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Chemical addressability of ultraviolet-inactivated viral nanoparticles (VNPs).

Rae C, Koudelka KJ, Destito G, Estrada MN, Gonzalez MJ, Manchester M - PLoS ONE (2008)

Bottom Line: Although CPMV has not been shown to replicate in mammalian cells, uptake in mammalian cells does occur in vitro and in vivo.Intermediate doses of 2.0-2.5 J/cm(2) were shown to maintain particle structure and chemical reactivity, and cellular binding properties were similar to CPMV-WT.These studies demonstrate that it is possible to inactivate CPMV infectivity while maintaining particle structure and function, thus paving the way for further development of CPMV nanoparticles for in vivo applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, and Center for Integrative Molecular Biosciences, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT

Background: Cowpea Mosaic Virus (CPMV) is increasingly being used as a nanoparticle platform for multivalent display of molecules via chemical bioconjugation to the capsid surface. A growing variety of applications have employed the CPMV multivalent display technology including nanoblock chemistry, in vivo imaging, and materials science. CPMV nanoparticles can be inexpensively produced from experimentally infected cowpea plants at high yields and are extremely stable. Although CPMV has not been shown to replicate in mammalian cells, uptake in mammalian cells does occur in vitro and in vivo. Thus, inactivation of the virus RNA genome is important for biosafety considerations, however the surface characteristics and chemical reactivity of the particles must be maintained in order to preserve chemical and structural functionality.

Methodology/principal findings: Short wave (254 nm) UV irradiation was used to crosslink the RNA genome within intact particles. Lower doses of UV previously reported to inactivate CPMV infectivity inhibited symptoms on inoculated leaves but did not prohibit systemic virus spread in plants, whereas higher doses caused aggregation of the particles and an increase in chemical reactivity further indicating broken particles. Intermediate doses of 2.0-2.5 J/cm(2) were shown to maintain particle structure and chemical reactivity, and cellular binding properties were similar to CPMV-WT.

Conclusions: These studies demonstrate that it is possible to inactivate CPMV infectivity while maintaining particle structure and function, thus paving the way for further development of CPMV nanoparticles for in vivo applications.

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Related in: MedlinePlus

UV-inactivation of CPMV infectivity.Primary leaves (panels A–F) were inoculated with CPMV and the presence of symptoms on these and secondary leaves (panels G–L) of the plant were monitored. Leaves were inoculated with CPMV that had been treated with the following doses of UV irradiation: 0 J/cm2 (positive control; panels A, G), 0.72 J/cm2 (panels B, H), 1.0 J/cm2 (panels C, I), 2.0 J/cm2 (panels D, J), 2.5 J/cm2 (panels E, K) or no infection (panels F and L). Insets in panels A and G show representative symptoms at approximately 3× magnification. Results shown are representative of five independent experiments with similar results.
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pone-0003315-g001: UV-inactivation of CPMV infectivity.Primary leaves (panels A–F) were inoculated with CPMV and the presence of symptoms on these and secondary leaves (panels G–L) of the plant were monitored. Leaves were inoculated with CPMV that had been treated with the following doses of UV irradiation: 0 J/cm2 (positive control; panels A, G), 0.72 J/cm2 (panels B, H), 1.0 J/cm2 (panels C, I), 2.0 J/cm2 (panels D, J), 2.5 J/cm2 (panels E, K) or no infection (panels F and L). Insets in panels A and G show representative symptoms at approximately 3× magnification. Results shown are representative of five independent experiments with similar results.

Mentions: CPMV particles are not sensitive to many standard methods of virus inactivation (e.g. hypochlorite or peptidase treatment [9], and methods that preserve capsid integrity such as formaldehyde treatment inhibit subsequent chemical bioconjugation. To determine the dose-response of inactivation by short-wave UV irradiation, samples of purified CPMV diluted to 2 mg/ml concentration were irradiated at various doses of 254 nm UV light as follows: 0 J/cm2 (control), 0.06 J/cm2, 0.12 J/cm2, 0.18 J/cm2, 0.36 J/cm2, 0.72 J/cm2, 1.0 J/cm2, 2.0 J/cm2, and 2.5 J/cm2, (Table 1). Following irradiation, virus samples were directly inoculated into bruised primary leaves of 9 day old cowpea plants and were monitored daily thereafter for the appearance of symptoms on inoculated primary and secondary leaves, as well as reduced growth in secondary leaves indicating systemic spread of CPMV. No reduction in symptoms compared to control was observed for those plants that were inoculated with CPMV receiving doses of 0.06 J/cm2, 0.12 J/cm2 and 0.18 J/cm2 of UV irradiation (Table 1). A decrease in the amount of lesions in primary leaves was observed in virus samples that received 0.36 J/cm2 and 0.72 J/cm2 UV irradiation, but both showed only a minimal reduction in the number of lesions in secondary leaves (Figure 1, Table 1). CPMV samples that received 1.0 J/cm2, 2.0 J/cm2, and 2.5 J/cm2 doses showed no lesions in inoculated leaves (Figure 1, Table 1). However, the sample receiving 1.0 J/cm2 produced a small amount of lesions and a decrease in size of secondary leaves (Figure 1I) whereas the sample receiving 2.0 J/cm2 or higher showed no signs of infection in any leaves with the plants displaying no symptoms (Figure 1D, 1J, Table 1).


Chemical addressability of ultraviolet-inactivated viral nanoparticles (VNPs).

Rae C, Koudelka KJ, Destito G, Estrada MN, Gonzalez MJ, Manchester M - PLoS ONE (2008)

UV-inactivation of CPMV infectivity.Primary leaves (panels A–F) were inoculated with CPMV and the presence of symptoms on these and secondary leaves (panels G–L) of the plant were monitored. Leaves were inoculated with CPMV that had been treated with the following doses of UV irradiation: 0 J/cm2 (positive control; panels A, G), 0.72 J/cm2 (panels B, H), 1.0 J/cm2 (panels C, I), 2.0 J/cm2 (panels D, J), 2.5 J/cm2 (panels E, K) or no infection (panels F and L). Insets in panels A and G show representative symptoms at approximately 3× magnification. Results shown are representative of five independent experiments with similar results.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2551747&req=5

pone-0003315-g001: UV-inactivation of CPMV infectivity.Primary leaves (panels A–F) were inoculated with CPMV and the presence of symptoms on these and secondary leaves (panels G–L) of the plant were monitored. Leaves were inoculated with CPMV that had been treated with the following doses of UV irradiation: 0 J/cm2 (positive control; panels A, G), 0.72 J/cm2 (panels B, H), 1.0 J/cm2 (panels C, I), 2.0 J/cm2 (panels D, J), 2.5 J/cm2 (panels E, K) or no infection (panels F and L). Insets in panels A and G show representative symptoms at approximately 3× magnification. Results shown are representative of five independent experiments with similar results.
Mentions: CPMV particles are not sensitive to many standard methods of virus inactivation (e.g. hypochlorite or peptidase treatment [9], and methods that preserve capsid integrity such as formaldehyde treatment inhibit subsequent chemical bioconjugation. To determine the dose-response of inactivation by short-wave UV irradiation, samples of purified CPMV diluted to 2 mg/ml concentration were irradiated at various doses of 254 nm UV light as follows: 0 J/cm2 (control), 0.06 J/cm2, 0.12 J/cm2, 0.18 J/cm2, 0.36 J/cm2, 0.72 J/cm2, 1.0 J/cm2, 2.0 J/cm2, and 2.5 J/cm2, (Table 1). Following irradiation, virus samples were directly inoculated into bruised primary leaves of 9 day old cowpea plants and were monitored daily thereafter for the appearance of symptoms on inoculated primary and secondary leaves, as well as reduced growth in secondary leaves indicating systemic spread of CPMV. No reduction in symptoms compared to control was observed for those plants that were inoculated with CPMV receiving doses of 0.06 J/cm2, 0.12 J/cm2 and 0.18 J/cm2 of UV irradiation (Table 1). A decrease in the amount of lesions in primary leaves was observed in virus samples that received 0.36 J/cm2 and 0.72 J/cm2 UV irradiation, but both showed only a minimal reduction in the number of lesions in secondary leaves (Figure 1, Table 1). CPMV samples that received 1.0 J/cm2, 2.0 J/cm2, and 2.5 J/cm2 doses showed no lesions in inoculated leaves (Figure 1, Table 1). However, the sample receiving 1.0 J/cm2 produced a small amount of lesions and a decrease in size of secondary leaves (Figure 1I) whereas the sample receiving 2.0 J/cm2 or higher showed no signs of infection in any leaves with the plants displaying no symptoms (Figure 1D, 1J, Table 1).

Bottom Line: Although CPMV has not been shown to replicate in mammalian cells, uptake in mammalian cells does occur in vitro and in vivo.Intermediate doses of 2.0-2.5 J/cm(2) were shown to maintain particle structure and chemical reactivity, and cellular binding properties were similar to CPMV-WT.These studies demonstrate that it is possible to inactivate CPMV infectivity while maintaining particle structure and function, thus paving the way for further development of CPMV nanoparticles for in vivo applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, and Center for Integrative Molecular Biosciences, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT

Background: Cowpea Mosaic Virus (CPMV) is increasingly being used as a nanoparticle platform for multivalent display of molecules via chemical bioconjugation to the capsid surface. A growing variety of applications have employed the CPMV multivalent display technology including nanoblock chemistry, in vivo imaging, and materials science. CPMV nanoparticles can be inexpensively produced from experimentally infected cowpea plants at high yields and are extremely stable. Although CPMV has not been shown to replicate in mammalian cells, uptake in mammalian cells does occur in vitro and in vivo. Thus, inactivation of the virus RNA genome is important for biosafety considerations, however the surface characteristics and chemical reactivity of the particles must be maintained in order to preserve chemical and structural functionality.

Methodology/principal findings: Short wave (254 nm) UV irradiation was used to crosslink the RNA genome within intact particles. Lower doses of UV previously reported to inactivate CPMV infectivity inhibited symptoms on inoculated leaves but did not prohibit systemic virus spread in plants, whereas higher doses caused aggregation of the particles and an increase in chemical reactivity further indicating broken particles. Intermediate doses of 2.0-2.5 J/cm(2) were shown to maintain particle structure and chemical reactivity, and cellular binding properties were similar to CPMV-WT.

Conclusions: These studies demonstrate that it is possible to inactivate CPMV infectivity while maintaining particle structure and function, thus paving the way for further development of CPMV nanoparticles for in vivo applications.

Show MeSH
Related in: MedlinePlus