RNA Binding of Ebola Virus VP30 Is Essential for Activating Viral Transcription

Abstract

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. IMPORTANCEEbola 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.

RNA binding specificity of Ebola virus transcription factor VP30

“…VP30 is shown as a trimer of dimers, taking into account that the protein's capability to form hexamers is essential for gel-resolvable RNA binding. 33 As a consequence, the RNA binding path is tentatively indicated at the interface between dimers. While short ssRNAs (»14 nt) rapidly dissociate from VP30, as inferred from the absence of gel-resolvable complexes in the case of a ssRNA 14-mer (Fig.…”

“…8C is based on the finding that VP30 forms hexamers 17 and that mutations in VP30 that prevent hexamerization of the protein, abolish RNA binding in the mobility shift assay. 33 Thus, we assume that VP30 hexamerization generates a composite RNA binding interface and is thus a prerequisite for stable (gel-resolvable) RNA binding.…”

“…3A. Two types of complexes were observed: the major fraction, defined here as C 1 complexes, migrated into the 4% PAA layer of the gel and was assigned to VP30:RNA complexes whose formation depends on the ability of VP30 to form hexamers; 33 and a minor fraction representing high molecular weight complexes, termed C HMW , (Fig. 3A).…”

“…17 VP30 8-272 supports viral transcription with equal efficiency as VP30_wt, and also the entire MBP-VP30 fusion protein activates viral transcription (twofold less efficiently than VP30_wt; 33 ). MBP-VP30 was overexpressed in Escherichia coli BL21 (DE3) cells and the VP30 moiety physically separated from the MBP-tag by TEV protease cleavage to rule out any steric interference of the MBP moiety with RNA binding (for more details, see the Supplementary material).…”

The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication

“…Although these effects are consistent with the infectious virus experiments in TRIM6 KO cells, in which virus replication is reduced but not completely abrogated, they suggest that ubiquitination of VP35 is a regulatory mechanism but is not essential for virus viability. A recent study showed that the interaction of VP35 with the EBOV transcription factor VP30 depends on the dsRNAbinding capacity of VP35, and mutations in the VP35 central basic patch (R305A, K309A, and R312A) reduced its ability to bind VP30 (55). Therefore, VP35 ubiquitination on K309 could affect VP35-VP30 interactions, thereby regulating the switch between virus transcription and replication.…”