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>>>>> Help ME Circle <<<<
>>>> 13 August 2011 <<<<
Editorship : j.van.roijen@chello.nl
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Thanks to "Kevin" <maitrikaruna@yahoo.com>
Below you will find the abstract and introduction of a very
interesting paper: *Broad-Spectrum Antiviral
Therapeutics*.
The full text can be found at: http://bit.ly/qSzlRj
For private members the full pdf file is attached, but can
also be found at: http://bit.ly/nd5wgK
~jvr
````
PLoS one
Research Article
Broad-Spectrum Antiviral Therapeutics
Todd H. Rider*, Christina E. Zook, Tara L. Boettcher,
Scott T. Wick, Jennifer S. Pancoast, Benjamin D. Zusman
Lincoln Laboratory, Massachusetts Institute of
Technology, Lexington, Massachusetts, United States of
America
Abstract
Currently there are relatively few antiviral therapeutics,
and most which do exist are highly pathogen-specific or
have other disadvantages.
We have developed a new broad-spectrum antiviral
approach, dubbed Double-stranded RNA
(dsRNA)Activated Caspase Oligomerizer (DRACO) that
selectively induces apoptosis in cells containing viral
dsRNA, rapidly killing infected cells without harming
uninfected cells.
We have created DRACOs and shown that they are
nontoxic in 11 mammalian cell types and effective against
15 different viruses, including dengue flavivirus, Amapari
and Tacaribe arenaviruses, Guama bunyavirus, and H1N1
influenza.
We have also demonstrated that DRACOs can rescue mice
challenged with H1N1 influenza.
DRACOs have the potential to be effective therapeutics or
prophylactics for numerous clinical and priority viruses,
due to the broad-spectrum sensitivity of the dsRNA
detection domain, the potent activity of the apoptosis
induction domain, and the novel direct linkage between
the two which viruses have never encountered.
Introduction
A serious threat is posed by viral pathogens, including
clinical viruses (HIV, hepatitis viruses, etc.), natural
emerging viruses (avian and swine influenza strains,
SARS, etc.), and viruses relevant to potential
bioterrorism (Ebola, smallpox, etc.).
Unfortunately, there are relatively few prophylactics or
therapeutics for these viruses, and most which do exist
can be divided into three broad categories]:
(1) Specific inhibitors of a virus-associated target (e.g.,
HIV protease inhibitors, RNAi) generally must be
developed for each virus or viral strain, are prone to
resistance if a virus mutates the drug target, are not
immediately available for emerging or engineered viral
threats, and can have unforeseen adverse effects.
(2) Vaccines also require a new vaccine to be developed
for each virus or viral strain, must be administered before
or in some cases soon after exposure to be effective, are
not immediately available for emerging or engineered viral
threats, can have unforeseen adverse effects, and are
difficult to produce for certain pathogens (e.g., HIV).
(3) Interferons and other pro- or anti-inflammatories are
less virus-specific, but still are only useful against certain
viruses, and they can have serious adverse effects
through their interactions with the immune and endocrine
systems.
To overcome these shortcomings of existing approaches,
we have developed and demonstrated a novel antiviral
approach that is effective against a very broad spectrum
of viruses, nontoxic in vitro andin vivo, and potentially
suitable for either prophylactic or therapeutic
administration.
Our approach, which we call a Double-stranded RNA
(dsRNA) Activated Caspase Oligomerizer (DRACO), is
designed to selectively and rapidly kill virus-infected cells
while not harming uninfected cells.
Our DRACO approach combines two natural cellular
processes.
The first process involves dsRNA detection in the
interferon pathway.
Most viruses have double- or single-stranded RNA (ssRNA)
genomes and produce long dsRNA helices during
transcription and replication; the remainder of viruses
have DNA genomes and typically produce long dsRNA via
symmetrical transcription [4]=96[5].
In contrast, uninfected mammalian cells generally do not
produce long dsRNA (greater than ~21=9623 base pairs)
[4]=96[5].
Natural cellular defenses exploit this difference in order to
detect and to attempt to counter viral infections [6]=96[7].
For example, protein kinase R (PKR) contains an N-terminal
domain with two dsRNA binding motifs (dsRBM 1 and 2)
and a C-terminal kinase domain [8]=96[9].
Binding of multiple PKR proteins to dsRNA with a length of
at least 30=9650 base pairs [5] activates the PKRs via
trans-autophosphorylation; activated PKR then
phosphorylates eIF-2 , thereby inhibiting translation of
viral (and cellular) proteins.
Other examples of proteins that detect viral dsRNA include
2 ,5 -oligoadenylate (2=965A) synthetases [10], RNase L
(activated via dimerization by 2=965A produced by 2=965A
synthetases in response to dsRNA [11]), TLR 3 [12],
interferon-inducible ADAR1 [13], and RIG-I and Mda-5
[6]=96[7].
The second natural process used by our approach is one
of the last steps in the apoptosis pathway[14], in which
complexes containing intracellular apoptosis signaling
molecules, such as apoptotic protease activating factor 1
(Apaf-1) [15]=96[16] or FLICE-activated death domain
(FADD) [17]=96[18], simultaneously bind multiple
procaspases.
The procaspases transactivate via cleavage, activate
additional caspases in the cascade, and cleave a variety
of cellular proteins [14], thereby killing the cell.
Many viruses attempt to counter these defenses.
A wide variety of viruses target dsRNA-induced signaling
proteins, including IPS-1, interferon response factors
(IRFs), interferons and interferon receptors, JAK/STAT
proteins, and eIF-2 [19]=96[20].
Some viral products attempt to sequester dsRNA (e.g.,
poxvirus E3L [21]) or to directly interfere with cellular
dsRNA binding domains (e.g., HIV TAR RNA[19]=96[20]).
Virtually all viruses that inhibit apoptosis do so by
targeting early steps in the pathway, for example by
inhibiting p53, mimicking anti-apoptotic Bcl-2, or
interfering with death receptor signaling[22]=96[23].
Among the few viral proteins that directly inhibit one or
more caspases are African swine fever virus A224L
(which inhibits caspase 3) [24], poxvirus CrmA (which
inhibits caspases 1, 8, and 10 but not others) [25], and
baculovirus p35 (which inhibits several caspases but is
relatively ineffective against caspase 9) [25].
Because PKR activation and caspase activation function in
similar ways and involve proteins that have separate
domains with well-defined functions, these two processes
can be combined to circumvent most viral blockades
[26]=96[27].
In its simplest form, a DRACO is a chimeric protein with
one domain that binds to viral dsRNA and a second
domain (e.g., a procaspase-binding domain or a
procaspase) that induces apoptosis when two or more
DRACOs crosslink on the same dsRNA.
If viral dsRNA is present inside a cell, DRACOs will bind to
the dsRNA and induce apoptosis of that cell. If viral
dsRNA is not present inside the cell, DRACOs will not
crosslink and apoptosis will not occur.
For delivery into cells in vitro or in vivo, DRACOs can be
fused with proven protein transduction tags, including a
sequence from the HIV TAT protein [28], the related
protein transduction domain 4 (PTD)[29], and
polyarginine (ARG) [30].
These tags have been shown to carry large cargo
molecules into both the cytoplasm and the nucleus of all
cell types in vitro and in vivo, even across the
blood-brain barrier.
~~~~=20
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