Summary auto-generated
This study demonstrates that bluetongue virus (BTV) core protein VP4 possesses nucleoside triphosphate phosphohydrolase (NTPase) activity in addition to its previously known guanylyltransferase function. Using purified recombinant VP4 protein and core-like particles, the researchers characterized this enzyme's properties. VP4 nonspecifically hydrolyzes all four ribonucleoside triphosphates (ATP, GTP, UTP, CTP) to their corresponding diphosphates, with preference for GTP over other substrates. The enzyme requires divalent cations (Mg2+, Mn2+, or Ca2+) for optimal activity, with peak activity at 4 mM Mg2+ or 6 mM Ca2+. The enzyme exhibits pH optimum between 7.5-8.0 and temperature optimum around 40°C. Kinetic analysis yielded a Km of 0.25 μM ATP and Vmax of 55 pmol ATP/min/μg protein. Polynucleotides had minimal stimulatory effects on NTPase activity, unlike some other viral NTPases. The authors propose that VP4's NTPase activity may function in mRNA processing or extrusion from viral cores during transcription.
Key findings
- BTV VP4 protein exhibits NTPase activity, hydrolyzing ribonucleoside triphosphates preferentially to diphosphates (GTP>ATP>UTP>CTP)
- VP4 NTPase activity requires divalent cations with optimal activity at 4 mM Mg2+ or 6 mM Ca2+
- Enzyme shows pH optimum of 7.5-8.0 and temperature optimum of approximately 40°C
- Kinetic parameters: Km = 0.25 μM ATP, Vmax = 55 pmol ATP hydrolyzed/min/μg protein
- Polynucleotides have minimal stimulatory effect on VP4 NTPase activity, suggesting it differs functionally from RNA-dependent NTPases
This summary was generated automatically from the article PDF and is not part of the original publication. Refer to the PDF for the authoritative text.
Abstract
The intact virion of bluetongue virus comprises ten segments of dsRNA enclosed in two concentric protein capsids. The core, which is transcriptionally active, includes three minor proteins (VP1, VP4 and VP6) which are considered to be the candidates for the core-associated enzymes that transcribe and modify full-length mRNA copies for each of the ten genome segments. Using purified recombinant VP4 protein and core-like particles containing VP4, in this report it is demonstrated that VP4 has nucleoside triphosphatase (NTPase) activity. VP4 is a nonspecific NTPase that hydrolyses four types of ribonucleoside triphosphate (NTP) to the corresponding nucleoside diphosphate. The substrate preference was GTP>ATP>UTP>CTP. NTP hydrolysis by VP4 was maximal when the Mg2+ or Ca2+ ion concentrations were 4 mM or 6 mM, respectively. The presence of single-stranded polynucleotides poly(A), poly(U) and poly(C) had little effect on the NTPase activity. Although the enzyme exhibited a broad temperature optimum around 40 degrees C, the pH optimum was sharp, between pH 7.5 and 8. The Km and Vmax of ATP hydrolysis were calculated to be 0.25+/-0.05 microM ATP and 55+/-4 pmol ATP hydrolysed min(-1) microg(-1), respectively. The Km was affected by the addition of poly(A) to only a small extent in contrast to the Vmax, which was increased by at least twofold.