PMID- 10600593
OWN - NLM
STAT- MEDLINE
DCOM- 20000202
LR  - 20220309
IS  - 0042-6822 (Print)
IS  - 0042-6822 (Linking)
VI  - 265
IP  - 2
DP  - 1999 Dec 20
TI  - Structural and functional analysis of the 5' untranslated region of 
      coxsackievirus B3 RNA: In vivo translational and infectivity studies of 
      full-length mutants.
PG  - 206-17
AB  - The lengthy 5' untranslated region (5'UTR) of coxsackievirus B3 (CVB3) forms a 
      highly ordered secondary structure, which plays an important role in controlling 
      viral transcription and translation. Our previous work has delineated the 
      internal ribosome entry site (IRES) by mutation of mono- and bicistronic plasmids 
      containing the 5'UTR and subsequent cell- free translation in rabbit reticular 
      lysate (D. Yang, J. E. Wilson, D. R. Anderson, L. Bohunek, C. Cordeiro, R. 
      Kandolf, and B. M. McManus. (1997). Virology 228, 63-73). To further identify the 
      sequence elements responsible for viral translation and infectivity in tissue 
      culture cells, >30 full-length mutants of CVB3 were constructed by mutations of 
      the IRES and its flanking regions. Viral RNAs were transcribed from these 
      constructs and transfected into HeLa cells. When the stem-loops G and H in the 
      putative IRES were deleted, viral infectivity was abolished and viral protein 
      translation was also undetectable by immunoblot analysis. However, when 
      stem-loops A and B were deleted or stem-loop E was partially deleted, viral 
      protein translation could be detected although cytopathic effect could not be 
      observed. The data suggest that the crucial sequence of the IRES is located at 
      stem-loops G and H. Further serial deletion mapping up and down stream of the 
      crucial sequence defined more accurately the 5' and 3' boundaries of the IRES, 
      located at nucleotides (nts) 309-432 and 639-670, respectively. These results 
      indicate that the core sequence of the IRES should be located at nts 432-639. 
      This IRES segment is much shorter and located closer to the initiation codon than 
      that of poliovirus. To further define critical nucleotides within the IRES core, 
      site-directed mutagenesis was conducted at the IRES core sequence by PCR. A 46-nt 
      deletion in the pyrimidine-rich tract of stem-loop G abolished viral translation 
      and infectivity. Interestingly, five single-nt substitutions in the 
      pyrimidine-rich tract aimed at destabilizing the base pairing between the viral 
      IRES and host 18S rRNA did not abolish CVB3 infectivity although viral protein 
      translation was significantly reduced. This finding suggests that ribosomal 
      internal initiation of translation and viral infectivity not only may require RNA 
      secondary structure but also may need tertiary structure and perhaps the 
      assistance of host protein factors.
CI  - Copyright 1999 Academic Press.
FAU - Liu, Z
AU  - Liu Z
AD  - Department of Pathology and Laboratory Medicine, University of British Columbia, 
      Vancouver, British Columbia, V6Z 1Y6, Canada.
FAU - Carthy, C M
AU  - Carthy CM
FAU - Cheung, P
AU  - Cheung P
FAU - Bohunek, L
AU  - Bohunek L
FAU - Wilson, J E
AU  - Wilson JE
FAU - McManus, B M
AU  - McManus BM
FAU - Yang, D
AU  - Yang D
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Virology
JT  - Virology
JID - 0110674
RN  - 0 (5' Untranslated Regions)
RN  - 0 (Pyrimidines)
RN  - 0 (RNA, Viral)
RN  - K8CXK5Q32L (pyrimidine)
SB  - IM
MH  - 5' Untranslated Regions/*chemistry/physiology
MH  - Binding Sites
MH  - Chromosome Mapping
MH  - Enterovirus B, Human/*genetics/pathogenicity
MH  - HeLa Cells
MH  - Humans
MH  - Mutagenesis, Site-Directed
MH  - *Nucleic Acid Conformation
MH  - *Protein Biosynthesis
MH  - Pyrimidines
MH  - RNA, Viral/*chemistry/physiology
MH  - Ribosomes/metabolism
MH  - Structure-Activity Relationship
EDAT- 1999/12/22 00:00
MHDA- 1999/12/22 00:01
CRDT- 1999/12/22 00:00
PHST- 1999/12/22 00:00 [pubmed]
PHST- 1999/12/22 00:01 [medline]
PHST- 1999/12/22 00:00 [entrez]
AID - S0042-6822(99)90048-X [pii]
AID - 10.1006/viro.1999.0048 [doi]
PST - ppublish
SO  - Virology. 1999 Dec 20;265(2):206-17. doi: 10.1006/viro.1999.0048.