Control of TANK-binding Kinase 1-mediated Signaling by the ��134.5 Protein of Herpes Simplex Virus 1

TANK-binding kinase 1 (TBK1) is a key component of Toll-like receptor-dependent and -independent signaling pathways. In response to microbial components, TBK1 activates interferon regulatory factor 3 (IRF3) and cytokine expression. Here we show that TBK1 is a novel target of the γ₁34.5 protein, a virulence factor whose expression is regulated in a temporal fashion. Remarkably, the γ₁34.5 protein is required to inhibit IRF3 phosphorylation, nuclear translocation, and the induction of antiviral genes in infected cells. When expressed in mammalian cells, the γ₁34.5 protein forms complexes with TBK1 and disrupts the interaction of TBK1 and IRF3, which prevents the induction of interferon and interferon-stimulated gene promoters. Down-regulation of TBK1 requires the amino-terminal domain. In addition, unlike wild type virus, a herpes simplex virus mutant lacking γ₁34.5 replicates efficiently in TBK1^-/- cells but not in TBK1^+/+ cells. Addition of exogenous interferon restores the antiviral activity in both TBK1^-/- and TBK^+/+ cells. Hence, control of TBK1-mediated cell signaling by the γ₁34.5 protein contributes to herpes simplex virus infection. These results reveal that TBK1 plays a pivotal role in limiting replication of a DNA virus.Herpes simplex virus 1 (HSV-1)³ is a large DNA virus that establishes latent or lytic infection, in which the virus triggers innate immune responses. In HSV-infected cells, a number of antiviral mechanisms operate in a cell type- and time-dependent manner (1). In response to double-stranded RNA (dsRNA), Toll-like receptor 3 (TLR3) recruits an adaptor TIR domain-containing adaptor inducing IFN-β and stimulates cytokine expression (2, 3). In the cytoplasm, RNA helicases, RIG-I (retinoid acid-inducible gene-I), and MDA5 (melanoma differentiation associated gene 5) recognize intracellular viral 5′-triphosphate RNA or dsRNA (2, 4). Furthermore, a DNA-dependent activator of IFN-regulatory factor (DAI) senses double-stranded DNA in the cytoplasm and induces cytokine expression (5). There is also evidence that viral entry induces antiviral programs independent of TLR and RIG-I pathways (6). While recognizing distinct viral components, these innate immune pathways relay signals to the two IKK-related kinases, TANK-binding kinase 1 (TBK1) and inducible IκB kinase (IKKi) (2).The IKK-related kinases function as essential components that phosphorylate IRF3 (interferon regulatory factor 3), as well as the closely related IRF7, which translocates to the nucleus and induces antiviral genes, such as interferon-α/β and ISG56 (interferon-stimulated gene 56) (7, 8). TBK1 is constitutively expressed, whereas IKKi is engaged as an inducible gene product of innate immune signaling (9, 10). IRF3 activation is attenuated in TBK1-deficient but not in IKKi-deficient cells (11, 12). Its activation is completely abolished in double-deficient cells (12), suggesting a partially redundant function of TBK1 and IKKi. Indeed, IKKi also negatively regulates the STAT-signaling pathway (13). TBK1/IKKi interacts with several proteins, such as TRAF family member-associated NF-κB activator (TANK), NAP1 (NAK-associated protein 1), similar to NAP1TBK1 adaptor (SINTBAD), DNA-dependent activator of IFN-regulatory factors (DAI), and secretory protein 5 (Sec5) in host cells (5, 14–18). These interactions are thought to regulate TBK1/IKKi, which delineates innate as well as adaptive immune responses.Upon viral infection, expression of HSV proteins interferes with the induction of antiviral immunity. When treated with UV or cycloheximide, HSV induces an array of antiviral genes in human lung fibroblasts (19, 20). Furthermore, an HSV mutant, with deletion in immediate early protein ICP0, induces ISG56 expression (21). Accordingly, expression of ICP0 inhibits the induction of antiviral programs mediated by IRF3 or IRF7 (21–23). However, although ICP0 negatively regulates IFN-β expression, it is not essential for this effect (24). In HSV-infected human macrophages or dendritic cells, an immediate early protein ICP27 is required to suppress cytokine induction involving IRF3 (25). In this context, it is notable that an HSV mutant, lacking a leaky late gene γ₁34.5, replicates efficiently in cells devoid of IFN-α/β genes (26). Additionally, the γ₁34.5 null mutant induces differential cytokine expression as compared with wild type virus (27). Thus, HSV modulation of cytokine expression is a complex process that involves multiple viral components. Currently, the molecular mechanism governing this event is unclear. In this study, we show that HSV γ₁34.5 targets TBK1 and inhibits antiviral signaling. The data herein reveal a previously unrecognized mechanism by which γ₁34.5 facilitates HSV replication.     

The Herpes Simplex Virus US11 Protein Effectively Compensates for the γ134.5 Gene if Present before Activation of Protein Kinase R by Precluding Its Phosphorylation and That of the α Subunit of Eukaryotic Translation Initiation Factor 2

In herpes simplex virus-infected cells, viral γ₁34.5 protein blocks the shutoff of protein synthesis by activated protein kinase R (PKR) by directing the protein phosphatase 1α to dephosphorylate the α subunit of eukaryotic translation initiation factor 2 (eIF-2α). The amino acid sequence of the γ₁34.5 protein which interacts with the phosphatase has high homology to a domain of the eukaryotic protein GADD34. A class of compensatory mutants characterized by a deletion which results in the juxtaposition of the α47 promoter next to U_S11, a γ₂ (late) gene in wild-type virus-infected cells, has been described. In cells infected with these mutants, protein synthesis continues even in the absence of the γ₁34.5 gene. In these cells, PKR is activated but eIF-2α is not phosphorylated, and the phosphatase is not redirected to dephosphorylate eIF-2α. We report the following: (i) in cells infected with these mutants, U_S11 protein was made early in infection; (ii) U_S11 protein bound PKR and was phosphorylated; (iii) in in vitro assays, U_S11 blocked the phosphorylation of eIF-2α by PKR activated by poly(I-C); and (iv) U_S11 was more effective if present in the reaction mixture during the activation of PKR than if added after PKR had been activated by poly(I-C). We conclude the following: (i) in cells infected with the compensatory mutants, U_S11 made early in infection binds to PKR and precludes the phosphorylation of eIF-2α, whereas U_S11 driven by its natural promoter and expressed late in infection is ineffective; and (ii) activation of PKR by double-stranded RNA is a common impediment countered by most viruses by different mechanisms. The γ₁34.5 gene is not highly conserved among herpesviruses. A likely scenario is that acquisition by a progenitor of herpes simplex virus of a portion of the cellular GADD34 gene resulted in a more potent and reliable means of curbing the effects of activated PKR. U_S11 was retained as a γ₂ gene because, like many viral proteins, it has multiple functions.The herpes simplex virus 1 (HSV-1) genome encodes two sets of functions. The first and paramount are functions related to viral gene expression, replication of viral DNA, synthesis of virion proteins, assembly, packaging, and egress of the virus from the infected cell. The second set of functions, no less important in the survival of the virus in the human population, is creation of the environment necessary to maximize the yield and spread of virus from cell to cell and from infected to uninfected individuals (reviewed in reference 38). Of these known genes, several play a significant role in abating or delaying a host response to infection. The earliest to be expressed is the U_L41 gene which encodes a protein that is introduced into the cell in virions during infection (26, 27). This protein reduces the synthesis of host proteins by causing the destruction of mRNA in a rather nonspecific manner and therefore could be expected to reduce the synthesis of cellular proteins deleterious to viral replication (26, 27, 44).A second and very different approach to blocking host defense mechanisms is exemplified by infected cell protein 47 (ICP47). Proteosomal degradation of viral proteins could be expected to produce antigenic peptides which, if presented on the cell surface, could provoke a cytotoxic cell response early in infection and thus reduce viral yield. ICP47, an α protein made immediately after infection, blocks the presentation of antigenic peptides on the surface of the infected cells (20).The focus of this laboratory has been on a third viral pathway designed to block cellular response to infection. In cells infected with most viruses, the synthesis of complementary mRNA leads to activation of double-stranded RNA-dependent protein kinase R (PKR). This enzyme phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF-2α) (23). A consequence of this phosphorylation is total shutoff of protein synthesis. This would be an example of a noble sacrifice of the infected cell for the sake of survival of the organism were it not for the fact that viruses, while activating the PKR kinase pathway by making double-stranded RNA, also express functions which block this host defense system (2–4, 6, 7, 10, 28, 30, 34). In the case of HSV-1, more than 50% of the viral DNA is represented late in infection in the form of cRNA (21, 25), and the gene whose product blocks the consequences of activation of PKR is γ₁34.5 (7). In the absence of the gene, eIF-2α is phosphorylated and protein synthesis is impaired beginning approximately 5 h after infection (7, 9). In its presence, protein synthesis continues unabated even though PKR is activated (9). Recent studies have shown that the carboxyl terminus of the γ₁34.5 gene binds to the protein phosphatase 1α (PP1) and redirects it to dephosphorylate eIF-2α (19). The effectiveness of the γ₁34.5-PP1 complex is apparent from the observation that the rate of dephosphorylation of eIF-2α in cells infected with wild-type virus is more than 1000 times that of uninfected cells or cells infected with the γ₁34.5⁻ virus (5, 19).The studies described in this report concern another aspect of virus-induced block of the consequence of activation of PKR. Briefly, Mohr and Gluzman reported that serial passage of a γ₁34.5⁻ mutant resulted in the selection of a compensatory mutation capable of sustained protein synthesis (35). A characteristic of the compensatory mutants isolated by Mohr and Gluzman is a deletion in the α47 gene resulting in the juxtaposition of the promoter of the α47 gene next to the 5′ end of U_S11, a late (γ₂) viral gene. Preliminary studies of those mutants revealed that PKR was activated in cells infected with either the wild-type parent or the γ₁34.5⁻ virus, but protein synthesis was unaffected in cells infected with wild-type virus or the mutant carrying the compensatory mutations (5, 18).In an attempt to define the phenotype of the virus carrying the compensatory mutation, we constructed a mutant lacking the γ₁34.5 and the U_S8 to -12 genes. This mutant, designated R5103, activated PKR and caused a shutoff of protein synthesis (5). We then inserted into the R5103 genome a DNA fragment consisting of the intact U_S10 gene and the U_S11 open reading frame fused to the α47 promoter. This virus, designated R5104, activated PKR but did not induce the shutoff of protein synthesis. Consistent with the conclusion of Mohr and Gluzman (35), the mutation maps in the domain inserted into the R5104 virus (5). Further studies yielded two significant observations. First, in stark contrast to lysates of cells infected with R5103 and other γ₁34.5⁻ mutants, the lysates of R5104 virus failed to phosphorylate the α subunit of eIF-2 (5). Second, in striking contrast to lysates of wild-type virus-infected cells, the phosphatase activity of lysates of R5104 virus-infected cells specific for eIF-2α could not be differentiated from that of mock-infected cells or those of cells infected with other γ₁34.5⁻ mutants (5). These results indicated that the compensatory mutation blocks PKR from phosphorylating eIF-2α.The studies summarized in this report focused on U_S11 protein. We report that in cells infected with the R5104 recombinant the U_S11 protein is made early in infection, that U_S11 protein interacts with PKR and blocks the phosphorylation of eIF-2α by activated PKR in in vitro assays, and that the effectiveness of the U_S11 protein is greater if the protein is present in the reaction before activation of PKR than if it is after PKR has been activated by the addition of poly(I-C). We also found that U_S11 is phosphorylated in the presence of activated PKR but not in its absence. We conclude that U_S11 may have been an ancient mechanism for blocking the effects of activated PKR and that it has been supplanted by acquisition of the carboxyl-terminal domain of the γ₁34.5 protein from a cellular gene. We also note that U_S11 protein made late in infection, after PKR has been activated, is ineffective.Relevant to this report are some of the properties of the U_S11 protein. U_S11 is one of the most abundant viral proteins expressed at late times in viral infection (22, 31). It binds mRNA in a sequence- and conformation-specific fashion (39–41). In HSV-1-infected cells, U_S11 suppresses the synthesis of a truncated RNA colinear with the 5′ domain of the U_L34 mRNA (40). The protein accumulates in nucleoli, in the cytoplasm in association with the 60S ribosomal subunit, and it is also packaged in virions (31, 37, 41). In newly infected cells, the U_S11 protein has been found associated with ribosomes (41).Recently a plethora of reports suggested that U_S11 may have novel functions not readily apparent from its localization in the infected cell. Thus, U_S11 protein has been reported to have functions similar to those of human immunodeficiency Tat and Rev proteins and has also been reported to complement Rev function in a Rev⁻ human immunodeficiency virus mutant (11). The U_S11 protein has been reported to confer thermotolerance and help restore protein synthesis in HeLa cells subjected to thermal injury (12).     

The C-Terminal α-Helix Domain of Apolipoprotein E Is Required for Interaction with Nonstructural Protein 5A and Assembly of Hepatitis C Virus

We have recently demonstrated that human apolipoprotein E (apoE) is required for the infectivity and assembly of hepatitis C virus (HCV) (K. S. Chang, J. Jiang, Z. Cai, and G. Luo, J. Virol. 81:13783-13793, 2007; J. Jiang and G. Luo, J. Virol. 83:12680-12691, 2009). In the present study, we have determined the molecular basis underlying the importance of apoE in HCV assembly. Results derived from mammalian two-hybrid studies demonstrate a specific interaction between apoE and HCV nonstructural protein 5A (NS5A). The C-terminal third of apoE per se is sufficient for interaction with NS5A. Progressive deletion mutagenesis analysis identified that the C-terminal α-helix domain of apoE is important for NS5A binding. The N-terminal receptor-binding domain and the C-terminal 20 amino acids of apoE are dispensable for the apoE-NS5A interaction. The NS5A-binding domain of apoE was mapped to the middle of the C-terminal α-helix domain between amino acids 205 and 280. Likewise, deletion mutations disrupting the apoE-NS5A interaction resulted in blockade of HCV production. These findings demonstrate that the specific apoE-NS5A interaction is required for assembly of infectious HCV. Additionally, we have determined that using different major isoforms of apoE (E2, E3, and E4) made no significant difference in the apoE-NS5A interaction. Likewise, these three major isoforms of apoE are equally compatible with infectivity and assembly of infectious HCV, suggesting that apoE isoforms do not differentially modulate the infectivity and/or assembly of HCV in cell culture.Hepatitis C virus (HCV) remains a major global health problem, chronically infecting approximately 170 million people worldwide, with severe consequences such as hepatitis, fibrosis/cirrhosis, and hepatocellular carcinoma (HCC) (2, 57). The current standard therapy for hepatitis C is pegylated alpha interferon in combination with ribavirin. However, this anti-HCV regimen has limited efficacy (<50% sustained antiviral response for the dominant genotype 1 HCV) and causes severe side effects (17, 39). Recent clinical studies on the HCV protease- and polymerase-specific inhibitors showed promising results but also found that drug-resistant HCV mutants emerged rapidly (3, 27), undermining the efficacy of specific antiviral therapy for hepatitis C. Therefore, future antiviral therapies for hepatitis C likely require a combination of several safer and more efficacious antiviral drugs that target different steps of the HCV life cycle. The lack of knowledge about the molecular details of the HCV life cycle has significantly impeded the discovery of antiviral drugs and development of HCV vaccines.HCV is a small enveloped RNA virus classified as a member of the Hepacivirus genus in the family Flaviviridae (46, 47). It contains a single positive-sense RNA genome that encodes a large viral polypeptide, which is proteolytically processed by cellular peptidases and viral proteases into different structural and nonstructural proteins in the order of C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (30, 31). Other novel viral proteins derived from the C-coding region have also been discovered (11, 13, 55, 59). The nucleotides at both the 5′ and 3′ untranslated regions (UTR) are highly conserved and contain cis-acting RNA elements important for internal ribosome entry site (IRES)-mediated initiation of protein translation and viral RNA replication (15, 16, 33, 56, 60).The success in the development of HCV replicon replication systems has made enormous contributions to the determination of the roles of the conserved RNA sequences/structures and viral NS proteins in HCV RNA replication (4, 5, 7, 32). However, the molecular mechanisms of HCV assembly, morphogenesis, and egression have not been well understood. A breakthrough advance has been the development of robust cell culture systems for HCV infection and propagation, which allow us to determine the roles of viral and cellular proteins in the HCV infectious cycle (9, 29, 54, 63). We have recently demonstrated that infectious HCV particles are enriched in apolipoprotein E (apoE) and that apoE is required for HCV infection and assembly (10, 23). apoE-specific monoclonal antibodies efficiently neutralized HCV infectivity. The knockdown of endogenous apoE expression by a specific small interfering RNA (siRNA) and the blockade of apoE secretion by microsomal triglyceride transfer protein (MTP) inhibitors remarkably suppressed HCV assembly (10, 23). More importantly, apoE was found to interact with the HCV NS5A in the cell and purified HCV particles, as determined by yeast two-hybrid and coimmunoprecipitation (co-IP) studies (6, 23). These findings suggest that apoE has dual functions in HCV infection and assembly via distinct interactions with cell surface receptors and HCV NS5A. To further understand the molecular mechanism of apoE in HCV assembly, we carried out a mutagenesis analysis of apoE and determined the importance of the apoE-NS5A interaction in HCV assembly. Progressive deletion mutagenesis analysis has mapped the NS5A-binding domain of apoE to the C-terminal α-helix region between amino acid residues 205 and 280. Mutations disrupting the apoE-NS5A interaction also blocked HCV production. Additionally, we have determined the effects of three major isoforms of apoE on HCV infection and assembly. Our results demonstrate that apoE isoforms do not determine the infectivity and assembly of infectious HCV in cell culture.     

The Spatiotemporal Dynamics of Chromatin Protein HP1α Is Essential for Accurate Chromosome Segregation during Cell Division

Heterochromatin protein 1α (HP1α) is involved in regulation of chromatin plasticity, DNA damage repair, and centromere dynamics. HP1α detects histone dimethylation and trimethylation of Lys-9 via its chromodomain. HP1α localizes to heterochromatin in interphase cells but is liberated from chromosomal arms at the onset of mitosis. However, the structural determinants required for HP1α localization in interphase and the regulation of HP1α dynamics have remained elusive. Here we show that centromeric localization of HP1α depends on histone H3 Lys-9 trimethyltransferase SUV39H1 activity in interphase but not in mitotic cells. Surprisingly, HP1α liberates from chromosome arms in early mitosis. To test the role of this dissociation, we engineered an HP1α construct that persistently localizes to chromosome arms. Interestingly, persistent localization of HP1α to chromosome arms perturbs accurate kinetochore-microtubule attachment due to an aberrant distribution of chromosome passenger complex and Sgo1 from centromeres to chromosome arms that prevents resolution of sister chromatids. Further analyses showed that Mis14 and perhaps other PXVXL-containing proteins are involved in directing localization of HP1α to the centromere in mitosis. Taken together, our data suggest a model in which spatiotemporal dynamics of HP1α localization to centromere is governed by two distinct structural determinants. These findings reveal a previously unrecognized but essential link between HP1α-interacting molecular dynamics and chromosome plasticity in promoting accurate cell division.     

Optimum Length and Flexibility of Reovirus Attachment Protein σ1 Are Required for Efficient Viral Infection

Reovirus attachment protein σ1 is an elongated trimer with head-and-tail morphology that engages cell-surface carbohydrate and junctional adhesion molecule A (JAM-A). The σ1 protein is comprised of three domains partitioned by two flexible linkers termed interdomain regions (IDRs). To determine the importance of σ1 length and flexibility at different stages of reovirus infection, we generated viruses with mutant σ1 molecules of altered length and flexibility and tested these viruses for the capacity to bind the cell surface, internalize, uncoat, induce protein synthesis, assemble, and replicate. We reduced the length of the α-helical σ1 tail to engineer mutants L1 and L2 and deleted midpoint and head-proximal σ1 IDRs to generate ΔIDR1 and ΔIDR2 mutant viruses, respectively. Decreasing length or flexibility of σ1 resulted in delayed reovirus infection and reduced viral titers. L1, L2, and ΔIDR1 viruses but not ΔIDR2 virus displayed reduced cell attachment, but altering σ1 length or flexibility did not diminish the efficiency of virion internalization. Replication of ΔIDR2 virus was hindered at a postdisassembly step. Differences between wild-type and σ1 mutant viruses were not attributable to alterations in σ1 folding, as determined by experiments assessing engagement of cell-surface carbohydrate and JAM-A by the length and IDR mutant viruses. However, ΔIDR1 virus harbored substantially less σ1 on the outer capsid. Taken together, these data suggest that σ1 length is required for reovirus binding to cells. In contrast, IDR1 is required for stable σ1 encapsidation, and IDR2 is required for a postuncoating replication step. Thus, the structural architecture of σ1 is required for efficient reovirus infection of host cells.     

Ns1 Is a Key Protein in the Vaccine Composition to Protect Ifnar(?/?) Mice against Infection with Multiple Serotypes of African Horse Sickness Virus

African horse sickness virus (AHSV) belongs to the genus Orbivirus. We have now engineered naked DNAs and recombinant modified vaccinia virus Ankara (rMVA) expressing VP2 and NS1 proteins from AHSV-4. IFNAR^(−/−) mice inoculated with DNA/rMVA-VP2,-NS1 from AHSV-4 in an heterologous prime-boost vaccination strategy generated significant levels of neutralizing antibodies specific of AHSV-4. In addition, vaccination stimulated specific T cell responses against the virus. The vaccine elicited partial protection against an homologous AHSV-4 infection and induced cross-protection against the heterologous AHSV-9. Similarly, IFNAR^(−/−) mice vaccinated with an homologous prime-boost strategy with rMVA-VP2-NS1 from AHSV-4 developed neutralizing antibodies and protective immunity against AHSV-4. Furthermore, the levels of immunity were very high since none of vaccinated animals presented viraemia when they were challenged against the homologous AHSV-4 and very low levels when they were challenged against the heterologous virus AHSV-9. These data suggest that the immunization with rMVA/rMVA was more efficient in protection against a virulent challenge with AHSV-4 and both strategies, DNA/rMVA and rMVA/rMVA, protected against the infection with AHSV-9. The inclusion of the protein NS1 in the vaccine formulations targeting AHSV generates promising multiserotype vaccines.     

Phospholipase C-related but Catalytically Inactive Protein Is Required for Insulin-induced Cell Surface Expression of ��-Aminobutyric Acid Type A Receptors

The γ-aminobutyric acid type A (GABA_A) receptors play a pivotal role in fast synaptic inhibition in the central nervous system. One of the key factors for determining synaptic strength is the number of receptors on the postsynaptic membrane, which is maintained by the balance between cell surface insertion and endocytosis of the receptors. In this study, we investigated whether phospholipase C-related but catalytically inactive protein (PRIP) is involved in insulin-induced GABA_A receptor insertion. Insulin potentiated the GABA-induced Cl⁻ current (I_GABA) by about 30% in wild-type neurons, but not in PRIP1 and PRIP2 double-knock-out (DKO) neurons, suggesting that PRIP is involved in insulin-induced potentiation. The phosphorylation level of the GABA_A receptor β-subunit was increased by about 30% in the wild-type neurons but not in the mutant neurons, which were similar to the changes observed in I_GABA. We also revealed that PRIP recruited active Akt to the GABA_A receptors by forming a ternary complex under insulin stimulation. The disruption of the binding between PRIP and the GABA_A receptor β-subunit by PRIP interference peptide attenuated the insulin potentiation of I_GABA. Taken together, these results suggest that PRIP is involved in insulin-induced GABA_A receptor insertion by recruiting active Akt to the receptor complex.     

The Pepper Extracellular Xyloglucan-Specific Endo-β-1,4-Glucanase Inhibitor Protein Gene,CaXEGIP1, Is Required for Plant Cell Death and Defense Responses

Plants produce various proteinaceous inhibitors to protect themselves against microbial pathogen attack. A xyloglucan-specific endo-β-1,4-glucanase inhibitor1 gene, CaXEGIP1, was isolated and functionally characterized in pepper (Capsicum annuum) plants. CaXEGIP1 was rapidly and strongly induced in pepper leaves infected with avirulent Xanthomonas campestris pv vesicatoria, and purified CaXEGIP1 protein significantly inhibited the hydrolytic activity of the glycoside hydrolase74 family xyloglucan-specific endo-β-1,4-glucanase from Clostridium thermocellum. Soluble-modified green fluorescent protein-tagged CaXEGIP1 proteins were mainly localized to the apoplast of onion (Allium cepa) epidermal cells. Agrobacterium tumefaciens-mediated overexpression of CaXEGIP1 triggered pathogen-independent, spontaneous cell death in pepper and Nicotiana benthamiana leaves. CaXEGIP1 silencing in pepper conferred enhanced susceptibility to virulent and avirulent X. campestris pv vesicatoria, accompanied by a compromised hypersensitive response and lowered expression of defense-related genes. Overexpression of dexamethasone:CaXEGIP1 in Arabidopsis (Arabidopsis thaliana) enhanced resistance to Hyaloperonospora arabidopsidis infection. Comparative histochemical and proteomic analyses revealed that CaXEGIP1 overexpression induced a spontaneous cell death response and also increased the expression of some defense-related proteins in transgenic Arabidopsis leaves. This response was also accompanied by cell wall thickening and darkening. Together, these results suggest that pathogen-inducible CaXEGIP1 positively regulates cell death-mediated defense responses in plants.Plant cell walls provide a physical barrier that separates challenging pathogens from the internal contents of plant cells. Additionally, the cell walls regulate cell expansion and differentiation (York et al., 2004; Flors et al., 2007; Cantu et al., 2008). Polysaccharides, such as cellulose, hemicellulose, and pectic polysaccharides, are the main components of primary cell walls. Xyloglucan (XG), the most abundant hemicellulose in the primary cell wall, plays a structural role by forming strong hydrogen bonds with cellulose microfibrils (Carpita and Gibeaut, 1993). The primary structure of XG contains a common β-(1→4)-d-glucan backbone, which is repeatedly substituted with α(1→6)-d-xylopyranosyl residues. Depolymerization of XG is proposed to play an important role during both cell wall expansion and pathogen invasion (Bourquin et al., 2002; Qin et al., 2003; Baumann et al., 2007). During cell wall expansion, plant XG endotransglycosylases cut and rejoin XG chains to allow the cellulose microfibrils to move apart. From a pathogen point of view, the carbon-rich complex represents a useful energy source for pathogen growth. To facilitate penetration into the plant tissues and to catabolize carbon sources, many plant pathogens secrete a mixture of cell wall-degrading enzymes, such as polygalacturonases, pectin methyl esterases, pectin/pecatae lyases, xylanases, and endoglucanases (Valette-Collet et al., 2003; DeBoy et al., 2008). Some microbial glycoside hydrolase (GH) family proteins, including GH5, GH12, and GH74, reportedly hydrolyze plant-derived XG (Martinez-Fleites et al., 2006; Gloster et al., 2007).To inhibit pathogen-derived cell wall-degrading enzymes, plants secrete a mixture of inhibitor proteins into the cell wall (Qin et al., 2003; An et al., 2008; Xie et al., 2008). Some of the best characterized inhibitor proteins are polygalacturonase-inhibiting proteins (PGIPs; Albersheim and Anderson, 1971; De Lorenzo and Ferrari, 2002; Federici et al., 2006). In bean (Phaseolus vulgaris), two pairs of PGIPs, PvPGIP1/PvPGIP2 and PvPGIP3/PvPGIP4, are present in the genome. These genes may have originated from independent gene duplication events (D’Ovidio et al., 2004a). PvPGIP2 strongly inhibits polygalacturonases from Fusarium phyllophilum and Aspergillus niger via three conserved Asp residues (Spinelli et al., 2009). PGIPs reduce the hydrolytic activity of polygalacturonases to favor the accumulation of long-chain oligogalacturonides, known as elicitors of a variety of defense responses (Côté and Hahn, 1994; D’Ovidio et al., 2004b). Furthermore, transgenic expression of pear (Pyrus communis) PGIP in transgenic tomato (Solanum lycopersicum) plants limited fungal colonization, suggesting a role of PGIPs in plant defense (Powell et al., 2000).The proteinaceous inhibitor of the cell wall-degrading enzyme xyloglucan-specific endo-β-1,4-glucanase (XEG) was identified from suspension-cultured tomatoes (Qin et al., 2003). The purified xyloglucan-specific endo-β-1,4-glucanase inhibitor protein (XEGIP) strongly inhibited XEG activity through the formation of a 1:1 protein:protein complex with XEG of Aspergillus aculeatus. More recently, two putative XEGIPs were isolated from Nicotiana benthamiana based on conserved regions found in plant XEGIP genes, and these genes were functionally characterized using virus-induced gene silencing (VIGS; Xie et al., 2008). VIGS of NbXEGIP1 strongly enhanced the wilting symptoms exhibited following infection by virulent Pseudomonas syringae pv tabaci. This finding supports the notion that NbXEGIP1 may act as an inhibitor of bacterial cell wall-degrading enzymes in N. benthamiana plants.Programmed cell death (PCD) has been extensively characterized in plants (Lam, 2004). The hypersensitive response (HR), a well-known form of plant PCD, is one of the most efficient and immediate resistance reactions of plants. The HR is characterized by the rapid death of cells in the local region surrounding an infection site. As a result, the growth and spread of the pathogen is restricted or confined. During HR cell death development, cell wall strengthening occurs. Histochemical analyses of cells involved in melon (Cucumis melo)-powdery mildew (Podosphaera fusca) interactions demonstrate the reinforcement of the cell wall compartment as part of HR cell death-mediated resistance (Romero et al., 2008). Treatment of suspension-cultured tobacco (Nicotiana tabacum) cells with cryptogein, a 10-kD protein secreted by the oomycete Phytophthora cryptogea, induces a HR on tobacco leaves, accompanied by induced strengthening of the cell wall (Kieffer et al., 2000). However, the role of cell wall strengthening in HR cell death is poorly understood. A second type of PCD is thought to be associated with the differentiation of procambium into tracheary elements in the xylem of vascular plants (Fukuda, 2000; Lam, 2004). During the early formation of mature tracheary elements, vacuoles accumulate degradation enzymes and the cell wall is remodeled into a highly reticulated form. A similar phenomenon occurs during some plant developmental processes, including senescence and aerenchyma formation in roots (Jones, 2001).In this study, we have isolated and functionally characterized a pepper (Capsicum annuum) xyloglucan-specific endo-β-1,4-glucanase inhibitor-protein1 gene (CaXEGIP1). Expression of CaXEGIP1 was strongly induced in pepper leaves infected with avirulent Xanthomonas campestris pv vesicatoria (Xcv) strain Bv5-4a. The purified CaXEGIP1 protein inhibited the hydrolytic activity of GH74 family XEG from the thermophilic bacterium Clostridium thermocellum. The soluble-modified GFP (smGFP)-fused CaXEGIP1 protein was localized in the external and intercellular regions of onion epidermal cells. Importantly, Agrobacterium tumefaciens-mediated transient expression of CaXEGIP1 induced the hypersensitive cell death response in pepper and N. benthamiana leaves. VIGS of CaXEGIP1 significantly enhanced the growth of virulent and avirulent Xcv in pepper leaves, accompanied by compromised HR cell death and lowered expression of CaPR1 (pathogenesis-related protein1 [PR1]) and CaDEF1 (defensin [DEF1]). We also investigated the role of CaXEGIP1 in plant cell death and defense responses using transgenic Arabidopsis (Arabidopsis thaliana) plants harboring the dexamethasone (DEX)-inducible CaXEGIP1 transgene. Overexpression of CaXEGIP1 triggered spontaneous cell death and modification of the cell wall compartment in Arabidopsis plants. Together, these results suggest that the pathogen-responsive CaXEGIP1 is involved in plant cell death-mediated defense signaling.     

Different Mechanisms of Inhibition of DNA Synthesis by (E)-5-(2-Bromovinyl)-2′-deoxyuridine in Cells Transfected with Gene for Thymidine Kinase of Herpes Simplex Virus Type 1 and in Cells Infected with the Virus

Abstract

The effect of (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) on deoxyribonucleoside 5′-triphosphate pools was studied in cells transfected with gene for thymidine kinase of herpes simplex virus type 1 and cells infected with the virus. When infected cells were treated with BVDU, the triphosphate form of the nucleoside analog was detected. When transfected cells were treated with BVDU, the triphosphate form was not detected and the pattern of changes in the pools was the same as after 5-fluoro-2′-deoxyuridine treatment. BVDU seems to inhibit DNA synthesis differently in the two cell lines and nucleotide metabolism in the transfected cells was not the same as in the infected cells.     

Importin-α-Mediated Nucleolar Localization of Potato Mop-Top Virus TRIPLE GENE BLOCK1 (TGB1) Protein Facilitates Virus Systemic Movement,Whereas TGB1 Self-Interaction Is Required for Cell-to-Cell Movement in Nicotiana benthamiana

Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-α family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-α paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-α in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-α-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-α interaction in plants, nucleolar targeting, and long-distance movement.Pomoviruses are causal agents of important diseases affecting potato (Solanum tuberosum), sugar beet (Beta vulgaris), and bean (Phaseolus vulgaris). Potato mop-top virus (PMTV), the type member of the genus Pomovirus, causes an economically important disease of potato called spraing, inducing brown lines and arcs internally and on the surface of tubers. PMTV is transmitted by the root- and tuber-infecting plasmodiophorid Spongospora subterranea (Jones and Harrison, 1969; Arif et al., 1995).The pomovirus genome is divided into three single-stranded RNA (ssRNA) segments of positive polarity. RNA-Rep encodes the putative RNA-dependent RNA polymerase, the replicase of the virus (Savenkov et al., 1999). RNA-CP encodes a coat protein (CP) and another protein called CP-RT or minor CP, which is produced by translational read-through of the CP stop codon (Sandgren et al., 2001). Whereas CP is the major structural protein of the virions, CP-RT is incorporated in one of the termini of the virus particles and a domain within the read-through region of the protein is needed for transmission of the virus by its vector (Reavy et al., 1998). Moreover, CP-RT, but not CP, interacts with the major movement protein TRIPLE GENE BLOCK1 (TGB1; Torrance et al., 2009), which is encoded by RNA-TGB. Besides encoding a triple gene block of movement proteins, TGB1, TGB2, and TGB3 (Zamyatnin et al., 2004), RNA-TGB also encodes a viral suppressor of RNA silencing, the 8K protein (Lukhovitskaya et al., 2013b).To establish a successful infection in the entire plant, viruses must be able to replicate and to move their genomic components between cells, tissues, and organs. Recently, it has become evident that PMTV utilizes a sophisticated mode of cell-to-cell and long-distance movement that involves two virus transport forms, one represented by the viral nucleoprotein complexes (vRNPs) consisting of virus RNA and the TGB1 protein and another represented by the polar virions containing CP-RT and TGB1 proteins attached to one extremity of virus particles (Torrance et al., 2009; for review, see Solovyev and Savenkov, 2014). Proteins implicated in PMTV cell-to-cell movement include TGB1, TGB2, and TGB3 (Zamyatnin et al., 2004; Haupt et al., 2005a). Indirect evidence suggests that CP-RT is required for the efficient systemic movement of intact virions through its interaction with TGB1 (Torrance et al., 2009).Early in infection, the vRNP is transported on the endoplasmic reticulum actomyosin network and targeted to plasmodesmata by TGB2 and TGB3. Later in infection, fluorescently labeled TGB1 is seen in the nucleus and accumulates in the nucleolus. Nucleolar TGB1 association has been shown to be necessary for long-distance movement (Wright et al., 2010).Two structurally distinct subdomains have been identified in the N terminus of TGB1 proteins of hordeiviruses and pomoviruses (Makarov et al., 2009), an N-terminal domain (NTD) comprising approximately 125 amino acids in PMTV (ssRNA in noncooperative and cooperative manners, respectively. The C-terminal half of TGB1 contains a nucleoside triphosphatase/helicase domain that displays cooperative RNA binding. Previously, Wright et al. (2010) reported that TGB1 expressed from a 35S promoter localizes in the cytoplasm and accumulates in the nucleus and nucleolus with occasional labeling of microtubules (MTs). The MT labeling was apparent behind the leading edge of infection when yellow fluorescent protein (YFP)-TGB1 was expressed from an infectious clone. Deletion of 84 amino acids from the N terminus of TGB1 (representing most of the NTD) resulted in the absence of MTs, and nucleolar labeling and fusion of these 84 N-terminal amino acids to GFP resulted in nucleolar enrichment of GFP but no labeling of MTs. Deletion of the 5′ proximal part of the TGB1 open reading frame (ORF), encoding this N-terminal 84 amino acids, in the virus clone abolished systemic but not cell-to-cell movement. However, such deletion had no effect on TGB1 interactions with the CP-RT or self-interaction (Wright et al., 2010).

Table I.

Structural features of the PMTV TGB1 proteinPositively charged amino acids are set in boldface type and underscored. NoD, Nucleolar localization sequence detector; NS, not shown.