Transcription of the highly pathogenic Ebola virus (EBOV) is dependent on VP30, a constituent of the viral nucleocapsid complex. Here we present evidence that phosphorylation of VP30, which takes place at six Nterminal serine residues and one threonine residue, is of functional significance. Replacement of the phosphoserines by alanines resulted in an only slightly phosphorylated VP30 (VP30 6A ) that is still able to activate EBOVspecific transcription in a plasmid-based minigenome system. VP30 6A , however, did not bind to inclusions that are induced by the major nucleocapsid protein NP. Three intracellular phosphatases (PP1, PP2A, and PP2C) have been determined to dephosphorylate VP30. The presence of okadaic acid (OA), an inhibitor of PP1 and PP2A, had the same negative effect on transcription activation by VP30 as the substitution of the six phosphoserines for aspartate residues. OA, however, did not impair transcription when VP30 was replaced by VP30 6A . In EBOV-infected cells, OA blocked virus growth dose-dependently. The block was mediated by the extensive phosphorylation of VP30, which is evidenced by the result that expression of VP30 6A , in trans, led to the progression of EBOV infection in the presence of OA. In conclusion, phosphorylation of VP30 was shown to regulate negatively transcription activation and positively binding to the NP inclusions. Ebola virus (EBOV),1 a filovirus, is notorious for its unpredictable sporadic outbreaks of a fatal hemorrhagic fever in Africa (1-4). To date, neither a vaccine nor a treatment of the EBOV infection is available.The enveloped EBOV particles are composed of seven structural proteins and the negative sense RNA genome. Four viral proteins NP, VP35, L, and VP30 are the constituents of the nucleocapsid. The main component of the nucleocapsid complex is NP, a heavily phosphorylated protein, that encapsidates the genomic RNA and forms intracellular inclusions upon recombinant expression (5-7). The NP inclusions are morphologically highly similar to the inclusions formed during EBOV infection of target cells. VP35 and L are the components of the viral polymerase. VP30 represents an EBOV-specific transcription activation factor (8).Most viruses of the order Mononegavirales contain three proteins, N (NP), P, and L, that drive the processes of replication (synthesis of genomic RNA) and transcription (synthesis of viral mRNAs). These proteins also constitute the respective viral nucleocapsid complex. N (or NP) represents the major nucleocapsid protein that encapsidates the viral genome. The encapsidated genome serves as a template for the viral polymerase complex, which is constituted by the catalytic subunit L and the cofactor P (9).A recently established minigenome-based reverse genetic system revealed that EBOV follows another strategy to synthesize the different RNA species. NP, VP35 (the P analogue), and L were sufficient for viral replication, similar to the other Mononegavirales. The fourth nucleocapsid protein VP30, although not influencing replication, drama...

The nucleocapsid protein VP30 of Ebola virus (EBOV), a member of the Filovirus family, is known to act as a transcription activator. By using a reconstituted minigenome system, the role of VP30 during transcription was investigated. We could show that VP30-mediated transcription activation is dependent on formation of a stem-loop structure at the first gene start site. Destruction of this secondary structure led to VP30-independent transcription. Analysis of the transcription products of bicistronic minigenomes with and without the ability to form the secondary structure at the first transcription start signal revealed that transcription initiation at the first gene start site is a prerequisite for transcription of the second gene, independent of the presence of VP30. When the transcription start signal of the second gene was exchanged with the transcription start signal of the first gene, transcription of the second gene also was regulated by VP30, indicating that the stem-loop structure of the first transcription start site acts autonomously and independently of its localization on the RNA genome. Our results suggest that VP30 regulates a very early step of EBOV transcription, most likely by inhibiting pausing of the transcription complex at the RNA structure of the first transcription start site.Ebola virus (EBOV) and the closely related Marburg virus (MBGV) are the only members of the family Filoviridae, which belongs to the order Mononegavirales.

In bluetongue virus (BTV)-infected cells, large cytoplasmic aggregates are formed, termed viral inclusion bodies (VIBs), which are believed to be the sites of viral replication and morphogenesis. The BTV nonstructural protein NS2 is the major component of VIBs. NS2 undergoes intracellular phosphorylation and possesses a strong single-stranded RNA binding activity. By changing phosphorylated amino acids to alanines and aspartates, we have mapped the phosphorylated sites of NS2 to two serine residues at positions 249 and 259. Since both of these serines are within the context of protein kinase CK2 recognition signals, we have further examined if CK2 is involved in NS2 phosphorylation by both intracellular colocalization and an in vitro phosphorylation assay. In addition, we have utilized the NS2 mutants to determine the role of phosphorylation on NS2 activities. The data obtained demonstrate that NS2 phosphorylation is not necessary either for its RNA binding properties or for its ability to interact with the viral polymerase VP1. However, phosphorylated NS2 exhibited VIB formation while unmodified NS2 failed to assemble as VIBs although smaller oligomeric forms of NS2 were readily formed. Our data reveal that NS2 phosphorylation controls VIBs formation consistent with a model in which NS2 provides the matrix for viral assembly.Protein phosphorylation is a ubiquitous protein modification that controls a number of intracellular processes. In eukaryotic systems, phosphorylation occurs almost exclusively on serine, threonine, or tyrosine residues (26). Also for RNA viruses, including vesicular stomatitis virus, ebola virus, human immunodeficiency virus type 1 (HIV-1), and rubella virus, protein phosphorylation has been shown to regulate vital processes such as virus transcription and replication, RNA binding activity, and virus assembly (9,22,28,34). The nonstructural protein 2 (NS2) is the only phosphorylated protein of the 10 viral proteins synthesized during a bluetongue virus (BTV) infection (25).BTV is the prototype of the Orbivirus genus in the Reoviridae family and hence characteristically possesses a doublestranded RNA genome enclosed by three consecutive capsid layers of multiple proteins. The BTV genome consists of 10 segments, each encoding one protein. There are seven structural proteins (viral polymerase 1 [VP1] through VP7), of which two are outer capsid proteins (VP2 and VP5) and the remaining five are associated with the BTV core. The viral core consists of a double-layered shell composed of VP3 and VP7, and within the core, there are three virus-encoded proteins (VP1, VP4, and VP6), all of which are enzymatically involved in viral transcription and replication. VP4 is the mRNA-capping enzyme, exhibiting guanyltransferase and methyltransferase activities, VP6 is a double-stranded RNA helicase, and the largest protein, VP1, is the viral RNA-dependent RNA polymerase (5,32,36,41). Core particles are transcriptionally active, producing and releasing mRNA. The remaining three BTV proteins are nonstructural...

Ebola virus VP30 is an essential activator of viral transcription. In viral particles, VP30 is closely associated with the nucleocapsid complex. A conspicuous structural feature of VP30 is an unconventional zinc-binding Cys 3 -His motif comprising amino acids 68 to 95. By using a colorimetric zinc-binding assay we found that the VP30-specific Cys 3 -His motif stoichiometrically binds zinc ions in a one-to-one relationship. Substitution of the conserved cysteines and the histidine within the motif led to a complete loss of the capacity for zinc binding. Functional analyses revealed that none of the tested mutations of the proposed zinc-coordinating residues influenced binding of VP30 to nucleocapsid-like particles but, concerning its role in activating viral transcription, all resulted in a protein that was inactive.Ebola virus (EBOV) and Marburg virus, the two members of the family Filoviridae, cause a severe hemorrhagic disease with an exceptionally high fatality rate in humans and monkeys (22). Due to their genome organization, filoviruses are grouped in the order Mononegavirales. The EBOV genome consists of a single-stranded nonsegmented negative-sense RNA molecule with a coding capacity for eight proteins. Four of the proteins, NP, VP35, VP30, and L, are associated with the viral genome forming the nucleocapsid complex (6). NP, VP35, and L are sufficient to mediate viral replication in a reconstituted replication and transcription system (21). Transcription, however, is strongly dependent on the presence of the fourth nucleocapsid protein, VP30 (21, 27). Recently it has been shown that VP30 acts as a transcription antitermination factor immediately after transcription initiation while transcription elongation is not affected by the protein. The function of VP30 as transcription activator has been found to be dependent on the formation of an RNA secondary structure at the transcription start site of the first gene (28). Furthermore, VP30 is heavily phosphorylated, and this posttranslational modification has been shown to regulate VP30 activity during transcription (18). However, the molecular mechanism by which VP30 activates viral transcription is presently unknown.Sequence analysis of filoviral VP30 revealed that it contains a motif similar to that of an unconventional Cys 3 -His zinc finger that was first characterized for Nup475, a mammalian nuclear protein (5). On the basis of nuclear magnetic resonance data, a structure of the Nup475 metal-binding domain was proposed in which the zinc ion is coordinated by the conserved cysteines and histidine (29). Comparable motifs have been discovered in various proteins from different eukaryotic and viral species (29). The suggestion that the Cys 3 -His motif within VP30 forms a zinc finger with biological relevance is supported by the fact that it is highly conserved among filoviruses (Fig. 1A). A similar motif was also identified in the M2-1 protein of pneumoviruses, which are closely related to filoviruses (12). Like VP30, the M2-1 protein of human respiratory sy...