Functional analysis of the non-coding regions of RNA segments and the nucleocapsid protein of the Uukuniemi virus

The Uukuniemi virus (UUKV) is a member of the Bunyaviridae family (genus Phlebovirus). The virus was isolated from Ixodes ricinus ticks from Uukuniemi, Finland in 1959 and was found to be non-pathogenic for humans. UUKV has served for more than four decades as an excellent model to study the molecular and cellular biology of the serious human pathogens that reside within this group. UUKV has a segmented, single-stranded RNA genome of negative polarity. The three RNA segments (S, M, and L) encode four structural proteins: a nucleocapsid (N) protein, two glycoproteins (Gn and Gc), and an RNA-dependent RNA polymerase (L protein). In addition, a non-structural protein (NSs) is encoded from the S segment using an ambisense coding strategy. At the termini of the RNA segments, there are non-coding regions, which contain regulatory elements for viral transcription and replication. The very terminal 5' and 3' ends within all non-coding regions are complementary to each other, and highly conserved within the genus. In order to function as templates for transcription and replication, all three RNA segments must be encapsidated by the N protein. The N protein forms oligomers, in which N protein molecules are bound to each other; this oligomer associates with RNA. This study focused on analyzing the function of the non-coding regions in the termini of the RNA segments, and on locating amino acid residues or domains of the UUKV N protein, which could potentially be involved in the oligomerization or RNA-binding. The function of the non-coding regions was studied using a minigenome system developed for UUKV, where the viral protein coding sequence is replaced by sequences encoding a reporter protein. The cells are transfected with the minigenomes and helper plasmids, and after replication and transcription of the minigenomes, the reporter protein expression can be measured. The non-coding regions of all three RNA segments were analyzed and promoter strengths and packaging efficiencies were compared. The results showed that the non-coding regions in all three RNA segments contain all the necessary signals for initiation of transcription and replication and encapsidation and packaging of the RNA segments. The strongest promoter strength was observed in M segment, followed by L and S segments. The role of the intergenic region, which is located between the N and NSs genes in the UUKV S segment was also analyzed and was found to regulate termination of transcription. To study the oligomerization and RNA-binding of UUKV N protein, a set of N protein mutants were generated based on 2D and 3D predictions of the N protein. The functionality of these mutants was analyzed using mammalian two-hybrid-, minigenome-, and virus-like particle-assays, which showed that both the N- and C-termini of the N protein are needed for the oligomerization. A specific structure in the N-terminal region plays an important role in the N-N interactions. Some putative RNA-binding residues were found, which severely affected the N protein functionality in all three assays. These residues were located within the proposed RNA-binding cavity in the predicted UUKV N protein models. These results are in agreement with observations with other bunyaviruses, and could help to better understand the molecular biology of bunyaviruses. Moreover, understanding the details of the oligomerization and RNA-binding of the N protein could help in design of potential antivirals for the pathogenic phleboviruses.

Grantor dc:publisher

Helsingin yliopisto

Year dc:date.issued

2012

Author dc:creator

• Katz, Anna

• virologia

Statement dc:rights

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Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/10138/33309

OAI record oai:identifier

https://helda.helsinki.fi/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Ahelda.helsinki.fi%3A10138%2F33309