Apoptosis Of Virus-infected Cells Research Articles

The Extended Cleavage Specificity of Channel Catfish Granzyme-like II, A Highly Specific Elastase, Expressed by Natural Killer-like Cells.

The extended cleavage specificity of catfish granzyme-like II has been characterized using substrate phage display. The preference for particular amino acids at and surrounding the cleavage site was further validated by using a panel of recombinant substrates. This serine protease, which has previously been isolated as cDNA from a catfish natural killer-like cell line showed a preference for Ala in the P1 position of the substrate, and for multiple basic amino acids N-terminally of the cleavage site. A closely related zebrafish serine protease (zebrafish esterase-like) showed a very similar cleavage specificity, indicating an evolutionary conservation of this protease specificity among various fish species. Two catfish serine proteases, originating from NK-like cells, have now been isolated and characterized. One of them is highly specific met-ase with similar characteristics as the mammalian granzyme M. This enzyme may be involved in the induction of apoptosis in virus-infected cells, with a potential target in (catfish) caspase 6. In contrast to catfish granzyme-like I, the second enzyme analyzed here does not seem to have a direct counterpart in mammalian NK cells, and its role in the immune function of catfish NK cells is, therefore, still not known. However, this enzyme seems to be able to cleave a number of cytoskeletal proteins, indicating a separate strategy to induce apoptosis in target cells. Both of these enzymes are very interesting targets for further studies of their roles in catfish immunity, as enzymes with similar specificities have also been identified in zebrafish.

Extended cleavage specificities of human granzymes A and K, two closely related enzymes with conserved but still poorly defined functions in T and NK cell-mediated immunity.

Granzymes A and K are two highly homologous serine proteases expressed by mammalian cytotoxic T cells (CTL) and natural killer cells (NK). Granzyme A is the most abundant of the different granzymes (gzms) expressed by these two cell types. Gzms A and K are found in all jawed vertebrates and are the most well conserved of all hematopoietic serine proteases. Their potential functions have been studied extensively for many years, however, without clear conclusions. Gzm A was for many years thought to serve as a key component in the defense against viral infection by the induction of apoptosis in virus-infected cells, similar to gzm B. However, later studies have questioned this role and instead indicated that gzm A may act as a potent inducer of inflammatory cytokines and chemokines. Gzms A and K form clearly separate branches in a phylogenetic tree indicating separate functions. Transcriptional analyses presented here demonstrate the presence of gzm A and K transcripts in both CD4+ and CD8+ T cells. To enable screening for their primary biological targets we have made a detailed analysis of their extended cleavage specificities. Phage display analysis of the cleavage specificity of the recombinant enzymes showed that both gzms A and K are strict tryptases with high selectivity for Arg over Lys in the P1 position. The major differences in the specificities of these two enzymes are located N-terminally of the cleavage site, where gzm A prefers small amino acids such as Gly in the P3 position and shows a relatively relaxed selectivity in the P2 position. In contrast, gzm K prefers large amino acids such as Phe, Tyr, and Trp in both the P2 and P3 positions and does not tolerate negatively charged residues in the P2 position. This major distinction in extended specificities is likely reflected also in preferred in vivo targets of these two enzymes. This information can now be utilized for high-precision screening of primary targets for gzms A and K in search of their highly conserved but still poorly defined functions in vertebrate immunity.

Extended cleavage specificity of a Chinese alligator granzyme B homologue, a strict Glu-ase in contrast to the mammalian Asp-ases

Granzyme B (GzmB) is primarily expressed by mammalian cytotoxic T cells and serves as one of the key components in the defense against viral infection by the induction of apoptosis in virus infected cells. By direct cell to cell contact and delivery into target cells by perforin, cytotoxic T cells activate apoptosis through the action of GzmB by both caspase-dependent and -independent pathways. In search for early ancestors of GzmB we have in the current study identified and characterized a GzmB homologue from a reptile, the Chinese alligator. This enzyme is encoded from the same locus as the mammalian counterparts, the chymase locus. Phage display analysis of the cleavage specificity of the recombinant alligator enzyme (named MCP1A-like) shows that it is a relatively strict Glu-ase, with strong preference for glutamic acid in the P1 position of a substrate. The majority of mammalian GzmB:s are, in marked contrast to the alligator enzyme, relatively strict Asp-ases. The alligator enzyme also showed strong preference for Ala, Pro and Gly in the P2 position and Val in the P3 position indicating that it has a narrow specificity, similar to the mammalian counterparts. Analysis of the three amino acids forming the substrate binding pocket (S1 pocket) in three amphibian homologues to MCP1A-like, from the frogs Xenopus laevis and Xenopus tropicalis, shows that these amphibian enzymes have similar substrate binding pocket as their mammalian counterparts. This finding, together with the apparent lack of GzmB homologs in fish, indicates that the ancestor of GzmB did appear with the amphibians at the base of tetrapod evolution. This study is a first step in a larger effort to understand the evolutionary processes involved in shaping anti-viral immunity in non-mammalian vertebrates.

Autophagy-competent mitochondrial translation elongation factor TUFM inhibits caspase-8-mediated apoptosis

Mitochondria support multiple cell functions, but an accumulation of dysfunctional or excessive mitochondria is detrimental to cells. We previously demonstrated that a defect in the autophagic removal of mitochondria, termed mitophagy, leads to the acceleration of apoptosis induced by herpesvirus productive infection. However, the exact molecular mechanisms underlying activation of mitophagy and regulation of apoptosis remain poorly understood despite the identification of various mitophagy-associated proteins. Here, we report that the mitochondrial translation elongation factor Tu, a mitophagy-associated protein encoded by the TUFM gene, locates in part on the outer membrane of mitochondria (OMM) where it acts as an inhibitor of altered mitochondria-induced apoptosis through its autophagic function. Inducible depletion of TUFM potentiated caspase-8-mediated apoptosis in virus-infected cells with accumulation of altered mitochondria. In addition, TUFM depletion promoted caspase-8 activation induced by treatment with TNF-related apoptosis-inducing ligand in cancer cells, potentially via dysregulation of mitochondrial dynamics and mitophagy. Importantly, we revealed the existence of and structural requirements for autophagy-competent TUFM on the OMM; the GxxxG motif within the N-terminal mitochondrial targeting sequences of TUFM was required for self-dimerization and mitophagy. Furthermore, we found that autophagy-competent TUFM was subject to ubiquitin-proteasome-mediated degradation but stabilized upon mitophagy or autophagy activation. Moreover, overexpression of autophagy-competent TUFM could inhibit caspase-8 activation. These studies extend our knowledge of mitophagy regulation of apoptosis and could provide a novel strategic basis for targeted therapy of cancer and viral diseases.

Evaluation on the antiviral effect of a hydroxycoumarin against infectious hematopoietic necrosis virus infection in vitro and in vivo

Infectious hematopoietic necrosis (IHN) caused by the viral pathogen infectious hematopoietic necrosis virus (IHNV) is a highly contagious disease of salmonid species, resulting in significant economic impact. The previous study showed a hydroxycoumarin derivative 7-[6-(2-methylimidazole) hexyloxy] coumarin (D5) significantly inhibited spring viraemia of carp virus (SVCV) infection, suggesting that D5 may be useful as a potential anti-IHNV agent. In this study, D5 at the concentration of up to 10 mg/L significantly inhibited IHNV replication in epithelioma papulosum cyprini (EPC) cells with a maximum inhibitory rate of >90%, maintained mitochondrial membrane potential (ΔΨm) levels, and decreased IHNV-induced apoptosis in virus-infected cells. As the consequence of protection on mitochondria, D5 enhanced antioxidant enzyme activities and decreased reactive oxygen species (ROS) to maintain the antioxidant-oxidant balance of IHNV-infected EPC cells. For in vivo study, D5 via intraperitoneal injection exhibited an anti-IHNV effect in the virus-infected fish by substantially enhancing the survival rate. Meanwhile, up-regulation of six interferon (IFN) related gene expressions demonstrated that D5 may activate IFN-related expressions for inhibiting IHNV replication during the early stage of viral infection, which is beneficial for the continuous antiviral action on controlling low viral loads in rainbow trout juvenile. Thus, D5 effective regulated IHNV-induced undesirable conditions to be an excellent potential therapeutic agent against IHNV infection.

RNase L Induces Expression of A Novel Serine/Threonine Protein Kinase, DRAK1, to Promote Apoptosis.

Apoptosis of virus-infected cells is an effective antiviral mechanism in addition to interferon induction to establish antiviral state to restrict virus spread. The interferon-inducible 2′–5′ oligoadenylate synthetase/RNase L pathway results in activation of RNase L in response to double stranded RNA and cleaves diverse RNA substrates to amplify interferon induction and promote apoptosis. Here we show that RNase L induces expression of Death-associated protein kinase-Related Apoptosis-inducing protein Kinase 1 (DRAK1), a member of the death-associated protein kinase family and interferon-signaling pathway is required for induction. Overexpression of DRAK1 triggers apoptosis in the absence of RNase L activation by activating c-Jun N-terminal kinase (JNK), translocation of BCL2 Associated X (Bax) to the mitochondria accompanied by cytochrome C release and loss of mitochondrial membrane potential promoting cleavage of caspase 3 and Poly(ADP-Ribose) Polymerase 1 (PARP). Inhibitors of JNK and caspase 3 promote survival of DRAK1 overexpressing cells demonstrating an important role of JNK signaling pathway in DRAK1-mediated apoptosis. DRAK1 mutant proteins that lack kinase activity or nuclear localization fail to induce apoptosis highlighting the importance of cellular localization and kinase function in promoting cell death. Our studies identify DRAK1 as a mediator of RNase L-induced apoptosis.

Regulation of Apoptosis during Flavivirus Infection.

Apoptosis is a type of programmed cell death that regulates cellular homeostasis by removing damaged or unnecessary cells. Its importance in host defenses is highlighted by the observation that many viruses evade, obstruct, or subvert apoptosis, thereby blunting the host immune response. Infection with Flaviviruses such as Japanese encephalitis virus (JEV), Dengue virus (DENV) and West Nile virus (WNV) has been shown to activate several signaling pathways such as endoplasmic reticulum (ER)-stress and AKT/PI3K pathway, resulting in activation or suppression of apoptosis in virus-infected cells. On the other hands, expression of some viral proteins induces or protects apoptosis. There is a discrepancy between induction and suppression of apoptosis during flavivirus infection because the experimental situation may be different, and strong links between apoptosis and other types of cell death such as necrosis may make it more difficult. In this paper, we review the effects of apoptosis on viral propagation and pathogenesis during infection with flaviviruses.

MicroRNA-548j inhibits type I interferon production by targeting ZBTB11 in patients with chronic hepatitis B

Type I IFNs play crucial roles in antiviral immune responses. By inducing cellular resistance to viral infection and apoptosis of virus-infected cells, they impair virus replication and eliminate the invading pathogens. However, in CHB patients, generation of type I IFN was significantly impaired and the underlying mechanisms have not been fully understood. MicroRNA-548 family has been implicated in regulating antiviral response upon various infections. Here we explored the potential role of miR-548j in modulating type I IFN production in CHB. We found that miR-548j expression increased in MoDCs from CHB patients than that from healthy volunteers and transient transfection of miR-548j mimic led to a marked decrease of IFN-α/β production in MoDCs from healthy volunteers while blockade of miR-548j by anti-miR-548j significantly restored IFN-α/β secretion in MoDCs from CHB patients. In addition, Zinc finger and BTB domain containing 11 (ZBTB11) was predicted and finally confirmed to be a target of miR-548j. Forced expression of ZBTB11 also restored IFN-α/β secretion in MoDCs from CHB patients. These results indicate the involvement of miR-548j in aberrant production of type I IFN in CHB patients, and provide a potential target for developing anti-HBV therapies.

Exploring the Possibility of Using Herpes Simplex Virus in Oncolytic Virotherapy of Multiple Myeloma

Introduction: Multiple myeloma (MM) is the second most prevalent hematologic malignancy. Approximately, 80,000 people have died of the disease in the United States and 25,000 new cases are registered every year. Majority of patients develop resistance to current therapeutic treatments and die within 5-10 years of diagnosis. Thus, need of novel therapeutic intervention is extremely urgent. Although the field of oncolytic virotherapy (OV) based on using viruses with natural or engineered tumor selective replication to intentionally infect and kill tumor cells has been extensively explored for the treatment of solid tumors, only few data are available for the treatment of hematopoietic malignancies. Our laboratory was one of the first to show that OV using Reovirus can be an effective therapeutic strategy for the treatment of MM in vitro and in MM patients. In this work we aim at exploring the possibility of using genetically engineered HSV1 (Herpes Simplex Virus) for the treatment of MM. HSV1 is an enveloped, double stranded DNA virus. Engineered HSV1 (HSVQ) has both copies of viral gene important in viral replication in normal cells viz., ICP 34.5 gene deleted and has one copy of GFP inserted into viral ICP6 gene. Such engineered virus has been used for cancer cell selective killing in preclinical and clinical studies for the treatment of several types of solid tumors including melanoma and glioblastoma multiforme. In this study, we investigated the biological and preclinical impact of HSVQ on MM cell in vitro and in vivo.Method: Recombinant HSVQ was amplified in African green monkey kidney epithelial Vero cells, purified by sucrose density gradient centrifugation and titrated by plaque assay on Vero cells. Several MM cell lines (MM1.S, U266, RPMI8226, L363, NIH-H929) were infected with HSVQ at Multiplicity of Infection (MOI) 0.01 to 5. Fluorescence microscopy and flow cytometry analysis were used to assess MM cell infectivity with the virus. RT-PCR was performed to detect presence of viral genome in MM cell lines. Viral replication assays were also performed. Cell proliferation and apoptotic assays including MTT Assay, Tryphan Blue exclusion test, LIVE/DEAD cell viability staining and Annexin/7-AAD assays were done to determine viability of virus infected MM cells. Western Blot analysis was carried out to determine endoplasmic reticulum (ER) stress response mediated by ERK, Hsp90, Bip/GRP78, Hsp40 and apoptosis in HSVQ treated MM cells. Total bone marrow (BM) cells obtained from MM patients were infected with HSVQ and multi parametric flow analysis was performed to determine infectivity and specific killing of CD138+ MM cells by the virus.To study in vivo anti-tumorigenic properties of HSVQ, 12.5 x106 GFP/Luc + MM1.S or NIH-H929 cells were subcutaneously injected into the right flank of 20 NOD-SCID mice. Two weeks after injection, mice with comparable size tumors were randomly divided (5 animal for each treatment group) and treated twice with 1x107PFU (Plaque Forming Unit) HSVQ for 2 weeks or with saline. Tumor growth was measured to determine anti tumorigenic effect of HSVQ on MM tumors.Results and Conclusion: Fluorescence microscopy and flow cytometry revealed that MM cell lines can be effectively infected with and killed by HSVQ even at MOI as low as 0.1. Under such conditions, Western Blot analysis revealed increased BAX expression, decreased BCL2 expression and cleavage of Caspase 3 and PARP indicating apoptosis of virus infected cells. Interestingly, multi parametric flow analysis revealed that HSVQ specifically infects and kills CD138+ MM plasma cells in a total population of BM cellular fraction isolated from MM patients. Moreover in vivo preclinical data show that HSVQ dramatically reduces tumor volume (p<0.001) in both MM.1S and NIH-H929 xenograft mouse models. Thus, from the preliminary observations, it can be concluded that HSVQ can selectively infect and induce apoptosis in myeloma cells. Mechanisms of HSVQ replication in MM cells and induced MM cell killing are being currently investigated. Here for the first time we are providing clear evidences that HSVQ can infect and specifically kill MM cells supporting the idea of the use of HSV for the treatment of MM. Moreover, since the backbone of HSVQ can be further engineered, it can be used to specifically deliver anti-angiogenic and anti-inflammatory genes to MM cells for the treatment of MM. DisclosuresHofmeister:Arno Therapeutics, Inc.: Research Funding; Celgene: Research Funding; Karyopharm Therapeutics: Research Funding; Incyte, Corp: Membership on an entity’s Board of Directors or advisory committees; Janssen: Pharmaceutical Companies of Johnson & Johnson: Research Funding; Signal Genetics, Inc.: Membership on an entity’s Board of Directors or advisory committees; Takeda Pharmaceutical Company: Research Funding; Teva: Membership on an entity’s Board of Directors or advisory committees.

Ubiquitination of the Transcription Factor IRF-3 Activates RIPA, the Apoptotic Pathway that Protects Mice from Viral Pathogenesis

The transcription factor IRF-3 mediates cellular antiviral response by inducing the expression of interferon and other antiviral proteins. In RNA-virus infected cells, IRF-3's transcriptional activation is triggered primarily by RIG-I-like receptors (RLR), which can also activate the RLR-induced IRF-3-mediated pathway of apoptosis (RIPA). Here, we have reported that the pathway of IRF-3 activation in RIPA was independent of and distinct from the known pathway of transcriptional activation of IRF-3. It required linear polyubiquitination of two specific lysine residues of IRF-3 by LUBAC, the linear polyubiquitinating enzyme complex, which bound IRF-3 in signal-dependent fashion. To evaluate the role of RIPA in viral pathogenesis, we engineered a genetically targeted mouse, which expressed a mutant IRF-3 that was RIPA-competent but transcriptionally inert; this single-action IRF-3 could protect mice from lethal viral infection. Our observations indicated that IRF-3-mediated apoptosis of virus-infected cells could be an effective antiviral mechanism, without expression of the interferon-stimulated genes.

Biochemical Analysis of Caspase-8-dependent Proteolysis of IRF3 in Virus-infected Cells.

Interferon regulatory factor 3 (IRF3) is a transcription factor, which is critical for the antiviral response against a wide range of viruses (Hiscott, 2007; Ikushima et al., 2013). It gets activated in virus- infected cells via Toll like receptors (TLRs), RIG-I (retinoic acid inducible gene 1) like receptors (RLRs), cyclic GMP-AMP synthase (cGAS) – stimulator of interferon genes (STING), which are sensors of viral components in the cells (Chattopadhyay and Sen, 2014a; 2014b; Hiscott, 2007). IRF3 is a cytoplasmic protein, upon activation by virally activated sensors it gets phosphorylated, translocated to the nucleus and binds to the interferon-sensitive response element (ISRE) of the gene promoters to induce their transcription (Hiscott, 2007). IRF3 has other functions, including direct stimulation of apoptosis in virus- infected cells. In this pathway, the transcriptional activity of IRF3 is not required (Chattopadhyay et al., 2013b; Chattopadhyay et al., 2016; Chattopadhyay et al., 2010; Chattopadhyay and Sen, 2010; Chattopadhyay et al., 2011). These pathways are negatively regulated by host factors as well as by viruses. Our studies indicate that IRF3 can be proteolytically processed by caspase-8-dependent cleavage (Sears et al., 2011). A specific site in IRF3 is targeted by caspase-8, activated in RNA or DNA virus-infected and dsRNA-stimulated cells (Sears et al., 2011). The direct involvement of caspase-8 was confirmed by in vitro cleavage assay using recombinant proteins and in vivo by virus activated caspase- 8. The proteolytic cleavage of IRF3 can be inhibited by chemical inhibition or genetic ablation of caspase-8. The cleavage of IRF3 removes the activated pool of IRF3 and thus can be used as a pro- viral mechanism (Figure 1). Using a C-terminally epitope-tagged human IRF3, we analyzed the cleavage of IRF3 in virus-infected cells. Moreover, we used recombinant proteins in vitro to conclude that IRF3 is a substrate of caspase-8 (Sears et al., 2011). In the current protocol, we have outlined a simple and detailed procedure to biochemically analyze the proteolysis of IRF3 in virus-infected cells and the specific role of caspase-8 in this process.

Evolutionary evidence of tumor necrosis factor super family members in the Japanese pufferfish (Takifugu rubripes): Comprehensive genomic identification and expression analysis

Tumor necrosis factor (TNF) and its superfamily (TNFSF) members are important inflammatory cytokines. Although fish have fourteen TNFSF genes, their genomic location and existence are yet to be described and confirmed in the Japanese pufferfish (Fugu) (Takifugu rubripes). Therefore, we conducted in silico identification, synteny analysis of TNFSF genes from Fugu with that of zebrafish and human TNFSF loci and their expression analysis in various tissues. We identified ten novel TNFSF genes, viz. TNFSF5 (CD40L), TNFSF6 (FasL), three TNFSF10 (TRAIL) (−1, 2 and 3), TNFSF11 (RANKlg), TNFSF12 (TWEAK), two TNFSF13B (BAFF) (1 and 2) and TNFSF14 (LIGHT) belonging to seven TNFSFs in Fugu. Several features such as existence of TNF family signature, conservation of genes in TNF loci with human and zebrafish chromosomes and phylogenetic clustering with other teleost TNFSF orthologs confirmed their identity. Fugu TNFSF genes were constitutively expressed in all eight different tissues with most of them expressed highly in liver. Fugu TNFSF10 gene has three homologs present on chromosomes 10 (TNFSF10-1), 8 (TNFSF10-2) and 2 (TNFSF10-3). Moreover, a phylogenetic analysis containing all available vertebrate (mammals, birds, reptiles, amphibians and fish) TNFSF10 orthologs showed that Fugu TNFSF10-1 and 10-3 are present in all vertebrates, whereas TNFSF10-2 was not related to any mammalian and avian orthologs. Viral double-stranded RNA mimic poly (I:C) caused an elevated expression of three Fugu TNFSF10 genes in head kidney cells at 4h indicating probable role of these genes to induce apoptosis in virus-infected cells. In conclusion, Fugu possesses genes belonging to nine TNFSFs including the newly identified seven and previously reported two, TNFSF New (TNF-N) and TNFSF2 (TNF-α). Our findings would add up information to TNFSF evolution among vertebrates.

Interferon regulatory factor-1 (IRF-1) is involved in the induction of phosphatidylserine receptor (PSR) in response to dsRNA virus infection and contributes to apoptotic cell clearance in CHSE-214 cell.

The phosphatidylserine receptor (PSR) recognizes a surface marker on apoptotic cells and initiates engulfment. This receptor is important for effective apoptotic cell clearance and maintains normal tissue homeostasis and regulation of the immune response. However, the regulation of PSR expression remains poorly understood. In this study, we determined that interferon regulatory factor-1 (IRF-1) was dramatically upregulated upon viral infection in the fish cell. We observed apoptosis in virus-infected cells and found that both PSR and IRF-1 increased simultaneously. Based on a bioinformatics promoter assay, IRF-1 binding sites were identified in the PSR promoter. Compared to normal viral infection, we found that PSR expression was delayed, viral replication was increased and virus-induced apoptosis was inhibited following IRF-1 suppression with morpholino oligonucleotides. A luciferase assay to analyze promoter activity revealed a decreasing trend after the deletion of the IRF-1 binding site on PSR promoter. The results of this study indicated that infectious pancreatic necrosis virus (IPNV) infection induced both the apoptotic and interferon (IFN) pathways, and IRF-1 was involved in regulating PSR expression to induce anti-viral effects. Therefore, this work suggests that PSR expression in salmonid cells during IPNV infection is activated when IRF-1 binds the PSR promoter. This is the first report to show the potential role of IRF-1 in triggering the induction of apoptotic cell clearance-related genes during viral infection and demonstrates the extensive crosstalk between the apoptotic and innate immune response pathways.

Role of Fas/FasL signaling in regulation of anti-viral response during HSV-2 vaginal infection in mice

Fas receptor–Fas ligand (FasL) signaling is involved in apoptosis of virus-infected cells but increasing evidence accumulates on Fas receptor as a mediator of apoptosis-independent processes such as induction of activating and pro-inflammatory signals. In this study, we examined the role of Fas/FasL pathway in regulation of anti-viral response to genital HSV-2 infection using a murine model of HSV-2 infection applied to C57BL6/J, B6. MRL-Faslpr/J and B6Smn.C3-Faslgld/J mice. HSV-2 infection of Fas- and FasL-deficient mice led to decreased migration of IFN-γ expressing NK cells and CD4+ T cells, but not of γδ T cells, into the vaginal tissue. The vaginal tissues of HSV-2 infected Fas- and FasL-deficient mice showed increased production of IL-10, followed by low expression of the early CD69 activation marker on CD4+ and CD8+ T cells and increased numbers of regulatory T cells (Tregs). Experiments in co-cultures of CD4+ T cells and bone marrow derived dendritic cells showed that lack of bilateral Fas–FasL signaling led to expansion of Tregs and increased production of IL-10 and TGF-β1. Our results demonstrate that Fas/FasL can regulate development of tolerogenic dendritic cells and expansion of Tregs early during HSV-2 infection, which further influences effective anti-viral response.

Inhibition of Bim Enhances Replication of Varicella-Zoster Virus and Delays Plaque Formation in Virus-Infected Cells

Programmed cell death (apoptosis) is an important host defense mechanism against intracellular pathogens, such as viruses. Accordingly, viruses have evolved multiple mechanisms to modulate apoptosis to enhance replication. Varicella-zoster virus (VZV) induces apoptosis in human fibroblasts and melanoma cells. We found that VZV triggered the phosphorylation of the proapoptotic proteins Bim and BAD but had little or no effect on other Bcl-2 family members. Since phosphorylation of Bim and BAD reduces their proapoptotic activity, this may prevent or delay apoptosis in VZV-infected cells. Phosphorylation of Bim but not BAD in VZV-infected cells was dependent on activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. Cells knocked down for Bim showed delayed VZV plaque formation, resulting in longer survival of VZV-infected cells and increased replication of virus, compared with wild-type cells infected with virus. Conversely, overexpression of Bim resulted in earlier plaque formation, smaller plaques, reduced virus replication, and increased caspase 3 activity. Inhibition of caspase activity in VZV-infected cells overexpressing Bim restored levels of virus production similar to those seen with virus-infected wild-type cells. Previously we showed that VZV ORF12 activates ERK and inhibits apoptosis in virus-infected cells. Here we found that VZV ORF12 contributes to Bim and BAD phosphorylation. In summary, VZV triggers Bim phosphorylation; reduction of Bim levels results in longer survival of VZV-infected cells and increased VZV replication.

Anticancer compound ABT-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice

ABT-263 and its structural analogues ABT-199 and ABT-737 inhibit B-cell lymphoma 2 (Bcl-2), BCL2L1 long isoform (Bcl-xL) and BCL2L2 (Bcl-w) proteins and promote cancer cell death. Here, we show that at non-cytotoxic concentrations, these small molecules accelerate the deaths of non-cancerous cells infected with influenza A virus (IAV) or other viruses. In particular, we demonstrate that ABT-263 altered Bcl-xL interactions with Bcl-2 antagonist of cell death (Bad), Bcl-2-associated X protein (Bax), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). ABT-263 thereby activated the caspase-9-mediated mitochondria-initiated apoptosis pathway, which, together with the IAV-initiated caspase-8-mediated apoptosis pathway, triggered the deaths of IAV-infected cells. Our results also indicate that Bcl-xL, Bcl-2 and Bcl-w interact with pattern recognition receptors (PRRs) that sense virus constituents to regulate cellular apoptosis. Importantly, premature killing of IAV-infected cells by ABT-263 attenuated the production of key pro-inflammatory and antiviral cytokines. The imbalance in cytokine production was also observed in ABT-263-treated IAV-infected mice, which resulted in an inability of the immune system to clear the virus and eventually lowered the survival rates of infected animals. Thus, the results suggest that the chemical inhibition of Bcl-xL, Bcl-2 and Bcl-w could potentially be hazardous for cancer patients with viral infections.

Validation of a novel secretion modification region (SMR) of HIV-1 Nef using cohort sequence analysis and molecular modeling

The HIV-1 accessory protein Nef plays an active role in the pathogenesis of AIDS by its numerous cellular interactions that facilitate the release of virus particles. This 27kDa protein is required for maintenance of the viral replication in HIV, and is also known to contribute to immune evasion, blocking of apoptosis in virus-infected cells and enhancement of virus infectivity. Nef has been shown to be secreted and is present on the surface of virus-infected cells. Recent studies from our laboratory have shown that the Nef protein is secreted from nef-transfected and HIV-1-infected cells in small exosome-like vesicles (40-100nm diam.) that do not contain virions. We have identified three amino-terminal domains of Nef as necessary for secretion: (i) the four arginine residues (17,19,21, 22) comprising the basic region; (ii) the phosphofurin acidic cluster sequence (PACS) composed of four glutamic acid residues (61-64); (iii) a previously unknown motif spanning amino acid residues 65-69 (VGFPV) which we named the secretion modification region (SMR). In this study, we have used population-based phylogeny data and sequence analysis to characterize the conservation of the Nef SMR domain that regulates vesicle secretion. We have performed in silico computational chemistry analysis involving molecular dynamic structure modeling of mutations in the SMR motif. Sequence analysis of Nef from HIV-1-infected patients, including slow progressors (SP), long term progressors (LTP) and long term non-progressors (LTNP) demonstrated 99% conservation of the Nef SMR motif. Computational analysis including modeling of wild-type HIV-1 Nef and V66A Nef SMR mutant using structural homology and molecular dynamics of ligand-associated interactions indicated significant structural changes in the Nef mutant, thus supporting the importance of the SMR domain for mediating Nef vesicle secretion.

Expression, purification and characterization of the interferon-inducible, antiviral and tumour-suppressor protein, human RNase L

The interferon (IFN)-inducible, 2',5'-oligoadenylate (2-5A)-dependent ribonuclease L (RNase L) plays key role in antiviral defense of mammalian cells. Induction by IFN and activation by double-stranded RNA lead to 2-5A cofactor synthesis, which activates RNase L by causing its dimerization. Active RNase L degrades single-stranded viral as well as cellular RNAs causing apoptosis of virus-infected cells. Earlier, we had reported that expression of recombinant human RNase L caused RNA-degradation and cell-growth inhibition in E. coli without the need for exogenous 2-5A. Expression of human RNase L in E. coli usually leads to problems of leaky expression, low yield and degradation of the recombinant protein, which demands number of chromatographic steps for its subsequent purification thereby, compromising its biochemical activity. Here, we report a convenient protocol for expression of full-length, soluble and biochemically active recombinant human RNase L as GST-RNase L fusion protein from E. coli utilizing a single-step affinity purification with an appreciable yield of the highly purified protein. Recombinant RNase L was characterized by SDS-PAGE, immunoblotting and MALDI-TOF analysis. A semi-quantitative agarose-gel-based ribonuclease assay was developed for measuring its 2-5A-dependent RNase L activity against cellular large rRNAs as substrates. The optimized expression conditions minimized degradation of the protein, making it a convenient method for purification of RNase L, which can be utilized to study effects of various agents on the RNase L activity and its protein-protein interactions.

Upregulation of Cellular Bcl-2 by the KSHV Encoded RTA Promotes Virion Production

Apoptosis of virus infected cells can restrict or dampen full blown virus propagation and this can serve as a protective mechanism against virus infection. Consequently, viruses can also delay programmed cell death by enhancing the expression of anti-apoptotic proteins. Human Bcl-2 is expressed on the surface of the mitochondrial membrane and functions as the regulator of the delicate balance between cell survival and apoptosis. In this report, we showed that the replication and transcription activator (RTA) encoded by KSHV ORF 50, a key regulator for KSHV reactivation from latent to lytic infection, upregulates the mRNA and protein levels of Bcl-2 in 293 cells, and TPA-induced KSHV-infected cells. Further analysis revealed that upregulation of the cellular Bcl-2 promoter by RTA is dose-dependent and acts through targeting of the CCN9GG motifs within the Bcl-2 promoter. The Bcl-2 P2 but not the P1 promoter is primarily responsive to RTA. The results of ChIP confirmed the direct interaction of RTA protein with the CCN9GG motifs. Knockdown of cellular Bcl-2 by lentivirus-delivered small hairpin RNA (shRNA) resulted in increased cell apoptosis and decreased virion production in KSHV-infected cells. These findings provide an insight into another mechanism by which KSHV utilizes the intrinsic apoptosis signaling pathways for prolonging the survival of lytically infected host cells to allow for maximum production of virus progeny.

Human T-lymphotropic type 1 virus p30 inhibits homologous recombination and favors unfaithful DNA repair

AbstractWhereas oncogenic retroviruses are common in animals, human T-lymphotropic virus 1 (HTLV-1) is the only transmissible retrovirus associated with cancer in humans and is etiologically linked to adult T-cell leukemia. The leukemogenesis process is still largely unknown, but relies on extended survival and clonal expansion of infected cells, which in turn accumulate genetic defects. A common feature of human tumor viruses is their ability to stimulate proliferation and survival of infected pretumoral cells and then hide by establishing latency in cells that have acquired a transformed phenotype. Whereas disruption of the DNA repair is one of the major processes responsible for the accumulation of genomic abnormalities and carcinogenesis, the absence of DNA repair also poses the threat of cell-cycle arrest or apoptosis of virus-infected cells. This study describes how the HTLV-1 p30 viral protein inhibits conservative homologous recombination (HR) DNA repair by targeting the MRE11/RAD50/NBS1 complex and favors the error-prone nonhomologous-end–joining (NHEJ) DNA-repair pathway instead. As a result, HTLV-1 p30 may facilitate the accumulation of mutations in the host genome and the cumulative risk of transformation. Our results provide new insights into how human tumor viruses may manipulate cellular DNA-damage responses to promote cancer.