Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli.

M L Howell,J Sanders-Loehr,T M Loehr,N A Roseman,C K Mathews,M B Slabaugh

doi:10.1016/s0021-9258(18)46003-6

M L Howell, J Sanders-Loehr + Show 4 more

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https://doi.org/10.1016/s0021-9258(18)46003-6

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Abstract

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.

Highlights

During itsinfectious cycle, vaccinia virus expresses Ribonucleotidereductase is an essential enzymein DNA a virus-encoded ribonucleotide reductase whichis dis- replication, providing the only source of de novo synthesized tinct from the host cellular enzyme
VVRB protein was shown to complement the activity of purified recombinant ribonucleotide reductaslearge subunit, with a specific activity that was similar to native VVRS from a virus-infected cell extract.A CD spectrum of the recombinant viral protesihnowed that like the mouse protein, the vaccinia virus protein has 50% a-helicalstructure.Likeotheriron-containing ribonucleotide reductase small subunits, recombinant VVRS protein contained a stable organic free radical that was detectable by EPR spectroscopy
A unit of enzyme activity is defined as the amounotf VVR2protein which catalyzes the formation of 1 nmol of dCDP in 1 min in the presence of an excess of complementary vaccinia virus ribonucleotide reductase large subunit (VVR1) protein at 30 "C

Summary

11 Present address

Dept. of Biology, Williams College, Williams- of ribonucleotide reductase, respectively; SDS, sodium dodecyl sultown, MA 01267. Comparison of the amino acid sequencesof the vaccinia gene products withother ribonucleotide reductase subunits revealed that the vaccinia virus enzyme has far greater similarittyo themammalian and yeast reductases than it does to the corresponding enzyme from E. coli, T4 bacteriophage, or the herpes viruses (Slabaugh et al, 1988).the vaccinia proteins share70-80% amino acid identity with the respective mouse subunits. In contrast to the herpes virus ribonucleotide reductase, which is unregulated (Averett et al, 1983), the vaccinia virus-induced enzyme exhibits the same pattern of allosteric regulation as the mammalian enzyme (Slabaugh et al, 1984). The regulatory and structural similarity of the VVR2 protein to the mammalian R2 subunits as well as thegenetic advantages offered by the readily manipulated nature of the vaccinia virus genome (Traktman, 1990) have led us to pursue study of the vaccinia virus ribonucleotide reductase. The radical content of the small subunit is quantitated, and thEe P R and CD spectra of recombinant proteinare compared with spectra of small subunit proteinsfrom other sources

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DISCUSSION