Ausencia de expresión de la proteína Core del virus de la hepatitis C en células dendríticas derivadas de monocitos humanos utilizando virus del Bosque de Semliki recombinante / Lack of expression of hepatitis c virus core protein in human monocyte-derived dendritic cells using recombinant semliki forest virus

ABSTRACT Hepatitis C Virus belongs to the Flaviviridae family. One proposed mechanism of HCV persistence in the ability to infect hematopoietic cells, including Dendritic cells (DCs). HCV infection of DCs could impair their functions that represent one of the mechanisms, thus hampering viral clearance by the host immune system. Among HCV-encoded proteins, the highly conserved Core protein has been suggested to be responsible for the immunomodulatory properties of this Hepacivirus. Recombinant viral vectors expressing the HCV Core protein and allowing its transduction and therefore the expression of the protein into DCs could be useful tools for the analysis of the properties of the Core protein. Vaccinia Virus and retrovirus have been used to transduce human DCs. Likewise, gene transfer into DCs using Semliki Forest Virus has been reported. This study aimed to express the HCV Core protein in human monocyte-derived DCs using an SFV vector, in which the subgenomic RNA encoding the structural proteins was replaced by the HCV Core sequence and then analyze the effects of its expression on DCs functions.

RESUMEN El virus de la Hepatitis C (VHC) pertenece a la familia Flaviviridae. Uno de los mecanismos propuestos de la persistencia del VHC es la capacidad de infectar células hematopoyéticas, incluidas las células dendríticas (DCs). La infección por VHC de DCs podría alterar sus funciones y corresponde a uno de los mecanismos que impiden el aclaramiento de la infección por VHC por el sistema inmunitario del hospedero. Entre las proteínas codificadas por el VHC, se ha sugerido que la proteína Core, altamente conservada, es responsable de las propiedades inmunomoduladoras de este Hepacivirus. Los vectores virales recombinantes que expresan la proteína Core y permiten su transducción a DCs podrían ser herramientas útiles para el análisis de las propiedades de esta proteína. El virus Vaccinia y el retrovirus se han utilizado para la transducción de DCs humanas. Del mismo modo, la transducción de DCs usando el virus del bosque de Semliki ha sido reportada. El objetivo de este estudio fue expresar la proteína Core de VHC en DCs derivadas de monocitos humanos utilizando un vector de SFV, en el que el ARN subgenómico que codifica las proteínas estructurales fue reemplazado por la secuencia Core del VHC y evaluar los efectos de su expresión en las funciones de DCs.

MxB is an interferon-induced restriction factor of human herpesviruses.

The type I interferon (IFN) system plays an important role in controlling herpesvirus infections, but it is unclear which IFN-mediated effectors interfere with herpesvirus replication. Here we report that human myxovirus resistance protein B (MxB, also designated Mx2) is a potent human herpesvirus restriction factor in the context of IFN. We demonstrate that ectopic MxB expression restricts a range of herpesviruses from the Alphaherpesvirinae and Gammaherpesvirinae, including herpes simplex virus 1 and 2 (HSV-1 and HSV-2), and Kaposi's sarcoma-associated herpesvirus (KSHV). MxB restriction of HSV-1 and HSV-2 requires GTPase function, in contrast to restriction of lentiviruses. MxB inhibits the delivery of incoming HSV-1 DNA to the nucleus and the appearance of empty capsids, but not the capsid delivery to the cytoplasm or tegument dissociation from the capsid. Our study identifies MxB as a potent pan-herpesvirus restriction factor which blocks the uncoating of viral DNA from the incoming viral capsid.

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis.

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

Tailored enrichment strategy detects low abundant small noncoding RNAs in HIV-1 infected cells.

BACKGROUND: The various classes of small noncoding RNAs (sncRNAs) are important regulators of gene expression across divergent types of organisms. While a rapidly increasing number of sncRNAs has been identified over recent years, the isolation of sncRNAs of low abundance remains challenging. Virally encoded sncRNAs, particularly those of RNA viruses, can be expressed at very low levels. This is best illustrated by HIV-1 where virus encoded sncRNAs represent approximately 0.1-1.0% of all sncRNAs in HIV-1 infected cells or were found to be undetected. Thus, we applied a novel, sequence targeted enrichment strategy to capture HIV-1 derived sncRNAs in HIV-1 infected primary CD4+ T-lymphocytes and macrophages that allows a greater than 100-fold enrichment of low abundant sncRNAs. RESULTS: Eight hundred and ninety-two individual HIV-1 sncRNAs were cloned and sequenced from nine different sncRNA libraries derived from five independent experiments. These clones represent up to 90% of all sncRNA clones in the generated libraries. Two hundred and sixteen HIV-1 sncRNAs were distinguishable as unique clones. They are spread throughout the HIV-1 genome, however, forming certain clusters, and almost 10% show an antisense orientation. The length of HIV-1 sncRNAs varies between 16 and 89 nucleotides with an unexpected peak at 31 to 50 nucleotides, thus, longer than cellular microRNAs or short-interfering RNAs (siRNAs). Exemplary HIV-1 sncRNAs were also generated in cells infected with different primary HIV-1 isolates and can inhibit HIV-1 replication. CONCLUSIONS: HIV-1 infected cells generate virally encoded sncRNAs, which might play a role in the HIV-1 life cycle. Furthermore, the enormous capacity to enrich low abundance sncRNAs in a sequence specific manner highly recommends our selection strategy for any type of investigation where origin or target sequences of the sought-after sncRNAs are known.

The cellular RNA helicase UAP56 is required for prevention of double-stranded RNA formation during influenza A virus infection.

The cellular DEAD box RNA helicase UAP56 plays a pivotal role in the efficient transcription/replication of influenza A virus. UAP56 is recruited by the nucleoprotein (NP) of influenza A viruses, and recent data revealed that the RNA helicase is required for the nuclear export of a subset of spliced and unspliced viral mRNAs. The fact that influenza viruses do not produce detectable amounts of double-stranded RNA (dsRNA) intermediates during transcription/replication suggests the involvement of cellular RNA helicases. Hence, we examined whether the RNA-unwinding activity of UAP56 or its paralog URH49 plays a role in preventing the accumulation of dsRNA during infection. First, our data showed that not only UAP56 but also its paralog URH49 can interact with NPs of avian and human influenza A viruses. The small interfering RNA (siRNA)-mediated depletion of either RNA helicase reduced the transport of M1 and hemagglutinin (HA) mRNAs and, to a lesser extent, NP and NS1 mRNAs into the cytoplasm. Moreover, we found that virus infection of UAP56-depleted cells leads to the rapid accumulation of dsRNA in the perinuclear region. In parallel, we observed a robust virus-mediated activation of dsRNA-dependent protein kinase R (PKR), indicating that the cellular RNA helicase UAP56 may be recruited by influenza virus to prevent dsRNA formation. The accumulation of dsRNA was blocked when actinomycin D or cycloheximide was used to inhibit viral transcription/replication or translation, respectively. In summary, we demonstrate that UAP56 is utilized by influenza A viruses to prevent the formation of dsRNA and, hence, the activation of the innate immune response.

Role of AF6 protein in cell-to-cell spread of Herpes simplex virus 1.

AF6 and its rat homologue afadin are multidomain proteins localized at cell junctions and involved in intercellular adhesion. AF6 interacts via its PDZ domain with nectin-1 at epithelial adherens junctions. Nectin-1 serves as a mediator of cell-to-cell spread for Herpes simplex virus 1 (HSV-1). We analyzed the role of AF6 protein in the viral spread and nectin-1 clustering at cell-cell contacts by knockdown of AF6 in epithelial cells. AF6 knockdown reduced efficiency of HSV-1 spreading, however, the clustering of nectin-1 at cell-cell contacts was not affected. Thus, AF6 protein is important for spreading of HSV-1 in epithelial cells, independently of nectin clustering, possibly by stabilization of the E-cadherin-dependent cell adhesion.

Anti-tumor activity of mesenchymal stem cells producing IL-12 in a mouse melanoma model.

Mesenchymal stem cells (MSCs) represent a new tool for delivery of therapeutic agents to tumor cells. In this study, we have evaluated the anti-tumor activity of human MSCs stably transduced with a retroviral vector expressing the cytokine interleukin-12 (IL-12) in a mouse melanoma model. Application of MSC(IL-12) but not control MSCs strongly reduced the formation of lung metastases of B16F10 melanoma cells. The activity of the MSC(IL-12) cells was dependent on the presence of natural killer (NK) cells in this experimental setting. Further, MSC(IL-12) cells elicited a pronounced retardation of tumor growth and led to prolonged survival when injected into established subcutaneous melanoma in a therapeutic regimen. The therapeutic effect of the MSC(IL-12) was in part mediated by CD8(+) T cells, while NK cells and CD4(+) T cells appeared to play a minor role. The anti-tumor effect of MSC(IL-12) cells was of similar efficiency as observed for application of naked plasmid DNA encoding IL-12. The presented data demonstrate that these two different strategies can induce a similar therapeutic anti-tumor efficacy in the mouse melanoma tumor model.

Analysis of antitumor activity elicited by vaccination with combinations of interleukin-12 DNA with gp100 DNA or the chemokine CCL21 in vivo.

The antitumor efficacy of human melanoma-associated antigen (hgp100) and chemokine CCL21 in combination with interleukin-12 (IL-12) was evaluated in a syngeneic melanoma mouse model. The rationale for this approach was based on previous studies showing that the efficacy of IL-12 therapy in melanoma patients correlated with the presence of antibodies against tumor-associated antigens. We have previously shown that application of xenogeneic human gp100 DNA (hgp100 DNA) is protective against mouse B16 melanoma. Furthermore, the chemokine CCL21 has the ability to chemoattract both dendritic cells (DCs) and T lymphocytes. We show here that intratumoral injection of IL-12-encoding DNA (IL-12 DNA) in combination with hgp100- encoding DNA (hgp100 DNA) into tumor-bearing mice led to a strong antitumor effect. Coapplication of IL- 12 DNA with CCL21-encoding DNA (CCL21 DNA) or recombinant CCL21 (recCCL21) protein also showed some efficacy. Triple therapy with IL-12 DNA, hgp100 DNA, and CCL21 DNA, however, showed less effect on tumor growth than double therapy with IL-12 DNA and hgp100 DNA. These findings open a new route of investigation of IL-12 and gp100 or other tumor-associated antigens in the immunotherapy of a variety of tumors.

Effect of ex vivo gene transfer with an adenoviral vector on human eye bank corneas.

BACKGROUND: Ex vivo gene transfer to donor corneas using adenoviral vectors has gained increasing attention. This study investigates the effect of adenovirus-mediated gene transfer on endothelial cell (EC) count in human eye bank corneas. METHODS: A replication-defective adenoviral vector containing the gene for green fluorescent protein was used to transduce organ-cultured normal human eye bank and porcine corneas. Transgene expression and EC count were assessed by light and fluorescence microscopy. RESULTS: The transgene was expressed earlier by porcine EC (27% of all EC on day 2) than by human EC (6% on day 2), but the maximal number of EC finally expressing the transgene was higher in human than in porcine corneas (45 vs. 31% of all EC on day 12). Gene transfer caused considerably less EC loss in human than in porcine corneas (2 vs. 60% EC loss after 10 days). CONCLUSIONS: Adenoviral vectors for ex vivo gene transfer are more efficient and less toxic in normal human eye bank corneas than in porcine corneas, but human EC require more time until expression of transgenic proteins.

Intratumoral injection of DNA encoding human interleukin 12 into patients with metastatic melanoma: clinical efficacy.

Plasmid DNA encoding human interleukin 12 (IL-12) was produced under GMP conditions and injected into lesions of nine patients with malignant melanoma (stage IV) previously treated with both standard and nonstandard therapies. The treatment was based on efficacy in preclinical studies with melanoma in mice and gray horses. The DNA was applied in cycles, three injections per cycle, for up to seven cycles. Three therapy arms comprised low (2 mg), medium (4 mg), and high (10 to 20 mg) amounts of total DNA. The therapy was well tolerated. Three of nine patients experienced a clinical response: two stable disease and one complete remission. One patient receiving a low dose of DNA experienced a long-lasting stabilization of the disease for more than 3 years, whereas the other two responders received high doses of DNA. All patients but one (patient 9) experienced a transient response at the intratumoral injection site. Immunohistochemical staining of responder sections showed local reduction of angiogenesis and lymphocyte infiltrations. All patients, in particular the clinical and local responders (patients 3, 7, and 8), exhibited an antigen-specific immune response against MAGE-1 and MART-1, which in some cases preexisted. Biopsies of responders showed some increase in IL-12, IP-10, and IFN-(). Serum levels revealed fluctuations. The results show that intratumoral injection of DNA produced some beneficial clinical effect. DNA encoding a cytokine may be useful as a therapeutic or adjuvant against various human cancers.

Antibodies from a DNA peptide vaccination decrease the brain amyloid burden in a mouse model of Alzheimer's disease.

The neuropathology of Alzheimer's disease(AD) is characterized by the accumulation of amyloid peptide Abeta in the brain derived from proteolytic cleavage of the amyloid precursor protein (APP). Vaccination of mice with plasmid DNA coding for the human Abeta42 peptide together with low doses of preaggregated peptide induced antibodies with detectable titers after only 2 weeks. One serum was directed against the four aminoterminal amino acids DAEF and differs from previously described ones. Both immune sera and monoclonal antibodies solubilized preformed aggregates of Abeta42 in vitro and recognized amyloid plaques in brain sections of mice transgenic for human APP. Passive immunization of transgenic AD mice caused a significant and rapid reduction in brain amyloid plaques within 24 h. The combined DNA peptide vaccine may prove useful for active immunization with few inoculations and low peptide dose which may prevent the recently described inflammatory reactions inpatients. The monoclonal antibodies are applicable for passive immunization studies and may lead to a therapy of AD.

IP-10-encoding plasmid DNA therapy exhibits anti-tumor and anti-metastatic efficiency.

We report here that the interferon-induced protein of 10 kDa (IP-10 or CXCL10) elicits strong anti-tumor and anti-metastatic responses in mice when administered by plasmid DNA. Intratumoral but not intramuscular IP-10 DNA inoculation resulted in reduced tumor formation of malignant melanoma (B16F10) and Lewis lung carcinoma (LL/2) in C57BL/6 mice. In addition, plasmid DNA-encoding IP-10 substantially reduced the establishment of metastases when injected systemically by the intramuscular route. In contrast to the primary tumor model, the anti-metastatic effect of DNA-encoding IP-10 was primarily mediated by NK cells. Compared to DNA-encoding interleukin-12 (IL-12), therapy with DNA-encoding IP-10 exhibits lower efficacy against primary melanoma tumors but equivalent efficacy against primary Lewis lung tumors and against B16F10 lung metastasis formation. Co-administration of DNA-encoding IP-10 and IL-12 enhanced the anti-tumor activity of IL-12 in the lung metastasis model but had little effect in the local treatment of established subcutaneous tumors. Interestingly, treatment of nude mice lacking T lymphocytes with DNA-encoding IP-10 or IL-12 still resulted in a pronounced reduction of tumor growth or metastasis formation.

Tumor regression of human and murine melanoma after intratumoral injection of IL-12-encoding plasmid DNA in mice.

DNA coding for murine interleukin 12 (IL-12) prevents the formation of B16-melanoma metastasis when administered intramuscularly. Here, the antitumor effect of IL-12-encoding DNA on established mouse B16 melanoma and human melanoma tumors was investigated in vivo using two animal models: B16 melanoma in C57B/6 mice and human melanoma in nude mice. In B16 melanoma, intratumoral injections of IL-12-encoding DNA resulted in highly significant growth retardation when compared with mice injected with control vector. In the case of the human melanoma model, treatment with DNA coding for IL-12 induced regression of tumors in all cases, with complete disappearance of the tumor in two out of five animals. DNA treatment did not induce systemic side-effects. In the animals injected with control vector the human melanoma tumors grew expansively. The therapeutic effect of the DNA injection was mediated in part by natural killer (NK) cells as shown by NK-depletion experiments. An antivascular effect of IL-12 treatment was evident in histological examination with endothelial thickening and abrupt changes in vessel diameters. These results suggest that intratumoral plasmid DNA coding for IL-12 holds some promise as a new therapeutic tool for accessible melanoma lesions and should be tested in clinical trial.