Rotaviral Enterotoxin Nonstructural Protein 4 Targets Mitochondria for Activation of Apoptosis during Infection

Viruses have evolved to encode multifunctional proteins to control the intricate cellular signaling pathways by using very few viral proteins. Rotavirus is known to express six nonstructural and six structural proteins. Among them, NSP4 is the enterotoxin, known to disrupt cellular Ca²⁺ homeostasis by translocating to endoplasmic reticulum. In this study, we have observed translocation of NSP4 to mitochondria resulting in dissipation of mitochondrial membrane potential during virus infection and NSP4 overexpression. Furthermore, transfection of the N- and C-terminal truncated NSP4 mutants followed by analyzing NSP4 localization by immunofluorescence microscopy identified the 61–83-amino acid region as the shortest mitochondrial targeting signal. NSP4 exerts its proapoptotic effect by interacting with mitochondrial proteins adenine nucleotide translocator and voltage-dependent anion channel, resulting in dissipation of mitochondrial potential, release of cytochrome c from mitochondria, and caspase activation. During early infection, apoptosis activation by NSP4 was inhibited by the activation of cellular survival pathways (PI3K/AKT), because PI3K inhibitor results in early induction of apoptosis. However, in the presence of both PI3K inhibitor and NSP4 siRNA, apoptosis was delayed suggesting that the early apoptotic signal is initiated by NSP4 expression. This proapoptotic function of NSP4 is balanced by another virus-encoded protein, NSP1, which is implicated in PI3K/AKT activation because overexpression of both NSP4 and NSP1 in cells resulted in reduced apoptosis compared with only NSP4-expressing cells. Overall, this study reports on the mechanism by which enterotoxin NSP4 exerts cytotoxicity and the mechanism by which virus counteracts it at the early stage for efficient infection.     

KSHV MicroRNAs Mediate Cellular Transformation and Tumorigenesis by Redundantly Targeting Cell Growth and Survival Pathways

Kaposi''s sarcoma-associated herpesvirus (KSHV) is causally linked to several human cancers, including Kaposi''s sarcoma, primary effusion lymphoma and multicentric Castleman''s disease, malignancies commonly found in HIV-infected patients. While KSHV encodes diverse functional products, its mechanism of oncogenesis remains unknown. In this study, we determined the roles KSHV microRNAs (miRs) in cellular transformation and tumorigenesis using a recently developed KSHV-induced cellular transformation system of primary rat mesenchymal precursor cells. A mutant with a cluster of 10 precursor miRs (pre-miRs) deleted failed to transform primary cells, and instead, caused cell cycle arrest and apoptosis. Remarkably, the oncogenicity of the mutant virus was fully restored by genetic complementation with the miR cluster or several individual pre-miRs, which rescued cell cycle progression and inhibited apoptosis in part by redundantly targeting IκBα and the NF-κB pathway. Genomic analysis identified common targets of KSHV miRs in diverse pathways with several cancer-related pathways preferentially targeted. These works define for the first time an essential viral determinant for KSHV-induced oncogenesis and identify NF-κB as a critical pathway targeted by the viral miRs. Our results illustrate a common theme of shared functions with hierarchical order among the KSHV miRs.     

Sequestration of Free Tubulin Molecules by the Viral Protein NSP2 Induces Microtubule Depolymerization during Rotavirus Infection

Microtubules, components of the cell cytoskeleton, play a central role in cellular trafficking. Here we show that rotavirus infection leads to a remodeling of the microtubule network together with the formation of tubulin granules. While most microtubules surrounding the nucleus depolymerize, others appear packed at the cell periphery. In microtubule depolymerization areas, tubulin granules are observed; they colocalize with viroplasms, viral compartments formed by interactions between rotavirus proteins NSP2 and NSP5. With purified proteins, we show that tubulin directly interacts in vitro with NSP2 but not with NSP5. The binding of NSP2 to tubulin is independent of its phosphatase activity. The comparison of three-dimensional (3-D) reconstructions of NSP2 octamers alone or associated with tubulin reveals electron densities in the positively charged grooves of NSP2 that we attribute to tubulin. Site-directed mutagenesis of NSP2 and competition assays between RNA and tubulin for NSP2 binding confirm that tubulin binds to these charged grooves of NSP2. Although the tubulin position within NSP2 grooves cannot be precisely determined, the tubulin C-terminal H12 α-helix could be involved in the interaction. NSP2 overexpression and rotavirus infection produce similar effects on the microtubule network. NSP2 depolymerizes microtubules and leads to tubulin granule formation. Our results demonstrate that tubulin is a viroplasm component and reveal an original mechanism. Tubulin sequestration by NSP2 induces microtubule depolymerization. This depolymerization probably reroutes the cell machinery by inhibiting trafficking and functions potentially involved in defenses to viral infections.Microtubules (MTs) are components of the cell cytoskeleton and play a major role in cellular trafficking. Molecular motors (dynein and kinesins) use MTs as tracks to address organelles to precise loci. Viruses are irreplaceable tools to study cellular processes; for example, many of them hijack cellular transport to reach the perinuclear region (for reviews, see references 27, 35, 39, and 40). Some viruses also modify the cell compartmentation and create viral inclusions where viral replication and virion assembly are performed (for a review, see reference 30). Both aspects are sometimes related; electron and fluorescence microscopy observations of reovirus-infected cells have shown that viral inclusions form an electron-dense coat surrounding the MTs (15, 32, 41). In the case of rotavirus, another member of the Reoviridae family, interactions between viral proteins and MTs remain unclear; some studies report an interaction between MTs and either NSP4 or VP4, whereas others did not detect these interactions (4, 19, 29, 51). Rotavirus is the leading agent of gastroenteritis in young children worldwide (31); studying its interactions with its host cell is thus of particular interest to identify new potential therapeutical targets.The rotavirus genome is composed of 11 double-stranded RNAs (dsRNA) surrounded by a triple-layer capsid. During rotavirus infection, punctuate cytoplasmic structures, named “viroplasms,” are formed; they are the sites of viral genome replication and virion assembly. These structures are made of several viral proteins and of viral mRNAs that serve as templates for genome replication. Two viral nonstructural proteins, NSP2 and NSP5, are crucial for viroplasm formation (10, 24, 38). Their coexpression in uninfected cells leads to the formation of punctuate cytoplasmic structures termed viroplasm-like structures (VLS) (18). NSP2 forms a doughnut-shaped octamer by tail-to-tail interaction of two tetramers; four positively charged grooves crossing the two tetramers have been identified (21). Structural and biochemical studies have revealed a histidine-triad (HIT)-like motif responsible for the nucleoside triphosphatase (NTPase), RNA triphosphatase (RTPase), and nucleoside diphosphate (NDP) kinase-like activities of NSP2 (21, 23, 42, 46). These catalytic activities are required for dsRNA synthesis but not for viroplasm formation (11, 43). NSP2 binds single-stranded RNA nonspecifically, has helix destabilizing activity (44), and undertakes conformational changes upon nucleotide binding (37). NSP2 might thus function as a molecular motor involved in genome replication and packaging. NSP5 is a dimeric O-linked glyco- and phosphoprotein, which exists as variously phosphorylated isoforms (1, 36, 48). A cryoelectron microscopy study pointed out that RNA and NSP5 compete for binding to the grooves of the NSP2 octamer (22). The function of NSP5 in rotavirus replication and the role of its phosphorylation remain unknown. No cellular partner of these two nonstructural proteins was known, until a possible association of both proteins with tubulin was reported (9).In the present report, we studied the interaction of rotavirus with tubulin and MTs. We focused on the cellular effects and the structural characterization of the interaction between tubulin and NSP2. Our results highlight that infection by the rotavirus RF strain disorganizes and depolymerizes the MT network of MA104 cells and that viroplasms colocalize with tubulin granules. Electron microscopy and biochemical experiments demonstrate that tubulin directly binds to the positively charged grooves of NSP2. Moreover, NSP2 overexpression induces MT depolymerization and tubulin granule formation. We thus propose that NSP2 sequesters tubulin in viroplasms during rotavirus infection. This sequestration induces the MT depolymerization observed during rotavirus infection and most probably modifies cellular trafficking.     

Knockdown of Cellular RNA Helicase DDX3 by Short Hairpin RNAs Suppresses HIV-1 Viral Replication Without Inducing Apoptosis

The targeting of a cellular co-factor, rather than the HIV-1-specific RNAs, by small interfering RNAs holds promise as the rapid mutational ability of the HIV-1 genome may obviate the potential clinical use of RNAi against this virus. The DEAD-box RNA helicase DDX3 is an essential Rev co-factor in the CRM1-Rev-RRE complex that promotes the export of unspliced and single-spliced HIV-1 RNAs from the nucleus to cytoplasm. In this report, human DDX3 was targeted by specific short hairpin RNAs, and the down-regulation of cell's endogenous DDX3 suppressed the nuclear export of unspliced HIV-1 RNAs but did not affect the cell viability. We further showed that the knockdown of cellular DDX3 could effectively inhibit the replication of HIV-1. Therefore, the current results suggest that the RNA helicase DDX3 may become a potential target by RNAi for future genetic therapy of HIV/AIDS.     

Endogenous Repair by the Activation of Cell Survival Signalling Cascades during the Early Stages of Rat Parkinsonism

Here we report a previously unknown self repair mechanism during extremely early stages of rat Parkinsonism. Two important cell survival signaling cascades, Phosphatidylinositol-3 kinases (PI3K)/Akt pathway and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway, could be responsible for this potential endogenous rescue system. In the 6-hydroxydopamine-lesioned rat, the phosphorylated p44/42 MAPK and its downstream target, the phosphorylated Bad at Ser 112, were up-regulated at post-lesion day 3 and lasted for a couple of weeks. Although the change in the phosphorylated Akt kinase was negligible throughout the studied period, its downstream target, the phosphorylated Bad at 136, was increased from post-lesion day 3 to post-lesion day 14. In the mean time, nestin-positive reactive astrocytes with low levels of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) appeared at post-lesion day 3 in 6-hydroxydopamine-lesioned rat. BDNF was expressed in both striatum and substantia nigra whereas GDNF was displayed in striatum only. At post-lesion day 14, nestin, BDNF and GDNF expressions were diminished. These neurotrophic factors were believed to initiate the above anti-apoptotic signal transduction cascades as we could see that their expression patterns were similar. The data strongly suggest that there is an endogenous repair effort by evoking the cell survival signaling and possibly via the releases of BDNF and GDNF from nestin-immunoreactive reactive astrocytes. ERK/MAPK pathway was proposed to be the key endogenous neuroprotective mechanisms, particularly in early stages of rat Parkinsonism. However, the self repair effort is only functional within an extremely short time window immediately after onset.     

Cellular Inhibitor of Apoptosis Protein cIAP2 Protects against Pulmonary Tissue Necrosis during Influenza Virus Infection to Promote Host Survival

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The Human Interferon-Induced MxA Protein Inhibits Early Stages of Influenza A Virus Infection by Retaining the Incoming Viral Genome in the Cytoplasm

The induction of an interferon-induced antiviral state is a powerful cellular response against viral infection that limits viral spread. Here, we show that a preexisting antiviral state inhibits the replication of influenza A viruses in human A549 cells by preventing transport of the viral genome to the nucleus and that the interferon-induced MxA protein is necessary but not sufficient for this process. This represents a previously unreported antiviral function of MxA against influenza A virus infection.     

Repression of Inflammasome by Francisella tularensis during Early Stages of Infection

Francisella tularensis is an important human pathogen responsible for causing tularemia. F. tularensis has long been developed as a biological weapon and is now classified as a category A agent by the Centers for Disease Control because of its possible use as a bioterror agent. F. tularensis represses inflammasome; a cytosolic multi-protein complex that activates caspase-1 to produce proinflammatory cytokines IL-1β and IL-18. However, the Francisella factors and the mechanisms through which F. tularensis mediates these suppressive effects remain relatively unknown. Utilizing a mutant of F. tularensis in FTL_0325 gene, this study investigated the mechanisms of inflammasome repression by F. tularensis. We demonstrate that muted IL-1β and IL-18 responses generated in macrophages infected with F. tularensis live vaccine strain (LVS) or the virulent SchuS4 strain are due to a predominant suppressive effect on TLR2-dependent signal 1. Our results also demonstrate that FTL_0325 of F. tularensis impacts proIL-1β expression as early as 2 h post-infection and delays activation of AIM2 and NLRP3-inflammasomes in a TLR2-dependent fashion. An enhanced activation of caspase-1 and IL-1β observed in FTL_0325 mutant-infected macrophages at 24 h post-infection was independent of both AIM2 and NLRP3. Furthermore, F. tularensis LVS delayed pyroptotic cell death of the infected macrophages in an FTL_0325-dependent manner during the early stages of infection. In vivo studies in mice revealed that suppression of IL-1β by FTL_0325 early during infection facilitates the establishment of a fulminate infection by F. tularensis. Collectively, this study provides evidence that F. tularensis LVS represses inflammasome activation and that F. tularensis-encoded FTL_0325 mediates this effect.     

Regulation of Lipid Signaling Pathways for Cell Survival and Apoptosis by bcl-2 in Prostate Carcinoma Cells

Compelling evidence indicates that activation of the JNK/SAPK signaling pathway is obligatory for apoptosis induction by multiple cell stresses that activate the sphingomyelin cycle. Moreover, ectopic expression of bcl-2 can impair apoptosis signaling by most of the cell stresses that activate the ceramide/JNK pathway. Here we show that enforced expression of bcl-2 protects prostate carcinoma cells against the induction of apoptosis by exogenous C₂-ceramide. Moreover, enforced bcl-2 expression blocked the capacity of C₂-ceramide to activate JNK1, indicating bcl-2 functions at the level of JNK1 or upstream of JNK1 in the ceramide/JNK pathway. The contribution of bcl-2 to the regulation of the arachidonate pathway for prostate carcinoma cell survival was also investigated using highly selective inhibitors of arachidonate metabolism. Our results indicate bcl-2 can protect cells against diminished availability of arachidonic acid, 12-HETE, and 15-HETE. Finally, arachidonic acid substantially suppresses the induction of apoptosis by C₂-ceramide, providing evidence for the opposing influences of these lipid signaling pathways in the mediation of prostate carcinoma cell survival. These results provide evidence for opposing influences of the ceramide and arachidonate signaling pathways in the mediation of cell death and cell survival, respectively, in prostate carcinoma cells and suggest a dual role for bcl-2 in this context.     

CSTP1, a Novel Protein Phosphatase,Blocks Cell Cycle,Promotes Cell Apoptosis,and Suppresses Tumor Growth of Bladder Cancer by Directly Dephosphorylating Akt at Ser473 Site

Akt/protein kinase B is a pivotal component downstream of phosphatidylinositol 3-kinase (PI3K) pathway, whose activity regulates the balance between cell survival and apoptosis. Phosphorylation of Akt occurs at two key sites either at Thr308 site in the activation loop or at Ser473 site in the hydrophobic motif. The phosphorylated form of Akt (pAkt) is activated to promote cell survival. The mechanisms of pAkt dephosphorylation and how the signal transduction of Akt pathway is terminated are still largely unknown. In this study, we identified a novel protein phosphatase CSTP1(complete s transactivated protein 1), which interacts and dephosphorylates Akt specifically at Ser473 site in vivo and in vitro, blocks cell cycle progression and promotes cell apoptosis. The effects of CSTP1 on cell survival and cell cycle were abrogated by depletion of phosphatase domain of CSTP1 or by expression of a constitutively active form of Akt (S473D), suggesting Ser473 site of Akt as a primary cellular target of CSTP1. Expression profile analysis showed that CSTP1 expression is selectively down-regulated in non-invasive bladder cancer tissues and over-expression of CSTP1 suppressed the size of tumors in nude mice. Kaplan-Meier curves revealed that decreased expression of CSTP1 implicated significantly reduced recurrence-free survival in patients suffered from non-invasive bladder cancers.     

Functional Characterization of Bovine Viral Diarrhea Virus Nonstructural Protein 5A by Reverse Genetic Analysis and Live Cell Imaging

Nonstructural protein 5A (NS5A) of bovine viral diarrhea virus (BVDV) is a hydrophilic phosphoprotein with RNA binding activity and a critical component of the viral replicase. In silico analysis suggests that NS5A encompasses three domains interconnected by two low-complexity sequences (LCSs). While domain I harbors two functional determinants, an N-terminal amphipathic helix important for membrane association, and a Zn-binding site essential for RNA replication, the structure and function of the C-terminal half of NS5A are still ill defined. In this study, we introduced a panel of 10 amino acid deletions covering the C-terminal half of NS5A. In the context of a highly efficient monocistronic replicon, deletions in LCS I and the N-terminal part of domain II, as well as in domain III, were tolerated with regard to RNA replication. When introduced into a bicistronic replicon, only deletions in LCS I and the N-terminal part of domain II were tolerated. In the context of the viral full-length genome, these mutations allowed residual virion morphogenesis. Based on these data, a functional monocistronic BVDV replicon coding for an NS5A variant with an insertion of the fluorescent protein mCherry was constructed. Live cell imaging demonstrated that a fraction of NS5A-mCherry localizes to the surface of lipid droplets. Taken together, this study provides novel insights into the functions of BVDV NS5A. Moreover, we established the first pestiviral replicon expressing fluorescent NS5A-mCherry to directly visualize functional viral replication complexes by live cell imaging.     

Immunization with a Recombinant Vaccinia Virus That Encodes Nonstructural Proteins of the Hepatitis C Virus Suppresses Viral Protein Levels in Mouse Liver

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29^(+/−)/MxCre^(+/−) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.