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

Lankinen, Hilkka; Everett, Roger D.; Cross, A. M.; Conner, Joe; Marsden, Howard S.

doi:10.1099/0022-1317-74-9-1871

Cited by 10 publications

(9 citation statements)

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“…In vitro studies have shown that aggressive tumor proliferation results in overamplification of RR Hurta and Wright, 1990). Expression of the RR gene of the herpes simplex virus is required for viral growth and neoplastic transformation (Luo et al, 1991;Desai et al, 1993;Jones et al, 1993;Lankinen et al, 1993). Deletion of viral RR can alter both the virulence and latency of the virus in culture systems (Luo et al, 1991;Howell et al, 1992;Desai et al, 1993;Jones et al, 1993;Lankinen et al, 1993;Salem et al, 1993).…”

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confidence: 99%

“…Expression of the RR gene of the herpes simplex virus is required for viral growth and neoplastic transformation (Luo et al, 1991;Desai et al, 1993;Jones et al, 1993;Lankinen et al, 1993). Deletion of viral RR can alter both the virulence and latency of the virus in culture systems (Luo et al, 1991;Howell et al, 1992;Desai et al, 1993;Jones et al, 1993;Lankinen et al, 1993;Salem et al, 1993). It has been shown in murine cell lines that signal transduction factors (such as cyclic AMP and protein kinase C) may be involved in the regulation of RR mRNA (Albert and Rozengurt, 1992).…”

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confidence: 99%

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Characterization of the human ribonucleotide reductase M2 subunit gene; genomic structure and promoter analyses

Zhou

Yen²

2001

Cytogenet Genome Res

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Ribonucleotide Reductase (RR) is a rate-limiting enzyme in DNA synthesis and repair consisting of two subunits, M1 and M2. RRM2 plays a role in cell proliferation, tumorgenicity, metastasis and drug resistance. To better understand the regulation of RRM2, we have sequenced the entire human RRM2 gene. Approximately 10.3 kb of genomic DNA was sequenced and deposited to GenBank (accession number AY032750). Intron/exon junctions were identified and the gene was found to consist of ten exons. Two transcription initiation sites were identified and correspond to mRNA transcripts of 3.4 kb and 1.65 kb. Deletion analysis of the 5′-flanking region showed that there was promoter activity consistent with the presence of two separate promoters driving expression of the two RRM2 transcripts. A thorough analysis of the genomic sequence and promoter regions of the RRM2 gene will provide insight into the processes involved in tumor transformation and drug resistance.

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confidence: 99%

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confidence: 99%

Characterization of the human ribonucleotide reductase M2 subunit gene; genomic structure and promoter analyses

Zhou

Yen²

2001

Cytogenet Genome Res

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“…In addition to their involvement in RR activity, the R1 subunits of herpes simplex virus (HSV) types 1 and 2 have been reported to possess a serine\threonine phosphokinase (PK) activity (R1PK) Paradis et al, 1991 ;Cooper et al, 1995) that is distinct from conventional eukaryotic kinases. The novel kinase activity is thought to be located within the N-terminal 310 amino acids, a domain which is unique to HSV R1 and is not required for ribonucleotide reduction (Conner et al, b, 1993Lankinen et al, 1993) ; also, its role in HSV replication\ pathogenesis is not yet established. Protein R1 is essential for virus pathogenicity as evidenced by the avirulence and failure to reactivate from latency of deletion mutants (Cameron et al, 1988 ;Jacobson et al, 1989 ;Yamada et al, 1991 ;Heineman & Cohen, 1994 ;deWind et al, 1993) which could reflect either the lack of RR or N-terminal activity or of both.…”

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The unique N terminus of herpes simplex virus type 1 ribonucleotide reductase large subunit is phosphorylated by casein kinase 2, which may have a homologue in Escherichia coli.

Conner

1999

Journal of General Virology

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Studies were performed to determine if the unique N-terminal domain of the R1 subunit from herpes simplex virus (HSV) type 1 ribonucleotide reductase is a substrate for casein kinase 2 (CK2). Transphosphorylation assays demonstrated that R1 was highly phosphorylated by this enzyme with multiple phosphorylation sites mapped to the N terminus between residues 1 and 245. Immunoprecipitation pull-down assays using R1-specific antisera failed to demonstrate a stable interaction between R1 and CK2 but residual amounts of CK2 present after immunoprecipitation efficiently transphosphorylated R1. Activity assays with a peptide substrate identified CK2 in R1 immunoprecipitated from infected-cell extracts but did not detect activity in R1 proteins immunoprecipitated from bacterial extracts. However, Western blotting identified potential E. coli homologues of the CK2 alpha and beta subunits. These results support conclusions that the N-terminal domain of HSV R1 is not a protein kinase and that all previous results can be explained by contaminating kinases, principally CK2.Conversion of ribonucleotides to the corresponding deoxyribonucleotides by ribonucleotide reductase (RR) is essential for the de novo synthesis of DNA in all living organisms (Reichard, 1993). Many herpesviruses encode their own RR and the active form is a tetramer of homodimeric R1 and R2 subunits (reviewed in Conner et al., 1994 a). RR activity is a target for antiviral chemotherapy and has provided a paradigm to study peptides which disrupt protein-protein interactions as a route to the development of antiviral drugs (Dutia et al.,

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“…Antibodies were detected by ELISA. Epitopes recognized by the MAbs were mapped using as antigens Rl-fl-galactosidase fusion proteins (Lankinen et al, 1993), 31 different N-terminally truncated R1 polypeptides and two defined C-terminally truncated R1 polypeptides .…”

Section: Methodsmentioning

confidence: 99%

“…The polyclonal antiserum 106, raised against purified dN245R1, was used as described in Conner et al (1993). Polyclonal antisera F1 and F3, raised against fusion proteins containing amino acids 3 to 181 and 282 to 436 of R1 respectively (Lankinen et al, 1993), were used as described in Conner et al (1992 b). Monoclonal antibodies (MAbs) were generated as described in Cross et al (1987) except that the BALB/c mice were immunized with dN245R1.…”

Section: Methodsmentioning

confidence: 99%

Identification of structural domains within the large subunit of herpes simplex virus ribonucleotide reductase

Conner¹,

Cross²,

Murray³

et al. 1994

Journal of General Virology

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The large subunit (R1) of herpes simplex virus (HSV) ribonucleotide reductase is a bifunctional protein consisting of a unique N-terminal protein kinase domain and a ribonucleotide reductase domain. Previous studies showed that the two functional domains are linked by a protease sensitive site. Here we provide evidence for two subdomains, of 30K and 53K, within the reductase domain. The two fragments, which were produced by limited proteolysis and were resistant to further degradation, remained tightly associated in a complex containing two molecules of each. They were capable of binding the R2 subunit of HSV ribonucleotide reductase with approximately the same affinity as the intact protein but the complex did not complement the small subunit (R2) to give an active enzyme. At low concentrations (0.4 lag/ml) of trypsin or V8 protease, cleavage between the subdomains was prevented by the presence of the Nterminal protein kinase domain. At higher protease concentrations (1 ~tg/ml) the N-terminal domain is extensively proteolysed and the 30K and 53K domains were generated. Identical results were obtained using purified R1 isolated from infected cell extracts or following expression in Escherichia coli. The origin of the two domains was investigated by N-terminal sequencing of the 53K fragment and by examining their reactivity with a panel of Rl-specific monoclonal antibodies which we isolated and epitope mapped for that purpose. The trypsin cleavage site was found to lie between arginine 575 and asparagine 576, and proteolysis in this region was not prevented by the presence of R2 or the nonapeptide YAGAVVNDL. We propose that the ribonucleotide reductase region of HSV R1 exists in a two domain structure, and that the interdomain linking region is protected by the unique N terminus.

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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

Cited by 10 publications

References 44 publications

Characterization of the human ribonucleotide reductase M2 subunit gene; genomic structure and promoter analyses

Characterization of the human ribonucleotide reductase M2 subunit gene; genomic structure and promoter analyses

The unique N terminus of herpes simplex virus type 1 ribonucleotide reductase large subunit is phosphorylated by casein kinase 2, which may have a homologue in Escherichia coli.

Identification of structural domains within the large subunit of herpes simplex virus ribonucleotide reductase

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