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Measles virus antisense sequences specifically cure cells persistently infected with measles virus
K Koschel1, U Brinckmann, V V Hoyningen-Huene
1Institut für Virologie, Universität Würzburg, Germany.
Abstract:
Vectors expressing antisense mRNAs complementary to the measles virus (MV) nucleoprotein N or hemagglutinin H genes were used to transfect MV-permissive Vero cells, MV-nonpermissive C6 rat glioma cells, and C6 cells persistently infected with measles/SSPE virus (C6/SSPE cells). Transfected Vero cells infected with MV showed a drastically reduced yield of infectious virus (90-99.99%). In plaque assays, plaque numbers and plaque size were significantly reduced compared with untransfected Vero cells. With an unrelated control virus, VSV, no effects were seen in the transfected Vero cells, underlining the specificity for MV. Following stable transfection with MV antisense vectors, C6 rat glioma cells, which are normally suitable to establish persistently MV-infected lines, can no longer be infected with the virus. In this case also, VSV infection was not influenced. Furthermore, antisense transfection of already persistently infected C6/SSPE cells leads to a loss of MV-specific immunofluorescence, concomitant with a disappearance of viral RNA. Single cell clones from the antisense-transfected C6/SSPE cells appear to be totally free of virus in cocultivation with Vero cells, suggesting that they are really cured. The effectiveness of even low amounts of antisense sequences suggests that they are good candidates for antisense oligonucleotide therapy in tissue culture and might eventually also be useful for in vivo application.
Insights
Antisense messenger RNA (mRNA) vectors targeting measles virus (MV) genes significantly reduced viral replication in cell cultures. This suggests potential for antisense oligonucleotide therapy against measles virus infections.
Area of Science:
- Virology
- Molecular Biology
- Gene Therapy
Background:
- Measles virus (MV) causes significant disease, and persistent infections are challenging to treat.
- Antisense technology offers a potential strategy for controlling viral replication by targeting viral genetic material.
Purpose of the Study:
- To investigate the efficacy of antisense mRNA vectors targeting measles virus (MV) nucleoprotein (N) and hemagglutinin (H) genes.
- To assess the potential of antisense therapy in both acute and persistent MV infections in cell culture models.
Main Methods:
- Transfection of MV-permissive Vero cells, MV-nonpermissive C6 rat glioma cells, and persistently infected C6/SSPE cells with antisense vectors.
- Infection of transfected cells with MV and analysis of viral yield, plaque formation, and viral RNA.
- Cocultivation of single-cell clones with permissive cells to assess viral clearance.
Main Results:
- Transfected Vero cells showed a 90-99.99% reduction in infectious MV yield and significantly smaller plaques.
- Antisense vectors prevented infection of C6 cells and led to clearance of MV from persistently infected C6/SSPE cells, evidenced by loss of viral RNA and immunofluorescence.
- Specificity was confirmed as unrelated vesicular stomatitis virus (VSV) infection was unaffected.
Conclusions:
- Antisense mRNA vectors are highly effective in inhibiting MV replication and clearing persistent infections in cell culture.
- Low-dose effectiveness suggests potential for antisense oligonucleotide therapy in vitro and possibly in vivo for measles virus.
- This approach shows promise for developing novel therapeutic strategies against measles virus.