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An assay for the presence of functional large (RR1) and small (RR2) subunits of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase has been developed. The system utilizes two temperature-sensitive mutants, ts1207, which has a lesion in RR1, and ts1222, which has a lesion in RR2. In cells infected with ts1207 at 39.5 degrees C, the defective RR1 is unable to associate with RR2 to form an active enzyme, and, as a result, a pool of functional RR2 and defective RR1 accumulates. Evidence presented in this paper suggest that cells infected with ts1222 at either 31 degrees C or 39.5 degrees C accumulate a pool of functional RR1, but do not contain detectable RR2. Virus-specific ribonucleotide reductase activity was produced in cells coinfected with both mutants at 39.5 degrees C, each virus contributing one functional subunit to the holoenzyme. No enzyme activity was detected in cells infected with each mutant alone at this temperature. When partially purified extracts of cells infected with ts1207 at the nonpermissive temperature were mixed with those from ts1222-infected cells, a fully functional enzyme was also formed. These results demonstrate that HSV-1 ribonucleotide reductase activity can be reconstituted both in vivo and in vitro from the nondefective subunits produced by ts1222 and ts1207.

Single temperature-sensitive (ts) mutants of a subgroup A strain of respiratory syncytial (RS) virus whose multiplication is restricted at 39 degrees C in MRC-6 cells and double ts mutants that are restricted at 38 degrees C, were obtained following mutagenesis using 5-fluorouracil and acridine-like compounds. Isolation and propagation of the parental RSS-2 strain of RS virus and its derived ts mutants were carried out entirely in MRC-5 human diploid cells. The immunogenicity and disease-producing ability of four of these mutants and the parental unmodified strain have been assessed by intranasal administration into groups of about 20 adult volunteers. The results of these trials indicate that the capacity of the parental RSS-2 strain to produce upper respiratory tract infection in adults was not diminished by limited propagation in MRC-5 cells. The mutants on the other hand were impaired in this respect to varying extents. The double mutant tslB in particular has characteristics that suggest that it may be suitable for further development as a live RS virus vaccine. It retained near normal immunogenicity and replicative ability in the upper respiratory tract, while exhibiting greatly reduced disease-producing potential.

35S-labeled cells infected with herpes simplex virus type 1 (HSV-1), temperature-sensitive (ts) mutant ts 1222 were used as a source of the large subunit of the viral ribonucleotide reductase (RR) to investigate the binding of the large (RR1) and small (RR2) subunits in the active enzyme. Mixing 35S-labeled RR1 from ts 1222 with unlabeled RR1/RR2 complex from wild type (wt) infected cells resulted in the formation of a complex between 35S-labeled RR1 and unlabeled RR2, indicating that the complex between the RR1 and RR2 subunits is dynamic and subunit dissociation/reassociation occurs during enzyme function. Similar results were obtained when unlabeled HSV-2 RR was substituted for HSV-1 RR, demonstrating that the holoenzyme can be formed the large subunit of HSV-1 RR and the small subunit of HSV-2.