Reconstitution of herpes simplex virus type 1 ribonucleotide reductase activity from the large and small subunits

Abstract

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.

The Unique N-terminal Domain of the Large Subunit of Herpes Simplex Virus Ribonucleotide Reductase Is Preferentially Sensitive to Proteolysis

“…Formation of this protein in extracts lacking 93K is always accompanied by degradation of RR2 and hence the loss of enzyme activity could not be unambiguously linked to loss of one or the other. In principle it should be possible to purify the 81K species and test whether it can complement the free small subunit to produce enzyme activity (Darling et al, 1988). We have not yet succeeded in doing this.…”

“…The herpes simplex virus (HSV)-coded ribonucleotide reductase (RR) (Cohen, 1972;Dutia, 1983 ;Preston et al, 1984) consists of two non-identical subunits (Frame et al, 1985;Bacchetti et al, 1986;Ingemarson & Lankinen, 1987;Darling et al, 1988) termed RR1 and RR2 (Frame et al, 1985) or H1 and H2 (Cohen et al, 1985). Sedimentation analysis of the enzyme suggests that both subunits are homodimers (Ingemarson & Lankinen, 1987) with the same basic structure as the Escherichia coli and mammalian reductases.…”

The unique N terminus of the herpes simplex virus type 1 large subunit is not required for ribonucleotide reductase activity

“…In HSV-I, the RR enzyme consists of a large subunit, R1, and a small subunit, R2, which have Mr values of 136K and 38K respectively (Frame et al, 1985;Bacchetti et al, 1986;Darling et al, 1988). The subunits exist as a tight complex in an (x2fl 2 structure similar to the RR enzymes of Escherichia coli and mammalian cells (Ingemarson & Lankinen, 1987).…”

A single amino acid substitution in the large subunit of herpes simplex virus type 1 ribonucleotide reductase which prevents subunit association

“…1) into wt HSV-1 DNA (Preston et al, 1984). The HSV-1 mutant ts1222 has been characterized Darling et al, 1988). Infections with ts1207 and ts1222 were carried out at the NPT of 39.5 °C.…”