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Journal of Virology, November 2004, p. 11726-11738, Vol. 78, No. 21
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.21.11726-11738.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Functional Analysis of the Noncoding Regions of the Uukuniemi Virus (Bunyaviridae) RNA Segments

Kirsten Flick,1,{dagger} Anna Katz,2,{dagger},{ddagger} Anna Överby,1,2,{dagger} Heinz Feldmann,1,3 Ralf F. Pettersson,2 and Ramon Flick1,2,3*

Special Pathogens Program, National Microbiology Laboratory, Health Canada, Canadian Science Centre for Human and Animal Health (CSCHAH),1 Department of Medical Microbiology, University of Manitoba, Winnipeg, Canada,3 Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institute, Stockholm, Sweden2

Received 21 April 2004/ Accepted 10 June 2004

The role of the variable portion of the noncoding regions (NCRs) of the three Bunyaviridae RNA segments (L, M, S) in transcription, replication, and packaging was studied using the recently developed plasmid-driven RNA polymerase I minigenome system for Uukuniemi (UUK) virus, genus Phlebovirus (11), as a model. Comparison of the different segments showed that all NCRs were sufficient to mediate transcription/replication of a minigenome but demonstrated decreased promoter strength in the order M > L > S. Chimeric minigenomes with flanking NCRs from different genome segments revealed that the number of total base pairs within the inverted, partially complementary ends was important for transcription and replication. Point mutations increasing the base-pairing potential produced increased reporter expression, indicating that complementarity between the 5' and 3' ends is crucial for promoter activity. The role of the intergenic region (IGR) located between the two open reading frames of the ambisense UUK virus S segment was analyzed by inserting this sequence element downstream of the reporter genes. The presence of the IGR was found to enhance reporter expression, demonstrating that efficient transcription termination, regulated by the IGR, is important for optimal minigenome mRNA translation. Finally, genome packaging efficacy varied for different NCRs and was strongest for L followed by M and S. Strong reporter gene activity was still observed after seven consecutive cell culture passages, indicating a selective rather than random genome-packaging mechanism. In summary, our results demonstrate that the NCRs from all three segments contain the necessary signals to initiate transcription and replication as well as packaging. Based on promoter strength, M-segment NCRs may be the preferred choice for the development of reverse genetics and minigenome rescue systems for bunyaviruses.

* Corresponding author. Present address: Department of Pathology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0609. Phone: (409) 747-2962. Fax: (409) 747-2437. E-mail: raflick{at}utmb.edu.

{dagger} K.F., A.K., and A.Ö. contributed equally to this work.

Journal of Virology, November 2004, p. 11726-11738, Vol. 78, No. 21
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.21.11726-11738.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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