Cytomegalovirus Immediate-early Promoter Research Articles

Generation of a DNA-launched classical swine fever virus infectious clone packaged in bacterial artificial chromosome

Reverse genetics system offers powerful tool for the research of RNA viruses. The infectious clones of classical swine fever virus (CSFV) were commonly constructed either in high- or low-copy number plasmids and transcribed to infectious RNA using phage RNA-polymerases. Herein, the full-length genome of CSFV Shimen strain, flanked by cytomegalovirus immediate-early (CMV) promoter (a eukaryotic RNA polymerase II promoter) sequence at the 5′-end and the hepatitis delta virus ribozyme along with the bovine growth hormone termination and polyadenylation signal sequences at the 3′-end, was packaged in bacterial artificial chromosome vector to establish a CSFV infectious clone pBAC-smCSFV. This infectious cDNA clone maintained stability after passaged 20 times in bacteria. Transfection of PK15 cells with this cDNA clone facilitated recovery of infectious progeny virus which was identical to parent virus as characterized by RT-qPCR, western blotting, indirect immunofluorescence assay, one-step growth kinetics analysis and nucleotide sequencing. Based on this CSFV infectious cDNA clone, the mCherry was inserted between viral Npro and C protein to develop reporter virus CSFV-mCherry. The mCherry was stably expressed after CSFV-mCherry was passaged 10 times in PK15 cells. Taken together, this present study develops a concise and efficient CSFV infectious cDNA clone and a reporter virus CSFV-mCherry. To the best of our knowledge, this is the first combination of CMV promoter and BAC system in construction of CSFV reverse genetics system. The CSFV infectious cDNA clone and the reporter virus will be useful in the study of CSFV virus biology, virulence determinants, molecular pathogenesis, vaccine development and virus-host interaction.

Elevated NTCP expression by an iPSC-derived human hepatocyte maintenance medium enhances HBV infection in NTCP-reconstituted HepG2 cells

BackgroundThe sodium taurocholate cotransporting polypeptide (NTCP) is a functional receptor for hepatitis B virus (HBV). NTCP-reconstituted human hepatoma cells support HBV infection, but the infection is suboptimal and no apparent HBV spread has been observed in this system.ResultsWe found that NTCP-reconstituted HepG2 cells were highly susceptible to HBV infection after cells were cultured in a commercial human inducible pluripotent stem cell (iPSC)-derived hepatocyte maintenance medium (HMM). The enhanced HBV infection coincided with increased NTCP expression, and was observed in six different clones of HepG2-NTCP cells. Promoter assays indicated that HMM activated the cytomegalovirus immediate-early (IE) promoter that drives the NTCP expression in the HepG2-NTCP cells. RNA-Seq analysis revealed that HMM upregulated multiple metabolic pathways. Despite highly upregulated NTCP expression by HMM, no obvious HBV spread was observed even in the presence of PEG 8000.ConclusionsOur data suggest that this particular medium could be used to enhance HBV infection in NTCP-reconstituted hepatocytes in vitro.

Design of synthetic promoters for controlled expression of therapeutic genes in retinal pigment epithelial cells.

Age-related macular degeneration (AMD) associated with dysfunction of retinal pigment epithelial (RPE) cells is the most common cause of untreatable blindness. To advance gene therapy as a viable treatment for AMD there is a need for technologies that enable controlled, RPE-specific expression of therapeutic genes. Here we describe design, construction and testing of compact synthetic promoters with a pre-defined transcriptional activity and RPE cell specificity. Initial comparative informatic analyses of RPE and photoreceptor (PR) cell transcriptomic data identified conserved and overrepresented transcription factor regulatory elements (TFREs, 8-19 bp) specifically associated with transcriptionally active RPE genes. Both RPE-specific TFREs and those derived from the generically active cytomegalovirus-immediate early (CMV-IE)promoter were then screened in vitro to identify sequence elements able to control recombinant gene transcription in model induced pluripotent stem (iPS)-derived and primary human RPE cells. Two libraries of heterotypic synthetic promoters varying in predicted RPE specificity and transcriptional activity were designed de novo using combinations of up to 20 discrete TFREs in series (323-602 bp) and their transcriptional activity in model RPE cells was compared to that of the endogenous BEST1 promoter (661 bp, plus an engineered derivative) and the highly active generic CMV-IE promoter (650 bp). Synthetic promoters with a highpredicted specificity, comprised predominantly of endogenous TFREs exhibited a range of activities up to 8-fold that of the RPE-specific BEST1 gene promoter. Moreover, albeit at a lower predicted specificity, synthetic promoter transcriptional activity in model RPE cells was enhanced beyond that of the CMV-IE promoter when viral elements were utilized in combination with endogenous RPE-specific TFREs, with a reduction in promoter size of 15%. Taken together, while our data reveal an inverse relationship between synthetic promoter activity and cell-type specificity, cell context-specific control of recombinant gene transcriptional activity may be achievable.

Improved adaptation to physical stress in mice overexpressing SUR2A is associated with changes in the pattern of Q-T interval

The purpose of this study was to determine whether increased expression of SUR2A, a regulatory subunit of sarcolemmal ATP-sensitive K+ (KATP) channels, improves adaptation to physical stress and regulates cardiac electrophysiology in physical stress. All experiments have been done on transgenic mice in which SUR2A expression was controlled by cytomegalovirus immediate-early (CMV) promoter (SUR2A) and their littermate wild-type controls (WT). The levels of mRNA in heart tissue were measured by real-time RT-PCR. Electrocardiogram (ECG) was monitored with telemetry. The physical adaptation to stress was elucidated using treadmill. We have found that SUR2A mice express 8.34 ± 0.20 times more myocardial SUR2A mRNA than WT (n = 8–18). The tolerated workload on exercise stress test was more than twofold higher in SUR2A than in WT (n = 5–7; P = 0.01). The pattern of Q-T interval from the beginning of the exercise test until drop point was as follows in the wild type: (1) increase in Q-T interval, (2) decrease in Q-T interval, (3) steady stage with a further decrease in Q-T interval, and (4) a sharp increase in Q-T interval. The pattern of Q-T interval was different in transgenic mice and the following stages have been observed: (1) increase in Q-T interval, (2) decrease in Q-T interval, and (3) prolonged steady-state stage with a slight decrease in Q-T interval. In SUR2A mice, no stage 4 (a sharp increase in Q-T interval) was observed. Based on the obtained results, we conclude that an increase in the expression of SUR2A improves adaptation to physical stress and physical endurance by increasing the number of sarcolemmal KATP channels and, by virtue of their channel activity, improving Ca2+ homeostasis in the heart.

Adeno-Associated Virus D-Sequence-Mediated Suppression of Expression of a Human Major Histocompatibility Class II Gene: Implications in the Development of Adeno-Associated Virus Vectors for Modulating Humoral Immune Response.

A 20-nt long sequence, termed the D-sequence, in the adeno-associated virus (AAV) inverted terminal repeat was observed to share a partial sequence homology with the X-box in the regulatory region of the human leukocyte antigen DRA (HLA-DRA) promoter of the human major histocompatibility complex class II (MHC-II) genes. The D-sequence was also shown to specifically interact with the regulatory factor binding to the X-box (RFX), binding of which to the X-box is a critical step in the MHC-II gene expression, suggesting that D-sequence might compete for RFX transcription factor binding, thereby suppressing expression from the MHC-II promoter. In DNA-mediated transfection experiments, using a reporter gene under the control of the HLA-DRA promoter, D-sequence oligonucleotides were found to inhibit expression of the reporter gene expression in HeLa and 293 cells by ∼93% and 96%, respectively. No inhibition was observed when nonspecific synthetic oligonucleotides were used. D-sequence oligonucleotides had no effect on expression from the cytomegalovirus immediate-early gene promoter. Interferon-γ-mediated activation of MHC-II gene expression was also inhibited by D-sequence oligonucleotides as well as after infection with either the wild-type AAV or transduction with recombinant AAV vectors. These studies suggest that the D-sequence-mediated downregulation of the MHC-II gene expression may be exploited toward the development of novel AAV vectors capable of dampening the host humoral response, which has important implication in the optimal use of these vectors in human gene therapy.

Rescue of Recombinant Zika Virus from a Bacterial Artificial Chromosome cDNA Clone.

The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines and/or antivirals have underscored the urgent need to develop ZIKV reverse genetic systems to facilitate the study of ZIKV biology and the development of therapeutic and/or prophylactic approaches. However, like with other flaviviruses, the generation of ZIKV full-length infectious cDNA clones has been hampered due to the toxicity of viral sequences during its amplification in bacteria. To overcome this problem, we have developed a nontraditional approach based on the use of bacterial artificial chromosomes (BACs). Using this approach, the full-length cDNA copy of the ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) is generated from four synthetic DNA fragments and assembled into the single-copy pBeloBAC11 plasmid under the control of the human cytomegalovirus (CMV) immediate-early promoter. The assembled BAC cDNA clone is stable during propagation in bacteria, and infectious recombinant (r)ZIKV is recovered in Vero cells after transfection of the BAC cDNA clone. The protocol described here provides a powerful technique for the generation of infectious clones of flaviviruses, including ZIKV, and other positive-strand RNA viruses, particularly those with large genomes that have stability problems during bacterialpropagation.

Transduction of a Neospora caninum antigen gene into mammalian cells using a modified Bombyx mori nucleopolyhedrovirus for antibody production

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can easily enter and transduce foreign genes into mammalian cells, but these functions are difficult for Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, we investigated the induction of antibody production in mice immunized with an engineered BmNPV. The GP64 of BmNPV (BmGP64) was replaced with the GP64 of AcMNPV (AcGP64); this construct, designated BmNPVΔbgp/AcGP64, displays AcGP64 on the surface of BmNPV. The Neospora caninum antigen (NcSRS2) expression cassette, consisting of the cytomegalovirus immediate-early promoter and NcSRS2 from N. caninum, was inserted into BmNPVΔbgp/AcGP64; this construct was designated BmNPVΔbgp/AcGP64/SRS2. For comparison, AcMNPV/SRS2, which contains the same NcSRS2 expression cassette as for BmNPVΔbgp/AcGP64, was also constructed. NcSRS2 was expressed in HEK293T cells when the engineered BmNPVs were transduced at a multiplicity of infection of 150. BmNPVΔbgp/AcGP64/SRS2 induced the production of NcSRS2-specific antibodies in mice, whereas AcMNPV/SRS2 and the control BmNPV did not. These results suggest that BmNPV prepared from silkworm hemolymph induces the production of antigen-specific antibodies in immunized mice and can be used for antibody production and vaccine development.

Bluetongue Virus NS4 Protein Is an Interferon Antagonist and a Determinant of Virus Virulence.

ABSTRACTBluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of ruminants with serious consequences to both animal health and the economy. The clinical outcome of BTV infection is highly variable and dependent on a variety of factors related to both the virus and the host. In this study, we show that the BTV nonstructural protein NS4 favors viral replication in sheep, the animal species most affected by bluetongue. In addition, NS4 confers a replication advantage on the virus in interferon (IFN)-competent primary sheep endothelial cells and immortalized cell lines. We determined that in cells infected with an NS4 deletion mutant (BTV8ΔNS4), there is increased synthesis of type I IFN compared to cells infected with wild-type BTV-8. In addition, using RNA sequencing (RNA-seq), we show that NS4 modulates the host IFN response and downregulates mRNA levels of type I IFN and interferon-stimulated genes. Moreover, using reporter assays and protein synthesis assays, we show that NS4 downregulates the activities of a variety of promoters, such as the cytomegalovirus immediate-early promoter, the IFN-β promoter, and a promoter containing interferon-stimulated response elements (ISRE). We also show that the NS4 inhibitory activity on gene expression is related to its nucleolar localization. Furthermore, NS4 does not affect mRNA splicing or cellular translation. The data obtained in this study strongly suggest that BTV NS4 is an IFN antagonist and a key determinant of viral virulence. IMPORTANCE Bluetongue is one of the main infectious diseases of ruminants and is caused by bluetongue virus (BTV), an arthropod-borne virus transmitted from infected to susceptible animals by Culicoides biting midges. Bluetongue has a variable clinical outcome that can be related to both virus and host factors. It is therefore critical to understand the interplay between BTV and the host immune responses. In this study, we show that a nonstructural protein of BTV (NS4) is critical to counteract the innate immune response of the host. Infection of cells with a BTV mutant lacking NS4 results in increased synthesis of IFN-β and upregulation of interferon-stimulated genes. In addition, we show that NS4 is a virulence factor for BTV by favoring viral replication in sheep, the animal species most susceptible to bluetongue.

553. Comparision of AAV Serotypes for Gene Delivery to iPSC Derived RPE

Inherited retinal degenerations, such as retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), are characterized by progressive impairment of visual function associated with degeneration of retinal pigment epithelium (RPE) and photoreceptors. These monogenic diseases are genetically heterogeneous due to mutations in genes expressed affecting those cells. Gene therapy holds great potential for the treatment of inherited retinal diseases for which there currently is none. Efficient retinal gene transfer has been achieved with several recombinant viral vectors, including those derived from adenovirus, retrovirus, herpesvirus, and adeno-associated virus (AAV). Among these, AAV vectors appear particularly amenable to retinal gene transfer. However the success of any preclinical study depends on the availability of relevant animal models. In situations where suitable animal models are unavailable recent studies have generated proof-of concept data using personalized cell models: induced pluripotent stem cells (iPSC) derived from affected individuals and un-affected controls. With the increasing number of retinal gene therapy paradigms and recombinant vectors, in vitro bioassays characterizing vector transduction efficiency and quality are becoming increasingly important. To date, most in vitro assays using recombinant vector transduction have targeted iPSCs. To elicit additional features relevant to the disease, we differentiated iPSCs to RPE and evaluated the transduction efficiencies of a panel of AAV serotypes. Characterization of iPSC-derived RPE by qRT-PCR, immunohistochemistry, western blot analysis and flow cytometry showed the expression of typical RPE markers, phagocytic ability and gene-expression patterns similar to those of native RPE. Comparison of transduction efficiencies of different AAVs in iPSC-derived RPE was carried out using an enhanced green-fluorescent protein (eGFP) reporter gene driven by the cytomegalovirus immediate-early (CMV) promoter. At 24 to 48 hours post transduction, cells expressing GFP were identified by Typhoon scanner, and flow cytometry. Relative GFP expression was evaluated by using the software, Image-J. Of the tested AAV serotypes, AAV2 transduced iPSC-derived RPE cells most efficiently, followed by AAV7m8, AAV1 and AAV6. Differentiation into retinal neuronal cells types may require use of alternative AAV serotypes in order to obtain transduction efficiencies relevant to determination of therapeutic efficacy.

556. Comparative In Vitro Transduction Efficiency of AAV Vector Serotypes 1-9 in Different Cellular Models

Recombinant viral vectors can be useful tools for expressing transgenes in neuronal cells to study disease mechanisms and to test for therapeutic effect. Recombinant adeno-associated virus (AAV), generated from a nonpathogenic parvovirus, has become the most widely used vector in gene therapy applications in human. However, lack of suitable animal models that accurately reflect the human disease phenotype often stalls the development of treatment strategies. To overcome this problem, research is focused on generating induced pluripotent stem cells (iPSCs) from affected human subjects as in vitro models to explore the potential of gene augmentation therapy. An advantage of iPSCs is that these cells can be differentiated along a retinal cell lineage. In this study, we differentiated iPSCs into Neural Progenitor Cells (NPC) and compared the in vitro transduction efficiency of AAV serotypes 1-9. Differentiated NPCs were characterized by immunocytochemistry with the expression of NPCs specific markers (Sox2, Nestin, Pax6). Experiments were carried out using different AAVs serotypes containing the same transgene cassette: an enhanced green-fluorescent protein (eGFP) reporter gene driven by the cytomegalovirus immediate-early (CMV) promoter. At 24 to 48 hours post transduction, positive GFP expressing cells are examined by laser scanning in Typhoon. Relative GFP expression was evaluated by using the software ImageJ. In addition to the laser scanning method, flow cytometry was used to evaluate GFP expression in the cell population. Live cell imaging and flow cytometry evaluation revealed that AAV2 is the most efficient vector for in vitro transduction in NPC followed by AAV1, AAV3 and AAV6 while AAV4, AAV5, AAV7, AAV8 and AAV9 showed no transduction or extremely low transduction efficiency.

In vivo microinjection and electroporation of mouse testis.

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis. The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV). For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red fluorescence, illustrating transfection success. Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

Development of a novel DNA-launched dengue virus type 2 infectious clone assembled in a bacterial artificial chromosome

Major progress in Dengue virus (DENV) biology has resulted from the use of infectious clones obtained through reverse genetics. The construction of these clones is commonly based on high- or low-copy number plasmids, yeast artificial chromosomes, yeast-Escherichia coli shuttle vectors, and bacterial artificial chromosomes (BACs). Prokaryotic promoters have consistently been used for the transcription of these clones. The goal of this study was to develop a novel DENV infectious clone in a BAC under the control of the cytomegalovirus immediate-early promoter and to generate a virus with the fusion envelope-green fluorescent protein in an attempt to track virus infection. The transfection of Vero cells with a plasmid encoding the DENV infectious clone facilitated the recovery of infectious particles that increased in titer after serial passages in C6/36 cells. The plaque size and syncytia phenotypes of the recombinant virus were similar to those of the parental virus. Despite the observation of autonomous replication and the detection of low levels of viral genome after two passages, the insertion of green fluorescent protein and Renilla luciferase reporter genes negatively impacted virus rescue. To the best of our knowledge, this is the first study using a DENV infectious clone under the control of the cytomegalovirus promoter to facilitate the recovery of recombinant viruses without the need for in vitro transcription. This novel molecular clone will be useful for establishing the molecular basis of replication, assembly, and pathogenesis, evaluating potential antiviral drugs, and the development of vaccine candidates for attenuated recombinant viruses.

Construction and Applications of Rabbit Hemorrhagic Disease Virus Replicon

The study of rabbit hemorrhagic disease virus (RHDV) has long been hindered by the absence of an in vitro culture system. In this study, using RHDV as a model, a series of DNA-based reporter replicons were constructed in which the firefly luciferase (Fluc) gene was fused in-frame with the open reading frame of the replicon. In this construct, the Fluc gene was inserted where the coding region of viral structural protein was deleted and was under the control of a minimal cytomegalovirus (CMV) immediate-early promoter. Fluc activity analysis showed that these reporter replicons replicate efficiently in mammalian cells. On the basis of the replicon, 5′non-coding regions (5′NCR) and genome-linked protein (VPg) were deleted, and the effect on the expression of replicon was analyzed. The results showed that the expression level of Fluc was reduced in the absence of 5′NCR and VPg, suggesting that the 5′NCR and VPg may play an important role in replication and/or translation of RHDV. To further verify the speculation, we also constructed a replication deficient mutant (pRHDV-luc/Δ3D), and the impact of 5′NCR and VPg deletion on viral translation efficiency was analyzed, our results indicated that both VPg and 5′NCR were involved in RHDV translation.

Heat shock activates transgene CFTR Cl channels

Loss of transgene expression is a common problem in gene therapy and has limited the effective correction of the cystic fibrosis‐linked CFTR anion channel. Transgene CFTR expression was measured in response to heat shock (BHT, 41–43°C for 60 min) or exposure to small molecule inhibitors of DNA methyltransferases (DNMTi) using CF bronchial epithelial cells (CFBE41o‐) stably expressing wild‐type CFTR under the regulation of the cytomegalovirus (CMV) immediate‐early gene promoter. After 6 hours, BHT induced a significant increase in CFTR mRNA (2.4‐fold), CFTR‐positive cells (5‐fold), and CFTR Cl currents (ICl, 3.6‐fold). Although CFTR mRNA returned to baseline after 24 h, CFTR‐positive cells and ICl remained elevated (3.9 and 2.5‐fold, respectively). Treatment with actinomycin D blunted BHT response suggesting effects on CFTR transcription. 5‐Aza‐dC, a DNMTi, increased CFTR‐positive cells and time‐dependently activated ICl similar to that observed by BHT. A structurally unrelated DNMTi, RG‐108, mimicked effects, whereas native ICl was not affected, suggesting specific regulatory effects on vector‐induced CFTR expression. These studies imply that methylation of DNA CpG sites (likely in the CMV promoter) is a key and reversible factor modulating transgene CFTR expression.

BacMam immunization partially protects pigs against sublethal challenge with African swine fever virus

Lack of vaccines and efficient control measures complicate the control and eradication of African swine fever (ASF). Limitations of conventional inactivated and attenuated virus-based vaccines against African swine fever virus (ASFV) highlight the need to use new technologies to develop efficient and safe vaccines against this virus. With this aim in mind, in this study we have constructed BacMam-sHAPQ, a baculovirus based vector for gene transfer into mammalian cells, expressing a fusion protein comprising three in tandem ASFV antigens: p54, p30 and the extracellular domain of the viral hemagglutinin (secretory hemagglutinin, sHA), under the control of the human cytomegalovirus immediate early promoter (CMVie). Confirming its correct in vitro expression, BacMam-sHAPQ induced specific T-cell responses directly after in vivo immunization. Conversely, no specific antibody responses were detectable prior to ASFV challenge. The protective potential of this recombinant vaccine candidate was tested by a homologous sublethal challenge with ASFV following immunization. Four out of six immunized pigs remained viremia-free after ASFV infection, while the other two pigs showed similar viremic titres to control animals. The protection afforded correlated with the presence of a large number of virus-specific IFNγ-secreting T-cells in blood at 17days post-infection. In contrast, the specific antibody levels observed after ASFV challenge in sera from BacMam-sHAPQ immunized pigs were indistinguishable from those found in control pigs. These results highlight the importance of the cellular responses in protection against ASFV and point towards BacMam vectors as potential tools for future vaccine development.

Genetic manipulation of myoblasts and a novel primary myosatellite cell culture system: comparing and optimizing approaches

The genetic manipulation of skeletal muscle cells in vitro is notoriously difficult, especially when using undifferentiated muscle cell lines (myoblasts) or primary muscle stem cells (myosatellites). We therefore optimized methods of gene transfer by overexpressing green fluorescent protein (GFP) in mouse C2C12 cells and in a novel system, primary rainbow trout myosatellite cells. A common lipid-based transfection reagent was used (Lipofectamine 2000) along with three different viral vectors: adeno-associated virus serotype 2 (AAV2), baculovirus (BAC) and lentivirus. Maximal transfection efficiencies of 49% were obtained in C2C12 cells after optimizing cell density and reagent : DNA ratio, although the GFP signal rapidly dissipated with proliferation and was not maintained with differentiation. The transduction efficiency of AAV2 was optimized to 65% by extending incubation time and decreasing cell density, although only 30% of cells retained expression after passing. A viral comparison revealed that lentivirus was most efficient at transducing C2C12 myoblasts as 97% of cells were transduced with only 10(6) viral genomes (vg) compared to 54% with 10(8) vg AAV2 and 23% with 10(9) vg BAC. Lentivirus also transduced 90% of primary trout myosatellites compared to 1-10% with AAV2 and BAC. The phosphoglycerate kinase 1 (pgk) promoter was 10-fold more active than the cytomegalovirus immediate-early promoter in C2C12 cells and both were effective in trout myosatellites. Maximal transduction of C2C12 myotubes was achieved by differentiating myoblasts previously transduced with lentivirus and the pgk promoter. Thus, our optimized protocol proved highly effective in diverse muscle cell systems and could therefore help overcome a common technological barrier.

The Size and Conservation of a Coiled-coil Structure in the Ectodomain of Human BST-2/Tetherin Is Dispensable for Inhibition of HIV-1 Virion Release

BST-2/CD317/tetherin is a host factor that inhibits HIV-1 release and is counteracted by HIV-1 Vpu. Structural studies indicate that the BST-2 ectodomain assumes a coiled-coil conformation. Here we studied the role of the BST-2 ectodomain for tethering function. First, we addressed the importance of the length and structure of the ectodomain by adding or substituting heterologous coiled-coil or non-coiled-coil sequences. We found that extending or replacing the BST-2 ectodomain using non-coiled-coil sequences resulted in loss of BST-2 function. Doubling the size of the BST-2 ectodomain by insertion of a heterologous coiled-coil motif or substituting the BST-2 coiled-coil domain with a heterologous coiled-coil motif maintained tethering function. Reductions in the size of the BST-2 coiled-coil domain were tolerated as well. In fact, deletion of the C-terminal half of the BST-2 ectodomain, including a series of seven consecutive heptad motifs did not abolish tethering function. However, slight changes in the positioning of deletions affecting the relative placing of charged or hydrophobic residues on the helix severely impacted the functional properties of BST-2. Overall, we conclude that the size of the BST-2 ectodomain is highly flexible and can be reduced or extended as long as the positioning of residues important for the stability of the dimer interface is maintained.

Reconstructed Adeno-Associated Virus with the Extracellular Domain of Murine PD-1 Induces Antitumor Immunity

The negative signaling provided by interactions of the co-inhibitory molecule, programmed death-1 (PD-1), and its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), is a critical mechanism contributing to tumor evasion; blockade of this pathway has been proven to enhance cytotoxic activity and mediate antitumor therapy. Here we evaluated the anti-tumor efficacy of AAV-mediated delivery of the extracellular domain of murine PD-1 (sPD-1) to a tumor site. An rAAV vector was constructed in which the expression of sPD-1, a known negative regulator of TCR signals, is driven by human cytomegalovirus immediate early promoter (CMV-P), using a triple plasmid transfection system. Tumor-bearing mice were then treated with the AAV/sPD1 construct and expression of sPD-1 in tumor tissues was determined by semi quantitative RT-PCR, and tumor weights and cytotoxic activity of splenocytes were measured. Analysis of tumor homogenates revealed sPD-1 mRNA to be significantly overexpressed in rAAV/sPD-1 treated mice as compared with control levels. Its use for local gene therapy at the inoculation site of H22 hepatoma cells could inhibit tumor growth, also enhancing lysis of tumor cells by lymphocytes stimulated specifically with an antigen. In addition, PD-1 was also found expressed on the surfaces of activated CD8+ T cells. This study confirmed that expression of the soluble extracellular domain of PD-1 molecule could reduce tumor microenvironment inhibitory effects on T cells and enhance cytotoxicity. This suggests that it might be a potential target for development of therapies to augment T-cell responses in patients with malignancies.

A new finding concerning adenoviral-mediated gene transfer: A high-level, cell-specific transgene expression in the neural stem cells of adult mice

Adenoviruses are highly efficient vectors for gene transfer into brain cells. Restricting transgene expression to specific cell types and maintaining long-term expression are major goals for adenoviral-mediated gene transfer in the central nervous system. Human adenovirus type 5 (Ad5) mediated transgene expression is described under the control of the murine cytomegalovirus (mCMV) immediate-early promoter. It was found that the neural stem cells in the dentate subgranular zone were transduced preferentially, minimal neurons were transduced in the granule cell layer of the dentate gyrus, no EGFP was detected in the pyramidal cell layers of CAl to CA3 area and EGFP activity can be detected for 2 months after infection. Therefore, the mCMV-adenoviral vectors can be used both for studying the function of various genes in the differentiation of neural stem cells and, ultimately, for gene therapy or to modulate specific gene expression.

C-terminal Hydrophobic Region in Human Bone Marrow Stromal Cell Antigen 2 (BST-2)/Tetherin Protein Functions as Second Transmembrane Motif

BST-2/CD317/HM1.24/tetherin is a host factor that inhibits the release of HIV-1 and other enveloped viruses. Structurally, tetherin consists of an N-terminal transmembrane (TM) region, a central coiled coil motif, and a putative C-terminal glycosylphosphatidylinositol (GPI) anchor motif. A current working model proposes that BST-2 inhibits virus release by physically tethering viral particles to the cell surface via its TM motif and GPI anchor. Here we analyzed the functional importance of the C-terminal GPI anchor motif in BST-2. We replaced the GPI anchor motif in BST-2 with the TM regions of several surface markers and found that the TM motifs of CD40 and transferrin receptor, but not that of CD45, could functionally substitute for a GPI anchor in BST-2. Conversely, replacing the TM region of CD4 by the putative GPI anchor signal of human BST-2 resulted in proper membrane targeting and surface expression of the chimeric protein, indicating that the BST-2 GPI anchor signal can function as a bona fide TM region. In fact, attempts to demonstrate GPI anchor modification of human BST-2 by biochemical methods failed. Our results demonstrate that the putative C-terminal GPI anchor motif in human BST-2 fulfills the requirements of a bona fide TM motif, leading us to propose that human BST-2 may in fact contain a second TM segment rather than a GPI anchor.