Limits...
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.

Show MeSH

Related in: MedlinePlus

UV-Crosslinking of CPMV genomic RNA.A. Total genomic RNA was isolated using phenol-chloroform extraction from purified CPMV particles either untreated (CPMV-WT) or treated with 2.0 J/cm2 of UV radiation (CPMV-UV2.0), and equal quantities of purified RNA were separated on an agarose gel matrix. Genomic RNA-1 (5.9 kb) and RNA-2 (3.6 kb) are indicated. M: molecular weight markers. B. RNA was extracted in triplicate from 20 micrograms of CPMV-WT or from three individually UV-inactivated samples of CPMV-UV2.0. Results are reported as total viral RNA isolated from phenol-chloroform extractions (mean+/−S.D.). Data are representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2551747&req=5

pone-0003315-g002: UV-Crosslinking of CPMV genomic RNA.A. Total genomic RNA was isolated using phenol-chloroform extraction from purified CPMV particles either untreated (CPMV-WT) or treated with 2.0 J/cm2 of UV radiation (CPMV-UV2.0), and equal quantities of purified RNA were separated on an agarose gel matrix. Genomic RNA-1 (5.9 kb) and RNA-2 (3.6 kb) are indicated. M: molecular weight markers. B. RNA was extracted in triplicate from 20 micrograms of CPMV-WT or from three individually UV-inactivated samples of CPMV-UV2.0. Results are reported as total viral RNA isolated from phenol-chloroform extractions (mean+/−S.D.). Data are representative of two independent experiments.

Mentions: To confirm the effect of CPMV inactivation, the integrity of the viral genomic RNA in irradiated samples was investigated. RNA was isolated from CPMV-UV2.0 and CPMV-WT whole virus particles by phenol-chloroform extraction and ethanol precipitation, and run on an agarose gel under RNAse-free conditions. RNA-protein crosslinking was observed. When viral protein was extracted from CPMV-UV2.0 using phenol-chloroform, RNA yield was reduced, however some viral RNAs remained that migrated with untreated CPMV RNAs (Figure 2A). Nevertheless, quantifying the viral RNA extracted from CPMV-WT and three independently irradiated sets of CPMV-UV2.0 resulted in an average 64% lower yield of viral genomic RNA when compared to non-irradiated samples, suggesting that such RNA-protein crosslinking had occurred (Figure 2B). Little is known about the structure of RNAs packaged inside CPMV aside from the fact that each particle can package a single RNA molecule [38]–[40], and in other virus systems RNA-RNA crosslinking has been primarily reported following UV treatment. It is possible inter- or intra- RNA crosslinking may also occur.


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

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

UV-Crosslinking of CPMV genomic RNA.A. Total genomic RNA was isolated using phenol-chloroform extraction from purified CPMV particles either untreated (CPMV-WT) or treated with 2.0 J/cm2 of UV radiation (CPMV-UV2.0), and equal quantities of purified RNA were separated on an agarose gel matrix. Genomic RNA-1 (5.9 kb) and RNA-2 (3.6 kb) are indicated. M: molecular weight markers. B. RNA was extracted in triplicate from 20 micrograms of CPMV-WT or from three individually UV-inactivated samples of CPMV-UV2.0. Results are reported as total viral RNA isolated from phenol-chloroform extractions (mean+/−S.D.). Data are representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003315-g002: UV-Crosslinking of CPMV genomic RNA.A. Total genomic RNA was isolated using phenol-chloroform extraction from purified CPMV particles either untreated (CPMV-WT) or treated with 2.0 J/cm2 of UV radiation (CPMV-UV2.0), and equal quantities of purified RNA were separated on an agarose gel matrix. Genomic RNA-1 (5.9 kb) and RNA-2 (3.6 kb) are indicated. M: molecular weight markers. B. RNA was extracted in triplicate from 20 micrograms of CPMV-WT or from three individually UV-inactivated samples of CPMV-UV2.0. Results are reported as total viral RNA isolated from phenol-chloroform extractions (mean+/−S.D.). Data are representative of two independent experiments.
Mentions: To confirm the effect of CPMV inactivation, the integrity of the viral genomic RNA in irradiated samples was investigated. RNA was isolated from CPMV-UV2.0 and CPMV-WT whole virus particles by phenol-chloroform extraction and ethanol precipitation, and run on an agarose gel under RNAse-free conditions. RNA-protein crosslinking was observed. When viral protein was extracted from CPMV-UV2.0 using phenol-chloroform, RNA yield was reduced, however some viral RNAs remained that migrated with untreated CPMV RNAs (Figure 2A). Nevertheless, quantifying the viral RNA extracted from CPMV-WT and three independently irradiated sets of CPMV-UV2.0 resulted in an average 64% lower yield of viral genomic RNA when compared to non-irradiated samples, suggesting that such RNA-protein crosslinking had occurred (Figure 2B). Little is known about the structure of RNAs packaged inside CPMV aside from the fact that each particle can package a single RNA molecule [38]–[40], and in other virus systems RNA-RNA crosslinking has been primarily reported following UV treatment. It is possible inter- or intra- RNA crosslinking may also occur.

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