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1.  Biology of IL-27 and its Role in the Host Immunity against Mycobacterium Tuberculosis 
IL-27, a heterodimeric cytokine of IL-12 family, regulates both innate and adaptive immunity largely via Jak-Stat signaling. IL-27 can induce IFN-γ and inflammatory mediators from T lymphocytes and innate immune cells. IL-27 has unique anti-inflammatory properties via both Tr1 cells dependent and independent mechanisms. Here the role and biology of IL-27 in innate and adaptive immunity are summarized, with special interest with immunity against Mycobacterium tuberculosis.
PMCID: PMC4279092  PMID: 25561899
IL-27; IL-27Rα; IL-12 family; Mycobacterium tuberculosis; Immunity
2.  Trans-translation mediates tolerance to multiple antibiotics and stresses in Escherichia coli 
Journal of Antimicrobial Chemotherapy  2013;68(11):2477-2481.
Trans-translation mediated by SsrA (tmRNA) and its associated protein SmpB plays an important role in rescuing stalled ribosomes and detoxifying toxic protein products under stress conditions. However, the role of SsrA and SmpB in bacterial persister survival has not been studied. The recent finding that pyrazinamide as a unique persister drug inhibits trans-translation in Mycobacterium tuberculosis prompted us to examine the role of trans-translation in persister survival.
Using Escherichia coli as a model, we constructed SsrA and SmpB mutants and assessed the susceptibility of the mutants to various antibiotics and stress conditions in MIC/MBC and persister assays.
We found that mutations in SsrA and SmpB caused a defect in persister survival as shown by their increased susceptibility to a variety of antibiotics, including gentamicin, streptomycin, amikacin, norfloxacin, trimethoprim and tetracycline, and also stresses, such as acid, weak acid salicylate, heat and peroxide. Additionally, the SsrA and SmpB mutants were 2–8-fold more susceptible than the parent strain to various antibiotics in MIC and MBC tests. The SmpB mutant was more susceptible to antibiotics and stresses than the SsrA mutant. A particularly interesting finding is the hypersusceptibility of the SmpB mutant and the SsrA mutant to trimethoprim. The defect of various SsrA and SmpB mutant phenotypes could be complemented by functional ssrA and smpB, respectively.
We conclude that SsrA and SmpB are important for persister survival and may serve as a good target for developing new antibiotics that kill persister bacteria for improved treatment of persistent bacterial infections.
PMCID: PMC3797643  PMID: 23812681
persisters; persister mechanisms; persistence; drug tolerance; drug target
3.  Cryptococcus inositol utilization modulates the host protective immune response during brain infection 
Cryptococcus neoformans is the most common cause of fungal meningitis among individuals with HIV/AIDS, which is uniformly fatal without proper treatment. The underlying mechanism of disease development in the brain that leads to cryptococcal meningoencephalitis remains incompletely understood. We have previously demonstrated that inositol transporters (ITR) are required for Cryptococcus virulence. The itr1aΔ itr3cΔ double mutant of C. neoformans was attenuated for virulence in a murine model of intra-cerebral infection; demonstrating that Itr1a and Itr3c are required for full virulence during brain infection, despite a similar growth rate between the mutant and wild type strains in the infected brain.
To understand the immune pathology associated with infection by the itr1aΔ itr3cΔ double mutant, we investigated the molecular correlates of host immune response during mouse brain infection. We used genome-wide transcriptome shotgun sequencing (RNA-Seq) and quantitative real-time PCR (qRT-PCR) methods to examine the host gene expression profile in the infected brain. Our results show that compared to the wild type, infection of mouse brains by the mutant leads to significant activation of cellular networks/pathways associated with host protective immunity. Most of the significantly differentially expressed genes (SDEG) are part of immune cell networks such as tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) regulon, indicating that infection by the mutant mounts a stronger host immune response compared to the wild type. Interestingly, a significant reduction in glucuronoxylomannan (GXM) secretion was observed in the itr1aΔ itr3cΔ mutant cells, indicating that inositol utilization pathways play a role in capsule production.
Since capsule has been shown to impact the host response during Cryptococcus-host interactions, our results suggest that the reduced GXM production may contribute to the increased immune activation in the mutant-infected animals.
Electronic supplementary material
The online version of this article (doi:10.1186/s12964-014-0051-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4172957  PMID: 25201772
Cryptococcus neoformans; Cryptococcal meningoencephalitis; Host immune response; Glucuronoxylomannan; Capsule production; Inositol transporters; Genome-wide transcriptome; Quantitative real-time PCR; Cellular networks; Immune pathways
4.  Mycobacterium tuberculosis PE_PGRS17 Promotes the Death of Host Cell and Cytokines Secretion via Erk Kinase Accompanying with Enhanced Survival of Recombinant Mycobacterium smegmatis 
Tuberculosis (TB) remains a serious threat to global public health, largely due to the successful manipulation of the host immunity by its etiological agent Mycobacterium tuberculosis. The PE_PGRS protein family of M. tuberculosis might be a contributing factor. To investigate the roles of PE_PGRS17, the gene of PE_PGRS 17 was expressed in nonpathogenic fast growing Mycobacterium smegmatis. We found that the recombinant strain survives better than the control in macrophage cultures, accompanied by more host cell death and a marked higher secretion of tumor necrosis factor-alpha by a recombinant strain compared with control. Blocking the action of Erk kinase by an inhibitor can abolish the above effects. In brief, our data showed that PE_PGRS 17 might facilitate pathogen survival and disserve the host cell via remodeling the macrophages immune niche largely consisting of inflammatory cytokines. This furnishes a novel insight into the immune role of this mycobacterium unique gene family.
PMCID: PMC3741429  PMID: 23663047
5.  Mycobacterium tuberculosis Rv3402c Enhances Mycobacterial Survival within Macrophages and Modulates the Host Pro-Inflammatory Cytokines Production via NF-Kappa B/ERK/p38 Signaling 
PLoS ONE  2014;9(4):e94418.
Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis, a process which depends on an array of virulence factors to colonize and replicate within the host. The M. tuberculosis iron regulated open reading frame (ORF) rv3402c, encoding a conserved hypothetical protein, was shown to be up-regulated upon infection in both human and mice macrophages. To explore the function of this ORF, we heterologously expressed the rv3402c gene in the non-pathogenic fast-growing Mycobacterium smegmatis strain, and demonstrated that Rv3402c, a cell envelope-associated protein, was able to enhance the intracellular survival of recombinant M. smegmatis. Enhanced growth was not found to be the result of an increased resistance to intracellular stresses, as growth of the Rv3402c expressing strain was unaffected by iron depletion, H2O2 exposure, or acidic conditions. Colonization of macrophages by M. smegmatis expressing Rv3402c was associated with substantial cell death and significantly greater amount of TNF-α and IL-1β compared with controls. Rv3402c-induced TNF-α and IL-1β production was found to be mediated by NF-κB, ERK and p38 pathway in macrophages. In summary, our study suggests that Rv3402c delivered in a live M. smegmatis vehicle can modify the cytokines profile of macrophage, promote host cell death and enhance the persistence of mycobacterium within host cells.
PMCID: PMC3983203  PMID: 24722253
6.  Prophage-like elements present in Mycobacterium genomes 
BMC Genomics  2014;15:243.
Prophages, integral components of many bacterial genomes, play significant roles in cognate host bacteria, such as virulence, toxin biosynthesis and secretion, fitness cost, genomic variations, and evolution. Many prophages and prophage-like elements present in sequenced bacterial genomes, such as Bifidobacteria, Lactococcus and Streptococcus, have been described. However, information for the prophage of Mycobacterium remains poorly defined.
In this study, based on the search of the complete genome database from GenBank, the Whole Genome Shotgun (WGS) databases, and some published literatures, thirty-three prophages were described in detail. Eleven of them were full-length prophages, and others were prophage-like elements. Eleven prophages were firstly revealed. They were phiMAV_1, phiMAV_2, phiMmcs_1, phiMmcs_2, phiMkms_1, phiMkms_2, phiBN42_1, phiBN44_1, phiMCAN_1, phiMycsm_1, and phiW7S_1. Their genomes and gene contents were firstly analyzed. Furthermore, comparative genomics analyses among mycobacterioprophages showed that full-length prophage phi172_2 belonged to mycobacteriophage Cluster A and the phiMmcs_1, phiMkms_1, phiBN44_1, and phiMCAN_1 shared high homology and could be classified into one group.
To our knowledge, this is the first systematic characterization of mycobacterioprophages, their genomic organization and phylogeny. This information will afford more understanding of the biology of Mycobacterium.
PMCID: PMC3986857  PMID: 24673856
Prophage; Mycobacterioprophage; Phylogeny; Comparative genomics
7.  Stellated Ag-Pt bimetallic nanoparticles: An effective platform for catalytic activity tuning 
Scientific Reports  2014;4:3969.
The usefulness of Pt-based nanomaterials for catalysis can be greatly enhanced by coupling morphology engineering to the strategic presence of a second or even third metal. Here we demonstrate the design and preparation of stellated Ag-Pt bimetallic nanoparticles where significant activity difference between the methanol oxidation reaction (MOR) and the oxygen reduction reaction (ORR) may be realized by relegating Ag to the core or by hollowing out the core. In particular the stellated Pt surface, with an abundance of steps, edges, corner atoms, and {111} facets, is highly effective for the ORR but is ineffective for MOR. MOR activity is only observed in the presence of a Ag core through electronic coupling to the stellated Pt shell. The bimetallic Ag-Pt stellates therefore demonstrate the feasibility of tuning a Pt surface for two very different structure sensitive catalytic reactions. Stellated bimetallics may therefore be an effective platform for highly tunable catalyst designs.
PMCID: PMC3913913  PMID: 24495979
8.  Assembly of Nanoions via Electrostatic Interactions: Ion-Like Behavior of Charged Noble Metal Nanoclusters 
Scientific Reports  2014;4:3848.
The assembly of ultrasmall metal nanoclusters (NCs) is of interest to both basic and applied research as it facilitates the determination of cluster structures and the customization of cluster physicochemical properties. Here we present a facile and general approach to assemble noble metal NCs by selectively inducing electrostatic interactions between negatively-charged metal NCs and divalent cations. The charged metal NCs, which have well-defined sizes, charges and structures; and behave similarly to multivalent anions, can be considered as nanoions. These nanoions exhibit step-like assembly behavior when interacting with the counter cations – assembly only occurs when the solubility product (Ksp) between the carboxylate ions on the NC surface and the divalent cations is exceeded. The assembly here is distinctively different from the random aggregation of colloidal particles by counter ions. The nanoions would assemble into fractal-like monodisperse spherical particles with a high order of regularity that mimic the assembly of ionic crystals.
PMCID: PMC3900922  PMID: 24457992
9.  Hierarchically Structured Co3O4@Pt@MnO2 Nanowire Arrays for High-Performance Supercapacitors 
Scientific Reports  2013;3:2978.
Here we proposed a novel architectural design of a ternary MnO2-based electrode – a hierarchical Co3O4@Pt@MnO2 core-shell-shell structure, where the complemental features of the three key components (a well-defined Co3O4 nanowire array on the conductive Ti substrate, an ultrathin layer of small Pt nanoparticles, and a thin layer of MnO2 nanoflakes) are strategically combined into a single entity to synergize and construct a high-performance electrode for supercapacitors. Owing to the high conductivity of the well-defined Co3O4 nanowire arrays, in which the conductivity was further enhanced by a thin metal (Pt) coating layer, in combination with the large surface area provided by the small MnO2 nanoflakes, the as-fabricated Co3O4@Pt@MnO2 nanowire arrays have exhibited high specific capacitances, good rate capability, and excellent cycling stability. The architectural design demonstrated in this study provides a new approach to fabricate high-performance MnO2–based nanowire arrays for constructing next-generation supercapacitors.
PMCID: PMC3797991  PMID: 24132040
10.  Nutlin-3 induces apoptosis, disrupts viral latency and inhibits expression of angiopoietin-2 in Kaposi sarcoma tumor cells 
Cell Cycle  2012;11(7):1393-1399.
Kaposi sarcoma (KS) tumors often contain a wild-type p53. However, the function of this tumor suppressor in KS tumor cells is inhibited by both MDM2 and latent nuclear antigen (LANA) of Kaposi sarcoma-associated herpes virus (KSHV). Here, we report that MDM2 antagonist Nutlin-3 efficiently reactivates p53 in telomerase-immortalized human umbilical vein endothelial cells (TIVE) that had been malignantly transformed by KSHV as well as in KS tumor cells. Reactivation of p53 results in a G1 cell cycle arrest, leading to inhibition of proliferation and apoptosis. Nutlin-3 inhibits the growth of “KS-like” tumors resulting from xenografted TIVE-KSHV cells in nude mice. In addition, Nutlin-3 strongly inhibits expression of the pro-angiogenic and pro-inflammatory cytokine angiopoietin-2 (Ang-2). It also disrupts viral latency by inducing expression of KSHV lytic genes. these results suggest that Nutlin-3 might serve as a novel therapy for KS.
PMCID: PMC3350880  PMID: 22421142
Kaposi sarcoma (KS); nutlin-3; p53; cell cycle arrest; apoptosis; angiopoietin-2
11.  Biology of a Novel Mycobacteriophage, SWU1, Isolated from Chinese Soil as Revealed by Genomic Characteristics 
Journal of Virology  2012;86(18):10230-10231.
Mycobacteriophage SWU1 is a newly isolated phage from a soil sample collected at Gongping village, Pingchang County, Sichuan Province, China, using Mycobacterium smegmatis mc2155 as a host. Plaques of SWU1 appear as a unique bull's-eye on an M. smegmatis lawn. In this paper, we report the complete genome sequence of SWU1 and some major findings from the analysis result.
PMCID: PMC3446572  PMID: 22923793
12.  Transcriptional analysis of the effect of exogenous decanoic acid stress on Streptomyces roseosporus 
Daptomycin is an important antibiotic against infections caused by drug-resistant pathogens. Its production critically depends on the addition of decanoic acid during fermentation. Unfortunately, decanoic acid (>2.5 mM) is toxic to daptomycin producer, Streptomyces roseosporus.
To understand the mechanism underlying decanoic tolerance or toxicity, the responses of S. roseosporus was determined by a combination of phospholipid fatty acid analysis, reactive oxygen species (ROS) measurement and RNA sequencing. Assays using fluorescent dyes indicated a sharp increase in reactive oxygen species during decanoic acid stress; fatty acid analysis revealed a marked increase in the composition of branched-chain fatty acids by approximately 10%, with a corresponding decrease in straight-chain fatty acids; functional analysis indicated decanoic acid stress has components common to other stress response, including perturbation of respiratory functions (nuo and cyd operons), oxidative stress, and heat shock. Interestingly, our transcriptomic analysis revealed that genes coding for components of proteasome and related to treholase synthesis were up-regulated in the decanoic acid –treated cells.
These findings represent an important first step in understanding mechanism of decanoic acid toxicity and provide a basis for engineering microbial tolerance.
PMCID: PMC3724488  PMID: 23432849
Daptomycin; Decanoic acid; Toxic; Tolerance; Streptomyces roseosporus
14.  Reproducibility and quantitation of amplicon sequencing-based detection 
The ISME Journal  2011;5(8):1303-1313.
To determine the reproducibility and quantitation of the amplicon sequencing-based detection approach for analyzing microbial community structure, a total of 24 microbial communities from a long-term global change experimental site were examined. Genomic DNA obtained from each community was used to amplify 16S rRNA genes with two or three barcode tags as technical replicates in the presence of a small quantity (0.1% wt/wt) of genomic DNA from Shewanella oneidensis MR-1 as the control. The technical reproducibility of the amplicon sequencing-based detection approach is quite low, with an average operational taxonomic unit (OTU) overlap of 17.2%±2.3% between two technical replicates, and 8.2%±2.3% among three technical replicates, which is most likely due to problems associated with random sampling processes. Such variations in technical replicates could have substantial effects on estimating β-diversity but less on α-diversity. A high variation was also observed in the control across different samples (for example, 66.7-fold for the forward primer), suggesting that the amplicon sequencing-based detection approach could not be quantitative. In addition, various strategies were examined to improve the comparability of amplicon sequencing data, such as increasing biological replicates, and removing singleton sequences and less-representative OTUs across biological replicates. Finally, as expected, various statistical analyses with preprocessed experimental data revealed clear differences in the composition and structure of microbial communities between warming and non-warming, or between clipping and non-clipping. Taken together, these results suggest that amplicon sequencing-based detection is useful in analyzing microbial community structure even though it is not reproducible and quantitative. However, great caution should be taken in experimental design and data interpretation when the amplicon sequencing-based detection approach is used for quantitative analysis of the β-diversity of microbial communities.
PMCID: PMC3146266  PMID: 21346791
metagenomics; amplicon sequencing; pyrosequencing; reproducibility; quantitation; random sampling
15.  Progress on RNAi-based molecular medicines 
RNA interference (RNAi) is a promising strategy to suppress the expression of disease-relevant genes and induce post-transcriptional gene silencing. Their simplicity and stability endow RNAi with great advantages in molecular medicine. Several RNAi-based drugs are in various stages of clinical investigation. This review summarizes the ongoing research endeavors on RNAi in molecular medicine, delivery systems for RNAi-based drugs, and a compendium of RNAi drugs in different stages of clinical development. Of special interest are RNAi-based drug target discovery and validation, delivery systems for RNAi-based drugs, such as nanoparticles, rabies virus protein-based vehicles, and bacteriophages for RNA packaging.
PMCID: PMC3418169  PMID: 22915846
RNA interference; delivery systems; liposome; nanoparticle; molecular medicines
16.  GeoChip-Based Analysis of the Functional Gene Diversity and Metabolic Potential of Microbial Communities in Acid Mine Drainage▿ †  
Acid mine drainage (AMD) is an extreme environment, usually with low pH and high concentrations of metals. Although the phylogenetic diversity of AMD microbial communities has been examined extensively, little is known about their functional gene diversity and metabolic potential. In this study, a comprehensive functional gene array (GeoChip 2.0) was used to analyze the functional diversity, composition, structure, and metabolic potential of AMD microbial communities from three copper mines in China. GeoChip data indicated that these microbial communities were functionally diverse as measured by the number of genes detected, gene overlapping, unique genes, and various diversity indices. Almost all key functional gene categories targeted by GeoChip 2.0 were detected in the AMD microbial communities, including carbon fixation, carbon degradation, methane generation, nitrogen fixation, nitrification, denitrification, ammonification, nitrogen reduction, sulfur metabolism, metal resistance, and organic contaminant degradation, which suggested that the functional gene diversity was higher than was previously thought. Mantel test results indicated that AMD microbial communities are shaped largely by surrounding environmental factors (e.g., S, Mg, and Cu). Functional genes (e.g., narG and norB) and several key functional processes (e.g., methane generation, ammonification, denitrification, sulfite reduction, and organic contaminant degradation) were significantly (P < 0.10) correlated with environmental variables. This study presents an overview of functional gene diversity and the structure of AMD microbial communities and also provides insights into our understanding of metabolic potential in AMD ecosystems.
PMCID: PMC3028740  PMID: 21097602
17.  Thiamin (Vitamin B1) Biosynthesis and Regulation: A Rich Source of Antimicrobial Drug Targets? 
Drug resistance of pathogens has necessitated the identification of novel targets for antibiotics. Thiamin (vitamin B1) is an essential cofactor for all organisms in its active form thiamin diphosphate (ThDP). Therefore, its metabolic pathways might be one largely untapped source of antibiotics targets. This review describes bacterial thiamin biosynthetic, salvage, and transport pathways. Essential thiamin synthetic enzymes such as Dxs and ThiE are proposed as promising drug targets. The regulation mechanism of thiamin biosynthesis by ThDP riboswitch is also discussed. As drug targets of existing antimicrobial compound pyrithiamin, the ThDP riboswitch might serves as alternative targets for more antibiotics.
PMCID: PMC3020362  PMID: 21234302
thiamin biosynthesis; riboswitch; Mycobacterium tuberculosis; drug targets
18.  Identification of Mannich Base as a Novel Inhibitor of Mycobacterium Tuberculosis Isocitrate by High-Throughput Screening 
Mycobacterium tuberculosis (MTB) remains one of the most significant human pathogens since its discovery in 1882. An estimated 1.5 million people died from tubercle bacillus (TB) in 2006, and globally, there were an estimated 9.27 million incident cases of TB in 2007. Glyoxylate bypass pathway occurs in a wide range of pathogens and plays a key role in the pathogenesis of Mycobacterium tuberculosis. Isocitrate lyase (ICL) can catalyses the first step of this pathway, and reversibly cleaves isocitrate into succinate and glyoxylate. So, ICL may represent a good drug target for the treatment of tuberculosis. ICL was cloned, expressed, and purified, and a high-throughput screen (HTS) developed to screen active molecule from a mannich base compounds library for inhibition of ICL. This assay had signal to noise (S/N) of 650.6990 and Z' factor of 0.8141, indicating that the assay was suitable for HTS. Screening of a collection of 124 mannich base compounds resulted in the identification of one mannich base compound, which has a significant inhibitory activity. So, a new family of compound was first reported to inhibit the activity of Mycobacterium tuberculosis ICL. This family of compound might offer new avenue to explore better anti-tuberculosis and fungi drugs.
PMCID: PMC3076504  PMID: 21494431
Isocitrate Lyase; Mycobacterium tuberculosis; Drug Target; High-Throughput Screening; mannich bases.
19.  Regulation of NF-κB inhibitor IκBα and viral replication by a KSHV microRNA 
Nature cell biology  2010;12(2):193-199.
Kaposi’s sarcoma-associated herpesvirus (KSHV) is causally linked to several acquired immune deficiency syndrome related malignancies including Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and a subset of multicentric Castleman’s disease1. Control of viral lytic replication is essential for KSHV latency, evasion of host immune system, and induction of tumors1. Here, we show that deletion of a cluster of 14 microRNAs (miRs) from KSHV genome significantly enhances viral lytic replication as a result of reduced NF-κB activity. The miR cluster regulates NF-κB pathway by reducing the expression of IκBα protein, an inhibitor of the NF-κB complexes. Computational and miR seed mutagenesis analyses identify KSHV miR-K1 that directly mediates IκBα ?protein level by targeting the 3’UTR of its transcript. Expression of miR-K1 is sufficient to rescue the NF-κB activity and inhibit viral lytic replication while inhibition of miR-K1 in KSHV-infected PEL cells has the opposite effects. Thus, KSHV encodes a miR to control viral replication by activating NF-κB pathway. These results illustrate an important role for KSHV miRs in regulating viral latency and lytic replication by manipulating a host survival pathway.
PMCID: PMC2815189  PMID: 20081837
KSHV; microRNA; viral latency and replication; NF-κB; reverse genetics
20.  Genome-wide identification of binding sites for Kaposi’s sarcoma-associated herpesvirus lytic switch protein, RTA 
Virology  2009;386(2):290-302.
Kaposi’s sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) encoded by ORF50 is a lytic switch protein for viral reactivation from latency. The expression of RTA activates the expression of downstream viral genes, and is necessary for triggering the full viral lytic program. Using chromatin immunoprecipitation assay coupled with a KSHV whole-genome tiling microarray (ChIP-on-chip) approach, we identified a set of 19 RTA binding sites in the KSHV genome in a KSHV-infected cell line BCBL-1. These binding sites are located in the regions of promoters, introns, or exons of KSHV genes including ORF8, ORFK4.1, ORFK5, PAN, ORF16, ORF29, ORF45, ORF50, ORFK8, ORFK10.1, ORF59, ORFK12, ORF71/72, ORFK14/ORF74, and ORFK15, the two origins of lytic replication OriLyt-L and OriLyt-R, and the microRNA cluster. We confirmed these RTA binding sites by ChIP and quantitative real-time PCR. We further mapped the RTA binding site in the first intron of the ORFK15 gene, and determined that it is RTA-responsive. The ORFK15 RTA binding sequence TTCCAGGAA TTCCTGGAA consists of a palindromic structure of two tandem repeats, of which each itself is also an imperfect inverted repeat. Reporter assay and electrophoretic mobility shift assay confirmed the binding of the RTA protein to this sequence in vitro. Sequence alignment with other RTA binding sites identified the RTA consensus binding motif as TTCCAGGAT(N)0–16TTCCTGGGA. Interestingly, most of the identified RTA binding sites contain only half or part of this RTA binding motif. These results suggest the complexity of RTA binding in vivo, and the involvements of other cellular or viral transcription factors during RTA transactivation of target genes.
PMCID: PMC2663009  PMID: 19233445
21.  Reactivation of Kaposi's sarcoma-associated herpesvirus from latency requires MEK/ERK, JNK and p38 multiple mitogen-activated protein kinase pathways 
Virology  2007;371(1):139-154.
Lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) promotes the progression of Kaposi's sarcoma (KS), a dominant malignancy in patients with AIDS. While 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced KSHV reactivation from latency is mediated by the protein kinase C δ and MEK/ERK mitogen-activated protein kinase (MAPK) pathways, we have recently shown that the MEK/ERK, JNK and p38 MAPK pathways modulate KSHV lytic replication during productive primary infection of human umbilical vein endothelial cells (Pan, H.Y., Xie, J.P., Ye, F.C., Gao, S.-J., 2006. J. Virol. 80, 5371-82). Here, we report that, besides the MEK/ERK pathway, the JNK and p38 MAPK pathways also mediate TPA-induced KSHV reactivation from latency. The MEK/ERK, JNK and p38 MAPK pathways were constitutively activated in latent KSHV-infected BCBL-1 cells. TPA treatment enhanced the levels of activated ERK and p38 but not those of activated JNK. Inhibitors of all three MAPK pathways reduced TPA-induced production of KSHV infectious virions in BCBL-1 cells in a dose-dependent fashion. The inhibitors blocked KSHV lytic replication at the early stage(s) of reactivation, and reduced the expression of viral lytic genes including RTA, a key immediate-early transactivator of viral lytic replication. Activation of MAPK pathways was necessary and sufficient for activating the promoter of RTA. Furthermore, we showed that the activation of RTA promoter by MAPK pathways was mediated by their downstream target AP-1. Together, these findings suggest that MAPK pathways might have general roles in regulating the life cycle of KSHV by mediating both viral infection and switch from viral latency to lytic replication.
PMCID: PMC2239004  PMID: 17964626
Kaposi's sarcoma; KSHV; reactivation; MAPK pathways; AP-1; RTA; TPA
22.  Kaposi's Sarcoma-Associated Herpesvirus Infection Promotes Invasion of Primary Human Umbilical Vein Endothelial Cells by Inducing Matrix Metalloproteinases▿  
Journal of Virology  2007;81(13):7001-7010.
Matrix metalloproteinases (MMPs) play important roles in cancer invasion, angiogenesis, and inflammatory infiltration. Kaposi's sarcoma is a highly disseminated angiogenic tumor of proliferative endothelial cells linked to infection by Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we showed that KSHV infection increased the invasiveness of primary human umbilical vein endothelial cells (HUVEC) in a Matrigel-based cell invasion assay. KSHV-induced cell invasion was abolished by an inhibitor of MMPs, BB-94, and occurred in both autocrine- and paracrine-dependent fashions. Analysis by zymography and Western blotting showed that KSHV-infected HUVEC cultures had increased secretion of MMP-1, -2, and -9. KSHV increased the secretion of MMP-2 within 1 h following infection without upregulating its mRNA expression level. In contrast, the secretion of MMP-1 and -9 was not increased until 6 h after KSHV infection and was correlated with the upregulation of their mRNA expression levels. Promoter analysis by reporter assays and electrophoretic mobility shift assays identified an AP-1 cis-element as the dominant KSHV-responsive site in the MMP-1 promoter. Together, these results suggest that KSHV infection modulates the production of multiple MMPs to increase cell invasiveness and thus contributes to the pathogenesis of KSHV-induced malignancies.
PMCID: PMC1933284  PMID: 17442715
23.  Modulation of Kaposi's Sarcoma-Associated Herpesvirus Infection and Replication by MEK/ERK, JNK, and p38 Multiple Mitogen-Activated Protein Kinase Pathways during Primary Infection 
Journal of Virology  2006;80(11):5371-5382.
Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma, a dominant AIDS-related tumor of endothelial cells, and several other lymphoproliferative malignancies. While activation of the phosphatidylinositol 3-kinase-protein kinase C-MEK-ERK pathway is essential for KSHV infection, we have recently shown that KSHV also activates JNK and p38 mitogen-activated protein kinase (MAPK) pathways during primary infection (J. Xie, H. Y. Pan, S. Yoo, and S.-J. Gao, J. Virol. 79:15027-15037, 2005). Here, we found that activation of both JNK and p38 pathways was also essential for KSHV infection. Inhibitors of all three MAPK pathways reduced KSHV infectivity in both human umbilical vein endothelial cells (HUVEC) and 293 cells. These inhibitory effects were dose dependent and occurred at the virus entry stage of infection. Consistently, inhibition of all three MAPK pathways with dominant-negative constructs reduced KSHV infectivity whereas activation of the ERK pathway but not the JNK and p38 pathways enhanced KSHV infectivity. Importantly, inhibition of all three MAPK pathways also reduced the yield of infectious virions during KSHV productive infection of HUVEC. While the reduction of infectious virions was in part due to the reduced infectivity, it was also the result of direct modulation of KSHV lytic replication by the MAPK pathways. Accordingly, KSHV upregulated the expression of RTA (Orf50), a master transactivator of KSHV lytic replication, and activated its promoter during primary infection. Furthermore, KSHV activation of RTA promoter during primary infection was modulated by all three MAPK pathways, predominantly through their downstream target AP-1. Together, these results indicate that, by modulating multiple MAPK pathways, KSHV manipulates the host cells to facilitate its entry into the cells and postentry productive lytic replication during primary infection.
PMCID: PMC1472170  PMID: 16699017
24.  Kaposi's Sarcoma-Associated Herpesvirus Induction of AP-1 and Interleukin 6 during Primary Infection Mediated by Multiple Mitogen-Activated Protein Kinase Pathways 
Journal of Virology  2005;79(24):15027-15037.
Kaposi's sarcoma is an angioproliferative disseminated tumor of endothelial cells linked to infection with Kaposi's sarcoma-associated herpesvirus (KSHV). AP-1 transcription factors are involved in diverse biological processes, including infection and replication of viruses, cell growth, oncogenesis, angiogenesis, and invasion of cancer cells. Here we show that KSHV activates AP-1 during primary infection. The activation of AP-1 at the early stage of KSHV infection is mainly mediated by virus entry events. Concurrently, KSHV infection strongly activates MEK, JNK, and to a lesser extent, p38 mitogen-activated protein kinase (MAPK) pathways. Specific inhibitors or dominant negative constructs of MEK and JNK completely abolish AP-1 activation by KSHV, while those of p38 reduce it by half. Furthermore, individual MAPK pathways differentially regulate KSHV activation of AP-1 components. KSHV activation of AP-1 leads to the transcriptional induction of interleukin 6 (IL-6), which is inhibited by inhibitors or dominant negative constructs of MAPK pathways. Together, these results demonstrate that KSHV induces AP-1 and IL-6 during primary infection by modulating multiple MAPK pathways. Because of the diverse roles of IL-6, AP-1, and MAPK pathways in viral infection and tumor induction and promotion, these results have important implications in the pathogenesis of KSHV-induced malignancies.
PMCID: PMC1316010  PMID: 16306573

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