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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

Surface chemical reactivity of CPMV-UV.CPMV-WT (squares) and CPMV-UV2.5 (triangles) samples were labeled with varying amounts of excess NHS-Fluorescein (ratio of dye molecules to asymmetric unit; x-axis), and following purification the dyes per particle were calculated (y-axis). Panel A shows labeling of CPMV-UV2.5 samples prior to FPLC fractionation. Panel B shows labeling of CPMV-UV2.5 where the 10 ml fraction (as in Figure 4) was first purified by FPLC prior to labeling. CPMV-F and CPMV-UV-F that had been labeled with NHS-fluorescein were run on SDS PAGE. Both large and small capsid subunits remain intact (C) and fluoresced under UV light (D).
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pone-0003315-g004: Surface chemical reactivity of CPMV-UV.CPMV-WT (squares) and CPMV-UV2.5 (triangles) samples were labeled with varying amounts of excess NHS-Fluorescein (ratio of dye molecules to asymmetric unit; x-axis), and following purification the dyes per particle were calculated (y-axis). Panel A shows labeling of CPMV-UV2.5 samples prior to FPLC fractionation. Panel B shows labeling of CPMV-UV2.5 where the 10 ml fraction (as in Figure 4) was first purified by FPLC prior to labeling. CPMV-F and CPMV-UV-F that had been labeled with NHS-fluorescein were run on SDS PAGE. Both large and small capsid subunits remain intact (C) and fluoresced under UV light (D).

Mentions: Having demonstrated that capsid integrity was essentially maintained following UV exposure, we then studied the ability of CPMV-UV2.5 particles to react with an amine-reactive fluorescent dye, which conjugates to exposed lysines on each asymmetric unit of the CPMV capsid. Lysine reactivity on the particle exterior surface is essential for a variety of techniques used for CPMV nanoblock chemistry including azide-alkyne [3+2] cycloaddition [28]. CPMV-UV2.5 samples were incubated with N-hydroxysuccinimide (NHS)-fluorescein at ratios of 1, 5, 10, 25, 50, 100, and 200 dyes molecules/asymmetric unit, in comparison to CPMV-WT labeled in the same manner. Following removal of unconjugated dye, the number of dyes/particle was calculated and compared with CPMV-WT. CPMV-UV2.5 samples demonstrated an increase in ability to react with the dye, producing dyes per particle values approximately two-fold greater than the untreated samples (Figure 4A).


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

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

Surface chemical reactivity of CPMV-UV.CPMV-WT (squares) and CPMV-UV2.5 (triangles) samples were labeled with varying amounts of excess NHS-Fluorescein (ratio of dye molecules to asymmetric unit; x-axis), and following purification the dyes per particle were calculated (y-axis). Panel A shows labeling of CPMV-UV2.5 samples prior to FPLC fractionation. Panel B shows labeling of CPMV-UV2.5 where the 10 ml fraction (as in Figure 4) was first purified by FPLC prior to labeling. CPMV-F and CPMV-UV-F that had been labeled with NHS-fluorescein were run on SDS PAGE. Both large and small capsid subunits remain intact (C) and fluoresced under UV light (D).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2551747&req=5

pone-0003315-g004: Surface chemical reactivity of CPMV-UV.CPMV-WT (squares) and CPMV-UV2.5 (triangles) samples were labeled with varying amounts of excess NHS-Fluorescein (ratio of dye molecules to asymmetric unit; x-axis), and following purification the dyes per particle were calculated (y-axis). Panel A shows labeling of CPMV-UV2.5 samples prior to FPLC fractionation. Panel B shows labeling of CPMV-UV2.5 where the 10 ml fraction (as in Figure 4) was first purified by FPLC prior to labeling. CPMV-F and CPMV-UV-F that had been labeled with NHS-fluorescein were run on SDS PAGE. Both large and small capsid subunits remain intact (C) and fluoresced under UV light (D).
Mentions: Having demonstrated that capsid integrity was essentially maintained following UV exposure, we then studied the ability of CPMV-UV2.5 particles to react with an amine-reactive fluorescent dye, which conjugates to exposed lysines on each asymmetric unit of the CPMV capsid. Lysine reactivity on the particle exterior surface is essential for a variety of techniques used for CPMV nanoblock chemistry including azide-alkyne [3+2] cycloaddition [28]. CPMV-UV2.5 samples were incubated with N-hydroxysuccinimide (NHS)-fluorescein at ratios of 1, 5, 10, 25, 50, 100, and 200 dyes molecules/asymmetric unit, in comparison to CPMV-WT labeled in the same manner. Following removal of unconjugated dye, the number of dyes/particle was calculated and compared with CPMV-WT. CPMV-UV2.5 samples demonstrated an increase in ability to react with the dye, producing dyes per particle values approximately two-fold greater than the untreated samples (Figure 4A).

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