TABLE 1.
Effects of mutations in MNV 3′ end on virus recovery, using reverse genetics
| Mutation | Description | Recoverya |
|---|---|---|
| ΔSL2 GA | Deletion of stem-loop 2 positions 7311 to 7320 | No |
| ΔSL3 | Deletion of stem-loop 3 position 7330 to poly(A) | No |
| ΔSL2 + 3 | Deletion of stem-loops 2 and 3 from position 7308 to poly(A) | No |
| SL2 ssm | Mutation disrupting secondary structure in SL2 stem and loop | No |
| SL3 ssm | Mutation disrupting secondary structure in SL2 stemb | No |
| SL3 ssm R | Secondary structure reversion mutant of SL3 ssm | No |
| SL3 GNRA | Substitution of polypyrimidine tract loop in SL3 with GNRA tetraloop | Yes |
| SL3 AAAA | Substitution of polypyrimidine tract loop in SL3 with AAAA tetraloop | Yes |
a
Virus recovery is defined as the ability to generate virus capable of generating clear cytopathic effect in RAW 264.7 cells by 5 days postinfection. The limit of detection of this assay was ∼50 TCID50/ml. Each recovery was repeated a minimum of three times and was scored as negative if no detectable virus was observed. The yields of wild-type, SL3 GNRA, and SL3 AAAA cDNA clones were typically >104 TCID50 per 35-mm dish.
b
A natural revertant of this virus (SL3 ssm NR) was generated following multiple blind passages of this virus in permissive cells (as described in the main text).