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Tip110 plays important roles for stem cell pluripotency and hematopoiesis. However, little is known about the regulatory mechanisms of Tip110 expression in this process. In this study, we first sho...

Tip110 expression regulates hematopoiesis, but the regulatory mechanisms during hematopoiesis are not fully understood. There are a number of putative microRNA (miRNA) binding sites identified within the Tip110 3' untranslated region (3'UTR). In this study, we determined the relationship among Tip110 miRNA, Tip110 expression and self-renewal and differentiation of human CD34+ hematopoietic cells. Using a Tip110 3UTR-based reporter gene assay, 11 miRNA showed the specific activity toward the Tip110 3'UTR and down-regulated constitutive Tip110 mRNA expression. When human cord blood CD34+ cells were differentiated, Tip110 mRNA expression showed significant decreases. Concurrently, five miRNA showed significant increases, five miRNA showed significant decreases, and one miRNA remained unchanged. To further assess the roles of miRNA in Tip110 expression and self-renewal and differentiation of human CD34+ hematopoietic cells, human cord blood CD34+ cells were transduced to express the full-length Tip110 3'UTR RNA. Expression of the Tip110 3'UTR RNA led to significant increases of Tip110 mRNA, and the number of hematopoietic stem cells and progenitor cells. Taken together, these results show important roles of Tip110 miRNA in Tip110 expression control and Tip110 regulation of hematopoiesis and offer a possibility of using Tip110 miRNA or 3'UTR as a strategy to maintain human CD34+ hematopoietic cells.

Astrocytes are susceptible to HIV infection and potential latent HIV reservoirs. Tat is one of three abundantly expressed HIV early genes in HIV-infected astrocytes and has been shown to be a major pathogenic factor for HIV/neuroAIDS. In this study, we sought to determine if and how Tat expression would affect HIV infection and latency in astrocytes. Using the glycoprotein from vesicular stomatitis virus-pseudotyped red-green HIV (RGH) reporter viruses, we showed that HIV infection was capable of establishing HIV latency in astrocytes. We also found that Tat expression decreased the generation of latent HIV-infected cells. Activation of latent HIV-infected astrocytes showed that treatment of GSK126, a selective inhibitor of methyltransferase enhancer of zeste homolog 2 (Ezh2) that is specifically responsible for tri-methylation of histone 3 lysine 27 (H3K27me3), led to activation of significantly more latent HIV-infected Tat-expressing astrocytes. Molecular analysis showed that H3K27me3, Ezh2, MeCP2, and Tat all exhibited a similar bimodal expression kinetics in the course of HIV infection and latency in astrocytes, although H3K27me3, Ezh2, and MeCP2 were expressed higher in Tat-expressing astrocytes and their expression were peaked immediately preceding Tat expression. Subsequent studies showed that Tat expression alone was sufficient to induce H3K27me3 expression, likely through its regulation of Ezh2 and MeCP2 expression. Taken together, these results showed for the first time that Tat expression induced H3K27me3 expression and contributed to HIV latency in astrocytes and suggest a new role and novel mechanism for Tat in HIV latency.

HIV-1 Nef is necessary and may be sufficient for HIV-1-associated AIDS pathogenicity, in that knockout of Nef alone can protect HIV-infected patients from AIDS. We therefore investigated the feasibility of physical knockout of Nef, using the host ubiquitin proteasome system in HIV-1-infected cells. Our co-immunoprecipitation analysis demonstrated that Nef interacted with ubiquitin specific protease 15 (USP15), and that USP15, which is known to stabilize cellular proteins, degraded Nef. Nef could also cause decay of USP15, although Nef-mediated degradation of USP15 was weaker than USP15-mediated Nef degradation. Direct interaction between Nef and USP15 was essential for the observed reciprocal decay of the proteins. Further, USP15 degraded not only Nef but also HIV-1 structural protein, Gag, thereby substantially inhibiting HIV-1 replication. However, Gag did not degrade USP15, indicating that the Nef and USP15 complex, in distinction to other viral proteins, play an integral role in coordinating viral protein degradation and hence HIV-1 replication. Moreover, Nef and USP15 globally suppressed ubiquitylation of cellular proteins, indicating that these proteins are major determinants for the stability of cellular as well as viral proteins. Taken together, these data indicate that Nef and USP15 are vital in regulating degradation of viral and cellular proteins and thus HIV-1 replication, and specific degradation of viral, not cellular proteins, by USP15 points to USP15 as a candidate therapeutic agent to combat AIDS by eliminating viral proteins from the infected cells via USP15-mediated proteosomal degradation.

Human immunodeficiency virus type 1 (HIV-1) Tat protein is a major pathogenic factor in HIV-associated neurological diseases; it exhibits direct neurotoxicity and indirect astrocyte-mediated neurotoxicity. We have shown that Tat alone is capable of activating glial fibrillary acidic protein (GFAP) expression and inducing astrocytosis involving sequential activation of early growth response protein 1 (Egr-1) and p300. In this study, we determined the roles of signal transducer and activator of transcription 3 (STAT3) in Tat-induced GFAP transactivation. STAT3 expression and phosphorylation led to significant increases in GFAP transcription and protein expression. Tat expression was associated with increased STAT3 expression and phosphorylation in Tat-expressing astrocytes and HIV-infected astrocytes. GFAP, Egr-1 and p300 transcription and protein expression all showed positive response to STAT3 and its phosphorylation. Importantly, knockdown of STAT3 resulted in significant decreases in Tat-induced GFAP and Egr-1 transcription and protein expression. Taken together, these findings show that STAT3 is involved in and acts upstream of Egr1 and p300 in the Tat-induced GFAP transactivation cascade and suggest important roles of STAT3 in controlling astrocyte proliferation and activation in the HIV-infected central nervous system.

Tip110 is a nuclear protein and has been shown to function in tumor antigenicity, regulation of gene transcription, pre-mRNA splicing, stem cell proliferation and differentiation, and embryonic development. To characterize the in vivo functions of Tip110, a transgene cassette expressing human Tip110 protein (hTip110) was used to generate hTip110 transgenic (Tg) mice. Unexpectedly, only Tip110 mRNA but not Tip110 protein was expressed in Tg MEF and tissues. Treatment of Tg MEF with proteasome inhibitors led to detection of hTip110 protein, which prompted us to investigate the regulatory mechanisms of Tip110 degradation in mouse cells. We found that hTip110 was more sensitive to ubiquitin-proteasome system (UPS)-mediated protein degradation than mouse Tip110 (mTip110), likely resulting from more hTip110 ubiquitination. Using affinity chromatography and proteomics, we identified USP15, a deubiquitinating enzyme, to be associated with Tip110. Tip110 expression led to re-distribution of USP15 from the cytoplasm to the nucleus and complete co-localization of Tip110 with USP15 in the nucleus, whereas USP15 expression resulted in hTip110 deubiquitination. Interestingly, USP15 knockdown restored hTip110 protein expression in Tg MEF and USP15 expression had little effects. Taken together, these results provide insights into the regulatory mechanism of human Tip110 degradation by USP15.

HIV-1 Tat-interacting protein of 110 kDa, Tip110, plays important roles in multiple biological processes. In this study, we aimed to characterize the function of Tip110 in embryonic development. Transgenic mice lacking expression of a functional Tip110 gene (Tip110–/–) died post-implantation, and Tip110–/– embryos exhibited developmental arrest between 8.5 and 9.5 days post coitum. However, in vitro cultures of Tip110–/–embryos showed that Tip110 loss did not impair embryo growth from the zygote to the blastocyst. Extended in vitro cultures of Tip110–/– blastocysts showed that Tip110 loss impaired both blastocyst outgrowth and self-renewal and survival of blastocyst-derived embryonic stem cells. Microarray analysis of Tip110–/– embryonic stem cells revealed that Tip110 loss altered differentiation, pluripotency, and cycling of embryonic stem cells and was associated with downregulation of several major stem cell factors including Nanog, Oct4, and Sox2 through a complex network of signaling pathways. Taken together, these findings document for the first time the lethal effects of complete loss of Tip110 on mammalian embryonic development and suggest that Tip110 is an important regulator of not only embryonic development but also stem cell factors.

HIV-1 Tat-interacting protein of 110kDa (Tip110), also referred to as squamous cell carcinoma antigen recognized by T cells 3 (Sart3), p110 or p110(nrb), was initially identified as a cDNA clone (KIAA0156) without annotated functions. Over the past twenty years, several functions have been attributed to this protein. The proposed biological functions include roles for Tip110 in pre-mRNA splicing, gene transcription, stem cell biology, and development. Dysregulation of Tip110 is also a contributing factor in the development of cancer and other human diseases. It is clear that our understanding of this protein is rapidly evolving. In this review, we aimed to provide a summary of all the existing literature on this gene/protein and its proposed biological functions.

Intracellular factors are involved in and essential for hematopoiesis. HIV-1 Tat-interacting protein of 110 kDa (TIP110; p110nrb/SART3/p110) is an RNA-binding nuclear protein implicated in the regulation of HIV-1 gene and host gene transcription, pre-mRNA splicing, and cancer immunology. In the present study, we demonstrate a role for TIP110 in the regulation of hematopoiesis. TIP110 was expressed in human CD34+ cells and decreased with differentiation of CD34+ cells. TIP110 mRNA was also expressed in phenotyped mouse marrow hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Using TIP110 transgenic (TIP110TG) and haploinsufficient (TIP110+/−) mice, we found that increased TIP110 expression enhanced HPC numbers, survival, and cell cycling, whereas decreased TIP110 expression had the opposite effects. Moreover, TIP110+/− bone marrow HPCs responded more effectively, and TIP110TG HPCs less effectively, than those of wild-type control mice to recovery from the cell-cycle–active drug 5-fluorouracil (5-FU). Unexplained sex differences were noted in HSC competitive repopulating ability, but not HPC numbers, in TIP110TG mice. Intracellularly, TIP110 regulated CMYC and GATA2 expression at the transcriptional level, and TIP110 and CMYC reciprocally regulated the expression of each other. These results demonstrate a role for TIP110 in the regulation of hematopoiesis, effects that are likely linked to TIP110 regulation of CMYC.

The order Mononegavirales (comprised of nonsegmented negative-stranded RNA viruses or NNSVs) contains many important pathogens. Parainfluenza virus 5 (PIV5), formerly known as simian virus 5, is a prototypical paramyxovirus and encodes a V protein, which has a cysteine-rich C terminus that is conserved among all paramyxoviruses. The V protein of PIV5, like that of many other paramyxoviruses, plays an important role in regulating viral RNA synthesis. In this work, we show that V interacts with Akt, a serine/threonine kinase, also known as protein kinase B. Both pharmacological inhibitors and small interfering RNA against Akt1 reduced PIV5 replication, indicating that Akt plays a critical role in PIV5 replication. Furthermore, treatment with Akt inhibitors also reduced the replication of several other paramyxoviruses, as well as vesicular stomatitis virus, the prototypical rhabdovirus, indicating that Akt may play a more universal role in NNSV replication. The phosphoproteins (P proteins) of NNSVs are essential cofactors for the viral RNA polymerase complex and require heavy phosphorylation for their activity. Inhibition of Akt activity reduced the level of P phosphorylation, suggesting that Akt is involved in regulating viral RNA synthesis. In addition, Akt1 phosphorylated a recombinant P protein of PIV5 purified from bacteria. The finding that Akt plays a critical role in replication of NNSV will lead to a better understanding of how these viruses replicate, as well as novel strategies to treat infectious diseases caused by NNSVs.

Paramyxoviruses include many important animal and human pathogens. The genome of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, encodes a V protein that inhibits viral RNA synthesis. In this work, the mechanism of inhibition was investigated. Using mutational analysis and a minigenome system, we identified regions in the N and C termini of the V protein that inhibit viral RNA synthesis: one at the very N terminus of V and the second at the C terminus of V. Furthermore, we determined that residues L16 and I17 are critical for the inhibitory function of the N-terminal region of the V protein. Both regions interact with the nucleocapsid protein (NP), an essential component of the viral RNA genome complex (RNP). Mutations at L16 and I17 abolished the interaction between NP and the N-terminal domain of V. This suggests that the interaction between NP and the N-terminal domain plays a critical role in V inhibition of viral RNA synthesis by the N-terminal domain. Both the N- and C-terminal regions inhibited viral RNA replication. The C terminus inhibited viral RNA transcription, while the N-terminal domain enhanced viral RNA transcription, suggesting that the two domains affect viral RNA through different mechanisms. Interestingly, V also inhibited the synthesis of the RNA of other paramyxoviruses, such as Nipah virus (NiV), human parainfluenza virus 3 (HPIV3), measles virus (MeV), mumps virus (MuV), and respiratory syncytial virus (RSV). This suggests that a common host factor may be involved in the replication of these paramyxoviruses. IMPORTANCE We identified two regions of the V protein that interact with NP and determined that one of these regions enhances viral RNA transcription via its interaction with NP. Our data suggest that a common host factor may be involved in the regulation of paramyxovirus replication and could be a target for broad antiviral drug development. Understanding the regulation of paramyxovirus replication will enable the rational design of vaccines and potential antiviral drugs.

MicroRNA (miR) regulates hematopoiesis through targeting different genes post-transcriptionally. We have recently shown that Tip110 expression is downregulated during hematopoietic stem cell differentiation. However, the underlying mechanisms are not known. In this study, we identified a conserved miR-124-binding site on the Tip110 3'-untranslated region (3'-UTR) and showed that Tip110 was downregulated by miR-124 through its 3'-UTR. We then examined the relationship among miR-124 and Tip110 expression and differentiation of human cord blood CD34(+) cells. We found that miR-124 was expressed in a low level in human cord blood CD34(+) cells, but it was considerably upregulated during culturing and differentiation of these cells. Moreover, we demonstrated that miR-124 expression decreased Tip110 expression and promoted differentiation of human cord blood CD34(+) cells, while miR-124 knockdown increased Tip110 expression, slowed down differentiation of human cord blood CD34(+) cells, and caused an expansion of hematopoietic progenitor cells in vitro. Finally, we used mouse embryonic fibroblasts derived from Tip110 transgenic mice, performed the exon array analysis, and found that Tip110 altered a number of genes in the hematopoiesis pathways. Dnmt3a as de novo methyltransferase was also significantly upregulated. That miR-124 was markedly upregulated during human cord blood CD34(+) cell differentiation could be the result of direct loss of its promoter methylation from Dnmt3a. Taken together, our study demonstrates that miR-124 regulates Tip110 expression and differentiation of human cord blood CD34(+) cells and suggests important roles of miR-124/Tip110 in hematopoiesis.

Hypoxia often occurs under various physiological and pathophysiological conditions, including solid tumors; it is linked to malignant transformation, metastatic progression, and treatment failure or resistance. Tip110 protein plays important roles in several known physiological and pathophysiological processes, including cancers. Thus, in the present study we investigated the regulation of Tip110 expression under hypoxia. Hypoxia led to Tip110 protein degradation through the ubiquitin-proteasome system. Under hypoxia, Tip110 stabilized p53, which in return destabilized Tip110. In addition, Tip110 regulated hypoxia-inducible factor 1α (HIF-1α), likely through enhancement of its protein stability. Furthermore, Tip110 upregulated p300, a known coactivator for both p53 and HIF-1α. Expression of a p53(22/23) mutant deficient in p300 binding accelerated Tip110 degradation under hypoxia. Tip110 knockdown resulted in the inhibition of cell proliferation and cell death in the presence of p53. Finally, significantly less Tip110, p53, and HIF-1α was detected in the hypoxic region of bone metastasis tumors in a mouse model of human melanoma cells. Taken together, these results suggest Tip110 is an important mediator in the cross talk between p53 and HIF-1α in response to hypoxic stress.

The severe acute respiratory syndrome coronavirus (SARS-CoV) is the major causative agent for the worldwide outbreak of SARS in 2003. The mechanism by which SARS-CoV causes atypical pneumonia remains unclear. The nuclear factor kappa B (NF-kappaB) is a key transcription factor that activates numerous genes involved in cellular immune response and inflammation. Many studies have shown that NF-kappaB plays an important role in the pathogenesis of lung diseases. In this study, we investigated the possible regulatory interaction between the SARS-CoV nucleocapsid (N) protein and NF-kappaB by luciferase activity assay. Our results showed that the SARS-CoV N protein can significantly activate NF-kappaB only in Vero E6 cells, which are susceptible to SARS-CoV infection, but not in Vero or HeLa cells. This suggests that NF-kappaB activation is cell-specific. Furthermore, NF-kappaB activation in Vero E6 cells expressing the N protein is dose-dependent. Further experiments showed that there is more than one function domain in the N protein responsible for NF-kappaB activation. Our data indicated the possible role of the N protein in the pathogenesis of SARS.

Severe acute respiratory syndrome (SARS) is an emerging infectious disease associated with a novel coronavirus (CoV) that was identified and molecularly characterized in 2003. Previous studies on various coronaviruses indicate that protein-protein interactions amongst various coronavirus proteins are critical for viral assembly and morphogenesis. It is necessary to elucidate the molecular mechanism of SARS-CoV replication and rationalize the anti-SARS therapeutic intervention. In this study, we employed an in vitro GST pull-down assay to investigate the interaction between the membrane (M) and the nucleocapsid (N) proteins. Our results show that the interaction between the M and N proteins does take place in vitro. Moreover, we provide an evidence that 12 amino acids domain (194-205) in the M protein is responsible for binding to N protein. Our work will help shed light on the molecular mechanism of the virus assembly and provide valuable information pertaining to rationalization of future anti-viral strategies.

A bioactive fraction (GLPG) was extracted and purified from the mycelia of Ganoderma lucidumby EtOH precipitation and DEAEcellulose column chromatography. GLPG was a proteoglycan and had a carbohydrate:protein ratio of 10.4:1. Its antiviral activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) were investigated by the cytopathic effect (CPE) inhibition assay in cell culture. This kind of polysaccharide inhibited the development of the cytopathic effect in dose-dependent manner in HSV-infected cells, moreover did not show any cytotoxic effects on cells even when a concentration was as high as 2000g/ml. In order to study the possible mode of action of the antiviral activity of GLPG, cells were treated with GLPG before, during and after infection, and the viral titers in the supernatant of cell culture 48 h post-infection were tested by TCID50 assay. The antiviral effects in pre-treated and treated during virus infection with GLPG were more remarkable than the treatment of post-infection. Although the precise mechanism has yet to be defined, our work suggested that GLPG inhibits viral replication by interfering with the early events of viral adsorption and entry into target cells. Thus, this proteoglycan seems to be a potential candidate for anti-HSV agents. © 2004 Elsevier Ireland Ltd. All rights reserved.

A novel coronavirus has been associated with a worldwide outbreak of atypical pneumonia referred to as Severe Acute Respiratory Syndrome (SARS-CoV). SARS-CoV nucleocapsid (N) protein has been cloned sequenced and expressed in Escherichia coli strain. Purified N protein was used to measure the SARS-CoV specific IgG antibodies from 16 SARS-CoV infected patients' sera and from 131 control subjects using ELISA assay. Specific antibody responses to the purified recombinant N protein after 10, 20, and 30 days of disease onset were observed in 13 of 16 (81.3%), 16 of 16 (100%) and 16 of 16 (100%) SARS patients sera, respectively. Comparison of detection results with a commercially available diagnostic kit coated with a mixture of SARS-CoV viral proteins showed 9 of 16 (56.3%), 13 of 16 (81.3%), and 15 of 16 (93.7%) positive responses, respectively. None of 131 control sera gave positive reaction in either assay. This data suggests that the N protein of SARS-CoV is immunodominant and this ELISA based test assay for detecting the SARS-CoV N antigen may hold a significant value for SARS diagnosis.

Transcription plays an important role in both HIV-1 gene expression and replication and mandates complicated but coordinated interactions between the host and virus. Our previous studies have shown that an HIV-1 Tat-interacting protein of 110 kDa, Tip110, binds to and enhances Tat function in Tat-mediated HIV-1 gene transcription and replication (Liu, Y., Li, J., Kim, B. O., Pace, B. S., and He, J. J. (2002) HIV-1 Tat protein-mediated transactivation of the HIV-1 long terminal repeat promoter is potentiated by a novel nuclear Tat-interacting protein of 110 kDa, Tip110. J. Biol. Chem. 277, 23854–23863). However, the underlying molecular mechanisms by which this takes place were not understood. In this study, we demonstrated that Tip110 bound to unphosphorylated RNA polymerase II (RNAPII) in a direct and specific manner. In addition, we detected Tip110 at the HIV-1 long terminal repeat (LTR) promoter and found that Tip110 expression was associated with increased phosphorylation of serine 2 of the heptapeptide repeats within the RNAPII C-terminal domain and increased recruitment of positive transcription elongation factor b to the LTR promoter. Consistent with these findings, we showed that Tip110 interaction with Tat directly enhanced transcription elongation of the LTR promoter. Taken together, these findings have provided additional and mechanistic evidence to support Tip110 function in HIV-1 transcription.

Background Tip110 plays important roles in tumor immunobiology, pre-mRNA splicing, expression regulation of viral and host genes, and possibly protein turnover. It is clear that our understanding of Tip110 biological function remains incomplete. Results Herein, we employed an immunoaffinity-based enrichment approach combined with protein mass spectrometry and attempted to identify Tip110-interacting cellular proteins. A total of 13 major proteins were identified to be complexed with Tip110. Among them was Y-box binding protein 1 (YB-1). The interaction of Tip110 with YB-1 was further dissected and confirmed to be specific and involve the N-terminal of both Tip110 and YB-1 proteins. A HIV-1 LTR promoter-driven reporter gene assay and a CD44 minigene in vivo splicing assay were chosen to evaluate the functional relevance of the Tip110/YB-1 interaction. We showed that YB-1 potentiates the Tip110/Tat-mediated transactivation of the HIV-1 LTR promoter while Tip110 promotes the inclusion of the exon 5 in CD44 minigene alternative splicing. Conclusions Tip110 and YB-1 interact to form a complex and mutually regulate each other’s biological functions.

HIV-1 Tat-interacting protein (Tip110), also known as squamous cell carcinoma antigen recognized by T cells 3 (SART3), is a multifaceted nuclear protein and has been shown to function in tumor antigenicity. Tip110 protein expression is very low in normal tissues and non-proliferating cells but becomes highly elevated in a number of malignant tumor cell lines and cancerous tissues, pointing to its possible role in tumorigenesis. Our previous studies have shown that Tip110 specifically interacted with oncogenic deubiquitinating enzyme, USP15, and that ectopic expression of the Tip110 protein led to re-distribution of USP15 from the cytoplasm to the nucleus. USP15 is known to inhibit TNF-α-induced NF-κB activity by modulation of IκB-α ubiquitination. In the current study, we demonstrated that Tip110 augmented TNF-α-induced transcription activity of NF-κB, and that deletion of the nuclear localization domain in Tip110 obliterated the observed enhancement of TNF-α-induced NF-κB activity, providing the very first evidence to support the specific role of Tip110 in NF-κB activation. In addition, we found that Tip110 expression modulated IκB-α phosphorylation and that activation of NF-κB by Tip110 was mediated through the p38/MAPK pathway. Moreover, we showed that ectopic expression of Tip110 induced translocation of NF-κB p65 from the cytoplasm to the nucleus, reminiscent of the aforementioned translocation of USP15, in the absence of direct interaction of Tip110 with either p65 or IκB-α. Taken together, these results indicate that opposing regulation of TNFα-induced NF-κB activity by Tip110 and USP15 could be pivotal for modulation of tumorigenesis. Further delineation of the underlying signaling cascades will likely provide new insights into development of anti-tumor agents. Citation Format: Khalid Amine Timani, Amanda Whitmill, Ying Liu, Johnny J. He. Tip110-mediated regulation of NF-κB activity is key to tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4557.

Abstract 2353 Embryonic stem cells (ESCs) are pluripotent, self-renew and can be differentiated into cells of all three germ layers, and nanog, Oct4 and Sox2, form a core of the self-renewal transcription network. Nanog expression is restricted to pluripotent cells and is down regulated upon differentiation; little is known about its regulation. Expression of the OCT4 gene maintains cell pluripotency via a stringent dose-dependent regulation with OCT4 levels above or below required dosages producing cellular differentiation; thus maintenance of a critical amount of OCT4 is necessary to prevent ESC differentiation. Sox2, a high-mobility group domain containing transcription factor, binds to the consensus motif CATTGTT. We recently reported in Blood that Tip110 is an essential gene expressed in earliest cells of adult bone marrow hematopoietic development. Increased TIP110 expression enhanced hematopoietic progenitor cell (HPC) numbers, survival, and cell cycling; decreased Tip110 expression manifested the opposite effect, demonstrating a role for TIP110 in regulation of hematopoiesis. Herein, we investigated TIP110 expression and actions in human (h)ESCs. Quantitative RT-PCR showed that TIP110, as well as Nanog, Oct4 and Sox2 were expressed in a hESC line. hESCs were removed from feeder layers and b-FGF for 5 days, to allow ESC differentiation. TIP110 expression levels were dramatically reduced (by 77%); this was associated with large decreases in expression of NANOG (82%), OCT4 (80%), and SOX2 (85%). We then assessed whether TIP110 might regulate hESC pluripotency. We exogenously over-expressed TIP110 in hESC cells. Feeder layers and b-FGF were withdrawn upon introducing the TIP110 vector and cells cultured for 5 days to test whether sustained TIP110 expression rendered ESCs less sensitive to differentiation. Compared with controls, TIP110 over-expressing cells stained positive for OCT4, NANOG and were negative for Tuji, SMA and AFP, demonstrating that over-expression of TIP110 rendered ESCs less responsive to differentiation. Next, we reduced TIP110 expression by transfection of the hESCs with TIP110 siRNA. Cells were cultured in mTeSR medium on Matrigel-coated dishes for an additional 5 days in order to maintain cells under undifferentiation conditions. TIP 110 siRNA vector expressing cells were negative for OCT4, NANOG, and positive for Tuji, SMA and AFP expression compared with control cells, demonstrating that enforced reduction of TIP110 expression in hESCs causes hESC differentiation. This demonstrated the importance of TIP110 in maintenance of ESC pluripotency. We speculated that TIP110 maintenance of hESC pluripotency might be through regulation of NANOG, OCT4 and SOX2. We silenced TIP110 expression in hESCs by transfection with a TIP110 siRNA vector, previously shown to reduce TIP110 expression by 70%. Cells were cultured in complete 20% KSR hESC medium for an additional 5 days. Expression of these three transcription factors was dramatically decreased, demonstrating that TIP110 is required for maintaining NANOG, OCT4 and SOX2 levels in this hESC line. Reduction of TIP110 expression caused hESC differentiation directly or indirectly through down-regulation of NANOG, OCT4 and SOX2 expression. Thus, TIP110 is preferentially expressed in the undifferentiated state in hESCs and plays a key role in regulating OCT4, SOX2, and NANOG, factors required to maintain pluripotency. Together, our present and previous studies suggest TIP110 expression as a useful marker to distinguish early from more differentiated cells. Modulating TIP110 expression in a controlled fashion may be relevant for cellular engineering and regenerative medicine. Disclosures: No relevant conflicts of interest to declare.

Parainfluenza virus 5 (PIV5) is a prototypical paramyxovirus. The V/P gene of PIV5 encodes two mRNA species through a process of pseudotemplated insertion of two G residues at a specific site during transcription, resulting in two viral proteins, V and P, whose N termini of 164 amino acid residues are identical. Previously it was reported that mutating six amino acid residues within this identical region results in a recombinant PIV5 (rPIV5-CPI−) that exhibits elevated viral protein expression and induces production of cytokines, such as beta interferon and interleukin 6. Because the six mutations correspond to the shared region of the V protein and the P protein, it is not clear whether the phenotypes associated with rPIV5-CPI− are due to mutations in the P protein and/or mutations in the V protein. To address this question, we used a minigenome system and recombinant viruses to study the effects of mutations on the functions of the P and V proteins. We found that the P protein with six amino acid residue changes (Pcpi−) was more efficient than wild-type P in facilitating replication of viral RNA, while the V protein with six amino acid residue changes (Vcpi−) still inhibits minigenome replication as does the wild-type V protein. These results indicate that elevated viral gene expression in rPIV5-CPI− virus-infected cells can be attributed to a P protein with an increased ability to facilitate viral RNA synthesis. Furthermore, we found that a single amino acid residue change at position 157 of the P protein from Ser (the residue in the wild-type P protein) to Phe (the residue in Pcpi−) is sufficient for elevated viral gene expression. Using mass spectrometry and 33 P labeling, we found that residue S157 of the P protein is phosphorylated. Based on these results, we propose that phosphorylation of the P protein at residue 157 plays an important role in regulating viral RNA replication.

To study the effects of hepatitis C virus (HCV) core and non-structural 5A (NS5A) proteins on nuclear factor-kappaB (NF-kappaB) activity for understanding their biological function on chronic hepatitis caused by HCV infection.Luciferase assay was used to measure the activity of NF-kappaB in three different cell lines cotransfected with a series of deletion mutants of core protein alone or together with NS5A protein using pNF-kappaB-Luc as a reporter plasmid. Western blot and indirect immunofluorescence assays were used to confirm the expression of proteins and to detect their subcellular localization, respectively. Furthermore, Western blot was also used to detect the expression levels of NF-kappaB/p65, NF-kappaB/p50, and inhibitor kappaB-a (IkappaB-a).The wild-type core protein (C191) and its mutant segments (C173 and C158) could activate NF-kappaB in Huh7 cells only and activation caused by (C191) could be enhanced by NS5A protein. Moreover, the full-length core protein and its different deletion mutants alone or together with NS5A protein did not enhance the expression level of NF-kappaB. The NF-kappaB activity was augmented due to the dissociation of NF-kappaB-IkappaB complex and the degradation of IkappaB-a.NF-kappaB is the key transcription factor that can activate many genes that are involved in the cellular immune response and inflammation. Coexpression of the full-length core protein along with NS5A can enhance the NF-kappaB activation, and this activation may play a significant role in chronic liver diseases including hepatocellular carcinoma associated with HCV infection.

SARS-associated coronavirus (SARS-CoV) causes inflammation and damage to the lungs resulting in severe acute respiratory syndrome. To evaluate the molecular mechanisms behind this event, we investigated the roles of SARS-CoV proteins in regulation of the proinflammatory factor, cyclooxygenase-2 (COX-2). Individual viral proteins were tested for their abilities to regulate COX-2 gene expression. Results showed that the COX-2 promoter was activated by the nucleocapsid (N) protein in a concentration-dependent manner. Western blot analysis indicated that N protein was sufficient to stimulate the production of COX-2 protein in mammalian cells. COX-2 promoter mutations suggested that activation of COX-2 transcription depended on two regulatory elements, a nuclear factor-kappa B (NF-kappaB) binding site, and a CCAAT/enhancer binding protein (C/EBP) binding site. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) demonstrated that SARS-CoV N protein bound directly to these regulatory sequences. Protein mutation analysis revealed that a Lys-rich motif of N protein acted as a nuclear localization signal and was essential for the activation of COX-2. In addition, a Leu-rich motif was found to be required for the N protein function. A sequence of 68 residuals was identified as a potential DNA-binding domain essential for activating COX-2 expression. We propose that SARS-CoV N protein causes inflammation of the lungs by activating COX-2 gene expression by binding directly to the promoter resulting in inflammation through multiple COX-2 signaling cascades.

A novel coronavirus (CoV) has recently been identified as the aetiological agent of severe acute respiratory syndrome (SARS). Nucleocapsid (N) proteins of the Coronaviridae family have no discernable homology, but they share a common nucleolar-cytoplasmic distribution pattern. There are three putative nuclear localization signal (NLS) motifs present in the N. To determine the role of these putative NLSs in the intracellular localization of the SARS–CoV N, we performed a confocal microscopy analysis using rabbit anti-N antisera. In this report, we show that the wild type N was distributed mainly in the cytoplasm. The N-terminal of the N, which contains the NLS1 (aa38–44), was localized to the nucleus. The C-terminus of the N, which contains both NLS2 (aa257–265) and NLS3 (aa369–390) was localized to the cytoplasm and the nucleolus. Results derived from analysis of various deletion mutations show that the region containing amino acids 226–289 is able to mediate nucleolar localization. The deletion of two hydrophobic regions that flanked the NLS3 recovered its activity and localized to the nucleus. Furthermore, deletion of leucine rich region (220-LALLLLDRLNRL) resulted in the accumulation of N to the cytoplasm and nucleolus, and when fusing this peptide to EGFP localization was cytoplasmic, suggesting that the N may act as a shuttle protein. Differences in nuclear/nucleolar localization properties of N from other members of coronavirus family suggest a unique function for N, which may play an important role in the pathogenesis of SARS.

By a cDNA array representing 2308 signal transduction-related genes, we studied the expression profiles of HeLa cells stably transfected by Hepatitis C virus nonstructural protein 4B (HCV-NS4B). The alterations of the expression of four genes were confirmed by real-time quantitative RTPCR; and the aldo-keto reductase family 1, member C1 (AKR1C1) enzyme activity was detected in HCV-NS4B transiently transfected HeLa cells and Huh-7, a human hepatoma cell line. Of the 2,308 genes we examined, 34 were up-regulated and 56 were down-regulated. These 90 genes involved oncogenes, tumor suppressors, cell receptors, complements, adhesions, transcription and translation, cytoskeleton and cellular stress. The expression profiling suggested that multiple regulatory pathways were affected by HCV-NS4B directly or indirectly. And since these genes are related to carcinogenesis, host defense system and cell homeostatic mechanism, we can conclude that HCV-NS4B could play some important roles in the pathogenesis mechanism of HCV.

The hepatitis C virus RNA polymerase (NS5B) is strictly required for viral replication and thus represents an attractive target for antiviral drug development. In this study, stable HeLa cell lines with an integrated NS5B gene were selected by G418 and then confirmed by genome PCR. Subsequently, transcription and expression of the integrated NS5B genes were demonstrated by RT-PCR and Western blot analysis. Further analysis demonstrated enzymatic activity of the expressed NS5B polymerase. The stable HeLa cell lines should be useful for the identification of NS5B inhibitors and for studying the mechanisms of HCV replication.