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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. 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.

SUBMITTER: John SP

PROVIDER: S-EPMC1951390 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Ebola virus VP30 is an RNA binding protein.

John Sinu P SP Wang Tan T Steffen Scott S Longhi Sonia S Schmaljohn Connie S CS Jonsson Colleen B CB

Journal of virology 20070613 17

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 exper ...[more]

PMID: 17567691

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Project description:The template for Ebola virus (EBOV) transcription and replication is the helical viral nucleocapsid composed of the viral negative-sense (-) RNA genome, which is complexed by the nucleoprotein (NP), VP35, polymerase L, VP24, and VP30. While viral replication is exerted by polymerase L and its cofactor VP35, EBOV mRNA synthesis is regulated by the viral nucleocapsid protein VP30, an essential EBOV-specific transcription factor. VP30 is a homohexameric phosphoprotein containing a nonconventional zinc finger. The transcriptional support activity of VP30 is strongly influenced by its phosphorylation state. We studied here how RNA binding contributed to VP30's function in transcriptional activation. Using a novel mobility shift assay and the 3'-terminal 154 nucleotides of the EBOV genome as a standard RNA substrate, we detected that RNA binding of VP30 was severely impaired by VP30 mutations that (i) destroy the protein's capability to form homohexamers, (ii) disrupt the integrity of its zinc finger domain, (iii) mimic its fully phosphorylated state, or (iv) alter the putative RNA binding region. RNA binding of the mutant VP30 proteins correlated strongly with their transcriptional support activity. Furthermore, we showed that the interaction between VP30 and the polymerase cofactor VP35 is RNA dependent, while formation of VP30 homohexamers and VP35 homotetramers is not. Our data indicate that RNA binding of VP30 is essential for its transcriptional support activity and stabilizes complexes of VP35/L polymerase with the (-) RNA template to favor productive transcriptional initiation in the presence of termination-active RNA secondary structures.Ebola virus causes severe fevers with unusually high case fatality rates. The recent outbreak of Ebola virus in West Africa claimed more than 11,000 lives and threatened to destabilize a whole region because of its dramatic effects on the public health systems. It is currently not completely understood how Ebola virus manages to balance viral transcription and replication in the infected cells. This study shows that transcriptional support activity of the Ebola virus transcription factor VP30 is highly correlated with its ability to bind viral RNA. The interaction between VP30 and VP35, the Ebola virus polymerase cofactor, is dependent on the presence of RNA as well. Our data contribute to the understanding of the dynamic interplay between nucleocapsid proteins and the viral RNA template in order to promote viral RNA synthesis.

Project description:Viral transcription and replication of Ebola virus (EBOV) is balanced by transcription factor VP30, an RNA binding protein. An RNA hairpin at the transcription start site (TSS) of the first gene (NP hairpin) in the 3'-leader promoter is thought to mediate the VP30 dependency of transcription. Here, we investigated the constraints of VP30 dependency using a series of monocistronic minigenomes with sequence, structure and length deviations from the native NP hairpin. Hairpin stabilizations decreased while destabilizations increased transcription in the absence of VP30, but in all cases, transcription activity was higher in the presence versus absence of VP30. This also pertains to a mutant that is unable to form any RNA secondary structure at the TSS, demonstrating that the activity of VP30 is not simply determined by the capacity to form a hairpin structure at the TSS. Introduction of continuous 3'-UN5 hexamer phasing between promoter elements PE1 and PE2 by a single point mutation in the NP hairpin boosted VP30-independent transcription. Moreover, this point mutation, but also hairpin stabilizations, impaired the relative increase of replication in the absence of VP30. Our results suggest that the native NP hairpin is optimized for tight regulation by VP30 while avoiding an extent of hairpin stability that impairs viral transcription, as well as for enabling the switch from transcription to replication when VP30 is not part of the polymerase complex.IMPORTANCE A detailed understanding is lacking how the Ebola virus (EBOV) protein VP30 regulates activity of the viral polymerase complex. Here, we studied how RNA sequence, length and structure at the transcription start site (TSS) in the 3'-leader promoter influence the impact of VP30 on viral polymerase activity. We found that hairpin stabilizations tighten the VP30 dependency of transcription but reduce transcription efficiency and attenuate the switch to replication in the absence of VP30. Upon hairpin destabilization, VP30-independent transcription - already weakly detectable at the native promoter - increases, but never reaches the same extent as in the presence of VP30. We conclude that the native hairpin structure involving the TSS (i) establishes an optimal balance between efficient transcription and tight regulation by VP30, (ii) is linked to hexamer phasing in the promoter, and (iii) favors the switch to replication when VP30 is absent.

Project description:Ebola virus (EBOV) VP30 regulates viral genome transcription and replication by switching its phosphorylation status. However, the importance of VP30 phosphorylation and dephosphorylation in other viral replication processes such as nucleocapsid and virion assembly is unclear. Interestingly, VP30 is predominantly dephosphorylated by cellular phosphatases in viral inclusions, while it is phosphorylated in the released virions. Thus, uncertainties regarding how VP30 phosphorylation in nucleocapsids is achieved and whether VP30 phosphorylation provides any advantages in later steps in viral replication have arisen. In the present study, to characterize the roles of VP30 phosphorylation in nucleocapsid formation, we used electron microscopic analyses and live cell imaging systems. We identified VP30 localized to the surface of protrusions surrounding nucleoprotein (NP)-forming helical structures in the nucleocapsid, suggesting the involvement in assembly and transport of nucleocapsids. Interestingly, VP30 phosphorylation facilitated its association with nucleocapsid-like structures (NCLSs). On the contrary, VP30 phosphorylation does not influence the transport characteristics and NCLS number leaving from and coming back into viral inclusions, indicating that the phosphorylation status of VP30 is not a prerequisite for NCLS departure. Moreover, the phosphorylation status of VP30 did not cause major differences in nucleocapsid transport in authentic EBOV-infected cells. In the following budding step, the association of VP30 and its phosphorylation status did not influence the budding efficiency of virus-like particles. Taken together, it is plausible that EBOV may utilize the phosphorylation of VP30 for its selective association with nucleocapsids, without affecting nucleocapsid transport and virion budding processes. IMPORTANCE Ebola virus (EBOV) causes severe fevers with unusually high case fatality rates. The nucleocapsid provides the template for viral genome transcription and replication. Thus, understanding the regulatory mechanism behind its formation is important for the development of novel therapeutic approaches. Previously, we established a live-cell imaging system based on the ectopic expression of viral fluorescent fusion proteins, allowing the visualization and characterization of intracytoplasmic transport of nucleocapsid-like structures. EBOV VP30 is an essential transcriptional factor for viral genome synthesis, and, although its role in viral genome transcription and replication is well understood, the functional importance of VP30 phosphorylation in assembly of nucleocapsids is still unclear. Our work determines the localization of VP30 at the surface of ruffled nucleocapsids, which differs from the localization of polymerase in EBOV-infected cells. This study sheds light on the novel role of VP30 phosphorylation in nucleocapsid assembly, which is an important prerequisite for virion formation.

Project description:UnlabelledEbola virus is the causative agent of a severe fever with high fatality rates in humans and nonhuman primates. The regulation of Ebola virus transcription and replication currently is not well understood. An important factor regulating viral transcription is VP30, an Ebola virus-specific transcription factor associated with the viral nucleocapsid. Previous studies revealed that the phosphorylation status of VP30 impacts viral transcription. Together with NP, L, and the polymerase cofactor VP35, nonphosphorylated VP30 supports viral transcription. Upon VP30 phosphorylation, viral transcription ceases. Phosphorylation weakens the interaction between VP30 and the polymerase cofactor VP35 and/or the viral RNA. VP30 thereby is excluded from the viral transcription complex, simultaneously leading to increased viral replication which is supported by NP, L, and VP35 alone. Here, we use an infectious virus-like particle assay and recombinant viruses to show that the dynamic phosphorylation of VP30 is critical for the cotransport of VP30 with nucleocapsids to the sites of viral RNA synthesis, where VP30 is required to initiate primary viral transcription. We further demonstrate that a single serine residue at amino acid position 29 was sufficient to render VP30 active in primary transcription and to generate a recombinant virus with characteristics comparable to those of wild-type virus. In contrast, the rescue of a recombinant virus with a single serine at position 30 in VP30 was unsuccessful. Our results indicate critical roles for phosphorylated and dephosphorylated VP30 during the viral life cycle.ImportanceThe current Ebola virus outbreak in West Africa has caused more than 28,000 cases and 11,000 fatalities. Very little is known regarding the molecular mechanisms of how the Ebola virus transcribes and replicates its genome. Previous investigations showed that the transcriptional support activity of VP30 is activated upon VP30 dephosphorylation. The current study reveals that the situation is more complex and that primary transcription as well as the rescue of recombinant Ebola virus also requires the transient phosphorylation of VP30. VP30 encodes six N-proximal serine residues that serve as phosphorylation acceptor sites. The present study shows that the dynamic phosphorylation of serine at position 29 alone is sufficient to activate primary viral transcription. Our results indicate a series of phosphorylation/dephosphorylation events that trigger binding to and release from the nucleocapsid and transcription complex to be essential for the full activity of VP30.

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Ebola virus VP30 is an RNA binding protein.

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Ebola virus VP30 is an RNA binding protein.

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