Human Cytomegalovirus Promoter Research Articles

Novel Enhanced Mammalian Cell Transient Expression Vector via Promoter Combination.

During the emergence of infectious diseases, evaluating the efficacy of newly developed vaccines requires antigen proteins. Available methods enhance antigen protein productivity; however, structural modifications may occur. Therefore, we aimed to construct a novel transient overexpression vector capable of rapidly producing large quantities of antigenic proteins in mammalian cell lines. This involved expanding beyond the exclusive use of the human cytomegalovirus (CMV) promoter, and was achieved by incorporating a transcriptional enhancer (CMV enhancer), a translational enhancer (woodchuck hepatitis virus post-transcriptional regulatory element), and a promoter based on the CMV promoter. Twenty novel transient expression vectors were constructed, with the vector containing the human elongation factor 1-alpha (EF-1a) promoter showing the highest efficiency in expressing foreign proteins. This vector exhibited an approximately 27-fold higher expression of enhanced green fluorescent protein than the control vector containing only the CMV promoter. It also expressed the highest level of severe acute respiratory syndrome coronavirus 2 receptor-binding domain protein. These observations possibly result from the simultaneous enhancement of the transcriptional activity of the CMV promoter and the human EF-1a promoter by the CMV enhancer. Additionally, the synergistic effect between the CMV and human EF-1a promoters likely contributed to the further enhancement of protein expression.

Construction of cytomegalovirus promoter-driven gene expression system in Laodelphax striatellus.

The small brown planthopper (SBPH, Laodelphax striatellus) is a significant rice pest, responsible for transmitting rice stripe virus (RSV) in a persistent and propagative manner. RSV is one of the most detrimental rice viruses, causing rice stripe disease, which results in considerable loss of rice grain yield. While RNA interference and gene knockout techniques have enabled gene downregulation in SBPH, no system currently exists for the overexpression of endogenous or exogenous genes. Consequently, the development of a protein expression system for SBPH is imperative to serve as a technical foundation for pest control and gene function investigations. This study aimed to construct an expression vector using the promoter of the constitutive-expressed tubulin gene of SBPH, and promoter of human cytomegalovirus (CMV). Fluorescence experiments demonstrated that both tubulin and CMV promoter could drive green fluorescent protein (GFP) expression in SBPH, and could also facilitate the expression of a nucleocapsid protein (NP) -GFP fusion protein containing viral NP with comparable efficiency. Through expression vector optimization, we have identified that the 3 tandem CMV promoters display a significantly higher promoter activity compared with both the 2 tandem CMV promoters and the single CMV promoter. In addition, the incorporation of Star polycation nanoparticles significantly enhanced the expression efficiency in SBPH. These results provide a promising technical platform for investigating gene functions in SBPH.

Local Multiple-site Injections of a Plasmid Encoding Human MnSOD Mitigate Radiation-induced Skin Injury by Inhibiting Ferroptosis.

Most patients who undergo radiotherapy develop radiation skin injury, for which effective treatment is urgently needed. MnSOD defends against reactive oxygen species (ROS) damage and may be valuable for treating radiation-induced injury. Here, we (i) investigated the therapeutic and preventive effects of local multiple-site injections of a plasmid, encoding human MnSOD, on radiation-induced skin injury in rats and (ii) explored the mechanism underlying the protective effects of pMnSOD. The recombinant plasmid (pMnSOD) was constructed with human cytomegalovirus (CMV) promoter and pUC-ori. The protective effects of pMnSOD against 20-Gy X-ray irradiation were evaluated in human keratinocytes (HaCaT cells) by determining cell viability, ROS levels, and ferroptosisrelated gene expression. In therapeutic treatment, rats received local multiple-site injections of pMnSOD on days 12, 19, and 21 after 40-Gy γ-ray irradiation. In preventive treatment, rats received pMnSOD injections on day -3 pre-irradiation and on day 4 post-irradiation. The skin injuries were evaluated based on the injury score and pathological examination, and ferroptosis-related gene expression was determined. In irradiated HaCaT cells, pMnSOD transfection resulted in an increased SOD2 expression, reduced intracellular ROS levels, and increased cell viability. Moreover, GPX4 and SLC7A11 expression was significantly upregulated, and erastin-induced ferroptosis was inhibited in HaCaT cells. In the therapeutic and prevention treatment experiments, pMnSOD administration produced local SOD protein expression and evidently promoted the healing of radiation-induced skin injury. In the therapeutic treatment experiments, the injury score in the high-dose pMnSOD group was significantly lower than in the PBS group on day 33 post-irradiation (1.50 vs. 2.80, P < 0.05). In the prevention treatment experiments, the skin injury scores were much lower in the pMnSOD administration groups than in the PBS group from day 21 to day 34. GPX4, SLC7A11, and Bcl-2 were upregulated in irradiated skin tissues after pMnSOD treatment, while ACSL4 was downregulated. The present study provides evidence that the protective effects of MnSOD in irradiated HaCaT cells may be related to the inhibition of ferroptosis. The multi-site injections of pMnSOD had clear therapeutic and preventive effects on radiation-induced skin injury in rats. pMnSOD may have therapeutic value for the treatment of radiation-induced skin injury.

Repression of the major immediate early promoter of human cytomegalovirus allows transcription from an alternate promoter.

Following infection, the human cytomegalovirus (HCMV) genome becomes rapidly associated with host histones which can contribute to the regulation of viral gene expression. This can be seen clearly during HCMV latency where silencing of the major immediate early promoter (MIEP), normally responsible for expression of the key lytic proteins IE72 and IE86, is mediated by histone methylation and recruitment of heterochromatin protein 1. Crucially, reversal of these histone modifications coupled with histone acetylation drives viral reactivation which can be blocked with specific histone acetyltransferase inhibitors (HATi). In lytic infection, a role for HATi is less clear despite the well-established enhancement of viral replication observed with histone deacetylase inhibitors. Here we report that a number of different broad-acting HATi have a minor impact on viral infection and replication during lytic infection with the more overt phenotypes observed at lower multiplicities of infection. However, specific analyses of the regulation of major immediate early (MIE) gene expression reveal that the HATi C646, which targets p300/CBP, transiently repressed MIE gene expression via inhibition of the MIEP but by 24 h post-infection MIE gene expression was rescued due to compensatory activation of an alternative IE promoter, ip2. This suggested that silencing of the MIEP promoted alternative ip2 promoter activity in lytic infection and, consistent with this, ip2 transcription is impaired in cells infected with a recombinant HCMV that does not auto-repress the MIEP at late times of infection. Furthermore, inhibition of the histone methyltransferases known to be responsible for auto-repression is similarly inhibitory to ip2 transcription in wild-type infected cells. We also observe that these discrete transcriptional activities of the MIEP and ip2 promoter are also reflected in reactivation; essentially in cells where the MIEP is silenced, ip2 activity is easier to detect at very early times post-reactivation whereas in cells where robust activation of the MIEP is observed ip2 transcription is reduced or delayed. Finally, we observe that inhibition of pathways demonstrated to be important for reactivation of HCMV in dendritic cells, e.g. in response to IL-6, are preferentially important for activation of the MIEP and not the ip2 promoter. Together, these data add to the hypothesis that the existence of multiple promoters within the MIE region of HCMV can drive reactivation in a cell type- and ligand-specific manner and also suggest that inter-dependent regulatory activity between the two promoters exists.

NFκB and Cyclic AMP Response Element Sites Mediate the Valproic Acid and UL138 Responsiveness of the Human Cytomegalovirus Major Immediate Early Enhancer and Promoter.

The major immediate early enhancer and promoter (MIEP) of human cytomegalovirus (HCMV) drives the transcription of the immediate early one (IE1) and IE2 genes, whose encoded proteins stimulate productive, lytic replication. The MIEP is activated by the virally encoded and tegument-delivered pp71 protein at the start of de novo lytic infections of fully differentiated cells. Conversely, the MIEP is silenced at the start of de novo latent infections within incompletely differentiated myeloid cells in part because tegument-delivered pp71 is sequestered in the cytoplasm in these cells, but also by viral factors that repress transcription from this locus, including the UL138 protein. During both modes of infection, MIEP activity can be increased by the histone deacetylase inhibitor valproic acid (VPA); however, UL138 inhibits the VPA-responsiveness of the MIEP. Here, we show that two families of cellular transcription factors, NF-κB and cAMP response element-binding protein (CREB), together control the VPA-mediated activation and UL138-mediated repression of the HCMV MIEP. IMPORTANCE Artificial regulation of the HCMV MIEP, either activation or repression, is an attractive potential means to target the latent reservoirs of virus for which there is currently no available intervention. The MIEP could be repressed to prevent latency reactivation or induced to drive the virus into the lytic stage that is visible to the immune system and inhibited by multiple small-molecule antiviral drugs. Understanding how the MIEP is regulated is a critical part of designing and implementing either strategy. Our revelation here that NF-κB and CREB control the responsiveness of the MIEP to the viral UL138 protein and the FDA-approved drug VPA could help in the formulation and execution of promoter regulatory strategies against latent HCMV.

Latency-associated upregulation of SERBP1 is important for the recruitment of transcriptional repressors to the viral major immediate early promoter of human cytomegalovirus during latent carriage.

Suppression of human cytomegalovirus (HCMV) major immediate early gene (IE) expression from the viral major immediate early promoter (MIEP) is known to be crucial for the establishment and maintenance of HCMV latency in myeloid progenitor cells and their undifferentiated derivatives. This suppression of the MIEP during latent infection is known to result from epigenetic histone modification imparting a repressive chromatin structure around the MIEP in undifferentiated myeloid cells. In contrast, reactivation, resulting from, e.g., myeloid cell differentiation, is associated with activatory chromatin marks around the MIEP. Recently, recruitment of the transcriptional repressor SETDB1, via KAP1, to latent HCMV genomes was shown to be involved in latency-associated MIEP suppression in CD34+ progenitor cells. KAP1 is also known to associate with Chromodomain-helicase-DNA-binding protein 3 (CHD3) as part of the NuRD complex which can aid transcriptional silencing. We now show that the cellular protein Plasminogen activator inhibitor 1 RNA-binding protein (SERBP1), a known interactor of CHD3, is significantly upregulated during HCMV latency and that this protein is required for MIEP suppression during latent infection of myeloid cells. We further show that SERBP1 mediates CHD3 association with the MIEP as well as KAP1 association with viral genomic DNA. We suggest that SERBP1 functions as a scaffold protein to recruit transcriptional repressors to the latent viral genome and to mediate transcriptional silencing of the MIEP during latent carriage.

Bioinformatic Design of Dendritic Cell-Specific Synthetic Promoters

Next-generation DNA vectors for cancer immunotherapies and vaccine development require promoters eliciting predefined transcriptional activities specific to target cell types, such as dendritic cells (DCs), which underpin immune response. In this study, we describe the de novo design of DC-specific synthetic promoters via in silico assembly of cis-transcription factor response elements (TFREs) that harness the DC transcriptional landscape. Using computational genome mining approaches, candidate TFREs were identified within promoter sequences of highly expressed DC-specific genes or those exhibiting an upregulated expression during DC maturation. Individual TFREs were then screened in vitro in a target DC line and off-target cell lines derived from skeletal muscle, fibroblast, epithelial, and endothelial cells using homotypic (TFRE repeats in series) reporter constructs. Based on these data, a library of heterotypic promoter assemblies varying in the TFRE composition, copy number, and sequential arrangement was constructed and tested in vitro to identify DC-specific promoters. Analysis of the transcriptional activity and specificity of these promoters unraveled underlying design rules, primarily TFRE composition, which govern the DC-specific synthetic promoter activity. Using these design rules, a second library of exclusively DC-specific promoters exhibiting varied transcriptional activities was generated. All DC-specific synthetic promoter assemblies exhibited >5-fold activity in the target DC line relative to off-target cell lines, with transcriptional activities ranging from 8 to 67% of the nonspecific human cytomegalovirus (hCMV-IE1) promoter. We show that bioinformatic analysis of a mammalian cell transcriptional landscape is an effective strategy for de novo design of cell-type-specific synthetic promoters with precisely controllable transcriptional activities.

Generation of duck Tembusu virus using a simple reverse genetic system in duck embryo fibroblast cells

Outbreaks of duck Tembusu virus (DTMUV) have caused serious economic losses in China since 2010. In this study, an infectious clone of the DTMUV BZ-2010strain, isolated from layer cherry duck in China, was constructed using the bacterium-free infectious subgenomic-amplicons method. The subgenomic-amplicons of the human cytomegalovirus promoter (pCMV) at the 5′ terminus of the first DNA fragment, the entire genome of DTMUV, and the hepatitis delta ribozyme followed by the simian virus 40 polyadenylation signal (HDR/SV40pA) at the 3′ terminus of the last DNA fragment were synthesized and amplified by PCR in three DNA fragments. The pCMV and HDR/SV40pA were used to drive the viral RNA transcription and generate a full-length RNA transcript of the virus, and were found to be effective in reassembling DTMUV in duck embryo fibroblast cells. The RNA transcripts from the infection clone were infectious in duck embryo fibroblast cells, generating the reconstituted DTMUV. This study provided a valuable reverse genetic tool for the further study DTMUV pathogenesis.

New function of a well‐known promoter: Enhancer activity of minimal CMV promoter enables efficient dual‐cassette transgene expression

Co-expression of multiple genes in single vectors has achieved varying degrees of success by employing two promoters and/or application of viral 2A-peptide or the internal ribosome entry-site (IRES). However, promoter interference, potential functional-interruption of expressed-proteins by 2A-generated residual peptides or weaker translation of IRES-mediated downstream genes has curtailed their utilization. Thus, there is the need for single vectors that robustly express multiple proteins for enhanced gene therapy applications. We engineered lentiviral-vectors for dual-cassette expression of green fluorescent protein and mCherry in uni- or bidirectional architectures using the short-version (Es) of elongation factor 1α (EF) promoter and simian virus 40 promoter (Sv). The regulatory function of a core fragment (cC) from human cytomegalovirus promoter was investigated with cell-lineage specificity in NIH3T3 (fibroblast) and hematopoietic cell lines U937 (monocyte/macrophage), LCL (lymphoid), DAMI (megakaryocyte) and MEL (erythroid). The cC element in reverse-orientation not only boosted upstream Es promoter to levels comparable to full-length EF in DAMI, U937 and 3T3 cells, but also blocked the suppression of downstream Sv promoter by Es in U937 and 3T3 cells with further improved Sv activity in DAMI cells. Such lineage-restricted up-regulation is likely attributed to two protein-binding domains of cC and diverse expression of related factors in different cell types for enhancer and terminator activities, but not spacing function. Such a newly developed dual-cassette vector could be advantageous, particularly in hematopoietic cell-mediated gene/cancer therapy, by allowing for independent and robust co-expression of therapeutic gene(s) and/or a selectable gene or imaging marker in the same cells.

High-throughput 5\u2032 UTR engineering for enhanced protein production in non-viral gene therapies

Despite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major technical challenge. Here, we develop a high-throughput strategy to design, screen, and optimize 5′ UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identify naturally occurring 5′ UTRs with high translation efficiencies and use this information with in silico genetic algorithms to generate synthetic 5′ UTRs. A total of ~12,000 5′ UTRs are then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identify three synthetic 5′ UTRs that outperform commonly used non-viral gene therapy plasmids in expressing protein payloads. In summary, we demonstrate that high-throughput screening of 5′ UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies.

Development of strategies to modulate gene expression of angiogenesis-related molecules in the retina.

Intravitreal anti-vascular endothelial growth factor agents are the gold standard treatment of ocular neovascular diseases. However, their short-term efficacy implies frequent intravitreal injections. Gene therapy has the ability to provide longer duration of the therapeutic effect. We have previously described the effectiveness of the self-replicating episomal vector, pEPito, in long-term gene expression in mouse retina. In this study, we evaluated different constructs to overexpress pigment epithelium-derived factor (PEDF), an angiogenesis inhibitor, and simultaneously, to silence placental growth factor (PlGF), a key player in neovascularization. We employed the human cytomegalovirus promoter to drive the expression of PEDF and PlGF shRNA, in conjunction with cis-acting ribozymes, using pEPito as expressing vector. Our results demonstrated that the non-viral systems were able to efficiently promote a sustained increase of the PEDF: PlGF ratio in the mice retina, decreased in pathological conditions. This innovative approach could open avenues for the development of new therapeutic strategies.

Lack of methylation on transgene leads to high level and persistent transgene expression in induced pluripotent stem cells

Short-lived therapeutic gene expression in mammalian cells by DNA methylation is one of the major challenges in gene therapy. In this study, we assessed the implication of DNA methylation on the duration of GFP expression in mouse embryonic stem (ES) and mouse induced pluripotent stem (iPS) cells. The cells were transduced with lentivirus (LV) carrying green fluorescent protein (GFP) driven by either human elongation factor (EF1α) or cytomegalovirus (CMV) promoter. Transduced iPS cells exhibited higher percentage of GFP+ cells with persistent mean fluorescent intensity than transduced ES cells. Analysis on the integrated copy of transgene in the population of the transduced cells demonstrated similar copy number. However, significant increase in GFP intensity following 5-azaC treatment was observed in transduced ES cells only, suggesting the influence of DNA methylation in transgene silencing. Subsequent DNA methylation analysis showed that the promoter and the GFP region of the provirus in iPS cells had negligible methylation profile compared to transduced ES cells. Interestingly, sustained transgene expression was observed upon directed differentiation of transduced iPS cells towards CD34+ CD45+ cells. Hence, this study has shown that favourable transgene activity from lentiviral transduced iPS cells was due to the lack of methylation at the proviral regions.

Transcriptional activities of human elongation factor-1α and cytomegalovirus promoter in transgenic dogs generated by somatic cell nuclear transfer.

Recent advances in somatic cell nuclear transfer (SCNT) in canines facilitate the production of canine transgenic models. Owing to the importance of stable and strong promoter activity in transgenic animals, we tested human elongation factor 1α (hEF1α) and cytomegalovirus (CMV) promoter sequences in SCNT transgenic dogs. After transfection, transgenic donor fibroblasts with the hEF1α-enhanced green fluorescence protein (EGFP) transgene were successfully isolated using fluorescence-activated cell sorting (FACS). We obtained four puppies, after SCNT, and identified three puppies as being transgenic using PCR analysis. Unexpectedly, EGFP regulated by hEF1α promoter was not observed at the organismal and cellular levels in these transgenic dogs. EGFP expression was rescued by the inhibition of DNA methyltransferases, implying that the hEF1α promoter is silenced by DNA methylation. Next, donor cells with CMV-EGFP transgene were successfully established and SCNT was performed. Three puppies of six born puppies were confirmed to be transgenic. Unlike hEF1α-regulated EGFP, CMV-regulated EGFP was strongly detectable at both the organismal and cellular levels in all transgenic dogs, even after 19 months. In conclusion, our study suggests that the CMV promoter is more suitable, than the hEF1α promoter, for stable transgene expression in SCNT-derived transgenic canine model.

Microfluidic generation of lipid nanoparticles to facilitate DNA entry into human mast cells

Abstract Mast cells are highly granulated immune cells that can be targeted for transfection using lipid nanoparticles (LNP). Lipid nanoparticles (LNPs) were composed of cholesterol and 1-Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, the ionizable lipid 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane, and the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene-glycol)-2000] using microfluidic mixing and used to encapsulate a plasmid encoding enhanced green fluorescent protein (EGFP) under the control of a human cytomegalovirus promoter (pEGFP-C1). Encapsulation efficiency was determined using PicoGreen and LNP size was measured using dynamic light scattering. Human mast cells-1 (HMC-1) were transfected with loaded or empty LNPs in the presence or absence of apolipoprotein E (ApoE) for 24, 48 or 72 hours in medium. Cells were analyzed by fluorescence microscopy and flow cytometry. LNPs were approximately 100 nm in diameter (ideal) and efficiently encapsulated plasmids pEGFP-C1 (75.38%). ApoE4 appeared to slightly augment (&amp;gt;10%) LNP uptake after 24 hr. Our study demonstrates that LNPs generated by microfluidic mixing efficiently encapsulate a plasmid payload and can be used to successfully transfect human mast cells. Carrier molecules such as ApoE can be used to augment LNP uptake by mast cells. Further functionalization and optimization of these LNPs can provide powerful new tools for targeting other immune cells.

Alternative promoters drive human cytomegalovirus reactivation from latency

Reactivation from latency requires reinitiation of viral gene expression and culminates in the production of infectious progeny. The major immediate early promoter (MIEP) of human cytomegalovirus (HCMV) drives the expression of crucial lytic cycle transactivators but is silenced during latency in hematopoietic progenitor cells (HPCs). Because the MIEP has poor activity in HPCs, it is unclear how viral transactivators are expressed during reactivation. It has been presumed that viral gene expression is reinitiated via de-repression of the MIEP. We demonstrate that immediate early transcripts arising from reactivation originate predominantly from alternative promoters within the canonical major immediate early locus. Disruption of these intronic promoters results in striking defects in re-expression of viral genes and viral genome replication in the THP-1 latency model. Furthermore, we show that these promoters are necessary for efficient reactivation in primary CD34+ HPCs. Our findings shift the paradigm for HCMV reactivation by demonstrating that promoter switching governs reactivation from viral latency in a context-specific manner.

Novel Promoters Derived from Chinese Hamster Ovary Cells via In Silico and In Vitro Analysis.

For the industrial production of recombinant proteins in mammalian cell lines, a high rate of gene expression is desired. Therefore, strong viral promoters are commonly used. However, these have several drawbacks as they override cellular responses, are not integrated into the cellular network, and thus can induce stress and potentially epigenetic silencing. Endogenous promoters potentially have the advantage of a better response to cellular state and thus a lower stress level by uncontrolled overexpression of the transgene. Such fine-tuning is typically achieved by endogenous enhancers and other regulatory elements, which are difficult to identify purely based on the genomic sequence. Here, Chinese hamster ovary (CHO) endogenous promoters and enhancers are identified using histone marks and chromatin states, ranked based on expression level and tested for normalized promoter strength. Successive truncation of these promoters at the 5'- and 3'-end as well as the combination with enhancers are identified in the vicinity of the promoter sequence further enhance promoter activity up to threefold. In an initial screen within stable cell lines, the strongest CHO promoter appears to be more stable than the human cytomegalovirus promoter with enhancer, making it a promising candidate for recombinant protein production and cell engineering applications. A deeper understanding of promoter functionality and response elements will be required to take full advantage of such promoters for cell engineering, in particular, for multigene network engineering applications.

ZB transposon and chicken vasa homologue (Cvh) promoter interact to increase transfection efficiency of primordial germ cells in vivo

ABSTRACT 1. In order to increase the efficiency of generating transgenic chicken, this trial focused on two points: primordial germ cells (PGCs）transfection in vivo and a germline-specific promoter. 2. In order to transfect PGCs in vivo, two plasmids (pZB-CAG-GFP, pCMV-ZB）were co-injected into chicken embryos via the subgerminal cavity at Hamburger and Hamilton (HH) stage 2–3 or via blood vessel at HH stage 13–14. Results showed that the percentage of GFP+ embryos, viability and hatching rate of embryos injected at HH stage 13–14 were significantly higher than that at HH stage 2–3. 3. Two plasmid transposon systems were used for chicken embryo micro-injections. The donor plasmid, with a green fluorescent protein (GFP) reporter gene, was mediated by the ZB transposon. The helper plasmid was a transposase expression vector driven by the promoter of the chicken vasa homologue (Cvh) gene or Human cytomegalovirus (CMV) promoter. Results showed that 60.98% of gonads in Cvh group expressed GFP, which was 52.50% higher than seen in the CMV group. Only gonad tissue from the Cvh group showed any GFP signal, whereas both gonads and other tissues in the CMV group showed green fluorescence. 4. The data suggested that ZB transposon-mediated gene transfer was efficient for transfecting PGCs in vivo; the Cvh promoter drove the transposase gene specifically in the germline and increased the efficiency of germline transmission. Blood vessels injection at HH stage 13–14 may be a more efficient route for PGCs transfection in vivo.

An engineered mouse embryonic stem cell model with survivin as a molecular marker and EGFP as the reporter for high throughput screening of embryotoxic chemicals in vitro.

Embryonic stem cell test (EST) is the only generally accepted in vitro method for assessing embryotoxicity without animal sacrifice. However, the implementation and application of EST for regulatory embryotoxicity screening are impeded by its technical complexity, long testing period, and limited endpoint data. In this study, a high throughput embryotoxicity screening based on mouse embryonic stem cells (mESCs) expressing enhanced green fluorescent protein (EGFP) driven by a human survivin promoter and a human cytomegalovirus promoter, respectively, was developed. These EGFP expressing mESCs were cultured in three-dimensional (3D) fibrous scaffolds in microbioreactors on a multiwell plate with EGFP fluorescence signals as cell responses to chemicals monitored noninvasively in a high throughput manner. Nine chemicals with known developmental toxicity were used to validate the survivin-based embryotoxicity assay, which showed that strongly embryotoxic compounds such as 5-fluorouracil, retinoic acid, and methotrexate downregulated survivin expression by more than 50% in 3 days, while weakly embryotoxic compounds such as boric acid, methoxyacetic acid, and tetracyclin showed modest downregulation effect and nonembryotoxic saccharin, penicillin G, and acrylamide had negligible downregulation effect on survivin expression, confirming that survivin can be used as a molecular endpoint for high throughput screening of embryotoxicants. The potential developmental toxicity of three Chinese herbal medicines were also evaluated using this assay, demonstrating its application in in vitro developmental toxicity test for drug safety assessment.

A double recombinant herpes virus of turkeys for the protection of chickens against Newcastle, infectious laryngotracheitis and Marek’s diseases

ABSTRACTA double recombinant strain of herpes virus of turkeys (HVT) was constructed that contains the fusion (F) gene from Newcastle disease virus (NDV) and the gD plus gI genes from infectious laryngotracheitis virus (ILTV) inserted into a non-essential region of the HVT genome. Expression of the F protein was controlled by a human cytomegalovirus promoter, whereas expression of gD plus gI was driven by an ILTV promoter. The double recombinant vaccine virus (HVT-NDV-ILT) was fully stable genetically and phenotypically following extended passage in cell culture and infection of chickens. Safety of the vaccine virus was confirmed by overdose and backpassage studies in specific-pathogen-free chickens. Chickens vaccinated with a single dose of HVT-NDV-ILT administered by the in ovo route were highly protected from challenge with the velogenic NDV (GB Texas), ILTV (LT 96-3) and Marek’s disease virus (GA 5) strains (97%, 94% and 97%, respectively). Similarly, chickens vaccinated with a single dose by subcutaneous (SC) route at 1 day of age were highly protected from challenge with the same three viruses (100%, 100%, and 88%, respectively). The protection level of a single dose given by in ovo or SC route against challenge with a virulent Marek’s disease virus strain demonstrates that insertion of multiple genes from two different pathogens within the HVT genome had no adverse effect on the capacity of HVT to protect against Marek’s disease. These results demonstrate that HVT-NDV-ILT is a safe and efficacious vaccine for simultaneous control of NDV, ILTV and Marek’s diseases.

Genome-wide analysis of regulatory G-quadruplexes affecting gene expression in human cytomegalovirus.

G-quadruplex (G4), formed by repetitive guanosine-rich sequences, is known to play various key regulatory roles in cells. Herpesviruses containing a large double-stranded DNA genome show relatively higher density of G4-forming sequences in their genomes compared to human and mouse. However, it remains poorly understood whether all of these sequences form G4 and how they play a role in the virus life cycle. In this study, we performed genome-wide analyses of G4s present in the putative promoter or gene regulatory regions of a 235-kb human cytomegalovirus (HCMV) genome and investigated their roles in viral gene expression. We evaluated 36 putative G4-forming sequences associated with 20 genes for their ability to form G4 and for the stability of G4s in the presence or absence of G4-stabilizing ligands, by circular dichroism and melting temperature analyses. Most identified sequences formed a stable G4; 28 sequences formed parallel G4s, one formed an antiparallel G4, and four showed mixed conformations. However, when we assessed the effect of G4 on viral promoters by cloning the 20 putative viral promoter regions containing 36 G4-forming sequences into the luciferase reporter and monitoring the expression of luciferase reporter gene in the presence of G4-stabilizing chemicals, we found that only 9 genes were affected by G4 formation. These results revealed promoter context-dependent gene suppression by G4 formation. Mutational analysis of two potential regulatory G4s also demonstrated gene suppression by the sequence-specific G4 formation. Furthermore, the analysis of a mutant virus incapable of G4 formation in the UL35 promoter confirmed promoter regulation by G4 in the context of virus infection. Our analyses provide a platform for assessing G4 functions at the genomic level and demonstrate the properties of the HCMV G4s and their regulatory roles in viral gene expression.