Assembly of Human Papillomavirus Type-16 Virus-Like Particles: Multifactorial Study of Assembly and Competing Aggregation

Hanslip, Simon J.; Zaccai, Nathan R.; Middelberg, Anton P. J.; Falconer, Robert J.

doi:10.1021/bp0502781

Cited by 33 publications

(23 citation statements)

References 20 publications

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“…The preparation was then diluted with increasing concentrations of CaCl 2 up to a final concentration of 5 mmol/L, with or without ZnCl 2 (10 nmol/L). ZnCl 2 was used because it has been reported that ZnCl 2 enhances the assembly of HPV capsomers into VLPs (29). Pseudovirions were then dialyzed against PBS 1Â overnight and stored at 4jC before use.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of cervical cancer cell growth by human papillomavirus virus–like particles packaged with human papillomavirus oncoprotein short hairpin RNAs

Bousarghin¹,

Touzé²,

Gaud³

et al. 2009

Molecular Cancer Therapeutics

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Overexpression of human papillomavirus (HPV E6 and HPV E7) oncogenes in human cervical cells results in the development of cancer, and E6 and E7 proteins are therefore targets for preventing cervical cancer progression. Here, we describe the silencing of E6 and E7 expression in cervical carcinoma cells by RNA interference. In order to increase the efficacy of the RNA interference, HPV pseudovirions coding for a short hairpin RNA (shRNA) sequence were produced. The results indicated the degradation of E6 and E7 mRNAs when shRNA against E6 or E7 were delivered by pseudovirions in HPV-positive cells (CaSki and TC1 cells). E6 silencing resulted in the accumulation of cellular p53 and reduced cell viability. More significant cell death was observed when E7 expression was suppressed. Silencing E6 and E7 and the consequences for cancer cell growth were also investigated in vivo in mice using the capacity of murine TC1 cells expressing HPV-16 E6 and E7 oncogenes to induce fast-growing tumors. Treatment with lentiviruses and HPV virus-like particle vectors coding for an E7 shRNA sequence both resulted in dramatic inhibition of tumor growth. These results show the ability of pseudovirion-delivered shRNA to produce specific gene suppression and provide an effective means of reducing HPV-positive tumor growth. [Mol Cancer Ther 2009; 8(2):357 -65]

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Section: Methodsmentioning

confidence: 99%

Inhibition of cervical cancer cell growth by human papillomavirus virus–like particles packaged with human papillomavirus oncoprotein short hairpin RNAs

Bousarghin¹,

Touzé²,

Gaud³

et al. 2009

Molecular Cancer Therapeutics

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“…For example, recombinant HPV VLP purified from yeast readily form fiber-like aggregate clusters during storage, especially at low salt and protein concentration conditions (Shi et al, 2005;Zhao et al, 2007). Also, experimental data (Dong et al, 1998;Hanslip et al, 2006;McCarthy et al, 1998;Salunke et al, 1989;Sun et al, 2007) have suggested empirically that non-native aggregation is often in competition with capsid assembly, resulting in polymorphic or amorphous aggregate structures. For instance, aggregation of structural protein or VLPs occurs during assembly of the HPV VLP, and is greatly increased when zinc is present in the assembly mixture (Hanslip et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

“…Also, experimental data (Dong et al, 1998;Hanslip et al, 2006;McCarthy et al, 1998;Salunke et al, 1989;Sun et al, 2007) have suggested empirically that non-native aggregation is often in competition with capsid assembly, resulting in polymorphic or amorphous aggregate structures. For instance, aggregation of structural protein or VLPs occurs during assembly of the HPV VLP, and is greatly increased when zinc is present in the assembly mixture (Hanslip et al, 2006). It has been shown recently that aggregation of already assembled VLPs during storage is exacerbated by surface adsorption of VLPs to storage vessels, thus can be minimized through optimization of salt and non-ionic surfactant levels (Shi et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

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Modeling the competition between aggregation and self‐assembly during virus‐like particle processing

Ding

Chuan

et al. 2010

Biotech & Bioengineering

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Understanding and controlling aggregation is an essential aspect in the development of pharmaceutical proteins to improve product yield, potency and quality consistency. Even a minute quantity of aggregates may be reactogenic and can render the final product unusable. Self-assembly processing of virus-like particles (VLPs) is an efficient method to quicken the delivery of safe and efficacious vaccines to the market at low cost. VLP production, as with the manufacture of many biotherapeutics, is susceptible to aggregation, which may be minimized through the use of accurate and practical mathematical models. However, existing models for virus assembly are idealized, and do not predict the non-native aggregation behavior of self-assembling viral subunits in a tractable nor useful way. Here we present a mechanistic mathematical model describing VLP self-assembly that accounts for partitioning of reactive subunits between the correct and aggregation pathways. Our results show that unproductive aggregation causes up to 38% product loss by competing favorably with the productive nucleation of self-assembling subunits, therefore limiting the availability of nuclei for subsequent capsid growth. The protein subunit aggregation reaction exhibits an apparent second-order concentration dependence, suggesting a dimerization-controlled agglomeration pathway. Despite the plethora of possible assembly intermediates and aggregation pathways, protein aggregation behavior may be predicted by a relatively simple yet realistic model. More importantly, we have shown that our bioengineering model is amenable to different reactor formats, thus opening the way to rational scale-up strategies for products that comprise biomolecular assemblies.

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“…A pH of 6.5 is more favorable for PCV2b VLP assembly. Coincidently, PH 6.5 is the most optimal pH for the effective assembly of HPV16-L1 VLPs [18,19]. We speculate that this is because pH 6.5 mimics the condition in the endoplasmic reticulum and Golgi apparatus, where the CP is physiologically self-assembled during the virus life cycle in mammals [12].…”

Section: Figmentioning

confidence: 99%

Detection of the Assembly and Disassembly of PCV2b Virus-Like Particles Using Fluorescence Spectroscopy Analysis

Fang

Diao²,

Dong³

et al. 2015

Intervirology

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Monitoring the assembly and disassembly of virus-like particles (VLPs) is important in developing effective VLP-based vaccines. We tried to establish a simple and rapid method to evaluate the status of VLP assembly using fluorescence spectroscopic analysis (FSA) while developing a VLP-based vaccine against porcine circovirus type 2b (PCV2b). We synthesized the gene coding for PCV2b capsid protein (CP). The CP was expressed in Escherichia coli in a soluble form, dialyzed into three different buffers, and assembled into VLPs. The immunogenicity of the VLPs was evaluated by an enzyme-linked immunosorbent assay using the sera of mice immunized with inactivated PCV2b. The VLP assembly was detected using transmission electron microscopy and FSA. The assembled VLPs showed a distinct FSA curve with a peak at 320 nm. We found that the assembly status was related to the immunogenicity, fluorescence intensity, and morphology of the VLP. The FSA assay was able to monitor the various denatured statuses of PCV2b VLPs treated with β-mercaptoethanol or β-mercaptoethanol plus urea. We have demonstrated that FSA can be used to detect the assembly of PCV2b VLPs produced in E. coli. This provides a simple solution for monitoring VLP assembly during the production of VLP-based vaccines.

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Assembly of Human Papillomavirus Type-16 Virus-Like Particles: Multifactorial Study of Assembly and Competing Aggregation

Cited by 33 publications

References 20 publications

Inhibition of cervical cancer cell growth by human papillomavirus virus–like particles packaged with human papillomavirus oncoprotein short hairpin RNAs

Inhibition of cervical cancer cell growth by human papillomavirus virus–like particles packaged with human papillomavirus oncoprotein short hairpin RNAs

Modeling the competition between aggregation and self‐assembly during virus‐like particle processing

Detection of the Assembly and Disassembly of PCV2b Virus-Like Particles Using Fluorescence Spectroscopy Analysis

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