Vaccinia virus-encoded ribonucleotide reductase: sequence conservation of the gene for the small subunit and its amplification in hydroxyurea-resistant mutants

Abstract

The vaccinia virus gene that encodes the small subunit of ribonucleotide reductase was localized to the Hindlll F fragment by using degenerate oligonucleotide probes. DNA sequencing revealed a leftward-reading open reading frame that predicted a protein of 37 kilodaltons whose amino acid sequence was much more homologous to the mouse and clam M2 sequences (-80%) than to the corresponding herpesvirus (-27%) or procaryotic (-19%) gene products. Vaccinia virus mutants selected for the ability to grow in high concentrations of a specific inhibitor of ribonucleotide reductase, hydroxyurea, amplified the M2 gene and harbored tandem arrays (2 to 15 copies) of the gene within the HindIII F region. RNA isolated at early times after infection with wild-type virus and probed with an internal fragment of the M2 gene indicated one major (1.2 kilobases) and two minor (4.0 and 2.1 kilobases) transcripts. Si nuclease analysis and primer extension experiments identified an RNA start site 12 nucleotides upstream of the putative initiation ATG codon.

Adaptive Gene Amplification As an Intermediate Step in the Expansion of Virus Host Range

“…Human immunodeficiency viruses, avian influenza viruses, and the recently described Middle East respiratory syndrome coronavirus exemplify the ongoing threat and potential of animal viruses to spread to and among humans, highlighting the urgent need to understand the mechanisms underlying cross-species transmission and adaptation to new hosts. One such mechanism, genetic locus amplification in response to selective pressure, has been observed in both viruses and bacteria [16][17][18]21]. Here we demonstrate that amplification of the exogenous gene rhtrs1 is sufficient to block potent PKR-mediated inhibition and improve VVDEDK+RhTRS1 replication in AGMderived PRO1190 cells.…”

“…Gene amplification as a mechanism of rapid adaptation in vaccinia virus has been well documented [17,18,21]. To determine whether gene amplification could account for the broadly improved replication of passaged VVDEDK+RhTRS1 we performed paired-end Illumina based deep sequencing (Short Read Archive #SRP033208).…”

“…Importantly, rhtrs1 amplification did not occur when the initial virus was passaged through human fibroblasts, suggesting that amplification in AGM cells was a necessary intermediate step to expand the virus host range. Gene amplification is a universal mechanism of rapid adaptation occurring in eukaryotes [14,15], prokaryotes [16], and viruses [17,18], enabling diverse adaptations including evasion of host restriction factors (Reviewed in [19]). Our results suggest that gene amplification in an intermediate host may be a risk factor for broad cross-species transmission independent of other adaptive events.…”

An active ribonucleotide reductase from Arabidopsis thaliana

“…Unfortunately R1 was not present in bacterial soluble extracts and was found exclusively in cell pellets during preparation of extracts, proving that R1 accumulated in insoluble inclusion bodies. The following conditions, alone or in combination, were used for preventing R1 from accumulating in the insoluble form: (a) a lower induction temperature (25 8C, 15 8C); (b) a lower level of inducer (40 mm); (c) a modified LB medium containing betaõ Ène and sorbitol [26]; (d) an addition of hydroxyurea during induction [27]. Only when induction was carried out at 15 8C with 40 mm IPTG for 18±20 h a significant fraction of protein R1 could be found in soluble form.…”

“…These optimal conditions were then routinely used even though they resulted into bacterial extracts in which protein R1 represented only 1% of the soluble protein. It should be noted that overexpression of protein R1 from vaccinia virus [27], herpes simplex virus [28] and mouse [29] in bacterial expression systems generate insoluble forms of the protein.…”