The members of the family Filoviridae, Marburg virus (MBGV) and Ebola virus (EBOV), are very similar in terms of morphology, genome organization, and protein composition. To compare the replication and transcription strategies of both viruses, an artificial replication system based on the vaccinia virus T7 expression system was established for EBOV. Specific transcription and replication of an artificial monocistronic minireplicon was demonstrated by reporter gene expression and detection of the transcribed and replicated RNA species. As it was shown previously for MBGV, three of the four EBOV nucleocapsid proteins, NP, VP35, and L, were essential and sufficient for replication. In contrast to MBGV, EBOV-specific transcription was dependent on the presence of the fourth nucleocapsid protein, VP30. When EBOV VP30 was replaced by MBGV VP30, EBOV-specific transcription was observed but with lower efficiency. Exchange of NP, VP35, and L between the two replication systems did not lead to detectable reporter gene expression. It was further observed that neither MBGV nor EBOV were able to replicate the heterologous minigenomes. A chimeric minigenome, however, containing the EBOV leader and the MBGV trailer was encapsidated, replicated, transcribed, and packaged by both viruses.

To study the mechanisms underlying the high pathogenicity of Ebola virus, we have established a system that allows the recovery of infectious virus from cloned cDNA and thus permits genetic manipulation. We created a mutant in which the editing site of the gene encoding envelope glycoprotein (GP) was eliminated. This mutant no longer expressed the nonstructural glycoprotein sGP. Synthesis of GP increased, but most of it accumulated in the endoplasmic reticulum as immature precursor. The mutant was significantly more cytotoxic than wild-type virus, indicating that cytotoxicity caused by GP is down-regulated by the virus through transcriptional RNA editing and expression of sGP.

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 this work we investigated the cis-acting signals involved in replication of Ebola virus (EBOV) genomic RNA. A set of mingenomes with mutant 3 ends were generated and used in a reconstituted replication and transcription system. Our results suggest that the EBOV genomic replication promoter is bipartite, consisting of a first element located within the leader region of the genome and a second, downstream element separated by a spacer region. While proper spacing of the two promoter elements is a prerequisite for replication, the nucleotide sequence of the spacer is not important. Replication activity was only observed when six or a multiple of six nucleotides were deleted or inserted, while all other changes in length abolished replication completely. These data indicate that the EBOV replication promoter obeys the rule of six, although the genome length is not divisible by six. The second promoter element is located in the 3 nontranslated region of the first gene and consists of eight UN 5 hexamer repeats, where N is any nucleotide. However, three consecutive hexamers, which could be located anywhere within the promoter element, were sufficient to support replication as long as the hexameric phase was preserved. By using chemical modification assays, we could demonstrate that nucleotides 5 to 44 of the EBOV leader are involved in the formation of a stable secondary structure. Formation of the RNA stem-loop occurred independently of the presence of the trailer, indicating that a panhandle structure is not formed between the 3 and 5 ends.Ebola virus (EBOV) and Marburg virus are members of the family Filoviridae, which belongs to the order Mononegavirales. Ebola viruses are divided into four genera, Zaire, Sudan, Ivory Coast, and Reston (26). All filoviruses cause severe hemorrhagic fevers in humans and nonhuman primates, with fatality rates of up to 90% (36), except for EBOV Reston, which seems to be apathogenic for humans (18,23). EBOV genomes consist of a nonsegmented, single-stranded RNA in negative orientation (NNS) of about 19 kb in length. Seven genes encoding eight proteins are arranged in a linear order. Short nontranscribed regions are located at the extreme 3Ј and 5Ј ends, called the leader and the trailer, respectively (Fig. 1A). Compared to other members of the Mononegavirales, filovirus (and henipavirus) genes have unusually long nontranslated regions (NTRs) flanking the open reading frames. Thus, the start codon of the first EBOV gene, the NP gene, is located at nucleotide positions 470 to 472, and the stop codon of the last gene, the L gene, is found 742 nucleotides upstream of the extreme 5Ј end. Naturally occurring defective interfering EBOV particles revealed that 155 nucleotides at the 3Ј terminus and 176 nucleotides of the 5Ј terminus are sufficient for replication (5).Four viral proteins, NP, VP35, VP30, and L, are associated with the viral RNA forming the nucleocapsid (1). While the nucleoprotein NP tightly encapsidates the viral RNA, the catalytic subunit of the viral polymerase, L, and t...