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Journal of Virology, August 2005, p. 10660-10671, Vol. 79, No. 16
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.16.10660-10671.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Ebola Virus Genomic Replication Promoter Is Bipartite and Follows the Rule of Six

Michael Weik,1,2,¶ Sven Enterlein,1,¶ Kathrin Schlenz,1,3 and Elke Mühlberger1*

Department of Virology, Philipps University Marburg, Robert-Koch-Str.17, 35037 Marburg, Germany,1 Dade Behring, Marburg GmbH, Marburg, Germany,2 Beiersdorf AG, Hamburg, Germany3

Received 22 March 2005/ Accepted 29 April 2005

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 UN5 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.

* Corresponding author. Mailing address: Department of Virology, Philipps University Marburg, Robert-Koch-Str.17, 35037 Marburg, Germany. Phone: 6421-2864525. Fax: 6421-2865482. E-mail: muehlber{at}staff.uni-marburg.de.

Both authors contributed equally to this work.

Journal of Virology, August 2005, p. 10660-10671, Vol. 79, No. 16
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.16.10660-10671.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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