Early Genes ICP4 Research Articles

Global exploration of the metabolic requirements of gallid alphaherpesvirus 1.

Although therapeutics targeting viral metabolic processes have been considered as promising strategies to treat herpesvirus infection, the metabolic requirements of gallid alphaherpesvirus 1 (ILTV), which is economically important to the poultry industry worldwide, remain largely unknown. Using the ILTV-susceptible but nonpermissive chicken cell line DF-1 and the ILTV-permissive chicken cell line LMH as models, the present study explored the metabolic requirements of ILTV by global transcriptome analysis and metabolome assays of ILTV infected cell lines in combination with a set of functional validations. The extensive metabolic exploration demonstrated that ILTV infection tended to promote a metabolic shift from glycolysis to fatty acid (FA) and nucleotide biosynthesis and utilizes glutamine independently of glutaminolysis, without significant general effect on the TCA cycle. In addition, different metabolic pathways were found to be required for distinct stages of ILTV replication. Glucose and glutamine were required for the transcription of viral immediate early gene ICP4 and subsequent steps of viral replication. However, FA synthesis was essential for assembly but not required for other upstream steps of ILTV replication. Moreover, the metabolic requirements of ILTV infection revealed in chicken cell lines were further validated in chicken primary cells isolated from chicken embryo kidneys and chicken embryo livers. The present study, to the best of our knowledge, provides the first global metabolic profile of animal herpesviruses and illustrates the main characteristics of the metabolic program of ILTV.

Antiviral Properties of R. tanguticum Nanoparticles on Herpes Simplex Virus Type I In Vitro and In Vivo.

Herpes simplex virus type 1 (HSV-1), an enveloped DNA virus, plays a key role in varieties of diseases including recurrent cold sores, keratoconjunctivitis, genital herpes and encephalitis in humans. Great efforts have been made in developing more effective and less side-effects anti-herpes simplex virus agents, including traditional Chinese herbal medicines. In the present study, we evaluated the antiviral efficacy of Rheum tanguticum nanoparticles against HSV-1 in vitro and in vivo. R. tanguticum nanoparticles could inactivate the HSV-1 virions and block the viral attachment and entry into cells. Time-of-addition assay indicated that R. tanguticum nanoparticles could interfere with the entire phase of viral replication. Besides, R. tanguticum nanoparticles showed the ability to inhibit the mRNA expression of HSV-1 immediate early gene ICP4 and early gene ICP8 as well as the expression of viral protein ICP4 and ICP8. Moreover, R. tanguticum nanoparticles have been proved to protect mice against HSV-1 induced lethality by decreasing the viral load and alleviated pathological changes in brain tissues. In conclusion, we demonstrated that R. tanguticum nanoparticles could inhibit HSV-1 infection through multiple mechanisms. These results suggest that R. tanguticum nanoparticles may have novel roles in the treatment of HSV-1 infection.

MicroRNAs of Gallid and Meleagrid herpesviruses show generally conserved genomic locations and are virus-specific

Many herpesviruses, including Marek's disease viruses (MDV1 and MDV2), encode microRNAs. In this study, we report microRNAs of two related herpesviruses, infectious laryngotracheitis virus (ILTV) and herpesvirus of turkeys (HVT), as well as additional MDV2 microRNAs. The genome locations, but not microRNA sequences, are conserved among all four of these avian herpesviruses. Most are clustered in the repeats flanking the unique long region (I/TR L), except in ILTV which lacks these repeats. Two abundant ILTV microRNAs are antisense to the immediate early gene ICP4. A homologue of host microRNA, gga-miR-221, was found among the HVT microRNAs. Additionally, a cluster of HVT microRNAs was found in a region containing two locally duplicated segments, resulting in paralogous HVT microRNAs with 96–100% identity. The prevalence of microRNAs in the genomic repeat regions as well as in local repeats suggests the importance of genetic plasticity in herpesviruses for microRNA evolution and preservation of function.

Engineering cell lines for production of replication defective HSV‐1 gene therapy vectors

Herpes simplex virus type 1 (HSV-1) represents an attractive vehicle for a variety of gene therapy applications. To render this virus safe for clinical use, its cytotoxic genes must be removed without losing its ability to express transgenes efficiently. Our vectors are deleted for the essential immediate early genes ICP4 and ICP27. These genes are controlled by unique promoters having enhancer elements responsive to a viral structural protein VP16. The expression of these genes occurs prior to the activation of all other lytic functions and is thus required to initiate and complete the virus replication cycle. For large scale manufacture of clinical grade vectors, efficient cell lines must be generated that express the essential viral gene products in trans during vector propagation. Here we describe methods for engineering HSV-1 production cell lines that improve vector growth by altering the kinetics of complementing gene expression. We examined the ability of Vero cells independently transduced with ICP4 and ICP27 under transcriptional control of their respective promoters to support the growth of a replication defective vector (JDTOZHE), deleted for ICP4, ICP27 and approximately 20 kb of internal elements that are not required for virus growth in Vero cells. Vector yield on this cell line was 3 logs lower than wild-type virus grown on Vero cells. To understand the mechanism underlying poor vector yield, we examined the expression of ICP4 and ICP27 during virus complementation. While ICP27 was expressed immediately on vector infection, the expression of ICP4 was considerably delayed by 8-10 h, suggesting that the ICP4 promoter was not adequately activated by VP16 delivered by the infectious vector particle. Use of the ICP0 promoter to express ICP4 from the cellular genome resulted in higher induction levels and faster kinetics of ICP4 expression and a 10-fold improvement in vector yield. This study suggests that vector complementation is highly dependent on the kinetics of complementing gene expression and can lead to large differences in vector yield.

Direct vaccination with tumor cells killed with ICP4-deficient HSVd120 elicits effective antitumor immunity

We tested whether tumor cells were killed by replication-incompetent recombinant herpes simplex virus (HSV) d120 lacking immediate early gene ICP4 and whether HSVd120-killed tumor cells could be used directly for tumor vaccination. Vaccine efficacy was tested in TC-1, a murine adenocarcinoma transformed with HPV16 E6 and E7, and ID8-Vegf, a murine epithelial ovarian cancer model. HSVd120 killed tumor cells by apoptosis. Tumor cells infected by HSVd120 were engulfed more avidly by immature DCs and induced DC maturation more efficiently than tumor cells killed by ultraviolet B (UVB) radiation. HSVd120 infection induced stronger upregulation of GRP94 than UVB in cells undergoing apoptosis. Immunization of mice with HSVd120-killed cells elicited stronger antitumor T cell response, including tumor reactive interferon-gamma secreting and cytotoxic T cells, and resulted in significantly stronger delay in tumor growth than immunization with UVB-killed tumor cells. Thus, the use of replication-incompetent HSV strains lacking ICP4 offers possible advantages in the preparation of whole tumor cell antigen for direct tumor vaccination.

1110. Engineering Cell Lines for the Production of Highly Replication Defective HSV-1 Vectors

In order to mass produce clinical grade highly replication defective HSV-1 vectors, efficient cell lines must be generated to complement missing viral functions while avoiding recombinant virus generation. The goal of this work is to increase the yield of replication defective virus per cell by engineering a cell line that would provide efficient timing and adequate expression levels of the immediate early gene ICP4. Conventional replication defective HSV-1 vectors have deletions in immediate early genes ICP4 and ICP27 and are propagated on complementing cell lines that stably express these genes in trans. Here a joint deleted ICP4, ICP22, and ICP27 mutant virus (JDTOZEH1) was used to examine the robustness of engineered cell lines during a low MOI (0.01) production. The ICP0 promoter was chosen to drive ICP4 complementation due to a higher concentration of viral activation elements (TAATGARAT sequences). This strategy lead to higher induction levels and faster kinetics of ICP4 expression which resulted in 10 fold increases in viral yield per cell. Quantitative PCR for ICP4 transcription revealed a 10-fold increase and faster kinetics of ICP4 RNA transcription suggesting this promoter is sufficient for ICP4 complementation. These results were verified by Western blotting for ICP4 showing both earlier and higher levels of expression. Additionally, stocks produced from this cell line contained no replication competent viruses since the fragments used to transfect the complementing cell lines have limited homology with the deletions of the viral genome. These cell lines will serve as building blocks for large scale production of consequently less toxic versions of therapeutic HSV-1 viral vectors.

214. Additive Effects of HSV Vectors Expressing Proenkephalin, Interleukin-4, and Tumor Necrosis Factor Alpha Soluble Receptor To Treat Bone Cancer Pain

Top of pageAbstract In order to determine whether increasing GABA in the spinal cord by glutamic acid decarboxylase 67 (GAD67) gene transfer to the dorsal root ganglion (DRG) would attenuate below-level central neuropathic pain resulting from spinal cord injury (SCI) we engineered a replication defective HSV based vector, deleted for the essential immediate early genes ICP4 and ICP27 and additionally deficient in expression of immediate early genes ICP22 and ICP47, to express GAD 67 driven by the HCMV IE promoter at the UL41 locus (vector QHGAD67). Transduction of DRG neurons in vitro with QHGAD67 resulted in the release of GABA measured by HPLC. Subcutaneous inoculation of the vector in the foot resulted in GAD67 expression and constitutive release of GABA in microdialysate of the dorsal horn. One week after left T13 spinal hemisection in rats, animals were inoculated subcutaneously into the plantar surface of both hindpaws with 30 microliters QHGAD67 (1x109 pfu/ml) or a lacZ-expressing control vector QOZHG. Mechanical allodynia and thermal hyperalgesia in both hindpaws was assessed with a series of calibrated von Frey filaments and Hargreaves heat apparatus, respectively. Animals inoculated with QHGAD67 demonstrated antiallodynic and antihyperalgesic effects that persisted for several weeks and could be effectively reestablished by reinoculation. The vector-mediated analgesic effects were reduced by intrathecal bicuculline or phaclofen administered at doses that produced neither mechanical allodynia nor thermal hyperalgesia. Vector-mediated GABA release attenuated the increase in spinal calcitonin gene related peptide (CGRP) caused by cord hemisection. These results demonstrate that transduction of DRG with a GAD-expressing HSV vector results in the release of GABA from nerve terminals in the spinal cord to provide an analgesic effect through actions at both GABAA and GABAB receptors, and suggests that gene therapy with a GAD67 expressing HSV based vector may prove useful in the treatment of central neuropathic pain after spinal cord injury.

Development of herpes simplex virus replication-defective multigene vectors for combination gene therapy applications.

Some gene therapy applications will require simultaneous expression of multiple gene products to achieve a therapeutic effect. In this study we describe the generation and characterization of replication incompetent herpes simplex virus type 1 (HSV-1) vectors (HX86Z or HX86G) carrying distinct and independently regulated expression cassettes for five transgenes (hIL-2, hGM-CSF, hB7.1, HSV-tk and lacZ or hIFN gamma). The transgenes, representing 12 kb of DNA sequence, were recombined into separate loci of a single mutant virus vector deleted for 11.6 kb of vector sequences representing portions of nine viral genes, ICP4, ICP22, ICP27, ICP47, UL24, UL41, UL44, US10 and US11. Deletion of the immediate--early genes ICP4, ICP22 and ICP27 substantially reduced vector cytotoxicity, prevented early and late viral gene expression and left intact MHC class I antigen expression. Simultaneous expression of multiple transgenes was obtained for up to 7 days in primary human melanoma cells with peak expression at 2-3 days after infection. The transgenes were chosen for their potential to function synergistically in tumor destruction and vaccine gene therapy applications, but the method and vector employed could be applied to other multigene therapy strategies. This study demonstrates the potential for engineering large transgene capacity DNA viruses such as HSV-1 for expression of multiple transgenes.

A latent, nonpathogenic HSV-1-derived vector stably expresses β-galactosidase in mouse neurons

A genetically engineered herpes simplex virus variant was constructed for use as a stable gene vector for neurons. To inhibit replication, the agent possessed a deletion in the immediate early gene ICP4, and to minimize reactivation from the latent state, the gene encoding the latency-associated transcript was deleted. The E. coli β-galactosidase gene under the control of the Maloney murine leukemia virus long terminal repeat promoter was inserted into the ICP4 region. When introduced into the peripheral nervous system, this virus established latent infections and stably expressed β-galactosidase in primary sensory neurons. Expression of β-galactosidase over a more limited time period was observed when the latent infection was established in motor neurons of the hypoglossal nucleus. Agents of this general design have considerable potential for use as gene vectors for studies of neuronal function and correction of genetic defects affecting neurons.