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Dogrammatzis C, Waisner H, Kalamvoki M. "Non-Essential" Proteins of HSV-1 with Essential Roles In Vivo: A Comprehensive Review. Viruses 2020; 13:E17. [PMID: 33374862 PMCID: PMC7824580 DOI: 10.3390/v13010017] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2020] [Revised: 12/17/2020] [Accepted: 12/18/2020] [Indexed: 12/19/2022] Open
Abstract
Viruses encode for structural proteins that participate in virion formation and include capsid and envelope proteins. In addition, viruses encode for an array of non-structural accessory proteins important for replication, spread, and immune evasion in the host and are often linked to virus pathogenesis. Most virus accessory proteins are non-essential for growth in cell culture because of the simplicity of the infection barriers or because they have roles only during a state of the infection that does not exist in cell cultures (i.e., tissue-specific functions), or finally because host factors in cell culture can complement their absence. For these reasons, the study of most nonessential viral factors is more complex and requires development of suitable cell culture systems and in vivo models. Approximately half of the proteins encoded by the herpes simplex virus 1 (HSV-1) genome have been classified as non-essential. These proteins have essential roles in vivo in counteracting antiviral responses, facilitating the spread of the virus from the sites of initial infection to the peripheral nervous system, where it establishes lifelong reservoirs, virus pathogenesis, and other regulatory roles during infection. Understanding the functions of the non-essential proteins of herpesviruses is important to understand mechanisms of viral pathogenesis but also to harness properties of these viruses for therapeutic purposes. Here, we have provided a comprehensive summary of the functions of HSV-1 non-essential proteins.
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Affiliation(s)
| | | | - Maria Kalamvoki
- Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, KS 66160, USA; (C.D.); (H.W.)
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Identification of Marek's Disease Virus VP22 Tegument Protein Domains Essential for Virus Cell-to-Cell Spread, Nuclear Localization, Histone Association and Cell-Cycle Arrest. Viruses 2019; 11:v11060537. [PMID: 31181775 PMCID: PMC6631903 DOI: 10.3390/v11060537] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2019] [Accepted: 06/05/2019] [Indexed: 12/30/2022] Open
Abstract
VP22 is a major tegument protein of alphaherpesviruses encoded by the UL49 gene. Two properties of VP22 were discovered by studying Marek's disease virus (MDV), the Mardivirus prototype; it has a major role in virus cell-to-cell spread and in cell cycle modulation. This 249 AA-long protein contains three regions including a conserved central domain. To decipher the functional VP22 domains and their relationships, we generated three series of recombinant MDV genomes harboring a modified UL49 gene and assessed their effect on virus spread. Mutated VP22 were also tested for their ability to arrest the cell cycle, subcellular location and histones copurification after overexpression in cells. We demonstrated that the N-terminus of VP22 associated with its central domain is essential for virus spread and cell cycle modulation. Strikingly, we demonstrated that AAs 174-190 of MDV VP22 containing the end of a putative extended alpha-3 helix are essential for both functions and that AAs 159-162 located in the putative beta-strand of the central domain are mandatory for cell cycle modulation. Despite being non-essential, the 59 C-terminal AAs play a role in virus spread efficiency. Interestingly, a positive correlation was observed between cell cycle modulation and VP22 histones association, but none with MDV spread.
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Mapping Murine Corneal Neovascularization and Weight Loss Virulence Determinants in the Herpes Simplex Virus 1 Genome and the Detection of an Epistatic Interaction between the UL and IRS/US Regions. J Virol 2016; 90:8115-31. [PMID: 27384650 PMCID: PMC5008079 DOI: 10.1128/jvi.00821-16] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2016] [Accepted: 06/22/2016] [Indexed: 01/14/2023] Open
Abstract
UNLABELLED Herpes simplex virus 1 (HSV-1) most commonly causes recrudescent labial ulcers; however, it is also the leading cause of infectious blindness in developed countries. Previous research in animal models has demonstrated that the severity of HSV-1 ocular disease is influenced by three main factors: host innate immunity, host immune response, and viral strain. We have previously shown that mixed infection with two avirulent HSV-1 strains (OD4 and CJ994) results in recombinants with a wide range of ocular disease phenotype severity. Recently, we developed a quantitative trait locus (QTL)-based computational approach (vQTLmap) to identify viral single nucleotide polymorphisms (SNPs) predicted to influence the severity of the ocular disease phenotypes. We have now applied vQTLmap to identify HSV-1 SNPs associated with corneal neovascularization and mean peak percentage weight loss (MPWL) using 65 HSV-1 OD4-CJ994 recombinants. The vQTLmap analysis using Random Forest for neovascularization identified phenotypically meaningful nonsynonymous SNPs in the ICP4, UL41 (VHS), UL42, UL46 (VP11/12), UL47 (VP13/14), UL48 (VP22), US3, US4 (gG), US6 (gD), and US7 (gI) coding regions. The ICP4 gene was previously identified as a corneal neovascularization determinant, validating the vQTLmap method. Further analysis detected an epistatic interaction for neovascularization between a segment of the unique long (UL) region and a segment of the inverted repeat short (IRS)/unique short (US) region. Ridge regression was used to identify MPWL-associated nonsynonymous SNPs in the UL1 (gL), UL2, UL4, UL49 (VP22), UL50, and ICP4 coding regions. The data provide additional insights into virulence gene and epistatic interaction discovery in HSV-1. IMPORTANCE Herpes simplex virus 1 (HSV-1) typically causes recurrent cold sores; however, it is also the leading source of infectious blindness in developed countries. Corneal neovascularization is critical for the progression of blinding ocular disease, and weight loss is a measure of infection severity. Previous HSV-1 animal virulence studies have shown that the severity of ocular disease is partially due to the viral strain. In the current study, we used a recently described computational quantitative trait locus (QTL) approach in conjunction with 65 HSV-1 recombinants to identify viral single nucleotide polymorphisms (SNPs) involved in neovascularization and weight loss. Neovascularization SNPs were identified in the ICP4, VHS, UL42, VP11/12, VP13/14, VP22, gG, US3, gD, and gI genes. Further analysis revealed an epistatic interaction between the UL and US regions. MPWL-associated SNPs were detected in the UL1 (gL), UL2, UL4, VP22, UL50, and ICP4 genes. This approach will facilitate future HSV virulence studies.
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Yu X, Wang Y, Xia Y, Zhang L, Yang Q, Lei J. A DNA vaccine encoding VP22 of herpes simplex virus type I (HSV-1) and OprF confers enhanced protection from Pseudomonas aeruginosa in mice. Vaccine 2016; 34:4399-405. [PMID: 27449680 DOI: 10.1016/j.vaccine.2016.07.017] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2016] [Revised: 07/12/2016] [Accepted: 07/13/2016] [Indexed: 01/28/2023]
Abstract
Pseudomonas aeruginosa antimicrobial resistance is a major therapeutic challenge. DNA vaccination is an attractive approach for antigen-specific immunotherapy against P. aeruginosa. We explored the feasibility of employing Herpes simplex virus type 1 tegument protein, VP22, as a molecular tool to enhance the immunogenicity of an OprF DNA vaccine against P. aeruginosa. Recombinant DNA vaccines, pVAX1-OprF, pVAX1-OprF-VP22 (encoding a n-OprF-VP22-c fusion protein) and pVAX1-VP22-OprF (encoding a n-VP22-OprF-c fusion protein) were constructed. The humoral and cellular immune responses and immune protective effects of these DNA vaccines in mice were evaluated. In this report, we showed that vaccination with pVAX1-OprF-VP22 induced higher levels of IgG titer, T cell proliferation rate. It also provided better immune protection against the P. aeruginosa challenge when compared to that induced by pVAX1-OprF or pVAX1-VP22-OprF DNA vaccines. Molecular mechanistic analyses indicated vaccination with pVAX1-OprF-VP22 triggered immune responses characterized by a preferential increase in antigen specific IgG2a and IFN-γ in mice, indicating Th1 polarization. We concluded that VP22 is a potent stimulatory molecular tool for DNA vaccination when fused to the carboxyl end of OprF gene. Our study provides a novel strategy for prevention and treatment of P. aeruginosa infection.
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Affiliation(s)
- Xian Yu
- Department of Pharmacy, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, PR China.
| | - Yan Wang
- Institute of Materia Medica, North Sichuan Medical College, Nanchong 637007, PR China
| | - Yifan Xia
- Department of Orthopaedics, Chongqing General Hospital, Chongqing 400014, PR China
| | - Lijuan Zhang
- Institute of Materia Medica, North Sichuan Medical College, Nanchong 637007, PR China
| | - Qin Yang
- Institute of Materia Medica, North Sichuan Medical College, Nanchong 637007, PR China
| | - Jun Lei
- Institute of Materia Medica, North Sichuan Medical College, Nanchong 637007, PR China
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Yu X, Lei J, Yang Q, Xu Z, Wang Y. Expression, purification and production of antisera against recombinant truncated VP22 protein. Exp Ther Med 2016; 11:1762-1766. [PMID: 27168799 PMCID: PMC4840775 DOI: 10.3892/etm.2016.3103] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2014] [Accepted: 02/11/2016] [Indexed: 11/11/2022] Open
Abstract
Cell-penetrating peptides (CPPs) are non-invasive vectors that can efficiently transport bioactive cargo across the cell membrane. Naturally occurring CPPs, such as the tegument protein VP22 of the Herpes simplex virus type 1, can potentiate protein-drug delivery into living cells. The aim of the present study was to construct anti-VP22 antibodies that can be used to detect VP22-fusion drugs. Therefore, 60- and 45-amino acid peptides corresponding to the N-terminus and C-terminus of VP22, respectively, were cloned, expressed and purified. Subsequently, polyclonal antisera against them were generated. The DNA sequence, cloned into the pGEX-5X-1 vector, was transformed into E. coli BL21 (DE3). After inducing expression with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 25°C for 4 h, the recombinant VP22 proteins were purified by electroelution. The high titers of polyclonal antisera obtained subsequent to immunization of mice with the purified recombinant truncated VP22 was confirmed by ELISA. Western blot and immunofluorescence analysis showed that the antisera detected both the truncated and full-length VP22 protein. Therefore, the polyclonal antisera against VP22 may be used in the detection of the intracellular location of VP22-fusion protein drugs.
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Affiliation(s)
- Xian Yu
- Department of Pharmacy, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, P.R. China
| | - Jun Lei
- Institute of Materia Medica, North Sichuan Medical College, Nanchong, Sichuan 637007, P.R. China
| | - Qin Yang
- Institute of Materia Medica, North Sichuan Medical College, Nanchong, Sichuan 637007, P.R. China
| | - Zhengmin Xu
- Institute of Materia Medica, North Sichuan Medical College, Nanchong, Sichuan 637007, P.R. China
| | - Yan Wang
- Institute of Materia Medica, North Sichuan Medical College, Nanchong, Sichuan 637007, P.R. China
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Yu X, Xu Z, Lei J, Li T, Wang Y. VP22 mediates intercellular trafficking and enhances the in vitro antitumor activity of PTEN. Mol Med Rep 2015; 12:1286-90. [PMID: 25816150 DOI: 10.3892/mmr.2015.3509] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2014] [Accepted: 02/03/2015] [Indexed: 11/06/2022] Open
Abstract
PTEN acts as a phosphatidylinositol phosphatase with a possible role in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. Mutations in PTEN are frequent and their presence is associated with poor prognosis in breast cancer, which is the most common type of non-cutaneous malignancy in females. Delivery of the tumor suppressor PTEN gene represents a powerful strategy for breast cancer therapy, but a present limitation of gene therapy is the ability to deliver sufficient quantities of active proteins to target cells. The capacity of HSV-1VP22 fusion proteins to spread from the primary transduced cell to surrounding cells could improve gene therapeutics, particularly in cancer. To assess the potential efficacy of VP22 as a gene therapy for breast cancer, expression vectors for N- and C-terminal PTEN-VP22 fusion proteins were constructed. VP22‑mediated intercellular transport and antitumor efficacy in BT549 (PTEN-null) breast tumor cells were investigated. The results showed that PTEN-VP22 has the same spreading abilities as VP22. In cell proliferation and apoptosis assays, PTEN-VP22 gene transfer induces a stronger anti-proliferative effect and apoptotic activity compared with PTEN gene transfer alone. In addition, VP22 enhanced the PTEN‑mediated decrease in the level of phosphorylated AKT. The results show that PTEN-VP22 can spread in vitro and PTEN-VP22 gene induces significantly greater antitumor activity than the PTEN gene alone. This study confirms the utility of VP22-mediated delivery in vitro and suggests that PTEN-VP22 may have applications in breast cancer gene therapy.
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Affiliation(s)
- Xian Yu
- Department of Pharmacy, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, P.R. China
| | - Zhengmin Xu
- Institute of Materia Medica, North Sichuan Medical College, Nanchong, Sichuan 637007, P.R. China
| | - Jun Lei
- Institute of Materia Medica, North Sichuan Medical College, Nanchong, Sichuan 637007, P.R. China
| | - Tingting Li
- Institute of Materia Medica, North Sichuan Medical College, Nanchong, Sichuan 637007, P.R. China
| | - Yan Wang
- Institute of Materia Medica, North Sichuan Medical College, Nanchong, Sichuan 637007, P.R. China
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Okada A, Kodaira A, Hanyu S, Izume S, Ohya K, Fukushi H. Intracellular localization of Equine herpesvirus type 1 tegument protein VP22. Virus Res 2014; 192:103-13. [PMID: 25192624 DOI: 10.1016/j.virusres.2014.08.006] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2014] [Revised: 08/14/2014] [Accepted: 08/14/2014] [Indexed: 11/22/2022]
Abstract
Intracellular localization of Equine herpesvirus type 1 (EHV-1) tegument protein VP22 was examined by using a plasmid that expressed VP22 fused with an enhanced green fluorescent protein (EGFP). Also a recombinant EHV-1 expressing VP22 fused with a red fluorescent protein (mCherry) was constructed to observe the localization of VP22 in infected cells. When EGFP-fused VP22 was overexpressed in the cells, VP22 localized in the cytoplasm and nucleus. Live cell imaging suggested that the fluorescently tagged VP22 also localized in the cytoplasm and nucleus. These results show that VP22 localizes in the cytoplasm and nucleus independently of other viral proteins. Experiments with truncation mutants of pEGFP-VP22 suggested that 154-188 aa might be the nuclear localization signal of EHV-1 VP22.
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Affiliation(s)
- Ayaka Okada
- Department of Applied Veterinary Sciences, United Graduated School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
| | - Akari Kodaira
- Department of Applied Veterinary Sciences, United Graduated School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
| | - Sachiko Hanyu
- Department of Applied Veterinary Sciences, United Graduated School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
| | - Satoko Izume
- Department of Applied Veterinary Sciences, United Graduated School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
| | - Kenji Ohya
- Department of Applied Veterinary Sciences, United Graduated School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan; Laboratory of Veterinary Microbiology, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
| | - Hideto Fukushi
- Department of Applied Veterinary Sciences, United Graduated School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan; Laboratory of Veterinary Microbiology, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
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Dewberry EJ, Dunkerley E, Duffy C. Purification of full-length VP22 from cells infected with HSV-1: A two-pronged approach for the solubilization and purification of viral proteins for use in biochemical studies. J Virol Methods 2012; 183:180-5. [PMID: 22569534 DOI: 10.1016/j.jviromet.2012.04.012] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2011] [Revised: 04/09/2012] [Accepted: 04/23/2012] [Indexed: 11/30/2022]
Abstract
VP22, encoded by the U(L)49 gene, is one of the most abundant proteins of the herpes simplex virus type 1 (HSV-1) tegument and has been shown to be important for virus replication and spread. However, the exact role(s) played by VP22 in the HSV-1 replication cycle have yet to be delineated. The lack of a procedure to purify full-length VP22 has limited molecular studies on VP22 function. A procedure was developed for the purification of soluble, full-length VP22 from cells infected with HSV-1. A recombinant virus encoding His-tagged VP22 was generated and found to express VP22 at levels comparable to the wild type virus upon infection of Vero cells. By experimenting with a wide variety of cell lysis buffer conditions, several buffers that promote the solubility of full-length VP22 were identified. Buffers that gave the highest levels of solubility were then used in immobilized metal ion affinity chromatography experiments to identify conditions that provided the greatest level of VP22 binding and recovery from cobalt and nickel affinity resins. Using this strategy soluble, full-length VP22 was purified from cells infected with HSV-1.
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Affiliation(s)
- Ebony J Dewberry
- Department of Biological Sciences, University of Alabama, 300 Hackberry Lane, Tuscaloosa, AL 35487, USA
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Host cell targets of tegument protein VP22 of herpes simplex virus 1. Arch Virol 2011; 156:1079-84. [DOI: 10.1007/s00705-011-0960-9] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2010] [Accepted: 02/21/2011] [Indexed: 10/18/2022]
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Schlegel EFM, Blaho JA. A conserved carboxy-terminal domain in the major tegument structural protein VP22 facilitates virion packaging of a chimeric protein during productive herpes simplex virus 1 infection. Virology 2009; 387:449-58. [PMID: 19307008 PMCID: PMC2674122 DOI: 10.1016/j.virol.2009.02.040] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2009] [Revised: 02/11/2009] [Accepted: 02/25/2009] [Indexed: 10/21/2022]
Abstract
Recombinant virus HSV-1(RF177) was previously generated to examine tegument protein VP22 function by inserting the GFP gene into the gene encoding VP22. During a detailed analysis of this virus, we discovered that RF177 produces a novel fusion protein between the last 15 amino acids of VP22 and GFP, termed GCT-VP22. Thus, the VP22 carboxy-terminal specific antibody 22-3 and two anti-GFP antibodies reacted with an approximately 28 kDa protein from RF177-infected Vero cells. GCT-VP22 was detected at 1 and 3 hpi. Examination of purified virions indicated that GCT-VP22 was incorporated into RF177 virus particles. These observations imply that at least a portion of the information required for virion targeting is located in this domain of VP22. Indirect immunofluorescence analyses showed that GCT-VP22 also localized to areas of marginalized chromatin during RF177 infection. These results indicate that the last fifteen amino acids of VP22 participate in virion targeting during HSV-1 infection.
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Affiliation(s)
- Elisabeth F. M. Schlegel
- Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029
| | - John A. Blaho
- Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029
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The presence of a deletion sequence in the BHV-1 UL49 homolog in a live attenuated vaccine for infectious bovine rhinotracheitis (IBR). Vaccine 2008; 26:477-85. [PMID: 18164789 DOI: 10.1016/j.vaccine.2007.11.037] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2006] [Revised: 10/10/2007] [Accepted: 11/18/2007] [Indexed: 11/24/2022]
Abstract
The difference between the attenuated live vaccine strain 758-43 and its parent virulent strain 758 was investigated genetically. These viruses were propagated in Madin-Darby bovine kidney cells, and viral DNA was obtained from the culture supernatants of the infected cells. Based on a previous report, a large deleted region would seem to exist in the Hind III J fragment located between nucleotide numbers 2439 and 11,270. Three pairs of primers were designed based on the complete BHV-1 DNA sequence. With one pair of primers used, the PCR products derived from strains 758 and LA resulted in fragment sizes of 1850 bp, whereas that from the vaccine strain was smaller than those from the virulent strains. The attenuated live vaccine strain, 758-43, lacked 652 bp in the PCR product region, accounting for approximately 84% of the coding region of the UL49 homolog gene of BHV-1. The present results provide a new and important information to distinguish the vaccine strain 758-43 clearly from wild-type BHV-1 isolates in Japan. The UL49 homolog gene seems to participate in pathogenicity in herpesvirus infections.
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Medina-Kauwe LK. "Alternative" endocytic mechanisms exploited by pathogens: new avenues for therapeutic delivery? Adv Drug Deliv Rev 2007; 59:798-809. [PMID: 17707545 PMCID: PMC2040389 DOI: 10.1016/j.addr.2007.06.009] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2007] [Accepted: 06/12/2007] [Indexed: 11/22/2022]
Abstract
Some pathogens utilize unique routes to enter cells that may evade the intracellular barriers encountered by the typical clathrin-mediated endocytic pathway. Retrograde transport and caveolar uptake are among the better characterized pathways, as alternatives to clathrin-mediated endocytosis, that are known to facilitate entry of pathogens and potential delivery agents. Recent characterization of the trafficking mechanisms of prion proteins and certain bacteria may present new paradigms for strategizing improvements in therapeutic spread and retention of therapy. This review will provide an overview of such endocytic pathways, and discuss current and future possibilities in using these routes as a means to improve therapeutic delivery.
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Lemken ML, Wolf C, Wybranietz WA, Schmidt U, Smirnow I, Bühring HJ, Mack AF, Lauer UM, Bitzer M. Evidence for intercellular trafficking of VP22 in living cells. Mol Ther 2007; 15:310-9. [PMID: 17235309 DOI: 10.1038/sj.mt.6300013] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
The intercellular trafficking property of the herpes simplex virus type 1 tegument protein VP22 makes it a promising tool for overcoming low transduction efficiencies in gene therapy. However, recent reports suggest not only that VP22 cannot facilitate intercellular spreading and that trafficking of VP22 fusion proteins results from artifacts of cell fixation only. To provide direct evidence for the presence or absence of VP22-mediated intercellular trafficking, we generated an adenoviral vector with a dual expression cassette for VP22 fused to green fluorescent protein (VP22 GFP) and DsRed under the control of distinct human cytomegalovirus immediate-early enhancer/promoter regions. Using this vector, we were able to distinguish clearly between primary transduced cells and cells taking up VP22GFP by intercellular trafficking. To our knowledge, for the first time, we could demonstrate by live-cell confocal fluorescence microscopy that VP22GFP can be found intracellularly in unfixed recipient cells. The extent of VP22 spread was similar in paraformaldehyde-fixed cells and unfixed cells as demonstrated by fluorescence-activated cell sorting analysis. We thus confirmed the ability of VP22-mediated intercellular trafficking in live unfixed cells.
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Affiliation(s)
- Marie-Luise Lemken
- Department of Internal Medicine I, University Clinic Tübingen, Tübingen, Germany
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Cilloniz C, Jackson W, Grose C, Czechowski D, Hay J, Ruyechan WT. The varicella-zoster virus (VZV) ORF9 protein interacts with the IE62 major VZV transactivator. J Virol 2006; 81:761-74. [PMID: 17079304 PMCID: PMC1797441 DOI: 10.1128/jvi.01274-06] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
The varicella-zoster virus (VZV) ORF9 protein is a member of the herpesvirus UL49 gene family but shares limited identity and similarity with the UL49 prototype, herpes simplex virus type 1 VP22. ORF9 mRNA is the most abundantly expressed message during VZV infection; however, little is known concerning the functions of the ORF9 protein. We have found that the VZV major transactivator IE62 and the ORF9 protein can be coprecipitated from infected cells. Yeast two-hybrid analysis localized the region of the ORF9 protein required for interaction with IE62 to the middle third of the protein encompassing amino acids 117 to 186. Protein pull-down assays with GST-IE62 fusion proteins containing N-terminal IE62 sequences showed that amino acids 1 to 43 of the acidic transcriptional activation domain of IE62 can bind recombinant ORF9 protein. Confocal microscopy of transiently transfected cells showed that in the absence of other viral proteins, the ORF9 protein was localized in the cytoplasm while IE62 was localized in the nucleus. In VZV-infected cells, the ORF9 protein was localized to the cytoplasm whereas IE62 exhibited both nuclear and cytoplasmic localization. Cotransfection of plasmids expressing ORF9, IE62, and the viral ORF66 kinase resulted in significant colocalization of ORF9 and IE62 in the cytoplasm. Coimmunoprecipitation experiments with antitubulin antibodies indicate the presence of ORF9-IE62-tubulin complexes in infected cells. Colocalization of ORF9 and tubulin in transfected cells was visualized by confocal microscopy. These data suggest a model for ORF9 protein function involving complex formation with IE62 and possibly other tegument proteins in the cytoplasm at late times in infection.
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Affiliation(s)
- Cristian Cilloniz
- Department of Microbiology, Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, SUNY, Buffalo, NY 14214, USA
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Abstract
Different approaches have been developed for the introduction of macromolecules, proteins and DNA into target cells. Viral (retroviruses, lentiviruses, etc.) and nonviral (liposomes, bioballistics etc.) vectors as well as lipid particles have been tested as DNA delivery systems. However, all of them share several undesirable effects that are difficult to overcome, such as unwanted immunoresponse and limited cell targeting. The discovery of the cell penetrating peptides (CPPs) showing properties of macromolecules carriers and enhancers of viral vectors, opened new opportunities for the delivery of biologically active cargos, including therapeutically relevant genes into various cells and tissues. This review summarizes recent data about the best characterized CPPs as well as those sharing cell-penetrating and cargo delivery properties despite differing in the primary sequence. The putative mechanisms of CPPs penetration into cells and interaction with intracellular structures such as chromosomes, cytoskeleton and centrioles are addressed. We further discuss recent developments in overcoming the lack of cells specificity, one of the main obstacles for CPPs application in gene therapy. In particular, we review a newly discovered affinity of CPPs to actively proliferating cells.
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Affiliation(s)
- Alexandre Kerkis
- Clínica e Centro de Pesquisa em Reprodução Humana Roger Abdelmassih, São Paulo, Brasil
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Mahley RW, Weisgraber KH, Huang Y. Apolipoprotein E4: a causative factor and therapeutic target in neuropathology, including Alzheimer's disease. Proc Natl Acad Sci U S A 2006; 103:5644-51. [PMID: 16567625 PMCID: PMC1414631 DOI: 10.1073/pnas.0600549103] [Citation(s) in RCA: 671] [Impact Index Per Article: 35.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
The premise of this review is that apolipoprotein (apo) E4 is much more than a contributing factor to neurodegeneration. ApoE has critical functions in redistributing lipids among CNS cells for normal lipid homeostasis, repairing injured neurons, maintaining synapto-dendritic connections, and scavenging toxins. In multiple pathways affecting neuropathology, including Alzheimer's disease, apoE acts directly or in concert with age, head injury, oxidative stress, ischemia, inflammation, and excess amyloid beta peptide production to cause neurological disorders, accelerating progression, altering prognosis, or lowering age of onset. We envision that unique structural features of apoE4 are responsible for apoE4-associated neuropathology. Although the structures of apoE2, apoE3, and apoE4 are in dynamic equilibrium, apoE4, which is detrimental in a variety of neurological disorders, is more likely to assume a pathological conformation. Importantly, apoE4 displays domain interaction (an interaction between the N- and C-terminal domains of the protein that results in a compact structure) and molten globule formation (the formation of stable, reactive intermediates with potentially pathological activities). In response to CNS stress or injury, neurons can synthesize apoE. ApoE4 uniquely undergoes neuron-specific proteolysis, resulting in bioactive toxic fragments that enter the cytosol, alter the cytoskeleton, disrupt mitochondrial energy balance, and cause cell death. Our findings suggest potential therapeutic strategies, including the use of "structure correctors" to convert apoE4 to an "apoE3-like" molecule, protease inhibitors to prevent the generation of toxic apoE4 fragments, and "mitochondrial protectors" to prevent cellular energy disruption.
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Affiliation(s)
- Robert W Mahley
- Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA 94158, USA.
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18
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Yi J, Gong WD, Wang L, Ling R, Chen JH, Yun J. VP22 fusion protein-based dominant negative mutant can inhibit hepatitis B virus replication. World J Gastroenterol 2005; 11:6429-32. [PMID: 16425411 PMCID: PMC4355781 DOI: 10.3748/wjg.v11.i41.6429] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the inhibitory effect of VP22 fusion protein-based dominant negative (DN) mutant on Hepatitis Bvrus (HBV) replication.
METHODS: Full-length or truncated fragment of VP22 was fused to C terminal of HBV core protein (HBc), and subcloned into pcDNA3.1 (-) vector, yielding eukaryotic expression plasmids of DN mutant. After transfection into HepG2.2.15 cells, the expression of DN mutant was identified by immunofluorescence staining. The inhibitory effect of DN mutant on HBV replication was indexed as the supernatant HBsAg concentration determined by RIA and HBV-DNA content by fluorescent quantification-PCR (FQ-PCR). Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry.
RESULTS: VP22-based DN mutants and its truncated fragment were expressed in HepG2.2.15 cells, and had no toxic effect on host cells. DN mutants could inhibit HBV replication and the transduction ability of mutant-bearing protein had a stronger inhibitory effect on HBV replication. DN mutants with full length of VP22 had the strongest inhibitory effect on HBV replication, reducing the HBsAg concentration by 81.94%, and the HBV-DNA content by 72.30%. MTT assay suggested that there were no significant differences in cell metabolic activity between the groups.
CONCLUSION: VP22-based DN mutant can inhibit HBV replication effectively.
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Affiliation(s)
- Jun Yi
- Department of general Surgery, Xijing Hospital, Fourth Military Medical University, Shaanxi Province, China
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19
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Greco O, Joiner MC, Doleh A, Scott SD. VP22-mediated intercellular transport for suicide gene therapy under oxic and hypoxic conditions. Gene Ther 2005; 12:974-9. [PMID: 15729368 DOI: 10.1038/sj.gt.3302482] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
During herpes simplex virus type 1 (HSV 1) infection, the tegument protein VP22 is exported from infected cells to the nuclei of surrounding uninfected cells. These intercellular transport characteristics have prompted the exploitation of VP22 fusion proteins for cancer gene therapy, with the goal of maximizing the bystander effect. Since solid tumors contain hypoxic cell populations that are often refractive to therapy, for efficient targeting, it would be optimal if VP22 functioned even at reduced oxygen concentrations. In the present work, VP22 activity under hypoxic conditions was examined for the first time. Plasmid-transfected human glioma U87-MG and U373-MG cells expressing VP22 fused to the green fluorescent protein (GFP) showed protein export to untransfected cells under tumor oxygenation conditions (0-5% O(2)). For suicide gene therapy, VP22 activity was demonstrated under hypoxia by coupling VP22 to the HSV thymidine kinase (HSVtk). In the presence of the prodrug ganciclovir, cell cultures expressing VP22-HSVtk showed a significant increase in toxicity compared with cells transfected with a construct containing HSVtk only, under all tested conditions. To allow effective suicide gene therapy and simultaneous visualization of therapeutic enzyme localization, a triple fusion protein GFP-HSVtk-VP22 was engineered. Functionality of all components was demonstrated under oxia and hypoxia.
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Affiliation(s)
- O Greco
- Department of Radiation Oncology, Wayne State University and Karmanos Cancer Institute, Hudson Webber CRC, Detroit, MI, USA
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20
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Sheridan PJ, Lawrie A, Crossman DC, Holt CM, Newman CM. VP22-mediated intercellular transport correlates with enhanced biological activity of MybEngrailed but not (HSV-I) thymidine kinase fusion proteins in primary vascular cells following non-viral transfection. J Gene Med 2005; 7:375-85. [PMID: 15543525 DOI: 10.1002/jgm.679] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
BACKGROUND The intercellular transport properties of the herpes simplex virus (HSV) protein VP22 have been harnessed to enhance the effectiveness of viral gene transfer. We investigated the intercellular transport and biological effects of VP22 fused with the dominant negative c-Myb chimera, MybEngrailed (MybEn) and HSV-I thymidine kinase (TK), in primary vascular smooth muscle cells (VSMC) following non-viral transfection. MATERIALS AND METHODS Porcine VSMC transfected with plasmids encoding MybEn, TK and their respective N- and C-terminal VP22 fusion proteins were assayed for the extent and distribution of transgene expression (by immunohistochemistry), culture growth and apoptosis. RESULTS The N-terminal MybEn fusion with VP22 (MybEnVP22) and both TK fusions, but not VP22MybEn, exhibited intercellular spread from primary transfected to up to 200 surrounding cells. pMybEnVP22-transfected cultures exhibited growth inhibition and apoptosis rates that were 10.6 +/- 3.6 and 3.2 +/- 1.0 fold higher than in pMybEn-transfected cultures; pVP22MybEn-transfected cultures showed no difference in these parameters. pTK-transfected cultures underwent 60-70% cell death in the presence of ganciclovir despite <2% primary transfection, which was not increased in cultures transfected with plasmids encoding VP22-TK fusions. CONCLUSIONS The close correlation between immunocytochemical and biological assays suggests that intercellular transport is crucial to the enhanced biological activity of the MybEnVP22 fusion. The "intrinsic" bystander activity of TK was 4-fold greater than was "engineered" by VP22 fusion, probably reflecting the abundance of gap junctions between VSMC. VP22 fusion may enhance the efficiency of non-viral gene delivery when combined with the appropriate therapeutic transgene, target tissue and transfection method.
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Affiliation(s)
- Paul J Sheridan
- Cardiovascular Research Unit, Division of Clinical Sciences (North), Clinical Sciences Centre, University of Sheffield, Northern General Hospital, Sheffield S5 7AU, UK.
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21
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Kueltzo LA, Middaugh CR. Nonclassical transport proteins and peptides: an alternative to classical macromolecule delivery systems. J Pharm Sci 2003; 92:1754-72. [PMID: 12949995 DOI: 10.1002/jps.10448] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
The number of peptides and proteins known to exhibit nonclassical transport activity has increased significantly in recent years. In most cases, these entities have been studied in relation to their ability to deliver high molecular weight compounds, including proteins and DNA, for the ultimate purpose of developing new drug delivery strategies. In this review, an overview of the various types of vectors is presented. The in vitro and in vivo delivery successes of this technology, as well as preliminary therapeutic efforts, are described. Although a comprehensive mechanism of nonclassical transport has not yet been clearly established, we propose a straightforward model based on the cationic nature of the vectors and the need for lack of highly organized structure. In this hypothesis we suggest that the movement of polycations is mediated by a network of extra- and intracellular polyanions while transport across the bilayer is facilitated by cation-pi interactions between the vectors' basic groups and aromatic amino acid side chains in the bilayer spanning helices of membrane proteins.
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Affiliation(s)
- Lisa A Kueltzo
- Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Ave., Lawrence, Kansas 66047, USA
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22
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Brignati MJ, Loomis JS, Wills JW, Courtney RJ. Membrane association of VP22, a herpes simplex virus type 1 tegument protein. J Virol 2003; 77:4888-98. [PMID: 12663795 PMCID: PMC152156 DOI: 10.1128/jvi.77.8.4888-4898.2003] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Tegument proteins of herpes simplex virus type 1 (HSV-1) are hypothesized to contain the functional information required for the budding or envelopment process proposed to occur at cytoplasmic compartments of the host cell. One of the most abundant tegument proteins of HSV-1 is the U(L)49 gene product, VP22, a 38-kDa protein of unknown function. To study its subcellular localization, a VP22-green fluorescent protein chimera was expressed in transfected human melanoma (A7) cells. In the absence of other HSV-1 proteins, VP22 localizes to acidic compartments of the cell that may include the trans-Golgi network (TGN), suggesting that this protein is membrane associated. Membrane pelleting and membrane flotation assays confirmed that VP22 partitions with the cellular membrane fraction. Through truncation mutagenesis, we determined that the membrane association of VP22 is a property attributed to amino acids 120 to 225 of this 301-amino-acid protein. The above results demonstrate that VP22 contains specific information required for targeting to membranes of acidic compartments of the cell which may be derived from the TGN, suggesting a potential role for VP22 during tegumentation and/or final envelopment.
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Affiliation(s)
- Michael J Brignati
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA
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23
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Boenicke L, Chu K, Pauls R, Tams C, Kruse ML, Kurdow R, Schniewind B, Böhle A, Kremer B, Kalthoff H. Efficient dose-dependent and time-dependent protein transduction of pancreatic carcinoma cells in vitro and in vivo using purified VP22-EGFP fusion protein. J Mol Med (Berl) 2003; 81:205-13. [PMID: 12682729 DOI: 10.1007/s00109-003-0421-3] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2001] [Accepted: 12/30/2002] [Indexed: 11/27/2022]
Abstract
We constructed a prokaryotic vector expressing a truncated VP22-EGFP gene and purified this fusion protein from Escherichia coli cultures using nickel resin. Application of purified VP22-EGFP protein to human pancreatic carcinoma cells showed a highly efficient time-dependent and dose-dependent uptake and resulted in green fluorescence predominantly located in the nuclei of treated cells. Purified VP22-EGFP efficiently translocated into deeper layers of pancreatic tumor cell spheroids. Homogeneous uptake into the whole tumor was observed after peritumoral injection in human pancreatic tumors in SCID mice. We conclude that the direct application of purified VP22 fusion proteins offers a new, peptide-mediated and potentially systemic therapy for pancreatic cancer. This opens the possibility of achieving specific antitumor effects induced by fused apoptosis-enhancing proteins.
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Affiliation(s)
- Lars Boenicke
- Molecular Oncology Research Laboratory, Clinic for General and Thoracic Surgery, UKSH, Campus Kiel, Arnold-Heller Strasse 7, 24105 Kiel, Germany
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24
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Zavaglia D, Favrot MC, Eymin B, Tenaud C, Coll JL. Intercellular trafficking and enhanced in vivo antitumour activity of a non-virally delivered P27-VP22 fusion protein. Gene Ther 2003; 10:314-25. [PMID: 12595890 DOI: 10.1038/sj.gt.3301904] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
VP22, a structural protein from herpes simplex virus type I, exhibits the unique property of intercellular trafficking. This protein is exported from primary expressing cells and subsequently imported into neighbouring cells. This property is conserved when VP22 is genetically fused to a protein, making it a promising tool to enhance the delivery of a gene product. We chose to study the intercellular transport and biological effect of a fusion protein between the putative tumour suppressor gene p27(Kip1) and VP22. We show that in vitro, P27VP22 is able to spread as efficiently as VP22. Functionality of the P27VP22 protein was demonstrated by its ability to inhibit cyclin/CDK2 complexes activity. In proliferation and clonogenicity assays, transfection with the P27VP22 plasmid resulted in a stronger cell growth inhibition when compared to transfection with the p27(Kip1) vector. In vivo, sub cutaneous tumours established in nude mice were injected with naked DNA encoding P27 or P27VP22. Our results show that P27VP22 can spread in vivo and that injections of the P27VP22 plasmid resulted in a significantly greater antitumour activity than injections of the P27 plasmid. This study confirms the usefulness of VP22-mediated delivery and suggests that P27VP22 may have applications in cancer gene therapy.
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Affiliation(s)
- D Zavaglia
- Groupe de Recherche sur le Cancer du Pumon, Equipe INSERM 9924, Institut Albert Bonnoit, La Tronche cedex, France
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25
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Abstract
Tissue regeneration in humans is limited and excludes vitals organs like heart and brain. Transformation experiments with oncogenes like T antigen have shown that retrodifferentiation of the respective cells is possible but hard to control. To bypass the risk of cancer formation a protein therapy approach has been developed. The transient delivery of proteins rather than genes could still induce terminally-differentiated cells to reenter the cell cycle. This approach takes advantage of protein-transducing domains that mediate the transfer of cargo proteins into cells. The goal of this brief review is to outline the basics of protein transduction and to discuss potential applications for tissue regeneration.
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26
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Bearer EL, Satpute-Krishnan P. The role of the cytoskeleton in the life cycle of viruses and intracellular bacteria: tracks, motors, and polymerization machines. CURRENT DRUG TARGETS. INFECTIOUS DISORDERS 2002; 2:247-64. [PMID: 12462128 PMCID: PMC3616324 DOI: 10.2174/1568005023342407] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Recent advances in microbiology implicate the cytoskeleton in the life cycle of some pathogens, such as intracellular bacteria, Rickettsia and viruses. The cellular cytoskeleton provides the basis for intracellular movements such as those that transport the pathogen to and from the cell surface to the nuclear region, or those that produce cortical protrusions that project the pathogen outwards from the cell surface towards an adjacent cell. Transport in both directions within the neuron is required for pathogens such as the herpesviruses to travel to and from the nucleus and perinuclear region where replication takes place. This trafficking is likely to depend on cellular motors moving on a combination of microtubule and actin filament tracks. Recently, Bearer et al. reconstituted retrograde transport of herpes simplex virus (HSV) in the giant axon of the squid. These studies identified the tegument proteins as the viral proteins most likely to recruit retrograde motors for the transport of HSV to the neuronal nucleus. Similar microtubule-based intracellular movements are part of the biological behavior of vaccinia, a poxvirus, and of adenovirus. Pathogen-induced surface projections and motility within the cortical cytoplasm also play a role in the life cycle of intracellular pathogens. Such motility is driven by pathogen-mediated actin polymerization. Virulence depends on this actin-based motility, since virulence is reduced in Listeria ActA mutants that lack the ability to recruit Arp2/3 and polymerize actin and in vaccinia virus mutants that cannot stimulate actin polymerization. Inhibition of intracellular movements provides a potential strategy to limit pathogenicity. The host cell motors and tracks, as well as the pathogen factors that interact with them, are potential targets for novel antimicrobial therapy.
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Affiliation(s)
- E L Bearer
- Department of Pathology and Laboratory Medicine, Brown University Medical School, Providence, RI 02912, USA.
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27
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Sciortino MT, Taddeo B, Poon APW, Mastino A, Roizman B. Of the three tegument proteins that package mRNA in herpes simplex virions, one (VP22) transports the mRNA to uninfected cells for expression prior to viral infection. Proc Natl Acad Sci U S A 2002; 99:8318-23. [PMID: 12060774 PMCID: PMC123065 DOI: 10.1073/pnas.122231699] [Citation(s) in RCA: 87] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
An earlier report has shown that herpes simplex virus 1 virions package RNA. Experiments designed to reveal the identity of the virion proteins capable of binding the RNA and to show whether the mRNA carried in the newly infected cells was expressed showed the following: (i) (32)P-labeled riboprobe generated by in vitro transcription of the U(S)8.5 ORF bound three proteins identified as the products of U(S)11, U(L)47, and U(L)49 (VP22) genes. (ii) Viral RNA was bound to U(L)47 or U(S)11 proteins immune precipitated from cells transduced with baculoviruses expressing U(L)47 or U(S)11 and then superinfected with HSV-1 under conditions that blocked DNA synthesis and assembly of virions. (iii) Virions were purified from cells transduced with a baculovirus encoding a U(S)8.5 protein fused to green fluorescent protein and superinfected with an HSV-1 mutant lacking the U(S)8-12 genes. HEp-2 cells infected with these virions expressed the chimeric protein in approximately 1% of infected cells. (iv) In mixed cultures, untreated Vero cells acquired the mRNA encoding the green fluorescent-U(S)8.5 chimeric protein from HEp-2 cells doubly transduced with the genes encoding VP22 and the chimeric protein. The transfer was RNase sensitive and VP22 dependent, indicating that the RNA encoded by the chimeric gene was transferred to Vero cells as mRNA. We conclude that (i) three virion proteins are capable of binding RNA; (ii) the packaged RNA can be expressed in newly infected cells; and (iii) the U(L)47 protein was earlier reported to shuttle from nucleus to the cytoplasm and may transport RNA. VP22 thus appears to be a member of a new class of viral proteins whose major function is to bind and transport infected cell mRNA to uninfected cells to create the environment for effective initiation of infection.
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Affiliation(s)
- Maria Teresa Sciortino
- The Marjorie B. Kovler Viral Oncology Laboratories, University of Chicago, 910 East 58th Street, Chicago, IL 60637, USA
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28
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Zender L, Kühnel F, Köck R, Manns M, Kubicka S. VP22-mediated intercellular transport of p53 in hepatoma cells in vitro and in vivo. Cancer Gene Ther 2002; 9:489-96. [PMID: 12032659 DOI: 10.1038/sj.cgt.7700465] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2002] [Indexed: 12/13/2022]
Abstract
The capacity of VP22 chimeric proteins to spread from the primary transduced cell to surrounding cells could improve gene therapy approaches, especially in cancer therapy. However, there are conflicting data about VP22-mediated intercellular trafficking in different studies. To assess the role of VP22 in gene therapy of hepatocellular carcinomas (HCCs) we constructed expression vectors for N- and C-terminal versions of VP22-p53 fusion proteins and investigated the VP22-mediated shuttle effect in hepatoma cells by cotransfection experiments. VP22-mediated trafficking was not detectable in hepatoma cells in vitro by fluorescence microscopy, but reporter gene transactivation assays demonstrated intercellular trafficking of functional VP22-p53 in vitro. For in vivo experiments, the recombinant adenoviruses Ad5CMVp53 and Ad5CMVp53-VP22 were constructed. In contrast to the in vitro experiments intercellular trafficking of VP22-p53 could be observed in subcutaneous tumors of hepatoma cells by fluorescence microscopy, indicating a stronger shuttle effect in solid tumors compared to cell culture experiments. Because spread of p53-VP22 in liver tumors was correlated with enhanced apoptosis of hepatoma cells VP22-mediated trafficking of potential therapeutic proteins may improve the results of gene therapy of HCCs.
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Affiliation(s)
- Lars Zender
- Department of Gastroenterology and Hepatology, Medical School Hannover, Hannover 30625, Germany
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29
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Martin A, O'Hare P, McLauchlan J, Elliott G. Herpes simplex virus tegument protein VP22 contains overlapping domains for cytoplasmic localization, microtubule interaction, and chromatin binding. J Virol 2002; 76:4961-70. [PMID: 11967313 PMCID: PMC136174 DOI: 10.1128/jvi.76.10.4961-4970.2002] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We have previously shown that the 301-amino-acid herpes simplex virus tegument protein VP22 exhibits a range of subcellular localization patterns when expressed in isolation from other virus proteins. By using live-cell analysis of cells expressing green fluorescent protein (GFP)-tagged VP22 we have shown that when VP22 is first expressed in the cell it localizes to the cytoplasm, where, when present at high enough concentrations, it can assemble onto microtubules, causing them to bundle and become highly stabilized. In addition we have shown that when a cell expressing VP22 enters mitosis, the cytoplasmic population of VP22 translocates to the nucleus, where it efficiently binds mitotic chromatin. Here we have investigated the specific regions of the VP22 open reading frame required for these properties. Using GFP-VP22 as our starting molecule, we have constructed a range of N- and C-terminal truncations and analyzed their localization patterns in live cells. We show that the C-terminal 242 residues of VP22 are sufficient to induce microtubule bundling. Within this subregion, the C-terminal 89 residues contain a signal for cytoplasmic localization of the protein, while a larger region comprising the C-terminal 128 residues of the VP22 protein is required for mitotic chromatin binding. Furthermore, a central 100-residue domain of VP22 maintains the ability to bind microtubules without inducing bundling, suggesting that additional regions flanking this microtubule binding domain may be required to alter the microtubule network. Hence, the signals involved in dictating the complex localization patterns of VP22 are present in overlapping regions of the protein.
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Affiliation(s)
- Ana Martin
- Virus Assembly Group, Marie Curie Research Institute, Oxted, Surrey RH8 0TL, United Kingdom
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30
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Hung CF, He L, Juang J, Lin TJ, Ling M, Wu TC. Improving DNA vaccine potency by linking Marek's disease virus type 1 VP22 to an antigen. J Virol 2002; 76:2676-82. [PMID: 11861834 PMCID: PMC135982 DOI: 10.1128/jvi.76.6.2676-2682.2002] [Citation(s) in RCA: 65] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We have previously employed an intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein to enhance DNA vaccine potency because DNA vaccines lack the intrinsic ability to amplify in cells. Recently, studies have demonstrated that the protein encoded by UL49 of Marek's disease virus type 1 (MDV-1) exhibits some degree of homology to the HSV-1 VP22 protein and features the property of intercellular transport. We therefore generated a DNA vaccine encoding MDV-1 VP22 linked to a model antigen, human papillomavirus type 16 E7. We demonstrated that compared with mice vaccinated with DNA encoding wild-type E7, mice vaccinated with MDV-1 VP22/E7 DNA exhibited a significant increase in number of gamma-interferon-secreting, E7-specific CD8(+)-T-cell precursors as well as stronger tumor prevention and treatment effects. Furthermore, our data indicated that the antitumor effect was CD8 dependent. These results suggested that the development of vaccines encoding VP22 fused to a target antigen might be a promising strategy for improving DNA vaccine potency.
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Affiliation(s)
- Chien-Fu Hung
- Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205, USA
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