed apoptosis is important for the replication of influenza A virus.The pathology of influenza A virus infection usually arises from acute lymphopenia and inflammation of the lungs and airway columnar epithelial cells (23, 38). Influenza A virus induces apoptotic death in infected epithelial, lymphocyte, and phagocytic cells, and apoptosis is a source of tissue damage during infection (3, 22, 33) and increased susceptibility to bacterial pathogens postinfection (31). While the induction of apoptosis by influenza A virus has been well documented (4, 19-21, 28, 33, 37), the mechanisms of this interaction are not well understood. Two viral proteins, NS1 and PB1-F2, have been associated with viral killing of cells. NS1, originally characterized as being proapoptotic (34), was later identified as being an interferon antagonist, inhibiting the activation of several key antiviral responses and restricting the apoptotic response to infection (1, 10, 15, 18, 35, 39, 46). In contrast, PB1-F2 induces apoptosis primarily by localizing to the outer mitochondrial membrane, promoting cytochrome c release, and triggering the apoptotic cascade (43). This effect, however, is typically restricted to infected monocytes, leading to the hypothesis that PB1-F2 induces apoptosis specifically to clear the landscape of immune responders (5, 44). Although PB1-F2 activity does not directly manipulate virus replication or virus-induced apoptosis, PB1-F2 localization to the mitochondrial membrane during infection potentiates the apoptotic response in epithelial and fibroblastic cells through tBID signaling with proapoptotic Bcl-2 family protein members Bax and Bak (22, 43, 44).The Bcl-2 protein family consists of both pro- and antiapoptotic members that regulate cytochrome c release during mitochondrion-mediated apoptosis through the formation of pore-like channels in the outer mitochondrial membrane (12, 16). During the initiation of mitochondrion-mediated apoptosis, cytoplasmic Bid is cleaved to form tBID. This, in turn, activates proapoptotic Bax and Bak (40), which drive cytochrome c release and subsequent caspase activation. Bak is constitutively associated with the mitochondrial membrane, whereas inactive Bax is primarily cytosolic, translocating to the outer mitochondrial membrane only after activation (6). The activation of Bax and Bak results in homo- and heterodimer formation at the outer mitochondrial membrane, generating pores that facilitate mitochondrial membrane permeabilization and cytochrome c release (14, 17), leading to caspase activation and the apoptotic cascade (8). Antiapoptotic members of the Bcl-2 protein family, including Bcl-2, inhibit the activation of proapoptotic Bax and Bak primarily by sequestering inactive Bax and Bak monomers via interactions between their BH3 homology domains (7).Bcl-2 expression has been linked to decreased viral replication rates (26). Bcl-2 overexpression inhibits influenza A virus-induced cell death and reduces the titer and spread of newly formed virions (29). The activation of caspase-3 in the absence of sufficient Bcl-2 is critical to the influenza A virus life cycle. Both Bcl-2 expression and the lack of caspase activation during infection lead to the nuclear accumulation of influenza virus ribonucleoprotein (RNP) complexes, thereby leading to the improper assembly of progeny virions and a marked reduction in titers of infectious virus (26, 41, 42, 45).Here we show that influenza A virus induces mitochondrion-mediated (intrinsic-pathway) apoptosis signaled specifically through Bax and that this Bax signaling is essential for the maximum efficiency of virus propagation. In contrast, Bak expression is strongly downregulated during infection. Cells lacking Bak (while expressing Bax) display a much more severe apoptotic phenotype in response to infection and produce infectious virions at a higher rate than the wild type (WT), suggesting that Bak, which can suppress viral replication, is potentially downregulated by the virus. Our results indicate essential and opposing roles for Bax and Bak in both the response of cells to influenza A virus infection and the ability of the virus to maximize its own replicative potential.