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A natural human retrovirus efficiently complements vectors based on murine leukemia virus.

Dong B, Silverman RH, Kandel ES - PLoS ONE (2008)

Bottom Line: Recently, an infectious gammaretrovirus designated XMRV has been identified in prostate cancer patients.We tested the ability of XMRV to complement replication-deficient MLV vectors upon co-infection of cultured human cells.We observed that XMRV can facilitate the spread of these vectors from infected to uninfected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States of America.

ABSTRACT

Background: Murine Leukemia Virus (MLV) is a rodent gammaretrovirus that serves as the backbone for common gene delivery tools designed for experimental and therapeutic applications. Recently, an infectious gammaretrovirus designated XMRV has been identified in prostate cancer patients. The similarity between the MLV and XMRV genomes suggests a possibility that the two viruses may interact when present in the same cell.

Methodology/principal findings: We tested the ability of XMRV to complement replication-deficient MLV vectors upon co-infection of cultured human cells. We observed that XMRV can facilitate the spread of these vectors from infected to uninfected cells. This functional complementation occurred without any gross rearrangements in the vector structure, and the co-infected cells produced as many as 10(4) infectious vector particles per milliliter of culture medium.

Conclusions/significance: The possibility of encountering a helper virus when delivering MLV-based vectors to human cells in vitro and in vivo needs to be considered to ensure the safety of such procedures.

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Related in: MedlinePlus

Determination of the titer of infectious LNCE in the presence of XMRV.The supernatant from DU145LNCE cells exposed to the medium from either DU145 (control medium) or DU145XMRV cells was applied in various dilutions to subconfluent cultures of naïve DU145 cells grown in 6-well plates. The treated cells were selected for G418 resistance and the surviving colonies were visualized by methylene blue staining. Neo-transducing particles were readily detectable in the supernatant of XMRV-exposed cells at 1,000-fold (panel A) and 10,000-fold (panel B), but not 100,000-fold (panel C) dilutions. The supernatant from DU145LNCE exposed to control medium was not toxic by itself (panel E), but it failed to transduce the resistance marker even when used without dilution (panel D).
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pone-0003144-g002: Determination of the titer of infectious LNCE in the presence of XMRV.The supernatant from DU145LNCE cells exposed to the medium from either DU145 (control medium) or DU145XMRV cells was applied in various dilutions to subconfluent cultures of naïve DU145 cells grown in 6-well plates. The treated cells were selected for G418 resistance and the surviving colonies were visualized by methylene blue staining. Neo-transducing particles were readily detectable in the supernatant of XMRV-exposed cells at 1,000-fold (panel A) and 10,000-fold (panel B), but not 100,000-fold (panel C) dilutions. The supernatant from DU145LNCE exposed to control medium was not toxic by itself (panel E), but it failed to transduce the resistance marker even when used without dilution (panel D).

Mentions: In order to test the ability of XMRV to mobilize MLV-based vectors we devised an experimental scheme (Figure 1A) that utilized LNCE, a previously described MLV-based construct (Figure 1B) [6], which carries the genes for neomycin resistance and green fluorescent protein. DU145 human prostate carcinoma cells were stably transduced with LNCE, and a population of G418-resistant cells was obtained. As expected, this pool of cells, designated DU145LNCE, was nearly uniformly positive for GFP expression (data not shown). These cells were exposed to the filtered supernatant of DU145XMRV (DU145 cells that were previously infected with a replication-competent XMRV) and cultured for additional week to allow a potential helper virus to propagate. If XMRV could serve as a helper virus for LNCE, one may expect doubly-infected cells to produce infectious LNCE particles. To test this prediction, we collected the supernatant from XMRV-infected DU145LNCE cells, filtered it and applied to fresh DU145 cells. Two days later, the freshly infected cells were subjected to G418 selection. We observed massive formation of G418-resistant colonies, most of which showed detectable GFP expression by fluorescent microscopy (data not shown). No G418-resistant colonies were obtained in control experiment in which fresh DU145 cells were exposed to DU145LNCE supernatant in the absence of XMRV (e.g. Figure 2d). These observations suggest that XMRV can act as a helper virus for MLV-based vectors.


A natural human retrovirus efficiently complements vectors based on murine leukemia virus.

Dong B, Silverman RH, Kandel ES - PLoS ONE (2008)

Determination of the titer of infectious LNCE in the presence of XMRV.The supernatant from DU145LNCE cells exposed to the medium from either DU145 (control medium) or DU145XMRV cells was applied in various dilutions to subconfluent cultures of naïve DU145 cells grown in 6-well plates. The treated cells were selected for G418 resistance and the surviving colonies were visualized by methylene blue staining. Neo-transducing particles were readily detectable in the supernatant of XMRV-exposed cells at 1,000-fold (panel A) and 10,000-fold (panel B), but not 100,000-fold (panel C) dilutions. The supernatant from DU145LNCE exposed to control medium was not toxic by itself (panel E), but it failed to transduce the resistance marker even when used without dilution (panel D).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2519784&req=5

pone-0003144-g002: Determination of the titer of infectious LNCE in the presence of XMRV.The supernatant from DU145LNCE cells exposed to the medium from either DU145 (control medium) or DU145XMRV cells was applied in various dilutions to subconfluent cultures of naïve DU145 cells grown in 6-well plates. The treated cells were selected for G418 resistance and the surviving colonies were visualized by methylene blue staining. Neo-transducing particles were readily detectable in the supernatant of XMRV-exposed cells at 1,000-fold (panel A) and 10,000-fold (panel B), but not 100,000-fold (panel C) dilutions. The supernatant from DU145LNCE exposed to control medium was not toxic by itself (panel E), but it failed to transduce the resistance marker even when used without dilution (panel D).
Mentions: In order to test the ability of XMRV to mobilize MLV-based vectors we devised an experimental scheme (Figure 1A) that utilized LNCE, a previously described MLV-based construct (Figure 1B) [6], which carries the genes for neomycin resistance and green fluorescent protein. DU145 human prostate carcinoma cells were stably transduced with LNCE, and a population of G418-resistant cells was obtained. As expected, this pool of cells, designated DU145LNCE, was nearly uniformly positive for GFP expression (data not shown). These cells were exposed to the filtered supernatant of DU145XMRV (DU145 cells that were previously infected with a replication-competent XMRV) and cultured for additional week to allow a potential helper virus to propagate. If XMRV could serve as a helper virus for LNCE, one may expect doubly-infected cells to produce infectious LNCE particles. To test this prediction, we collected the supernatant from XMRV-infected DU145LNCE cells, filtered it and applied to fresh DU145 cells. Two days later, the freshly infected cells were subjected to G418 selection. We observed massive formation of G418-resistant colonies, most of which showed detectable GFP expression by fluorescent microscopy (data not shown). No G418-resistant colonies were obtained in control experiment in which fresh DU145 cells were exposed to DU145LNCE supernatant in the absence of XMRV (e.g. Figure 2d). These observations suggest that XMRV can act as a helper virus for MLV-based vectors.

Bottom Line: Recently, an infectious gammaretrovirus designated XMRV has been identified in prostate cancer patients.We tested the ability of XMRV to complement replication-deficient MLV vectors upon co-infection of cultured human cells.We observed that XMRV can facilitate the spread of these vectors from infected to uninfected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States of America.

ABSTRACT

Background: Murine Leukemia Virus (MLV) is a rodent gammaretrovirus that serves as the backbone for common gene delivery tools designed for experimental and therapeutic applications. Recently, an infectious gammaretrovirus designated XMRV has been identified in prostate cancer patients. The similarity between the MLV and XMRV genomes suggests a possibility that the two viruses may interact when present in the same cell.

Methodology/principal findings: We tested the ability of XMRV to complement replication-deficient MLV vectors upon co-infection of cultured human cells. We observed that XMRV can facilitate the spread of these vectors from infected to uninfected cells. This functional complementation occurred without any gross rearrangements in the vector structure, and the co-infected cells produced as many as 10(4) infectious vector particles per milliliter of culture medium.

Conclusions/significance: The possibility of encountering a helper virus when delivering MLV-based vectors to human cells in vitro and in vivo needs to be considered to ensure the safety of such procedures.

Show MeSH
Related in: MedlinePlus