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

Abstract

The herpes simplex virus type 1 temperature-sensitive (ts) mutant ts1207 does not induce detectable levels of ribonucleotide reductase activity at the non-permissive temperature (NPT, 39.5 °C). The ts lesion prevents the association of the enzyme's large (RR1) and small (RR2) subunits to give an active holoenzyme and maps within the gene specifying RR1. Here, it is shown that the ts mutant phenotype is due to the substitution of an asparagine for the wild-type (wt) serine at RR1 position 961, which is located within a region highly conserved between herpesviral and cellular RR 1 subunit polypeptides. This ts1207 asparagine is predicted to alter a wt c~-helix to a #-strand. We have used synthetic oligopeptides, corresponding to the wt amino acid sequence of the mutation site, and antisera raised against them to determine whether this region is involved in subunit association. Neither the oligopeptides nor the antisera inhibit the enzyme activity, or the reconstituted activity formed by mixing intact RR2 and RR1 subunits present in partially purified extracts of cells infected at the N.PT with ts1207 or ts1222 (an HSV-1 mutant with a lesion in the RR2 subunit), respectively. We infer from these results that the site of the mutation is unlikely to be positioned at the surface of RR1 and hence is probably not directly involved in subunit association. We suggest that the mutation site identifies an important RR1 region whose alteration in tsl207 changes the structure of a contact region(s) positioned at the RR1/RR2 interface.

Purification and characterization of the herpes simplex virus type 1 ribonucleotide reductase small subunit following expression in Escherichia coli

“…A region involved directly in the R1-R2 interaction has been identified (Cohen et al, 1986a;Dutia et al, 1986) as has a further region in R1 which, though not in direct contact, is nevertheless important (Nikas et al, 1990). The crystal structure of the E. coli R2 has now been determined and an interactive surface proposed (Nordlund et al, 1990).…”

Identification of ribonucleotide reductase mutation causing temperature‐sensitivity of herpes simplex virus isolates from whitlow by deep sequencing

“…Both RR1 ( α 2) and RR2 ( β 2) are homodimers. HSV RR is the complex between the large (RR1) and small (RR2) subunits for enzyme function . Mutation in the RR1 or RR2 subunit is responsible for temperature‐sensitivity .…”

“…Ts mutants of herpes simplex virus (HSV) have been generated or found in the laboratory and reported to have a mutation in UL36 of HSV-1 [2], ICP4 of HSV-1 [3][4][5][6][7][8], UL15 of HSV-1 [9], HSV-1 protease of HSV-1 [10], UL9 of HSV-1 [11], UL28 of HSV-1 [12], gB of HSV-1 [3,13], ribonucleotide reductase (RR) large subunit of HSV-1 [14,15] and HSV-2 [15] and small subunit of HSV-1 [14,16], UL11 of HSV-1 [17], Vmw65 of HSV-1 [18], ICP27 of HSV-1 [19], ICP8 of HSV-1 [6], DNA polymerase [6], and virion-associated host shutoff protein [20]. Thus, ts mutants have been analyzed to understand their gene functions, but all mutants examined were laboratory strains, making it difficult to predict the location of the This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.…”

Epitope mapping identifies an exposed loop between the unique amino- and conserved carboxy-domains of the large subunit of herpes simplex virus type 1 ribonucleotide reductase

“…Antisera. MAb 1026 specific for R1 has been described (Nikas et al, 1990). Antipeptide antiserum C2 was raised against a peptide corresponding to residues 959 to 970 of HSV R1 (Lankinen et al, 1989).…”

“…Proteins containing fl-galactosidase fused to fragments of R1 have been produced. We describe mapping of the epitope recognized by an Rl-specific monoclonal antibody (MAb), 1026 (Nikas et al, 1990), using the R1 fragment/fl-galactosidase fusion proteins. Fine mapping of the epitope with synthetic peptides revealed that the unique N-terminal region forms a discrete domain which is linked to the conserved carboxy region by an exposed loop.…”