John SP, Wang T, Steffen S, Longhi S, Schmaljohn CS, Jonsson CB.
J Virol. 2007 Sep;81(17):8967-76. Epub 2007 Jun 13.
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. Zn(2+), but not Mg(2+), 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.
Free PMC Article
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. Zn(2+), but not Mg(2+), 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.
Fabozzi G, Nabel CS, Dolan MA, Sullivan NJ.
J Virol. 2011 Mar;85(6):2512-23. doi: 10.1128/JVI.01160-10. Epub 2011 Jan 12.
Abstract
Cellular RNA
interference (RNAi) provides a natural response against viral infection,
but some viruses have evolved mechanisms to antagonize this form of
antiviral immunity. To determine whether Ebolavirus (EBOV) counters RNAi
by encoding suppressors of RNA silencing (SRSs), we screened all EBOV
proteins using an RNAi assay initiated by exogenously delivered small
interfering RNAs (siRNAs) against either an EBOV or a reporter gene.
In addition to viral protein 35 (VP35), we found that VP30 and VP40 independently act as SRSs. Here, we present the molecular mechanisms of VP30 and VP35. VP30 interacts with Dicer independently of siRNA and with one Dicer partner, TRBP, only in the presence of siRNA. VP35 directly interacts with Dicer partners TRBP and PACT in an siRNA-independent fashion and in the absence of effects on interferon (IFN). Taken together, our findings elucidate a new mechanism of RNAi suppression that extends beyond the role of SRSs in double-stranded RNA (dsRNA) binding and IFN antagonism. The presence of three suppressors highlights the relevance of host RNAi-dependent antiviral immunity in EBOV infection and illustrates the importance of RNAi in shaping the evolution of RNA viruses.
In addition to viral protein 35 (VP35), we found that VP30 and VP40 independently act as SRSs. Here, we present the molecular mechanisms of VP30 and VP35. VP30 interacts with Dicer independently of siRNA and with one Dicer partner, TRBP, only in the presence of siRNA. VP35 directly interacts with Dicer partners TRBP and PACT in an siRNA-independent fashion and in the absence of effects on interferon (IFN). Taken together, our findings elucidate a new mechanism of RNAi suppression that extends beyond the role of SRSs in double-stranded RNA (dsRNA) binding and IFN antagonism. The presence of three suppressors highlights the relevance of host RNAi-dependent antiviral immunity in EBOV infection and illustrates the importance of RNAi in shaping the evolution of RNA viruses.
2.
Martínez MJ, Biedenkopf N, Volchkova V, Hartlieb B, Alazard-Dany N, Reynard O, Becker S, Volchkov V.
J Virol. 2008 Dec;82(24):12569-73. doi: 10.1128/JVI.01395-08. Epub 2008 Oct 1.
Abstract
VP30 is a
phosphoprotein essential for the initiation of Ebola virus
transcription. In this work, we have studied the effect of mutations in
VP30 phosphorylation sites on the ebolavirus replication cycle by using a
reverse genetics system. We demonstrate that VP30 is involved in
reinitiation of gene transcription and that this activity is affected by
mutations at the phosphorylation sites.
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