Sindbis virus tagged with EGF as a cancer targeting vector

Hongsheng Dai

Table of Contents:
Page
Abstract 2
Research Plan:
A.Specific Aims 3
B.Background and significance 4-6
C.Preliminary Studies N/A
D.Research Design and Methods 7-11
Human Subjects N/A
Vertebrate Animals N/A
Literature Cited 12

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Abstract:
The overall goal of this research is to create Sindbis virus specifically infecting certain cancer cells and
not harming normal tissue cells. We hypothesized that a Sindbis virus displaying EGF on the surface can
specifically recognize those EGFR positive cancer cells and kill them. To reach this aim, firstly, I will
insert EGF at different position in Sindbis E2 glycoprotein, these mutation will help identify tolerable
domain on Sindbis E2 proteins for insertion of EGF tag. Second, optimize Sindbis E2-EGF recombination
virus through in vitro evolution. Third, screen Sindbis virus which specifically infect EGFR+ cells through
RNAi based high-throughput method. And finally validate Sindbis vector targeting to EGFR+ cells. Two
novel methods will be developed: optimizing virus vector via in vitro evolution and high-throughput
screening of receptor-specific viruse. The results of this project will help establish protocols for refining
targeting cancer or infectious disease.

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Research plan
Specific Aims
Gene therapy has been widely used to treat inherited genetic disorders, cardiovascular disease, cancer and
infectious diseases. However, the successful use of gene therapy is impeded by the paucity of highly
efficient cell-specific gene delivery system. All kinds of virus vectors with their intrinsic advantages and
disadvantages have been employed to transiently (Herpes simplex virus, adenovirus, adeo-associated
virus, vaccinia virus) or permanently( Lentivirus and retrovirus) express therapeutic genes. The most
important concern about virus-based gene therapy is the safety of the virus vectors. Therefore, clearly
understanding the host-virus interaction and developing virus vector having well-defined target are
critical to optimize the application of virus vector for treating diseases. In terms of cancer therapy, it
would be ideal to have virus vectors that specifically replicate in tumor cells and do not infect normal
tissue cells. Recent advance in modification of the virus envelop make it possible to built up viruses
which can recognize tumor specific marker and detarget from nature receptor.

Several factors contribute to the sindbis potential utility for cancer gene therapy. First, Sindbis genome is
very simple and full length Sindbis cDNA clone virus has been established for decades. Second Sindbis is
blood-borne virus and suitable for systemic administration. Third, Sindbis replication complex can take
over cellular ribosomes and shut down cellular protein synthesis and thus sindbis infection is highly
apoptotic in mammalian cells, especially cancer cells which are defective in production of interferon.
Other characteristics including efficiency of gene transfer and the growth of the chimeric vector to high
titer also make Sindbis a good oncolytic candidate.

Our long term goal is to create Sindbis virus specifically infecting certain cancer cells and not harming
normal tissue cells. We hypothesized that a Sindbis virus displaying EGF on the surface can specifically
recognize those EGFR positive cancer cells and kill them.

the specific aims of this research are:

1 Identify tolerable domain on Sindbis E2 proteins for insertion of EGF tag, which will facilitate Sindbis
virus specifically infect (cancer) cells expressing EGF receptor;

2 Construct a in vitro evolution library of Sindbis E2-EGF recombination virus;

3 High-throughput screen Sindbis virus with high affinity and specificity to EGFR;

4 Functionally validate Sindbis vector targeting to EGFR+ cells.

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Background and Significance
Sindbis virus, a member of the Alphavirus genus in the Togaviridae family, is an enveloped spherical
positive-sense ssRNA virus. Sindbis genome contains 11703 nucleotides, which is capped at the 5’ end
and has polyA tail at the 3’ end, encoding three structural proteins Capsid(C), E2 and E1 and 4
nonstructural proteins nSP1, nSP 2, nSP 3 and nSP 4. E2 has been established as the receptor binding
protein and E1 has been found to be critical for the membrane fusion. Nonstructural proteins do not
incorporate into virus particles, their primary function is to form replication complex, shut down cellular
macromolecular synthesis and facilitate translation of viral proteins(1).

nSP1 has Guanine-7-methyltransferase and guanylyltransferase activities and it is necessary for mRNA
and genome RNA capping. nSP1’s capping activity is different from cellular capping enzymes in
substrate preference, which is why. nSP2 is composed of about 800 amino acid residues and is the largest
of the replication proteins. Its N-terminal domain of has helicase, nucleoside triphosphatase, and RNA
triphosphatase activities. The C-terminal of nSP2 functions as a proteinase. nsP3 N-terminal is highly
conserved among alphaviruses while C-terminal is rich in Threonine and Serine. Although genetic
analyses indicate that it plays a role in RNA synthesis and neurovirulence, nSP3’s function is yet to be
defined. nSP4 is the RNA-dependent RNA polymerase (RdRP) and it is also the core of the virus
replication complex. The majority of the protein from the C-terminus constitutes the RdRP domain based
on homology with other polymerases and predicted secondary structures. A short region exists at the N-
terminus that lacks a counterpart in other viral polymerases, and it has been suggested that it might be a
binding domain for the other nSPs(2).

Sindbis virus has a very broad host tropism not only in terms of susceptible host species but also in terms
of cell types. Sindbis virus is transmitted via a complex cycle of infection involving an insect vector
(usually mosquito), a mammalian reservoir with incidental dead-end hosts. Within a host, Sindbis are
capable of infecting many different tissues. Sindbis viral RNA has been extracted from blood, brown fat,
cardiac and smooth muscle, and the brain. Sindbis’ entry into cells begins with attachment of E2
glycoprotein with proteinous receptors expressed on cell surface. Two hypotheses are proposed to explain
the broad host range of Sindbis virus. First, the virus E2 glycoprotein may contain multiple receptor-
binding epitopes so E2 can bind different receptors on different susceptible cells through different
receptor-binding sites. The alternative explanation is that Sindbis virus uses a ubiquitous receptor which
is highly conserved across species and expressed on both mammals and mosquitoes. Current research
actually support both hypothesis, and it is likely that a combination of the two is the true(1).

Receptor-bound Sindbis viruses are endocytosed into clathrin coated vesicles. The vesicles are
subsequently acidified, and the resultant acidic pH dissociates the E1-E2 heterodimer. The dissociation of
the proteins results in the exposure of the fusion peptide that is found on the distal tip of E1. The fusion
peptide of E1 inserts into the target membrane followed by the trimerization of E1. A large
conformational change in E1 results in the viral and cell membranes being brought into close opposition.
The formation of E1 trimers promotes membrane deformation and membrane mixing. Finally, a fusion
pore will form as the two membranes complete the process. Following the fusion of the viral and cellular
membranes, the nucleocapsid core is released into the cytoplasm and the viral genome is exposed in
cytoplasm and ready for translation and transcription(2).

Sindbis virus produce three species of RNAs: genome plus strand RNA, complementary minus strand
RNA, and a 26S subgenomic mRNA. The genome RNA serves as mRNA for the synthesis of the
nonstructural proteins. Dependant on whether cellular translation machinery reads through or not the opal
codon between nSP3 and nSP4, nonstructural proteins are synthesized as P1234 or P123 polypeptides.
nSP2 protease posttranslationaly cleaves these polypeptides into individual nSP1, nSP2, nSP3 and nSP4,
which together form the membrane-associated viral replication complex. Complementary minus strand
RNA is the template for replicated genomic RNA. The subgenomic RNA is the template for the

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translation of the structural proteins in the sequence 5′ C-PE2-6K-E1 3′ . The structural polyprotein is
proteolytically cleaved by an autocatalytic activity within the capsid protein, cellular signalase proteins,
and a furin-like protease. Following autoproteolysis, the capsid protein rapidly and efficiently assembly
into a core particle appears to be both rapid and efficient. E3 is released from most alphavirus particles
following cleavage of its PE2 precursor. The E1 and E2 glycoproteins form a heterodimer in the ER, PE2
oligomerizes with a partially folded intermediate of E1 and that this oligomerization is sufficient for the
proteins to exit the ER. The final stage of the virus life cycle is the effective interaction between the
capsid protein and the glycoproteins to promote virus budding(2).

Sindbis virion is T=4 icosahedral structure with a diameter of 70nm. The virion envelop is composed of
two transmembrane glycoproteins E2 and E1, a lipid membrane derived host cells. 240 copies of E1 and
E2 heteodimers form 80 spikes and embed into the lipid bilayer. The outer glycoprotein shell is held
together with the inner core through non covalent interactions of the E2 endodomain with the
nucleocapsid. E2 is a glycoprotein with 423 amino acid residues. The x-ray crystal structure of E2 is yet
to be determined. Cryo-EM reconstruction and biochemistry experiment have shown the first 260 amino
acids of E2 constitute the ectodomain, followed by about 100 amino acids that form the stem region
before entering the lipid bilayer, crossed by a 30 amino acid-long helix. The E2 transmembrane helix
enters the outer lipid leaflet at residue His363 and emerges past the inner phospholipid leaflet at Cys390.
The 33 amino acids in the C terminal form cytoplasmic domain and interact with capsid protein. Deletion
one or more of these residues affect virus assembly(3).

Retroviruses, lentiviruses, adenovirus, adeno-associated viruses and herpes simplex virus (HSV) are viral
vectors traditionally used in gene therapy. Retroviruses and lentiviruses can selectively infect actively
proliferating cells and thus, have been widely used for the gene therapy of tumors. One of the major
drawbacks of retrovirus therapy is in producing sufficient quantities of the virus because of the low titers
of retroviruses. Besides, because of their genome integration properties, potential cell transforming is a
major issue for both retroviruses and lentiviruses. Although adenoviruses can produce high titers
viruses(>1011 pfu/ml) but the strong immune response elicited by the virus is a major concern for
adenovirus-based gene therapy protocols. Adeno-associated viruses is less immunogenic, but their
relatively small size and low yields limit their usefulness. HSV vectors have the largest loading capacity,
However, the potential latency and neurotoxicity of HSV is a significant biosafety issue. (4)

Recently, Sindbis virus has received increasing attention to be a promising viral vectors for gene therapy.
Several factors contribute to the sindbis potential utility for cancer gene therapy. First, Sindbis genome is
very simple and full length Sindbis cDNA clone virus has been established for decades. Sindbis full
length cDNA clone made it very easy to investigate the function of almost each amino acid residues and
each domain in all viral proteins through genomic manipulation. Second Sindbis is transmitted to
mammals by mosquito bites and subsequently spreads throughout the body via the bloodstream. blood-
borne attribute are suitable for systemic administration. Third, Sindbis replication complex can take over
cellular ribosomes and shut down cellular protein synthesis and thus sindbis infection is highly apoptotic
in mammalian cells, especially cancer cells which are defective in production of interferon. Fourth,
Sindbis is a RNA virus, the lacke of DNA intermediate in the whole life cycle exclude the possibility for
Sindbis to integrate into cellular genome and transform cells. Other characteristics including efficiency of
gene transfer and the growth of the chimeric vector to high titer also make Sindbis a good oncolytic
candidate.

However, the obstacle that Sindbis has to overcome to be a successful oncolytic vector lies in its broad
host spectrum. Binding of E2 to many different protein render Sindbis unable to target specific cells. A
range of chemical and genetic engineering strategies have been tested to retarget the cell entry of
enveloped viruses through designated cancer-cell-specific receptors, but certain approaches have been
challenging. In particular, the structural constraints of the icosahedral symmetry of Sindbis viruses often
makes displaying specificity domains incompatible with efficient particle assembly. In addition, the
biological characteristics of ligands and viruses can sometimes be incompatible, making it difficult to

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combine certain ligands with some viruses. Dubuisson and Rice identified a 15 amino acid insertion
between residues 69 and 74 of E2mutations in the SINV glycoproteins that had a block in virus entry at
the level of attachment but was normal in all other respects(5).

For targeting SINV to a cancer specific or receptor, a appropriate targeting ligand needs to be
incorporated into the virus structure at a surface-accessible location. The 53-amino-acid human epidermal
growth factor (EGF) was chosen. This molecule has been successfully used as a targeting domain for a
number of retroviruses, adenoviruses and measles virus. Human EGF is recognized by the epidermal
growth factor receptor (EGFR) and upon receptor binding the EGF-EGFR complex is internalized
through clathrin-mediated endocytosis pathway, which conveys the complex to endosomes for
degradation or reuse. This entry pathway is ideal for SINV, because the entry of SINV into cells is also
dependent on the clathrin-mediated endocytic pathway(Chanakha Navaratnarajah, thesis). The family of
ERBB has four members: ERBB1/EGFR, ERBB2, ERBB3 and ERBB4. EGFR and ERBB2 are
implicated in the development of many types of cancer, and EGFR was the first tyrosine-kinase receptor
to be linked directly to human tumours. It has been shown various alteration of ERBB receptors involved
in human tumours. Gene amplification, constitutive activation and structural rearrangements of EGFR are
often found in human cancers. As such they are excellent candidates for selective anticancer therapies(6).
The goal of this research is to create Sindbis virus specifically infecting certain cancer cells and not
harming normal tissue cells.

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Research Design and Methods
Specific aim 1: Identify tolerable domain on Sindbis E2 proteins for insertion of EGF tag.
In order to design an accurately targeted vector while minimizing effects on infectivity, alternate insertion
sites and strategies should be explored. Chanakha et al has tried to put EGF in the N terminal of E2,
which is surface accessible. This targeted SINV vector showed low levels of enhanced infectivity in cells
expressing the targeted receptor, however it was not able to completely stop SINV interaction with its
native receptor and, furthermore, viral infectivity was adversely affected(Chanakha, unpublished data).
This suggested that the site used for the insertion of EGF is not optimal or EGF insertion is not enough to
abolish Sindbis native receptor binding ability. In this part, we try to explore all possible exposed
subregions on E2 surface tolerable for construction of a recombinant SINV vector expressing the hEGF
targeting moiety.

Figure 1: Surface accessibility of Sindbis E2 glycoprotein(only shown are first 289 amino acids, which
form ectodomain of E2). PredictProtein was used to analysis the secondary structure of E2. pH_sec means
probability' for assigning helix (1=high, 0=low), pE_sec probability' for assigning strand and pL_sec
probability' for assigning neither helix, nor strand. An amino acid residue assigned a higher pL_sec score
correspondingly has higher relative solvent accessibility and thus, this residue is less likely being buried
inside. In other words, amino acids with high pL-sec are more likely exposed on the surface and more
tolerable for inserting peptide fragment.

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Chanakha et al through using transposon mediated random insertion mutation has identified several wild-
type-like mutants and identify regions that accommodate insertions without perturbing virus
production(7). The regions they found can be used to insert EGF moieties to direct SINV to EGF
receptors( Chanakha Navaratnarajah, thesis). To explore other possible insertion sites, PredictProtein was
used to calculate the exposure and surface accessibility of E2(Figure 1)(8). PredictProteins result has
show me 23 possible exposure, after reviewing research papers, ten position were chosen for further
research(Figure 2).

Figure 2: To minimize the disturbance of insertion moiety on E2 folding and traffic, EGF was flanked
with flexible linkers(FL). Beside on cryo-EM result, mAb neutralization experiment and trasposon
mutagenesis library research on E2 and the predicted secondary structure, ten positions 71Q, 89G,106P,
146H,162T, 192P, 205D, 232D, 242D and 274P were selected as potential tolerable insertion sites into
which an EGF tag is inserted.Corresponding GFP and Luciferase reporter constructs are also produced to
facilitate virus detection and quantification.

To characterize the resultant recombinant Sindbis virus. Various assay will be done to see the virus
infectivity, E2 expression and traffic, virus proliferation and virus stability. In order to ensure EGF
insertion does not affect the virus, the plaque phenotype of EGF tagged Sindbis virus will be compared
with wild type Sindbis virus. The plaque size and the plaque morphology of the recombinant virus; A one-
step growth analysis will be performed to investigate the infectivity of Sindbis-EGF and
immunofluorescence assay will be done on BHK cells infected with Sindbis-EGF virus. Especially,
flowcytometry and western blotting will be used to detect the expression of EGF and the surface exposure
and accessibility of this small moiety.

After finishing above experiment, we can find positions is tolerable for insertion of EGF, that means even
with a EGF tag is accessible to cellular receptors and may also interfere with native receptor binding
while causing the least perturbation to the virus. The ideal positions should ensure that the targeting
ligand is accessible to cellular receptors and may also interfere with native receptor binding while causing
the least perturbation to the virus.

Specific aim2: construct a Sindbis E2-EGF library.

Theoretically, a chimeric E2 protein with a specific targeting ligand attached to it should retarget the virus
to a specific receptor. However, this strategy had several disadvantages and it is unlikely that this strategy
will completely ablate native receptor binding, because it has been shown that SINV has multiple
antireceptor sites. Besides, the structural constraints of the icosahedral symmetry of Sindbis viruses often
makes displaying specificity domains incompatible with efficient particle assembly. In addition, the
biological characteristics of ligands and viruses can sometimes be incompatible, making it difficult to

8
combine certain ligands with some viruses. Therefore, once finding out suitable insertion sites for tagging
Sindbis E2, it is needed to find a way to optimize the chimerical E2-EGF protein.

The experimental design of in vitro evolution studies is straightforward: First, random mutations are
introduced in the DNA by in vitro or in vivo techniques. Subsequently, the corresponding genes are
expressed in a microbial host, and clones producing proteins with improved function are isolated by
screening or, in special cases, using a biological selection.
In vitro evolution is a powerful tool for elucidating protein structure function relationships and for
modifying proteins to improve or alter their characteristics. This technology is used to evolve genes in
vitro through an iterative process consisting of recombinant generation. It has led to a number of
remarkable successes in the engineering of proteins with improved function or stability. In vitro evolution
has been used to improve the binding affinity of a number of antibodies. Coupled with the development of
powerful high-throughput screening or selection methods, this technique would definitely solve problems
in viral targeting. There are many methods to generate genetic diversity by random mutagenesis and to
create combinatorial libraries. This can be achieved by treating DNA or whole bacteria with various
chemical mutagens, by passing cloned genes through mutator strains, by "error-prone" PCR mutagenesis,
by rolling circle error-prone PCR, or by saturation mutagenesis. Error prone PCR is a random
mutagenesis technique for generating amino acid substitutions in proteins by introducing mutations into a
gene during PCR. Mutations are deliberately introduced through the use of error prone DNA polymerases
and/or reaction conditions(9).
To make E2-EGF in vitro evolution library, appropriate E2-EGF fusion acquired from step1 will be
amplified by Mutazyme II DNA polymerase(Strategene). Mutation frequency, the product of DNA
polymerase error rate and number of duplication, will be precisely controlled. PCR products with high
mutation ratio(9-16mutation/Kb), middle mutation(4.5-9mutation/Kb) and low mutation (1-
4.5mutation/Kb) will be pooled and cloned into Sindbis full length cDNA construct.
Random mutagenesis allows researchers to identify beneficial mutations in the absence of structural
information, or when such mutations are difficult to predict from protein structure.
Specific aim 3: High-throughput screen Sindbis virus with high affinity and specificity to
EGFR;
To screen the in vitro evolution library, a high-throughput screening platform is needed(Figure 3). RNA
interference (RNAi) is a sequence-specific gene silencing mechanism in most eukaryotic cells and plant
cells, which is triggered by 21–25-nucleotide (nt) small interfering RNA (siRNA). In this process,
siRNAs are incorporated into a multi-protein complex known as the RNA-induced silencing complex
(RISC), which ultimately leads to homologous mRNA degradation. RNA silence has been successfully
used to inhibit Semliki virus, which is also an alpha virus(10). RNA interference (RNAi) gene silencing
can be achieved by delivering vectors that transcribe short hairpin RNA (shRNA), which stably express
small interfering RNA in target cells. Therefore, shRNA is of potential therapeutic use for inhibiting
cancer cells and Virus infection in which aberrant expression of certain mRNA's causes problems.
pLKO.1 is a replication-incompetent lentiviral vector chosen by the TRC for expression of shRNAs.
pLKO.1 can be introduced into cells via direct transfection, or can be converted into lentiviral particles
for subsequent infection of a target cell line. Once transfected, the puromycin resistance marker encoded
in pLKO.1 allows for convenient stable selection(Addgene, protocol). In this project, pLKO vector is
modified to contain two U6 promoter and one H1 promoter, therefore the pLKO plasmid can express
three shRNA targeting different genome sequence of Sindbis virus at the same time. Simultaneously
expression of three shRNA against Sindbis genome is to ensure any Sindbis RNA emerging in the cells
would be degraded and no virus replication occurs.

The virus high-throughput system mainly constitute of two BHK cell lines, one persistently producing
shRNAs which recognize and hydrolysis Sindbis genome, one presenting EGFR on the surface. EGFR+
BHK cells are transfected by in-vitro transcription product of Sind-EGF mutation library. 8h

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posttransfection, EGFR+ BHK are incubated with Sind ShRNA BHK cells but in different compartments
separated by filter membrane. Filter membrane functions to block cell contact and thus stop the spread of
RNA from EGFR+ BHK to Sind shRNA BHK cells. In this system, the size of aperture(0.2um) on the
filter membrane is so large that viruses can freely traffic between two compartments. EGFR+ BHK does
not express shRNA, so Sindbis virus with EGFR specificity can replicate and proliferate; while Sind
ShRNA BHK cells will eliminate all viruses entering into these cells. The virus eliminated by ShRNA
BHK cells are mostly not able to recognize EGFR specifically. Final result is EGFR specific Sindbis
viruses are left in the system.

Figure3: A high-throughput screening system for selecting receptor specific virus. Two BHK cell lines
were established, one persistently producing shRNAs which recognize and hydrolysis Sindbis genome,
one presenting EGFR on the surface. EGFR+ BHK cells are transfected by in-vitro transcription product
of Sind-EGF mutation library. 8h posttransfection, EGFR+ BHK are incubated with Sind ShRNA BHK
cells but in different compartments separated by filter membrane. Filter membrane functions to block cell
contact and thus stop the spread of RNA from EGFR+ BHK to Sind shRNA BHK cells. In this system, the
size of aperture(0.2um) on the filter membrane is so large that viruses can freely traffic between two
compartments. ShRNA BHK cells will eliminate viruses not able to recognize EGFR specifically.

Specific aim 4: Functionally validate Sindbis vector targeting to EGFR+ cells.

Once successfully isolated EGF tagged Sindbis viruses from step three, we need to confirm these viruses
do specifically use the EGF ligand to infect cells. HeLa cells expresse the human EGF receptor and HeLa
cells also express the native SINV receptor(s). In order to perform this assay a monoclonal antibody
(MAb) that can neutralize both the wild-type virus and the hEGF tagged virus is required. Prior to
infection of HeLa monolayers, equal plaque-forming units of Sindbis virus and Sindbis-EGF are
incubated with MAb 202, which blocks Sindbis native receptor. The neutralizing MAb will prevent the
infection of HeLa cells with wild type Sindbis virus, but it should not prevent infection with Sindbis-EGF
if this recombinant virus is in fact able to use the hEGF ligand to infect cells.

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On the other side, EGF should block the infection of Sindbis-EGF but not the wild type Sindbis virus. If
using RNAi to knockdown the expression of EGFR on Hela cells, the infection of the EGFR specific
Sindbis virus will be largely inhibited, while there is no reduction on the infection of wild type.

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