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

Cell-surface binding and uptake of CPMV-WT and CPMV-UV2.0.A. CPMV-WT and the three CPMV-UV2.0 samples used in figure 2B were incubated separately with the plasma membrane enriched fraction of HeLa cells and virus binding to an established CPMV binding protein (CPMV-BP) was visualized through VOPBA. No binding was seen when CCMV was used in place of CPMV. B. CPMV-WT (green bars) and CPMV-UV2.0 (red bars) particles were incubated with HeLa cells for 2 hours at 4°C to measure cellular binding or at 37°C to measure binding and internalization. Respective disrupted particles were incubated at 37°C as well. Percentage of cells with CPMV signal were detected by flow cytometry are indicated on the y-axis and data are reported as mean+/−S.D. of triplicate samples.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2551747&req=5

pone-0003315-g005: Cell-surface binding and uptake of CPMV-WT and CPMV-UV2.0.A. CPMV-WT and the three CPMV-UV2.0 samples used in figure 2B were incubated separately with the plasma membrane enriched fraction of HeLa cells and virus binding to an established CPMV binding protein (CPMV-BP) was visualized through VOPBA. No binding was seen when CCMV was used in place of CPMV. B. CPMV-WT (green bars) and CPMV-UV2.0 (red bars) particles were incubated with HeLa cells for 2 hours at 4°C to measure cellular binding or at 37°C to measure binding and internalization. Respective disrupted particles were incubated at 37°C as well. Percentage of cells with CPMV signal were detected by flow cytometry are indicated on the y-axis and data are reported as mean+/−S.D. of triplicate samples.

Mentions: Previous studies indicated that UV irradiation of the picornaviruses poliovirus and hepatitis A virus abolishes their binding to cell surface receptors [41]. For CPMV, studies have demonstrated that wild-type and fluorophore-labeled CPMVs are endocytosed in a variety of cell types both in vitro and in vivo [9]–[11]. In addition CPMV is known to bind to a 54 kilodalton mammalian surface protein (CPMV-BP) [42]. Using a Virus Overlay Protein Blot Assay (VOPBA), binding of CPMV-UV2.0 and CPMV-WT particles to CPMV-BP from HeLa human epithelial cell plasma membranes was tested indicating no difference in binding capacity (Figure 5A). When cowpea chlorotic mottle virus (CCMV), a similarly structured virus that infects the same plant host, was used in place of CPMV no binding in the 54 kilodalton region was observed (Figure 5A) [42]. Triplicate samples of HeLa cells were also incubated with 105 particles of CPMV-WT (green bars) or CPMV- UV2.0 (red bars) per cell for two hours at 4°C to illustrate binding or at 37°C to show binding and internalization (Figure 5B). Heat-disrupted capsids showed little binding and uptake when incubated with HeLa cells at 37°C (Figure 5B). UV-inactivated particles are still capable of near wildtype cellular binding and uptake in vitro. These data further indicate that unlike other picornaviruses, the CPMV capsid remains stable and intact, and maintains surface reactivities and cellular binding properties following UV irradiation.


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

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

Cell-surface binding and uptake of CPMV-WT and CPMV-UV2.0.A. CPMV-WT and the three CPMV-UV2.0 samples used in figure 2B were incubated separately with the plasma membrane enriched fraction of HeLa cells and virus binding to an established CPMV binding protein (CPMV-BP) was visualized through VOPBA. No binding was seen when CCMV was used in place of CPMV. B. CPMV-WT (green bars) and CPMV-UV2.0 (red bars) particles were incubated with HeLa cells for 2 hours at 4°C to measure cellular binding or at 37°C to measure binding and internalization. Respective disrupted particles were incubated at 37°C as well. Percentage of cells with CPMV signal were detected by flow cytometry are indicated on the y-axis and data are reported as mean+/−S.D. of triplicate samples.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003315-g005: Cell-surface binding and uptake of CPMV-WT and CPMV-UV2.0.A. CPMV-WT and the three CPMV-UV2.0 samples used in figure 2B were incubated separately with the plasma membrane enriched fraction of HeLa cells and virus binding to an established CPMV binding protein (CPMV-BP) was visualized through VOPBA. No binding was seen when CCMV was used in place of CPMV. B. CPMV-WT (green bars) and CPMV-UV2.0 (red bars) particles were incubated with HeLa cells for 2 hours at 4°C to measure cellular binding or at 37°C to measure binding and internalization. Respective disrupted particles were incubated at 37°C as well. Percentage of cells with CPMV signal were detected by flow cytometry are indicated on the y-axis and data are reported as mean+/−S.D. of triplicate samples.
Mentions: Previous studies indicated that UV irradiation of the picornaviruses poliovirus and hepatitis A virus abolishes their binding to cell surface receptors [41]. For CPMV, studies have demonstrated that wild-type and fluorophore-labeled CPMVs are endocytosed in a variety of cell types both in vitro and in vivo [9]–[11]. In addition CPMV is known to bind to a 54 kilodalton mammalian surface protein (CPMV-BP) [42]. Using a Virus Overlay Protein Blot Assay (VOPBA), binding of CPMV-UV2.0 and CPMV-WT particles to CPMV-BP from HeLa human epithelial cell plasma membranes was tested indicating no difference in binding capacity (Figure 5A). When cowpea chlorotic mottle virus (CCMV), a similarly structured virus that infects the same plant host, was used in place of CPMV no binding in the 54 kilodalton region was observed (Figure 5A) [42]. Triplicate samples of HeLa cells were also incubated with 105 particles of CPMV-WT (green bars) or CPMV- UV2.0 (red bars) per cell for two hours at 4°C to illustrate binding or at 37°C to show binding and internalization (Figure 5B). Heat-disrupted capsids showed little binding and uptake when incubated with HeLa cells at 37°C (Figure 5B). UV-inactivated particles are still capable of near wildtype cellular binding and uptake in vitro. These data further indicate that unlike other picornaviruses, the CPMV capsid remains stable and intact, and maintains surface reactivities and cellular binding properties following UV irradiation.

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