PDF电子书：Antiviral RNAi - Concepts, Methods, and Applications 2011

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) F- T, w4 `) X& D9 K0 Q& pPreface7 L( W5 e8 @/ v1 s/ h: `6 U0 S
Little over a decade ago, Andrew Fire, Craig Mello, and colleagues demonstrated  that
; |) ]( U% \/ u6 U: o, C. Udouble-stranded  (ds)RNA  induces  sequence-specifc  gene  silencing  in  the  nematode 4 p/ I0 l. y, E' ]/ D& V0 _
Caenorhabditis elegans (RNA interference, RNAi). This work converged with research in 1 E2 z- G  R6 G9 a  z
plants, in which related RNA-based silencing processes were known to exist. Ever since,
; c9 M8 C# ?; U4 @" e# A* ^; Tresearch in the feld has progressed at an astonishing rate, resulting in our appreciation of % G, {8 t" k7 C; t9 L. T# |" j' r
small silencing RNAs as central regulators of gene expression, as guards of genome integ-8 |) O/ l! X6 q$ \0 X7 a
rity,  and  as  essential mediators  of  antiviral  defense. The  discovery  that  synthetic  small " f7 T; U. Y+ Z' C( b% O5 B% d8 C
interfering RNA (siRNA) induces gene silencing in mammals, by Thomas Tuschl and col-7 J1 }  |( U; V5 _
leagues in 2001, has further boosted the development of novel therapeutics and experi-
3 j' {& E! o1 w1 ?9 S. R7 k+ xmental tools based on RNAi technology.
5 ^+ [4 R- e8 l( `: D, p  NViruses and RNAi share an intricate relationship at many levels. Early work in plants
$ _9 a# q' l" `7 Iindicated that viruses can be both inducers and targets of RNA-based post-transcriptional 2 o- s' k. w( j( r3 a, v& `. A- j
gene silencing (which we now know as RNAi or RNA silencing). The concept of RNAi as & \- l4 C4 O" ~, J& x( `
an  antiviral  defense mechanism  is  now well-established  in  plants  and  other  organisms, 5 T' m2 D. h/ R- C2 p
including insects. In vertebrates, viruses also interact with a related RNA silencing mecha-
) t" i8 G" e& }+ v" e( `nism, the microRNA (miRNA) pathway. Many nuclear DNA viruses encode their own set
" _1 {' ]8 ?# K8 f" [/ Zof miRNAs, by which they regulate viral or host gene expression and modify, for example,
4 t$ c  |6 T# `1 pthe transition from latent to lytic infection and the recognition of infected cells by the host
3 T- b0 }9 b* U  X. R. |immune system. Furthermore, cellular miRNAs likely regulate expression of many genes
! o) A9 M6 m- _" f5 k' Athat are important for virus biology, but they have been suggested to directly target viral
- [. s0 ^+ I( C& \. ORNA as well.* N8 k% m, @! B) g% j2 J3 ^
The therapeutic potential of RNAi-based antiviral drugs was recognized early on. It is
! _: J. O* P+ f+ G( Y: Unow clear  that  replication of many,  if not all, mammalian viruses can be  suppressed by / T# [, H  n" z6 ?7 y# r
RNAi  in  cell  culture. While  these  results have  raised  considerable optimism  about  the
& g; g( P4 `! U# ~2 R* l8 mpotential of RNAi-based drugs, important hurdles remain, including issues related to the % E/ {7 F6 |5 r& L
delivery and stability of siRNAs and the risk of viral escape.7 m! p  Q' H7 J4 q' Y) p
From this brief overview it will be apparent that a great — and increasing — number
+ _' g/ D9 h4 }  eof  tools and  techniques are available  for  those  interested  in  the  interface of viruses and # g5 P+ O) d9 Y2 }
RNAi. Antiviral RNAi: concepts, methods, and applications provides a collection of proto-
7 D1 z% P& N9 [( b, z7 xcols for the analysis of natural antiviral RNAi responses and viral miRNAs, as well as for
: K& U4 X  f' |( fthe development and optimization of RNAi-based antiviral drugs. As RNAi  is a central - l0 K4 s# X0 T8 K" R! I. R
regulatory mechanism in the cell, the methods in this volume can also be applied out of
# `6 e9 C% `  `' S& g) rthe context of a virus infection. In the established tradition of the Methods in Molecular
& L% f+ R& W3 z1 J5 s6 N) n, _Biology series, Antiviral RNAi: concepts, methods, and applications provides detailed step-
0 P. G% ~  o" q/ Lby-step protocols and extra tools and tricks that should be useful to those new to the feld 6 {0 Z, s* U5 a6 n4 P% A4 M
and experienced scientists alike.
- e) j1 Y3 @6 o) z7 g8 S# [This volume consists of fve parts. Part 1 reviews important basic concepts in the feld + ?: _) |/ `# Z7 o% B( k8 }8 S
of antiviral RNAi. Part 2 provides experimental and bio-informatic tools for the analysis of + q* B' N; }. x( a' y
small silencing RNAs. Part 3 covers methods to biochemically dissect RNAi-based antivi-  N* g; T- A0 {8 z/ f- T
ral defense and viral counter-defense mechanisms. Part 4 describes methods for the design,   b- B8 }$ g: K9 P9 P
expression,  and delivery of  therapeutic  antiviral  siRNAs. Part 5  presents  genome-wide
0 I" a" |$ n% _1 m. ~" J9 s8 FRNAi approaches for the identifcation of factors involved in virus replication, which may
5 Q+ i% k# X. f$ O1 wrepresent novel targets for antiviral therapy.
0 u! b" [, G4 R- zI am grateful to all authors for providing their outstanding contributions and to John
3 t6 S7 a8 P. f' F, lWalker  for guidance while  editing  this  volume.  I  thank members of my  lab,  especially   M' h( m0 G* u7 w, ?9 P" d* C
Walter Bronkhorst, Koen van Cleef, Marius van den Beek, and Joël van Mierlo, for discus-# d/ q9 n7 p7 s: l
sions. I am thankful to Raul Andino for having been a great mentor and for introducing
2 ]+ e9 S/ |5 G! g# ~* u" Q& _4 Ame to this exciting feld of research. Finally, I would like to apologize for doing little jus-
) h# n2 V! @; @0 \tice to the seminal work in plants; space limitations forced me to focus this volume on the
' O" c/ B* t. Nanimal system.9 w0 ~" p$ T) {
Nijmegen, The Netherlands  Ronald P. van Rij

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