Summary.
Minor structural protein VP6 is the putative helicase of African horse sickness virus (AHSV), of the genus Orbivirus in the Reoviridae family. We investigated how the protein interacts with double-stranded (ds) RNA and other nucleic acids. Binding was assayed using an electrophoretic migration retardation assay and a nucleic acid overlay protein blot assay. VP6 bound double and single stranded RNA and DNA in a NaCl concentration sensitive reaction. Of six truncated VP6 peptides investigated, two partially overlapping peptides were found to bind dsRNA at pH 7.0, while other peptides with the same overlap did not. The distinction between the peptides appeared to be the pI which ranged from more than 8.0 to just above 6.0. Changing the pH of the binding buffer modified the binding activity. Regardless of assay conditions, only peptides with a specific region of amino acids in common, showed evidence of binding activity. No sequence homology was identified with other binding domains, however, the presence of charged amino acids are assumed to be important for binding activity. The results suggested dsRNA binding in the blot assay was strongly affected by the net charge on the peptide.
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References
CW Bremer H Huismans AA Van Dijk (1990) ArticleTitleCharacterization and cloning of the African horse sickness virus genome. J Gen Virol 71 793–799 Occurrence Handle2324709
CH Calisher PP Mertens (1998) ArticleTitleTaxonomy of African horse sickness viruses. Arch Virol Suppl 14 3–11 Occurrence Handle9785490
A Fernandez JA Garcia (1996) ArticleTitleThe RNA helicase CI from plum pox potyvirus has two regions involved in binding to RNA. FEBS Lett 388 206–210 Occurrence Handle10.1016/0014-5793(96)00571-6 Occurrence Handle8690088
A Fukusho Y Yu S Yamaguchi P Roy (1989) ArticleTitleCompletion of the sequence of bluetongue virus serotype 10 by the characterization of a structural protein, VP6, and a non-structural protein, NS2. J Gen Virol 70 1677–1689 Occurrence Handle2544660
P Gouet JM Diprose JM Grimes R Malby JN Burroughs S Zientara DI Stuart PP Mertens (1999) ArticleTitleThe highly ordered double-stranded RNA genome of bluetongue virus revealed by crystallography. Cell 97 481–490 Occurrence Handle10.1016/S0092-8674(00)80758-8 Occurrence Handle10338212
JM Grimes JN Burroughs P Gouet JM Diprose R Malby S Zientara PP Mertens DI Stuart (1998) ArticleTitleThe atomic structure of the bluetongue virus core. Nature 395 470–478 Occurrence Handle10.1038/26694 Occurrence Handle9774103
SH Hassan C Wirblich M Forzan P Roy (2001) ArticleTitleExpression and functional characterization of bluetongue virus VP5 protein: role in cellular permeabilization. J Virol 75 8356–8367 Occurrence Handle10.1128/JVI.75.18.8356-8367.2001 Occurrence Handle11507181
SS Hassan P Roy (1999) ArticleTitleExpression and functional characterization of bluetongue virus VP2 protein: role in cell entry. J Virol 73 9832–9842 Occurrence Handle10559295
E Hayama JK Li (1994) ArticleTitleMapping and characterization of antigenic epitopes and the nucleic acid-binding domains of the VP6 protein of bluetongue viruses. J Virol 68 3604–3611 Occurrence Handle7514678
JA House (1993) ArticleTitleAfrican horse sickness. Vet Clin North Am Equine Pract 9 355–364 Occurrence Handle8358648
AK Kar P Roy (2003) ArticleTitleDefining the structure-function relationships of bluetongue virus helicase protein VP6. J Virol 77 11347–11356 Occurrence Handle10.1128/JVI.77.21.11347-11356.2003 Occurrence Handle14557620
S-K Lee DL Hacker (2001) ArticleTitleIn vitro analysis of an RNA binding site within the N-terminal 30 amino acids of the Southern cowpea mosaic virus coat protein. Virology 286 317–327 Occurrence Handle10.1006/viro.2001.0979 Occurrence Handle11485399
VA Luckow SC Lee GF Barry PO Olins (1993) ArticleTitleEfficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol 67 4566–4579 Occurrence Handle8392598
WE Mears SA Rice (1996) ArticleTitleThe RGG box motif of the herpes simplex virus ICP27 protein mediates an RNA-binding activity and determines in vivo methylation. J Virol 70 7445–7453 Occurrence Handle8892862
PS Mellor J Boorman (1995) ArticleTitleThe transmission and geographical spread of African horse sickness and bluetongue viruses. Ann Trop Med Parasitol 89 1–15 Occurrence Handle7741589
PS Mellor C Hamblin (2004) ArticleTitleAfrican horse sickness. Vet Res 35 445–466 Occurrence Handle10.1051/vetres:2004021 Occurrence Handle15236676
PP Mertens J Diprose (2004) ArticleTitleThe bluetongue virus core: a nano-scale transcription machine. Virus Res 101 29–43 Occurrence Handle10.1016/j.virusres.2003.12.004 Occurrence Handle15010215
P Roy A Adachi T Urakawa TF Booth CP Thomas (1990) ArticleTitleIdentification of bluetongue virus VP6 protein as a nucleic acid-binding protein and the localization of VP6 in virus-infected vertebrate cells. J Virol 64 1–8 Occurrence Handle2152806
P Roy PP Mertens I Casal (1994) ArticleTitleAfrican horse sickness virus structure. Comp Immunol Microbiol Infect Dis 17 243–273 Occurrence Handle10.1016/0147-9571(94)90046-9 Occurrence Handle8001348
G Sachetto-Martins LO Franco DE de Oliveira (2000) ArticleTitlePlant glycine-rich proteins: a family or just proteins with a common motif? Biochim Biophys Acta 1492 1–14 Occurrence Handle10858526
N Stäuber J Martinez-Costas G Sutton K Monastyrskaya P Roy (1997) ArticleTitleBluetongue virus VP6 protein binds ATP and exhibits an RNA-dependent ATPase function and a helicase activity that catalyze the unwinding of double-stranded RNA substrates. J Virol 71 7220–7226 Occurrence Handle9311795
J Theron JM Uitenweerde H Huismans LH Nel (1994) ArticleTitleComparison of the expression and phosphorylation of the non-structural protein NS2 of three different orbiviruses: evidence for the involvement of an ubiquitous cellular kinase. J Gen Virol 75 3401–3411 Occurrence Handle7527835
PJ Turnbull SB Cormack H Huismans (1996) ArticleTitleCharacterization of the gene encoding core protein VP6 of two African horse sickness virus serotypes. J Gen Virol 77 1421–1423 Occurrence Handle8757982
JM Uitenweerde J Theron MA Stoltz H Huismans (1995) ArticleTitleThe multimeric nonstructural NS2 proteins of bluetongue virus, African horse sickness virus, and epizootic hemorrhagic disease virus differ in their single-stranded RNA-binding ability. Virology 209 624–632 Occurrence Handle10.1006/viro.1995.1294 Occurrence Handle7539971
Q Wang J Bergeron T Mabrouk G Lemay (1996) ArticleTitleSite-directed mutagenesis of the double-stranded RNA binding domain of bacterially-expressed sigma 3 reovirus protein. Virus Res 41 141–151 Occurrence Handle10.1016/0168-1702(96)01281-6 Occurrence Handle8738173
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de Waal, P., Huismans, H. Characterization of the nucleic acid binding activity of inner core protein VP6 of African horse sickness virus. Arch Virol 150, 2037–2050 (2005). https://doi.org/10.1007/s00705-005-0547-4
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DOI: https://doi.org/10.1007/s00705-005-0547-4