EPICENTRE Forum
Peter Nagy
University of Kentucky
Peter Nagy’s lab at the University of Kentucky uses defective
interfering (DI) RNAs to study regulatory elements involved in the
replication of the positive-strand RNA viral genome of tomato
bushy stunt virus (TBSV). DI RNAs are naturally occurring deletion mutants of a parent virus. These modified RNA genomes
usually have deletions in the viral replicase gene and cannot
replicate without the parent virus acting as a helper virus. DI
RNAs retain viral replication signals, but are significantly smaller than the parent genome, so they replicate more efficiently and
provide good model systems for studying viral replication.
Genome replication of plus-strand RNA viruses requires the production of some negative-strand RNA to provide the template for
the plus strand. However, viral replication produces about 100fold less negative-strand RNA compared to positive-strand
genomic RNA. Pogany et al.1 designed experiments to study an
RNA-RNA interaction at the 3’-end of the plus-strand RNA for its
role in down regulating the replication of negative-strand RNA.
Hiroshi Takaku
Chiba Institute of Technology
Hiroshi Takaku’s lab at the Chiba Institute of Technology in
Japan is investigating the use of RNA interference (RNAi) as a
potential therapy against HIV-1. RNAi uses double-stranded
RNA (dsRNA) to target the elimination of specific mRNAs in the
cell. Short dsRNAs (21 to 23 nucleotides) combine with a protein complex to form RNA-induced silencing complexes
(RISCs), which recognize and degrade the targeted mRNA.
To determine how effectively RNAi can inhibit HIV-1 gene
expression, Park et al.2 designed four short dsRNAs against different, central regions of the HIV-1 env gene. To produce the
dsRNAs, they used double-stranded oligonulcleotide DNA templates that contained T7 promoter sequences. Individual singlestranded RNAs were transcribed from each DNA template in
vitro using an AmpliScribe™ T7 High Yield Transcription Kit.
Resulting sense and antisense RNA molecules were annealed to
form dsRNAs, or left single stranded, and cotransfected with an
HIV-1 proviral expression vector into COS, HeLa-CD+, and
peripheral blood mononuclear cells (natural target cells for HIV
infection). The production of HIV-1 virus was determined by
assaying for the viral p24 Gag protein. Two of the four dsRNAs
very effectively inhibited HIV-1 gene expression, as well as viral
replication, and were significantly more effective than the single-stranded antisense RNAs.
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After extensive in vitro experiments studying and characterizing
this novel cis-acting “replication silencer”, they tested DI RNA
replication silencer mutants and compensatory mutants in vivo
in cucumber protoplasts. An AmpliScribe™ T7 High Yield
Transcription Kit was used to prepare RNA transcripts of the
mutants to inoculate the protoplasts, which were co-infected
with full-length TBSV helper virus. Results showed that in vivo
the mutant replication silencers significantly reduced all replication activity, while compensatory mutants restored some of
that activity, indicating that the cis-acting replication silencer
plays an important role in viral replication.
For more information on this work by Pogany et al.,
see reference1.
For information on Peter Nagy’s lab go to:
www.ca.uky.edu/agcollege/plantpathology/Nagy/Nagy.html.
For more information on AmpliScribe™ T7 or T7-Flash™
Transcription Kits go to:
www.epicentre.com/ampliscribe.asp or
www.epicentre.com/flash.asp.
Park et al. suggest that RNAi could prove to be an effective gene
therapy method that would counteract the common problem of
drug-resistant viruses. They further suggest that T7 in vitro transcription could provide a simple and economical method to
produce therapeutic quantities of dsRNA for RNAi.
For more information on this work by Park et al., see reference.
For more information on AmpliScribe™ T7 or T7-Flash™
Transcription Kits go to:
www.epicentre.com/ampliscribe.asp or
www.epicentre.com/flash.asp.
References
1. Pogany, J. et al. (2003) EMBO J. 22(20), 5602.
2. Park, W-S. et al. (2003) Gene Ther. 10, 2046.
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