Broad-Spectrum Antiviral Therapeutics

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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

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http://bit.ly/qSzlRj

PLoS one

Research Article

Broad-Spectrum Antiviral Therapeutics

Todd H. Rider*, E. Zook, Tara L. Boettcher,

T. Wick, S. Pancoast, 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]–[5].

In contrast, uninfected mammalian cells generally do not

produce long dsRNA (greater than ~21–23 base pairs)

[4]–[5].

Natural cellular defenses exploit this difference in order to

detect and to attempt to counter viral infections [6]–[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]–[9].

Binding of multiple PKR proteins to dsRNA with a length of

at least 30–50 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 (2–5A) synthetases [10], RNase L

(activated via dimerization by 2–5A produced by 2–5A

synthetases in response to dsRNA [11]), TLR 3 [12],

interferon-inducible ADAR1 [13], and RIG-I and Mda-5

[6]–[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]–[16] or FLICE-activated death domain

(FADD) [17]–[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]–[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]–[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]–[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]–[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.