Are reovirus ts mutants with defined blocks in viral replication capable of inducing apoptosis?

METHODS

1. HeLa cells were infected with temperature sensitive (ts) mutants Reovirus mutants containing defects in either synthesis of dsRNA, assembly of core particles, or cell entry of progeny virions. at an MOI of 10 PFU per cell and incubated at either the permissive temperature (32°C) or the nonpermissive temperature (39°C).
   
   
   

   Mutant	Gene	Protein	Replication block
   tsA201	M2	µ1	Entry of ts progeny
   tsB352	L2	l2	Assembly of outer capsid
   tsC447	S2	s2	Assembly of core particles, dsRNA synthesis
   tsD357	L1	l3	dsRNA synthesis
   tsE320	S3	sNS	dsRNA synthesis
   tsG453	S4	s3	Assembly of outer capsid

   
   
   
2. Viral titers were determined at 0 h and 48 h after infection, and the viral yield was calculated by dividing the viral titer at 0 h by the viral titer at 48 h.
   
   
3. HeLa cells were infected with six of the ts mutants at an MOI of 100 PFU per cell and incubated at both 32°C and 39°C.
   
   
4. Cells were tested for apoptosis using acridine orange staining 48 h after infection.
   
   
5. An immunoblot assay testing the presence of the 89 kD PARP cleavage product PARP is cleaved by caspases 12 h after reovirus infection. was performed.
   
   
6. Nuclear extracts were prepared 24 h after infection and 50µg of protein were run on an SDS-PAGE gel.
   
   
7. The gel was transferred to nitrocellulose and blotted using a PARP-specific antiserum.
   
   
   

RESULTS

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