Functional analysis of the tick-borne encephalitis virus cyclization elements indicates major differences between mosquito-borne and tick-borne flaviviruses.
The linear, positive-stranded RNA genome of flaviviruses is thought to adopt a circularized conformation via interactions of short complementary sequence elements located within its terminal regions. This process of RNA cyclization is a crucial precondition for RNA replication. In the case of mosquito-borne flaviviruses, highly conserved cyclization sequences (CS) have been identified, and their functionality has been experimentally confirmed. Here, we provide an experimental identification of CS elements of tick-borne encephalitis virus (TBEV). These elements, termed 5'-CS-A and 3'-CS-A, are conserved among various tick-borne flaviviruses, but they are unrelated to the mosquito-borne CS elements and are located at different genomic positions. The 5'-CS-A element is situated upstream rather than downstream of the AUG start codon and, in contrast to mosquito-borne flaviviruses, it was found that the entire protein C coding region is not essential for TBEV replication. The complementary 3'-CS-A element is located within the bottom stem rather than upstream of the characteristic 3'-terminal stem-loop structure, implying that this part of the proposed structure cannot be formed when the genome is in its circularized conformation. Finally, we demonstrate that the CS-A elements can also mediate their function when the 5'-CS-A element is moved from its natural position to one corresponding to the mosquito-borne CS. The recognition of essential RNA elements and their differences between mosquito-borne and tick-borne flaviviruses has practical implications for the design of replicons in vaccine and vector development.
Clinical Institute of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria.
Regina M
RM
Kofler
Verena M
VM
Hoenninger
Caroline
C
Thurner
Christian W
CW
Mandl
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D009033:Culicidae / Q000821:virology*; D004669:Encephalitis Viruses, Tick-Borne / Q000235:genetics*; D005416:Flavivirus / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry; D012093:Replicon
D012367:RNA, Viral