Release of Dengue Virus Genome Induced by a Peptide Inhibitor

Dengue virus infects approximately 100 million people annually, but there is no available therapeutic treatment. The mimetic peptide, DN59, consists of residues corresponding to the membrane interacting, amphipathic stem region of the dengue virus envelope (E) glycoprotein. This peptide is inhibitory to all four serotypes of dengue virus, as well as other flaviviruses. Cryo-electron microscopy image reconstruction of dengue virus particles incubated with DN59 showed that the virus particles were largely empty, concurrent with the formation of holes at the five-fold vertices. The release of RNA from the viral particle following incubation with DN59 was confirmed by increased sensitivity of the RNA genome to exogenous RNase and separation of the genome from the E protein in a tartrate density gradient. DN59 interacted strongly with synthetic lipid vesicles and caused membrane disruptions, but was found to be non-toxic to mammalian and insect cells. Thus DN59 inhibits flavivirus infectivity by interacting directly with virus particles resulting in release of the genomic RNA.     

Increased Phosphorylation of Vimentin in Noninfiltrative Meningiomas

Background

Tissue invasion or tissue infiltration are clinical behaviors of a poor-prognosis subset of meningiomas. We carried out proteomic analyses of tissue extracts to discover new markers to accurately distinguish between infiltrative and noninfiltrative meningiomas.

Methodology/Principal Findings

Protein lysates of 64 different tissue samples (including two brain-invasive and 32 infiltrative tumors) were submitted to SELDI-TOF mass spectrometric analysis. Mass profiles were used to build up both unsupervised and supervised hierarchical clustering. One marker was found at high levels in noninvasive and noninfiltrative tumors and appeared to be a discriminative marker for clustering infiltrative and/or invasive meningiomas versus noninvasive meningiomas in two distinct subsets. Sensitivity and specificity were 86.7% and 100%, respectively. This marker was purified and identified as a multiphosphorylated form of vimentin, a cytoskeletal protein expressed in meningiomas.

Conclusions/Significance

Specific forms of vimentin can be surrogate molecular indicators of the invasive/infiltrative phenotype in tumors.     

Cellular Vimentin Regulates Construction of Dengue Virus Replication Complexes through Interaction with NS4A Protein

Dengue virus (DENV) interacts with host cellular factors to construct a more favorable environment for replication, and the interplay between DENV and the host cellular cytoskeleton may represent one of the potential antiviral targeting sites. However, the involvement of cellular vimentin intermediate filaments in DENV replication has been explored less. Here, we revealed the direct interaction between host cellular vimentin and DENV nonstructural protein 4A (NS4A), a known component of the viral replication complex (RC), during DENV infection using tandem affinity purification, coimmunoprecipitation, and scanning electron microscopy. Furthermore, the dynamics of vimentin-NS4A interaction were demonstrated by using confocal three-dimensional (3D) reconstruction and proximity ligation assay. Most importantly, we report for the first time the discovery of the specific region of NS4A that interacts with vimentin lies within the first 50 amino acid residues at the cytosolic N-terminal domain of NS4A (N50 region). Besides identifying vimentin-NS4A interaction, vimentin reorganization and phosphorylation by calcium calmodulin-dependent protein kinase II occurs during DENV infection, signifying that vimentin reorganization is important in maintaining and supporting the DENV RCs. Interestingly, we found that gene silencing of vimentin by small interfering RNA induced a significant alteration in the distribution of RCs in DENV-infected cells. This finding further supports the crucial role of intact vimentin scaffold in localizing and concentrating DENV RCs at the perinuclear site, thus facilitating efficient viral RNA replication. Collectively, our findings implicate the biological and functional significance of vimentin during DENV replication, as we propose that the association of DENV RCs with vimentin is mediated by DENV NS4A.     

Mechanism of Enhanced Immature Dengue Virus Attachment to Endosomal Membrane Induced by prM Antibody

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Vimentin is secreted by activated macrophages

Vimentin is a widely expressed intermediate filament protein thought to be involved mainly in structural processes, such as wound healing. We now demonstrate that activated human macrophages secrete vimentin into the extracellular space. The maturation of blood-derived monocytes into macrophages involves several signalling pathways. We show that secretion of vimentin, which is phosphorylated at serine and threonine residues, is enhanced by the phosphatase inhibitor okadaic acid and blocked by the specific protein kinase C inhibitor GO6983. These findings are consistent with previous observations that phosphorylation of vimentin affects its intracellular localization and that vimentin is a substrate for protein kinase C (PKC). We also show that the anti-inflammatory cytokine interleukin-10 (IL-10), which inhibits PKC activity, blocks secretion of vimentin. In contrast, the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-alpha) can trigger secretion of vimentin. Finally, we found that extracellular vimentin is involved in bacterial killing and the generation of oxidative metabolites, two important functions of activated macrophages. These data establish that vimentin is secreted by macrophages in response to pro-inflammatory signalling pathways and is probably involved in immune function.     

Dengue Virus Impairs Mitochondrial Fusion by Cleaving Mitofusins

Mitochondria are highly dynamic subcellular organelles participating in many signaling pathways such as antiviral innate immunity and cell death cascades. Here we found that mitochondrial fusion was impaired in dengue virus (DENV) infected cells. Two mitofusins (MFN1 and MFN2), which mediate mitochondrial fusion and participate in the proper function of mitochondria, were cleaved by DENV protease NS2B3. By knockdown and overexpression approaches, these two MFNs showed diverse functions in DENV infection. MFN1 was required for efficient antiviral retinoic acid-inducible gene I–like receptor signaling to suppress DENV replication, while MFN2 participated in maintaining mitochondrial membrane potential (MMP) to attenuate DENV-induced cell death. Cleaving MFN1 and MFN2 by DENV protease suppressed mitochondrial fusion and deteriorated DENV-induced cytopathic effects through subverting interferon production and facilitating MMP disruption. Thus, MFNs participate in host defense against DENV infection by promoting the antiviral response and cell survival, and DENV regulates mitochondrial morphology by cleaving MFNs to manipulate the outcome of infection.     

The Stem Region of Premembrane Protein Plays an Important Role in the Virus Surface Protein Rearrangement during Dengue Maturation

Newly assembled dengue viruses (DENV) undergo maturation to become infectious particles. The maturation process involves major rearrangement of virus surface premembrane (prM) and envelope (E) proteins. The prM-E complexes on immature viruses are first assembled as trimeric spikes in the neutral pH environment of the endoplasmic reticulum. When the virus is transported to the low pH environment of the exosomes, these spikes rearrange into dimeric structures, which lie parallel to the virus lipid envelope. The proteins involved in driving this process are unknown. Previous cryoelectron microscopy studies of the mature DENV showed that the prM-stem region (residues 111–131) is membrane-associated and may interact with the E proteins. Here we investigated the prM-stem region in modulating the virus maturation process. The binding of the prM-stem region to the E protein was shown to increase significantly at low pH compared with neutral pH in ELISAs and surface plasmon resonance studies. In addition, the affinity of the prM-stem region for the liposome, as measured by fluorescence correlation spectroscopy, was also increased when pH is lowered. These results suggest that the prM-stem region forms a tight association with the virus membrane and attracts the associated E protein in the low pH environment of exosomes. This will lead to the surface protein rearrangement observed during maturation.     

Protection by Immunoglobulin Dual-Affinity Retargeting Antibodies against Dengue Virus

Dengue viruses are the most common arthropod-transmitted viral infection, with an estimated 390 million human infections annually and ∼3.6 billion people at risk. Currently, there are no approved vaccines or therapeutics available to control the global dengue virus disease burden. In this study, we demonstrate the binding, neutralizing activity, and therapeutic capacity of a novel bispecific dual-affinity retargeting molecule (DART) that limits infection of all four serotypes of dengue virus.     

应用DNA-RNA斑点杂交法检测登革病毒核酸

新近制备了大量纯化的pEH920 DNA,该质粒DNA插入了登革病毒2型核酸片段的互补DNA。以[a-~(32)P]dCTP按缺口转译法标记pEH 920 DNA作为探针,以感染病毒的蚊细胞c_6/36培养上清作标本,应用DNA-RNA斑点杂交法检测了登革病毒核酸。结果显示同位素标记探针(pEH 920)与登革病毒2型标本反应最强,具有一定的型特异性。但与其它血清型登革病毒也呈一定交叉反应。初步探讨了探针的敏感性,至少可检出TCID_(50)625的登革病毒2型核酸。     

mTOR／4E—BP1参与诺考达唑诱导的Dami细胞多倍体化调控

目的：多倍性是物种形成的重要机制,决定一些重要器官细胞产生的数量和功能,而且与某些病理过程（如恶性肿瘤）的发生有密切关系。我们通过建立相对同步化的多倍体细胞模型,已经证实mTOR／S6K1参与多倍体细胞周期的调控。本课题主要研究roTOR下游的另一个重要信号分子4E-BP1是否也参与细胞的倍体化调控。方法：诺考达唑诱导Dami细胞建立相对同步化的多倍体细胞模型,Western-blot分析多倍体细胞模型中mTOR／4E—BP1通路信号分子表达和磷酸化修饰位点的变化,流式细胞仪双荧光分析4E—BP1不同结构域磷酸化位点修饰与细胞周期各时相的关系。结果：诺考达唑诱导的Dami细胞可作为相对同步化的多倍体细胞周期模型,在二倍体和多倍体细胞周期中,mTOR表达增加及第2448位丝氨酸位点磷酸化发生在G1期进入S期,4E—BP1的第37,46位苏氨酸和第65位丝氨酸位点磷酸化发生在G2／M期。结论：mTOR／4E-BP1通路参与多倍体细胞周期的调控。     

mTOR/4E-BP1参与诺考达唑诱导的Dami细胞多倍体化调控

目的:多倍性是物种形成的重要机制,决定一些重要器官细胞产生的数量和功能,而且与某些病理过程(如恶性肿瘤)的发生有密切关系.我们通过建立相对同步化的多倍体细胞模型,已经证实mTOR/S6K1参与多倍体细胞周期的调控.本课题主要研究mTOR下游的另一个重要信号分子4E-BP1是否也参与细胞的倍体化调控.方法:诺考达唑诱导Dami细胞建立相对同步化的多倍体细胞模型,Western-blot分析多倍体细胞模型中mTOR/4E-BP1通路信号分子表达和磷酸化修饰位点的变化,流式细胞仪双荧光分析4E-BP1不同结构域磷酸化位点修饰与细胞周期各时相的关系.结果:诺考达唑诱导的Dami细胞可作为相对同步化的多倍体细胞周期模型,在二倍体和多倍体细胞周期中,mTOR表达增加及第2448位丝氨酸位点磷酸化发生在G1期进入S期,4E-BP1的第37,46位苏氨酸和第65位丝氨酸位点磷酸化发生在G2/M期.结论:mTOR/4E-BP1通路参与多倍体细胞周期的调控.     

应用RT-PCR制备登革病毒诊断基因芯片探针

根据GenBank数据库中的生物信息,利用BLAST免费分析软件找出4种型别登革病毒的保守序列及各型特异性序列,针对上述序列设计引物经RT-PCR扩增登革病毒的特异片段,利用此RT-PCR法收集探针是一种快速、简便制备基因芯片探针的实用方法。     

Vimentin Is Involved in Peptidylarginine Deiminase 2-Induced Apoptosis of Activated Jurkat Cells

Peptidylarginine deiminase type 2 (PADI2) deiminates (or citrullinates) arginine residues in protein to citrulline residues in a Ca²⁺-dependent manner, and is found in lymphocytes and macrophages. Vimentin is an intermediate filament protein and a well-known substrate of PADI2. Citrullinated vimentin is found in ionomycin-induced macrophage apoptosis. Citrullinated vimentin is the target of anti-Sa antibodies, which are specific to rheumatoid arthritis, and play a critical role in the pathogenesis of the disease. To investigate the role of PADI2 in apoptosis, we generated a Jurkat cell line that overexpressed the PADI2 transgene from a tetracycline-inducible promoter, and used a combination of 12-O-tetradecanoylphorbol-13-acetate and ionomycin to activate Jurkat cells. We found that PADI2 overexpression reduced the cell viability of activated Jurkat cells in a dose- and time-dependent manner. The PADI2-overexpressed and -activated Jurkat cells presented typical manifestations of apoptosis, and exhibited greater levels of citrullinated proteins, including citrullinated vimentin. Vimentin overexpression rescued a portion of the cells from apoptosis. In conclusion, PADI2 overexpression induces apoptosis in activated Jurkat cells. Vimentin is involved in PADI2-induced apoptosis. Moreover, PADI2-overexpressed Jurkat cells secreted greater levels of vimentin after activation, and expressed more vimentin on their cell surfaces when undergoing apoptosis. Through artificially highlighting PADI2 and vimentin, we demonstrated that PADI2 and vimentin participate in the apoptotic mechanisms of activated T lymphocytes. The secretion and surface expression of vimentin are possible ways of autoantigen presentation to the immune system.     

Homologous Interference Induced by Sindbis Virus

Homologous interference during Sindbis virus infection has been investigated. Prior infection of either chicken embryo fibroblast or BHK(21) cell cultures results in reduced yields of progeny virions of the superinfecting genotype. This reduction in yield results from a reduction in the number of cells in the cultures capable of producing the superinfecting genotype. The development of interference parallels the attachment kinetics of Sindbis virus. Interference requires an active viral genome since the activity is sensitive to inactivation by ultraviolet light, and an RNA(-) mutant, ts-24, fails to induce interference under nonpermissive conditions. However, ts-6, an RNA(-) mutant belonging to a different complementation group, and the RNA(+) mutants, ts-2 and ts-20, interfere at both permissive and nonpermissive temperatures.     

Phorbol Myristate Acetate and 8-Bromo-Cyclic AMP-Induced Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin in Astrocytes: Comparison of Phosphorylation Sites

Both the protein kinase C (PK-C) activator, phorbol 12-myristate 13-acetate (PMA), and the cyclic AMP-dependent protein kinase (PK-A) activator, 8-bromo-cyclic AMP (8-BR), have been shown to increase 32P incorporation into glial fibrillary acidic protein (GFAP) and vimentin in cultured astrocytes. Also, treatment of astrocytes with PMA or 8-BR results in the morphological transformation of flat, polygonal-shaped cells into stellate, process-bearing cells, suggesting the possibility that signals mediated by these two kinase systems converge at the level of protein phosphorylation to elicit similar changes in cell morphology. Therefore, studies were conducted to determine whether treatment with PMA and 8-BR results in the phosphorylation of the same tryptic peptide fragments on GFAP and vimentin in astrocytes. Treatment with PMA increased 32P incorporation into all the peptide fragments that were phosphorylated by 8-BR on both vimentin and GFAP; however, PMA also stimulated phosphorylation of additional fragments of both proteins. The phosphorylation of vimentin and GFAP resulting from PMA or 8-BR treatment was restricted to serine residues in the N-terminal domain of these proteins. Studies were also conducted to compare the two-dimensional tryptic phosphopeptide maps of GFAP and vimentin from intact cells treated with PMA and 8-BR with those produced when the proteins were phosphorylated with purified PK-C or PK-A. PK-C phosphorylated the same fragments of GFAP and vimentin that were phosphorylated by PMA treatment. Additionally, PK-C phosphorylated some tryptic peptide fragments of these proteins that were not observed with PMA treatment in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca~(2 )-Calmodulin is Involved in Betacyanin Accumulation Induced by Dark in C_3 Halophyte Suaeda salsa

The C_3 halophyte Suaeda salsa was used to investigate the roles of Ca~(2 ),Ca~(2 )channels,and calmodulin(CAM)in betacyaninmetabolism.Seeds of S.salsa were cultured in both the dark and light for 3 days.The fresh weight and betacyanin contentwere much higher in S.salsa seedlings formed in the dark than in seedlings formed in the light.The addition of Ca~(2 )tothe half-strength MS nutrient solution promoted betacyanin accumulation in the dark,whereas Ca~(2 )depletion by EGTAsuppressed the dark-induced betacyanin accumulation in shoots of S.salsa.The Ca~(2 )channel blocker LaCl_3 also inhibiteddark-induced betacyanin accumulation.The highest activity of CaM and the maximum betacyanin content decreased by51% and 45%,respectively,in shoots of S.salsa seedlings treated with the potent CaM antagonist chlorpromazine in thedark.Furthermore,the other CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide(W-7)also inhibited theactivity of CaM and dark-dependent betacyanin accumulation,whereas its less active structural analog N-(6-aminohexyl)-1-naphthalenesulfonamide(W-5)had little effect on the responses to dark of S.salsa seedlings.These results suggest thatCa~(2 ),Ca~(2 )-regulated ion channels,and CaM play an important role in dark-induced betacyanin accumulation in the shootsof the C_3 halophyte S.salsa.