Ebola virus VP30 is an RNA binding protein

Sinu P. John, Tan Wang, Scott Steffen, Sonia Longhi, Connie S. Schmaljohn, Colleen Jonsson

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn2+, but not Mg2+, enhanced activity. Enhancement of transcription by VP30 requires a RNA stem-loop located within nucleotides 54 to 80 of the leader region. VP30 showed low binding affinity to the predicted stem-loop alone or to double-stranded RNA but showed a good binding affinity for the stem-loop when placed in the context of upstream and downstream sequences. To map the region responsible for interacting with RNA, we constructed, purified, and assayed a series of N-terminal deletion mutations of VP30 for RNA binding. The key amino acids supporting RNA binding activity map to residues 26 to 40, a region rich in arginine. Thus, we show for the first time the direct interaction of EBOV VP30 with RNA and the importance of the N-terminal region for binding RNA.

Original languageEnglish (US)
Pages (from-to)8967-8976
Number of pages10
JournalJournal of Virology
Volume81
Issue number17
DOIs
StatePublished - Sep 1 2007

Fingerprint

Ebolavirus
RNA-binding proteins
RNA-Binding Proteins
RNA
Viral RNA
transcription (genetics)
stems
Double-Stranded RNA
Sequence Deletion
Computational Biology
double-stranded RNA
Arginine
crosslinking
Proteins
bioinformatics
Nucleotides

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

John, S. P., Wang, T., Steffen, S., Longhi, S., Schmaljohn, C. S., & Jonsson, C. (2007). Ebola virus VP30 is an RNA binding protein. Journal of Virology, 81(17), 8967-8976. https://doi.org/10.1128/JVI.02523-06

Ebola virus VP30 is an RNA binding protein. / John, Sinu P.; Wang, Tan; Steffen, Scott; Longhi, Sonia; Schmaljohn, Connie S.; Jonsson, Colleen.

In: Journal of Virology, Vol. 81, No. 17, 01.09.2007, p. 8967-8976.

Research output: Contribution to journalArticle

John, SP, Wang, T, Steffen, S, Longhi, S, Schmaljohn, CS & Jonsson, C 2007, 'Ebola virus VP30 is an RNA binding protein', Journal of Virology, vol. 81, no. 17, pp. 8967-8976. https://doi.org/10.1128/JVI.02523-06
John SP, Wang T, Steffen S, Longhi S, Schmaljohn CS, Jonsson C. Ebola virus VP30 is an RNA binding protein. Journal of Virology. 2007 Sep 1;81(17):8967-8976. https://doi.org/10.1128/JVI.02523-06
John, Sinu P. ; Wang, Tan ; Steffen, Scott ; Longhi, Sonia ; Schmaljohn, Connie S. ; Jonsson, Colleen. / Ebola virus VP30 is an RNA binding protein. In: Journal of Virology. 2007 ; Vol. 81, No. 17. pp. 8967-8976.
@article{dc3ea4e05e6845368923339f00550d06,
title = "Ebola virus VP30 is an RNA binding protein",
abstract = "The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90{\%} homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn2+, but not Mg2+, enhanced activity. Enhancement of transcription by VP30 requires a RNA stem-loop located within nucleotides 54 to 80 of the leader region. VP30 showed low binding affinity to the predicted stem-loop alone or to double-stranded RNA but showed a good binding affinity for the stem-loop when placed in the context of upstream and downstream sequences. To map the region responsible for interacting with RNA, we constructed, purified, and assayed a series of N-terminal deletion mutations of VP30 for RNA binding. The key amino acids supporting RNA binding activity map to residues 26 to 40, a region rich in arginine. Thus, we show for the first time the direct interaction of EBOV VP30 with RNA and the importance of the N-terminal region for binding RNA.",
author = "John, {Sinu P.} and Tan Wang and Scott Steffen and Sonia Longhi and Schmaljohn, {Connie S.} and Colleen Jonsson",
year = "2007",
month = "9",
day = "1",
doi = "10.1128/JVI.02523-06",
language = "English (US)",
volume = "81",
pages = "8967--8976",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "17",

}

TY - JOUR

T1 - Ebola virus VP30 is an RNA binding protein

AU - John, Sinu P.

AU - Wang, Tan

AU - Steffen, Scott

AU - Longhi, Sonia

AU - Schmaljohn, Connie S.

AU - Jonsson, Colleen

PY - 2007/9/1

Y1 - 2007/9/1

N2 - The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn2+, but not Mg2+, enhanced activity. Enhancement of transcription by VP30 requires a RNA stem-loop located within nucleotides 54 to 80 of the leader region. VP30 showed low binding affinity to the predicted stem-loop alone or to double-stranded RNA but showed a good binding affinity for the stem-loop when placed in the context of upstream and downstream sequences. To map the region responsible for interacting with RNA, we constructed, purified, and assayed a series of N-terminal deletion mutations of VP30 for RNA binding. The key amino acids supporting RNA binding activity map to residues 26 to 40, a region rich in arginine. Thus, we show for the first time the direct interaction of EBOV VP30 with RNA and the importance of the N-terminal region for binding RNA.

AB - The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn2+, but not Mg2+, enhanced activity. Enhancement of transcription by VP30 requires a RNA stem-loop located within nucleotides 54 to 80 of the leader region. VP30 showed low binding affinity to the predicted stem-loop alone or to double-stranded RNA but showed a good binding affinity for the stem-loop when placed in the context of upstream and downstream sequences. To map the region responsible for interacting with RNA, we constructed, purified, and assayed a series of N-terminal deletion mutations of VP30 for RNA binding. The key amino acids supporting RNA binding activity map to residues 26 to 40, a region rich in arginine. Thus, we show for the first time the direct interaction of EBOV VP30 with RNA and the importance of the N-terminal region for binding RNA.

UR - http://www.scopus.com/inward/record.url?scp=34548161584&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34548161584&partnerID=8YFLogxK

U2 - 10.1128/JVI.02523-06

DO - 10.1128/JVI.02523-06

M3 - Article

VL - 81

SP - 8967

EP - 8976

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 17

ER -