The Experts below are selected from a list of 1836 Experts worldwide ranked by ideXlab platform
Youchun Wang - One of the best experts on this subject based on the ideXlab platform.
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establishment and validation of a Pseudovirus neutralization assay for sars cov 2
Emerging microbes & infections, 2020Co-Authors: Qianqian Li, Chenyan Zhao, Li Zhang, Weijin Huang, Jiajing Wu, Meng Wang, Qiong Lu, Xiaoyu Li, Miao Xu, Youchun WangAbstract:Pseudoviruses are useful virological tools because of their safety and versatility, especially for emerging and re-emerging viruses. Due to its high pathogenicity and infectivity and the lack of effective vaccines and therapeutics, live SARS-CoV-2 has to be handled under biosafety level 3 conditions, which has hindered the development of vaccines and therapeutics. Based on a VSV Pseudovirus production system, a Pseudovirus-based neutralization assay has been developed for evaluating neutralizing antibodies against SARS-CoV-2 in biosafety level 2 facilities. The key parameters for this assay were optimized, including cell types, cell numbers, virus inoculum. When tested against the SARS-CoV-2 Pseudovirus, SARS-CoV-2 convalescent patient sera showed high neutralizing potency, which underscore its potential as therapeutics. The limit of detection for this assay was determined as 22.1 and 43.2 for human and mouse serum samples respectively using a panel of 120 negative samples. The cutoff values were set as 30 and 50 for human and mouse serum samples, respectively. This assay showed relatively low coefficient of variations with 15.9% and 16.2% for the intra- and inter-assay analyses respectively. Taken together, we established a robust Pseudovirus-based neutralization assay for SARS-CoV-2 and are glad to share Pseudoviruses and related protocols with the developers of vaccines or therapeutics to fight against this lethal virus.
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development and application of a bioluminescent imaging mouse model for chikungunya virus based on Pseudovirus system
Vaccine, 2017Co-Authors: Jiajing Wu, Weijin Huang, Chenyan Zhao, Youchun WangAbstract:Abstract Chikungunya virus (CHIKV) is an arthropod-borne virus that is transmitted to humans primarily via the bite of an infected mosquito. Infection of humans by CHIKV can cause chikungunya fever which is an acute febrile illness associated with severe, often debilitating polyarthralgias. Since a re-emergence of CHIKV in 2004, the virus has spread into novel locations in nearly 40 countries including non-endemic regions and has led to millions of cases of disease throughout countries. Handling of CHIKV is restricted to the high-containment Biosafety Level 3 (BSL-3) facilities, which greatly impede the research progress of this virus. In this study, an envelope-pseudotyped virus expressing the firefly luciferase reporter protein (pHIV–CHIKV–Fluc) was generated. An in vitro sensitive neutralizing assay and an in vivo bioluminescent-imaging-based mouse infection model had been developed based on the CHIKV Pseudovirus. Utilizing the platform, protection effect of DNA vaccine was evaluated. Therefore, this study provides a safe, sensitive and visualizing model for evaluating vaccines and antiviral therapies against CHIKV in low containment BSL-2 laboratories.
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a bioluminescent imaging mouse model for marburg virus based on a Pseudovirus system
Human Vaccines & Immunotherapeutics, 2017Co-Authors: Li Zhang, Weijin Huang, Qianqian Li, Youchun WangAbstract:Marburg virus (MARV) can cause lethal hemorrhagic fever in humans. Handling of MARV is restricted to high-containment biosafety level 4 (BSL-4) facilities, which greatly impedes research into this virus. In this study, a high titer of MARV Pseudovirus was generated through optimization of the HIV backbone vectors, the ratio of backbone vector to MARV glycoprotein expression vector, and the transfection reagents. An in vitro neutralization assay and an in vivo bioluminescent imaging mouse model for MARV were developed based on the Pseudovirus. Protective serum against MARV was successfully induced in guinea pigs, which showed high neutralization activity in vitro and could also protect Balb/c mice from MARV Pseudovirus infection in vivo. This system could be a convenient tool to enable the evaluation of vaccines and therapeutic drugs against MARV in non-BSL-4 laboratories.
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development of in vitro and in vivo rabies virus neutralization assays based on a high titer Pseudovirus system
Scientific Reports, 2017Co-Authors: Xiaohong Wu, Weijin Huang, Xuguang Li, Yuhua Li, Youchun WangAbstract:Pseudoviruses are useful virological tools because of their safety and versatility; however the low titer of these viruses substantially limits their wider applications. We developed a highly efficient Pseudovirus production system capable of yielding 100 times more rabies Pseudovirus than the traditional method. Employing the high-titer Pseudoviruses, we have developed robust in vitro and in vivo neutralization assays for the evaluation of rabies vaccine, which traditionally relies on live-virus based assays. Compared with current rapid fluorescent focus inhibition test (RFFIT), our in vitro Pseudovirus-based neutralization assay (PBNA) is much less labor-intensive while demonstrating better reproducibility. Moreover, the in vivo PBNA assay was also found to be superior to the live virus based assay. Following intravenous administration, the Pseudovirus effectively infected the mice, with dynamic viral distributions being sequentially observed in spleen, liver and brain. Furthermore, data from in vivo PBNA showed great agreement with those generated from the live virus model but with the experimental time significantly reduced from 2 weeks to 3 days. Taken together, the effective Pseudovirus production system facilitated the development of novel PBNA assays which could replace live virus-based traditional assays due to its safety, rapidity, reproducibility and high throughput capacity.
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Study of the relationship between antibody level and protection induced by human papillomavirus(HPV) L2-peptide
Chinese journal of microbiology and immunology, 2010Co-Authors: Xueling Wu, Chuntao Zhang, Shu-fang Meng, Youchun WangAbstract:Objective To detect the protection induced by HPV-58 L2 11-200 AA in animal, and analyze the relationship between antibody or neutralizing antibody titers and the protection generated by the immunizmg agent. Methods The peptide of HPV-58 L2 11-200 AA was expressed in E. coli and the mice were immunized with the peptide after purification and adsorption with aluminum adjuvant. The protection provided by different immunizing doses was detected in the mouse model against the challenge of the pseud-ovirions of human papiilomavirus types 58. The total antibodies and neutralizing antibody titers of serum were tested with ELISA and neutralization assay against HPV-58 Pseudovirus, respectively. The total antibodies or neutralizing antibody titers that can protect the mouse from infection were analyzed. Results The mice can be protected from the challenge with HPV Pseudovirus when the immunizing dose was 8 μg. The neutralizing antibody can not be detected in the immune serum by neutralization assay against Pseudovirus. The total anti-body level has a corresponding relationship with the protection showed in mouse model. The results of total antibodies detected by ELISA showed that when the titer of total antibodies was ≥25 000, luminescent signal can not be detected and the mice can be protected from Pseudovirus infection. Conclusion HPV-58 L2 11-200 AA peptide can protect mice from Pseudovirus infection. L2 peptide has a promising perspective to be a candidate vaccine and the level of total antibodies in the immune serum can be used as a surrogate for the evaluation of protection against HPV infection. Key words: Human papillomavirus; L2-peptide; Pseudovirus; Infection model; Neutralizing anti-bodies
John T. Schiller - One of the best experts on this subject based on the ideXlab platform.
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immunogenic human papillomavirus Pseudovirus mediated suicide gene therapy for bladder cancer
International Journal of Molecular Sciences, 2016Co-Authors: Rim Hojeij, Marianne Nkosi, Dalila Gharbi, John T. Schiller, Sonia Domingospereira, Laurent Derre, Patrice Jichlinski, Denise NardellihaefligerAbstract:Bladder cancer is the second most common urological malignancy in the world. In 70% of cases it is initially diagnosed as non-muscle-invasive bladder cancer (NMIBC) and it is amenable to local treatments, with intravesical (IVES) Bacillus-Calmette-Guerin (BCG) immunotherapy being routinely used after transurethral resection of the lesion. However, this treatment is associated with significant side-effects and treatment failures, highlighting the necessity of novel strategies. One potent approach is the suicide-gene mediated therapy/prodrug combination, provided tumor-specificity can be ensured and anti-tumor immune responses induced. Using the mouse syngeneic orthotopic MB49-bladder tumor model, here we show that IVES human papillomavirus non-replicative pseudovirions (PsV) can pseudoinfect tumors with a ten-fold higher efficacy than normal bladders. In addition, PsV carrying the suicide-gene herpes-simplex virus thymidine kinase (PsV-TK) combined to Ganciclovir (GCV) led to immunogenic cell-death of tumor cells in vitro and to MB49-specific CD8 T-cells in vivo. This was associated with reduction in bladder-tumor growth and increased mice survival. Altogether, our data show that IVES PsV-TK/GCV may be a promising alternative or combinatory treatment for NMIBC.
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vaccination with human papillomavirus Pseudovirus encapsidated plasmids targeted to skin using microneedles
PLOS ONE, 2015Co-Authors: Rhonda C Kines, Kizzmekia S Corbett, John Nicewonger, Teresa R Johnson, Man Chen, Yuk Ying S Pang, Vladimir Zarnitsyn, Anu Gangopadhyay, Mark R Prausnitz, John T. SchillerAbstract:Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.
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identification of a role for the trans golgi network in human papillomavirus 16 Pseudovirus infection
Journal of Virology, 2013Co-Authors: Cynthia D Thompson, Douglas R Lowy, Rachel M Schowalter, John T. SchillerAbstract:Human papillomavirus 16 (HPV16) enters its host cells by a process that most closely resembles macropinocytosis. Uncoating occurs during passage through the endosomal compartment, and the low pH encountered in this environment is essential for infection. Furin cleavage of the minor capsid protein, L2, and cyclophilin B-mediated separation of L2 and the viral genome from the major capsid protein, L1, are necessary for escape from the late endosome (LE). Following this exodus, L2 and the genome are found colocalized at the ND10 nuclear subdomain, which is essential for efficient pseudogenome expression. However, the route by which L2 and the genome traverse the intervening cytoplasm between these two subcellular compartments has not been determined. This study extends our understanding of this phase in PV entry in demonstrating the involvement of the Golgi complex. With confocal microscopic analyses involving 5-ethynyl-2′-deoxyuridine (EdU)-labeled pseudogenomes and antibodies to virion and cellular proteins, we found that the viral pseudogenome and L2 travel to the trans-Golgi network (TGN) following exit from the LE, while L1 is retained. This transit is dependent upon furin cleavage of L2 and can be prevented pharmacologically with either brefeldin A or golgicide A, inhibitors of anterograde and retrograde Golgi trafficking. Additionally, Rab9a and Rab7b were determined to be mediators of this transit, as expression of dominant negative versions of these proteins, but not Rab7a, significantly inhibited HPV16 Pseudovirus infection.
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murine skin and vaginal mucosa are similarly susceptible to infection by pseudovirions of different papillomavirus classifications and species
Virology, 2012Co-Authors: Alessandra Handisurya, Christopher B Buck, Cynthia D Thompson, Douglas R Lowy, Kihyuck Kwak, Richard B S Roden, John T. SchillerAbstract:Abstract Depending upon viral genotype, productive papillomavirus infection and disease display preferential tropism for cutaneous or mucosal stratified squamous epithelia, although the mechanisms are unclear. To investigate papillomavirus entry tropism, we used reporter pseudovirions based on various cutaneous and mucosal papillomavirus species, including the recently identified murine papillomavirus. Pseudovirus transduction of BALB/c mice was examined using an improved murine skin infection protocol and a previously developed cervicovaginal challenge model. In the skin, HPV5, HPV6, HPV16, BPV1 and MusPV1 pseudovirions preferentially transduced keratinocytes at sites of trauma, similar to the genital tract. Skin infection, visualized by in vivo imaging using a luciferase reporter gene, peaked between days 2–3 and rapidly diminished for all pseudovirion types. Murine cutaneous and genital tissues were similarily permissive for pseudovirions of HPV types 5, 6, 8, 16, 18, 26, 44, 45, 51, 58 and animal papillomaviruses BPV1 and MusPV1, implying that papillomavirus' tissue and host tropism is governed primarily by post-entry regulatory events in the mouse.
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mucosal delivery of human papillomavirus Pseudovirus encapsidated plasmids improves the potency of dna vaccination
Mucosal Immunology, 2010Co-Authors: Barney S Graham, Rhonda C Kines, Kizzmekia S Corbett, John Nicewonger, Teresa R Johnson, Man Chen, Daaimah Lavigne, Jeffrey N Roberts, Nicolas Cuburu, John T. SchillerAbstract:Mucosal delivery of human papillomavirus Pseudovirus-encapsidated plasmids improves the potency of DNA vaccination
T M Wilson - One of the best experts on this subject based on the ideXlab platform.
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expression of tobacco mosaic virus coat protein and assembly of Pseudovirus particles in escherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Dukju Hwang, I M Roberts, T M WilsonAbstract:The bidirectional self-assembly of tobacco mosaic virus (TMV, common or U1 strain) has been studied extensively in vitro. Foreign single-stranded RNA molecules containing the TMV origin-of-assembly sequence (OAS, 75-432 nt in length) are also packaged by TMV coat protein (CP) in vitro to form helical Pseudovirus particles. To study virus assembly in vivo requires an easily manipulated model system, independent of replication in plants. The TMV assembly machinery also provides a convenient means to protect and recover chimeric gene transcripts of almost any length or sequence for a variety of applications. Native TMV CP expressed in and purified from Escherichia coli formed nonhelical, stacked aggregates after dialysis into pH 5 buffer and was inactive for in vitro assembly with TMV RNA. U1 CP derivatives in which the second amino acid was changed from Ser to Ala or Pro, nonacetylated N termini found in two natural strains of the virus, failed to remediate these anomalous properties. However, in vivo coexpression of CP and single-stranded RNAs (up to approximately 2 kb) containing the TMV OAS gave high yields of helical Pseudovirus particles of the predicted length (up to 7.4 +/- 1.4 micrograms/mg of total bacterial protein). If the OAS-containing RNA was first recruited into bacterial polyribosomes, elongation of Pseudovirus assembly was blocked. In vivo, E. coli expression of a full-length cDNA clone of the TMV genome (6.4 kb) resulted in high, immunodetectable levels of CP and assembly of sufficient intact genomic RNA to initiate systemic infection of susceptible tobacco plants.
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assembly of tobacco mosaic virus and tmv like Pseudovirus particles in escherichia coli
Archives of virology. Supplementum, 1994Co-Authors: Dukju Hwang, I M Roberts, T M WilsonAbstract:High-level expression of plant viral proteins, including coat protein (CP), is possible in Escherichia coli. Native tobacco mosaic virus (TMV) CP expressed in E. coli remains soluble but has a non-acetylated N-terminal Ser residue and, following extraction, is unable to package TMV RNA in vitro under standard assembly conditions. Changing the Ser to Ala or Pro by PCR-mutagenesis did not confer assembly competence in vitro, despite these being non-acetylated N-termini present in two natural strains of TMV. All TMV CPs made in E. coli formed stacked cylindrical aggregates in vitro at pH 5.0 and failed to be immunogold-labelled using a mouse monoclonal antibody specific for helically assembled TMV CP. TMV self-assembly has been studied extensively in vitro, and an origin of assembly sequence (OAS) mapped internally on the 6.4kb ssRNA genome. Pseudovirus particles can be assembled mono- or bi-directionally in vitro using virus-derived CP and chimeric ssRNAs containing the cognate TMV OAS, but otherwise of unlimited length and sequence. Studies on plant virus assembly in vivo would be facilitated by a model system amenable to site-directed mutagenesis and rapid recovery of progeny particles. When chimeric transcripts containing the TMV OAS were co-expressed with TMV CP in vivo for 2–18h, helical TMV-like ribonucleoprotein particles of the predicted length were formed in high yield (up to 7.4µg/mg total bacterial protein). In addition to providing a rapid, inexpensive and convenient system to produce, protect and recover chimeric gene transcripts of any length or sequence, this E. coli system also offers a rapid approach for studying the molecular requirements for plant virus “self-assembly” in vivo. Transcription of a full-length cDNA clone of TMV RNA also resulted in high levels of CP expression and assembly of sufficient intact genomic RNA to initiate virus infection of susceptible tobacco plants.
Shibo Jiang - One of the best experts on this subject based on the ideXlab platform.
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a safe and convenient Pseudovirus based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus mers cov
Virology Journal, 2013Co-Authors: Guangyu Zhao, Lanying Du, Ye Li, Lin Li, Vincent K M Poon, Lili Wang, Fei Yu, Bojian Zheng, Shibo JiangAbstract:Background: Evidence points to the emergence of a novel human coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), which causes a severe acute respiratory syndrome (SARS)-like disease. In response, the development of effective vaccines and therapeutics remains a clinical priority. To accomplish this, it is necessary to evaluate neutralizing antibodies and screen for MERS-CoV entry inhibitors. Methods: In this study, we produced a Pseudovirus bearing the full-length spike (S) protein of MERS-CoV in the Env-defective, luciferase-expressing HIV-1 backbone. We then established a Pseudovirus-based inhibition assay to detect neutralizing antibodies and anti-MERS-CoV entry inhibitors. Results: Our results demonstrated that the generated MERS-CoV Pseudovirus allows for single-cycle infection of a variety of cells expressing dipeptidyl peptidase-4 (DPP4), the confirmed receptor for MERS-CoV. Consistent with the results from a live MERS-CoV-based inhibition assay, the antisera of mice vaccinated with a recombinant protein containing receptor-binding domain (RBD, residues 377–662) of MERS-CoV S fused with Fc of human IgG exhibited neutralizing antibody response against infection of MERS-CoV Pseudovirus. Furthermore, one small molecule HIV entry inhibitor targeting gp41 (ADS-J1) and the 3-hydroxyphthalic anhydride-modified human serum albumin (HP-HSA) could significantly inhibit MERS-CoV Pseudovirus infection. Conclusion: Taken together, the established MERS-CoV inhibition assay is a safe and convenient Pseudovirus-based alternative to BSL-3 live-virus restrictions and can be used to rapidly screen MERS-CoV entry inhibitors, as well as evaluate vaccine-induced neutralizing antibodies against the highly pathogenic MERS-CoV.
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receptor binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation dependent epitopes that induce highly potent neutralizing antibodies
Journal of Immunology, 2005Co-Authors: Yuxian He, Hong Lu, Pamela Siddiqui, Yusen Zhou, Shibo JiangAbstract:The spike (S) protein of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is a major antigenic determinant capable of inducing protective immunity. Recently, a small fragment on the SARS-CoV S protein (residues 318–510) was characterized as a minimal receptor-binding domain (RBD), which mediates virus binding to angiotensin-converting enzyme 2, the functional receptor on susceptible cells. In this study, we demonstrated that a fusion protein containing RBD linked to human IgG1 Fc fragment (designated RBD-Fc) induced high titer of RBD-specific Abs in the immunized mice. The mouse antisera effectively neutralized infection by both SARS-CoV and SARS Pseudovirus with mean 50% neutralization titers of 1/15,360 and 1/24,737, respectively. The neutralization determinants on the RBD of S protein were characterized by a panel of 27 mAbs isolated from the immunized mice. Six groups of conformation-dependent epitopes, designated as Conf I–VI, and two adjacent linear epitopes were identified by ELISA and binding competition assays. The Conf IV and Conf V mAbs significantly blocked RBD-Fc binding to angiotensin-converting enzyme 2, suggesting that their epitopes overlap with the receptor-binding sites in the S protein. Most of the mAbs (23 of 25) that recognized the conformational epitopes possessed potent neutralizing activities against SARS Pseudovirus with 50% neutralizing dose ranging from 0.005 to 6.569 μg/ml. Therefore, the RBD of SARS S protein contains multiple conformational epitopes capable of inducing potent neutralizing Ab responses, and is an important target site for developing vaccines and immunotherapeutics.
Dukju Hwang - One of the best experts on this subject based on the ideXlab platform.
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expression of tobacco mosaic virus coat protein and assembly of Pseudovirus particles in escherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Dukju Hwang, I M Roberts, T M WilsonAbstract:The bidirectional self-assembly of tobacco mosaic virus (TMV, common or U1 strain) has been studied extensively in vitro. Foreign single-stranded RNA molecules containing the TMV origin-of-assembly sequence (OAS, 75-432 nt in length) are also packaged by TMV coat protein (CP) in vitro to form helical Pseudovirus particles. To study virus assembly in vivo requires an easily manipulated model system, independent of replication in plants. The TMV assembly machinery also provides a convenient means to protect and recover chimeric gene transcripts of almost any length or sequence for a variety of applications. Native TMV CP expressed in and purified from Escherichia coli formed nonhelical, stacked aggregates after dialysis into pH 5 buffer and was inactive for in vitro assembly with TMV RNA. U1 CP derivatives in which the second amino acid was changed from Ser to Ala or Pro, nonacetylated N termini found in two natural strains of the virus, failed to remediate these anomalous properties. However, in vivo coexpression of CP and single-stranded RNAs (up to approximately 2 kb) containing the TMV OAS gave high yields of helical Pseudovirus particles of the predicted length (up to 7.4 +/- 1.4 micrograms/mg of total bacterial protein). If the OAS-containing RNA was first recruited into bacterial polyribosomes, elongation of Pseudovirus assembly was blocked. In vivo, E. coli expression of a full-length cDNA clone of the TMV genome (6.4 kb) resulted in high, immunodetectable levels of CP and assembly of sufficient intact genomic RNA to initiate systemic infection of susceptible tobacco plants.
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assembly of tobacco mosaic virus and tmv like Pseudovirus particles in escherichia coli
Archives of virology. Supplementum, 1994Co-Authors: Dukju Hwang, I M Roberts, T M WilsonAbstract:High-level expression of plant viral proteins, including coat protein (CP), is possible in Escherichia coli. Native tobacco mosaic virus (TMV) CP expressed in E. coli remains soluble but has a non-acetylated N-terminal Ser residue and, following extraction, is unable to package TMV RNA in vitro under standard assembly conditions. Changing the Ser to Ala or Pro by PCR-mutagenesis did not confer assembly competence in vitro, despite these being non-acetylated N-termini present in two natural strains of TMV. All TMV CPs made in E. coli formed stacked cylindrical aggregates in vitro at pH 5.0 and failed to be immunogold-labelled using a mouse monoclonal antibody specific for helically assembled TMV CP. TMV self-assembly has been studied extensively in vitro, and an origin of assembly sequence (OAS) mapped internally on the 6.4kb ssRNA genome. Pseudovirus particles can be assembled mono- or bi-directionally in vitro using virus-derived CP and chimeric ssRNAs containing the cognate TMV OAS, but otherwise of unlimited length and sequence. Studies on plant virus assembly in vivo would be facilitated by a model system amenable to site-directed mutagenesis and rapid recovery of progeny particles. When chimeric transcripts containing the TMV OAS were co-expressed with TMV CP in vivo for 2–18h, helical TMV-like ribonucleoprotein particles of the predicted length were formed in high yield (up to 7.4µg/mg total bacterial protein). In addition to providing a rapid, inexpensive and convenient system to produce, protect and recover chimeric gene transcripts of any length or sequence, this E. coli system also offers a rapid approach for studying the molecular requirements for plant virus “self-assembly” in vivo. Transcription of a full-length cDNA clone of TMV RNA also resulted in high levels of CP expression and assembly of sufficient intact genomic RNA to initiate virus infection of susceptible tobacco plants.
