Analysis of the effects of alterations in the tick-borne encephalitis virus 3'-noncoding region on translation and RNA replication using reporter replicons.
The 3'-noncoding region (3'-NCR) of the flavivirus genome includes a variable region that tolerates the insertion of heterologous genetic information. Natural isolates of tick-borne encephalitis virus (TBEV) have particularly long variable regions, which, for some strains, include an internal poly(A) tract. We constructed luciferase reporter replicons of TBEV to analyze the impact of various manipulations of the 3'-NCR on viral RNA translation and replication. The choice of the reporter gene, its position and processing within the viral polyprotein, and the choice of standards were found to be important for obtaining a sensitive and reliable test system. We observed that truncation or complete removal of the internal poly(A) tract, or even the entire variable region, had no significant impact on translation and replication of the RNA in mammalian cell culture. Substitution of the variable region with foreign genetic elements impaired RNA replication to various degrees but generally had no influence on viral translation. Expression cassettes driven by an IRES element inhibited RNA replication more strongly than did repetitive protein-binding elements derived from a bacteriophage, even when the ligand that binds these elements was co-expressed in the cells. Previously identified mutations in the IRES partially relieved this inhibition when introduced into the reporter replicon but provided no evidence for intramolecular competition for translation factors. Impairment of replication appeared to depend more on the type of foreign insert than on its length. These results provide a rational basis for the construction of TBEV-based vectors or vaccines as well as molecular tools for studying flavivirus replication.
Clinical Institute of Virology, Medical University of Vienna, Austria.
Verena M
VM
Hoenninger
Harald
H
Rouha
Klaus K
KK
Orlinger
Lisa
L
Miorin
Alessandro
A
Marcello
Regina M
RM
Kofler
Christian W
CW
Mandl
D020413:3' Untranslated Regions / Q000235:genetics / Q000494:pharmacology*; D004669:Encephalitis Viruses, Tick-Borne / Q000235:genetics* / Q000502:physiology; D017930:Genes, Reporter / Q000187:drug effects*; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology*; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis / Q000187:drug effects* / Q000502:physiology; D012367:RNA, Viral / Q000494:pharmacology*; D012093:Replicon; D014779:Virus Replication / Q000187:drug effects* / Q000502:physiology
D020413:3' Untranslated Regions; D012367:RNA, Viral