Cloning and expression of the Nodamura virus RNA-dependent RNA polymerase Poster presentation at Society for the Advancement of Chicanos and Native Americans in Science (SACNAS) National Conference, October 2012, Seatltle, WA

1. Cloning and expression of the
Nodamura virus RNA-dependent RNA
polymerase
Debarko Banerji1, Alexandra Navarro1,
Vincent U. Gant, Jr.1, and Kyle L.
Johnson1,2
1Department of Biological Sciences and 2Border
Biomedical Research Center, The University of Texas at
El Paso, El Paso, TX 79968
www.linkedin.com/pub/debarko-banerji/7/799/80a/

2. Nodamura virus (NoV) is the type species of the Alphanodavirus Genus of the Family Nodaviridae, which
also includes Flock House virus (FHV). These are small icosahedral viruses with bipartite, positive-strand
RNA genomes. Nodaviruses can replicate their genomes in a wide variety of cells, including baby hamster
kidney (BHK21) cells and cells of the yeast Saccharomyces cerevisiae, which makes them an attractive
model system for RNA replication studies. NoV is unique among the nodaviruses in that it can infect both
insects and mammals, resulting in hind segment/limb paralysis and death (1). Genome replication and
capsid functions are divided onto separate genome segments: RNA 1 and RNA 2, respectively. NoV RNA 1
encodes Protein A (hereafter referred to as NA), a 110 kDa RNA-dependent RNA polymerase (RdRp) that is
essential for infectivity. The NoV and FHV RdRps, which share only 44% sequence identity (2), show unique
template specificity in that the two RdRps do not replicate one another’s RNA templates.
Currently, the mechanism of initiation of NA RNA synthesis is unknown, i.e. we do not know how NA
recognizes the template RNA. NA has yet to be purified to homogeneity, however preliminary data from
our lab has shown that NA can be purified using affinity chromatography. Purified NA will allow us to
conduct in vitro RNA replication studies (3,4,5) as well as elucidate the 3D structure. We hypothesize that
membrane targeted expression of NA using the SRP-SecY pathway in E. coli will yield membrane-soluble
RdRp that can then be purified to homogeneity to carry out further studies (6). In this project we are
developing a recombinant DNA based protocol for the overexpression and purification of NA. from E. coli.
INTRODUCTION

3. The purpose of this project is to develop a
reliable protocol for the overexpression and
purification of recombinant NA (NoV RdRp) to
be used for downstream studies of NA
enzymatic activity.
PURPOSE

4. Our laboratory studies the mechanism of viral RNA replication, using Nodamura virus
(NoV) as a model, due to its genetic simplicity, tremendous levels of RNA amplification,
and ability to replicate in a wide variety of host cells. NoV contains a bipartite positive-
strand RNA genome. The larger segment (RNA1) encodes the RNA-dependent RNA
polymerase (RdRp) that catalyzes replication of the viral genome, while RNA2, encodes
the viral capsid protein precursor, which is dispensable for RdRp function.
We are developing an in vitro replication system to study the specific mechanism by
which the viral RdRp recognizes its RNA template. As a first step, we will express
recombinant RdRp with an N-terminal 6-HIS tag in E. coli from an IPTG-inducible T7
promoter. We used PCR to introduce an NcoI site, a start codon, and a 6-HIS tag to the
5’-end of the RdRp ORF and to introduce a 3’-terminal HindIII site after the RdRp stop
codon. The PCR products were subcloned into pGEM-T-Easy vectors and confirmed by
DNA sequencing. The 5’ and 3’ terminal fragments isolated from these subclones will be
ligated to an internal fragment containing the majority of the RdRp ORF and introduced
into the pET45b(+) vector. The vector will be used to transform competent T7-EXPRESS
E. coli cells and pilot studies will be performed to determine the optimal expression
conditions. The expressed protein will be purified by affinity chromatography and
assayed for RdRp activity. The purified protein will allow us to determine the replication
initiation mechanism used by this enzyme.
ABSTRACT

5. Construction of pET45b(+)-SRP-NA expression plasmid.
5' and 3' Insert Fragments. The 5' and 3' fragments of the insert sequence of DNA were PCR-amplified from
parental plasmid pTXB1-SRP-NoV1, which contains the gene for recombinant NA as follows: N-terminal E. coli
SRP signal (required to target protein synthesis to the membrane) followed by a Factor X cleavage site. In
amplifying the 5' fragment, primers were designed so as to introduce on the 5' end of the recombinant gene, a
BbsI restriction site which upon digestion would leave an NcoI overhang (BbsI is a remote cutter) to allow
ligation into the destination vector, followed by an AUG start codon, followed by the sequence for a 6X
histidine tag (to enable purification via Ni2+-NTA affinity chromatography) (3). In amplifying the 3' fragment,
primers were designed so as to introduce a HindIII restriction site on the 3' end to allow ligation into the
destination vector. To ensure that these modifications were successful, the 5' and 3' fragments were ligated
into the pGEM-T-Easy “hotel” vector and sequence-confirmed.
Subsequently, the fragments were digested out. The NA5' fragment was generated by digesting with BbsI to
leave the designed NcoI overhang on the 5' end and a non-specific sticky end on the 3' end. The NA3' fragment
was generated by digesting with BamHI leaving a 5' BamHI sticky end, PCR-purifying and then digesting with
HindIII to leave a 3' HindIII sticky end. The 348 bp NA5’ fragment and 306 bp NA3’ fragment were gel-isolated
and purified.
Internal Insert Fragment. The internal fragment was generated by digesting parental plasmid pT7R-NoV1,
which contains the NA cDNA sequence, with BbsI to leave a 5' non-specific sticky end that is complementary to
the NA5' fragment's 3' sticky end, PCR-purifying and then digesting with BamHI to leave a 3' BamHI sticky end.
These ends would allow the internal fragment's ligation with the NA5' and NA3' fragments. The 2583 bp
fragment was gel-isolated and purified.
MATERIALS & METHODS

6. Vector Fragments. The expression vector, pET45b(+) was chosen as the destination plasmid. It drives transcription of a
gene-of-interest under the control of a T7 promoter. It contains an ampicillin resistance gene (AmpR), allowing for
propagation in selective ampicillin media. pET45b(+) was first linearized via PvuI digestion, which cuts in the middle of
AmpR. After PCR-purification, digestion with NcoI and HindIII, both of which cut in the Multiple Cloning Site (MCS),
liberated two fragments of sizes 3933 bp and 1217 bp respectively. The vector fragments were gel-isolated and purified.
The rationale for cutting in AmpR is that it acts to facilitate positive clone selection. Only those cells containing properly
ligated plasmid with functioning AmpR will be ampicillin-resistant and grow on selective ampicillin plates.
Plasmid Construction and Propagation. All five fragments were then resolved on a 1.2% TBE agarose gel and analyzed to
confirm correct size, and to ascertain purity and adequate quantity. The fragments were ligated overnight with T4 DNA
ligase (Invitrogen/Life Technologies, Carlsbad, CA) in the presence of T4 DNA ligase buffer (Invitrogen/Life Technologies),
after which the enzyme was heat-inactivated at 60°C for 20 minutes.
Transformation and Cell Culture. The final construct was propagated in NEB 10-β E. coli cells (New England Biolabs,
Ipswich, MA). Cells were transformed according to manufacturer's protocol. Transformed cells were amplified in 1mL
SOC media (New England Biolabs) at 37°C for 1 hour, then plated on 2X Yeast Extract/Tryptone (2X YT) plates containing
ampicilin (10g/L Yeast Extract, 16g/L Tryptone, 5g/L NaCl [pH 7.4], 15g/L Agar, 100mg/L Ampicillin). Plates were
incubated at 37°C overnight. Ampicillin-resistant colonies were amplified in 2mL 2X YT at 37°C overnight, small-scale
plasmid preparations were performed (QIAprep Spin Miniprep Kit, Qiagen, Hilden, Germany) and positive clones were
screened by restriction profiling.
DNA Gel Electrophoresis and Analysis. Running Buffer: Tris Borate EDTA (TBE) (12.1g/L Tris base, 5.1g/L Boric acid,
0.37g/L Disodium EDTA [pH 8.3]), 1.2% (wt/vol) TBE agarose gel: 50mL TBE, 0.6g agarose, 25μg Ethidium Bromide
(BioRad, Hercules, CA). Gels were visualized using Gel Doc XR (BioRad) automated UV transilluminator.
Digests and DNA Purification. All restriction digests were set up using enzymes and appropriate buffers from New
England Biolabs according to manufacturer’s protocols. PCR Purification and Gel Purification Kits by Qiagen.
MATERIALS & METHODS (Cont.)

7. RESULTS
Fig. 1: pET45b(+)-SRP-NA construction strategy. All new elements (restriction sites at 5’ and 3’
ends to facilitate ligation into destination vector, start codon, and 6X His tag) were introduced
into the NA ORF at the primer design stage. Ligation into pGTE subclone vectors facilitated
sequence confirmation of the newly modified NA5’ and NA3’ fragments.
Purpose of each element: The SRP (Signal Recognition Particle) element will target protein
synthesis at the E. coli cell membrane allowing us to obtain soluble protein using membrane-
solubilization methods. The crucial introduction of the 6X His tag on the 5’ end of the SRP-NA
ORF will allow for Ni2+-NTA affinity purification of the recombinant protein. Cleavage with Factor
X at the advantageously positioned Factor X Cleavage Site (Fac X) will remove NA from the
bound His-tag yielding pure wild-type NA.
Note on the generation of vector fragments: The destination vector was first cut in the middle
of the ampicillin resistance gene (AmpR) with PvuI and then with NcoI and HindIII to yield the
two vector fragments. This cut in the AmpR gene acts as a measure to further facilitate the
selection of positive clones; only those cells that are transformed with properly ligated plasmid,
containing a functioning AmpR gene, will grow on selective ampicillin plates.

10. Fig. 2: TBE agarose gel electrophoresis of
DNA fragments. Cloned DNA fragments
for pET45b(+)-SRP-NA construction were
analyzed for size, purity and quantity on a
1.2% TBE agarose gel. Lanes:
1. 1 kbp ladder
2. pET45b(+) [PvuI-NcoI] vector fragment
(3933 bp)
3. pET45b(+) [PvuI-HindIII] vector
fragment (1217 bp)
4. pT7R-Nov1 [BbsI-BamHI] NA internal
fragment (2583 bp)
5. 100 bp ladder
6. pGTE-NA5’ [BbsI(NcoI)-BbsI] NA5’
fragment (348 bp)
7. pGTE-NA3’ [BamHI-HindIII] NA3’
fragment (306 bp).
Gel was visualized on a Gel Doc XR
(BioRad). All fragments are of desired
size, purity and quantity.
1 2 3 4 5 6 7

11. Fig. 3: Final construct
pET45b(+)-SRP-NA.
Restriction sites used
to ligate insert
fragments into vector
are shown. Ampicillin
resistance gene
(AmpR) enables
propagation of
positive clones on
selective media.
Transcription of
recombinant SRP-NA
is under control of T7
promoter. Also, origin
of replication (Ori) is
shown.
pET-45b(+)-SRP-NA
(8380 bp)
BbsI
BamHI
HindIII
NcoI
AmpR
Ori
T7 Promoter
6xHis-SRP-FacX-
Nov RdRp

12. Endogenou
s Inducible
Plasmid
pET45b(+)
-SRP-NA
T7 RNA
Pol
IPTG
RN
A
SRP X NA6X
His
Factor X
Cleavage
Site
pET45b(+)
-SRP-NA
Transform
Cell Membrane
T7 EXPRESS E. coli
(NEB)
FUTURE WORK

13. Fig. 4: Protein Induction. T7 EXPRESS E. coli will be transformed with the
newly constructed plasmid, pET45b(+)-SRP-NA and grown in culture to an
optimal optical density. The cells, which carry endogenous plasmid
encoding T7 polymerase under regulation of the lac operon, will be
induced with isopropyl-β-D-thio-galactoside (IPTG) to synthesize high
levels of the T7 polymerase. This will drive transcription of the
recombinant SRP-NA, which is under control of a T7 promoter.
Synthesized protein will be targeted to the cell membrane via the SRP-
SecY pathway due to the presence of the N-terminal SRP signal. Protein
will then be harvrested.
FUTURE WORK

14. We will transform competent T7 EXPRESS E. coli (New England BioLabs) cells
with the newly cloned pET45b(+)-SRP-NA. We will use individual colonies to
inoculate 2X YT/Ampicillin cultures and allow growth until the optimal optical
density for protein induction. We will induce expression of the recombinant
protein with IPTG (3). The SRP tag will target protein expression to the cell
membrane (6). Subsequently we will follow a lysis procedure from which we will
harvest the membrane fraction via ultracentrifugation. The membranes will be
solubilized using detergents to release the recombinant protein in soluble form.
The solubilized membrane proteins will be loaded onto a Ni2+-NTA column and
purified via affinity chromatography. The 6X His tag will enable binding onto the
column. After several washes, on-column cleavage of the NA protein will be
performed with FacX to obtain purified protein (3).
The purified NA will then be used to conduct in vitro studies on the replication
initiation mechanism of the NoV RNA-dependent RNA-polymerase (3,4,5). It will
also be used to obtain a crystal structure.

15. REFERENCES
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5. Sun J.H., Adkins S., Faurote G., Kao C.C. (1996). Initiation of (–)-Strand RNA
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16. We thank the UTEP BBRC DNA Analysis Core Facility. This
project was supported by grant number 5G12RR008124 from
the National Center for Research Resources (NCRR), a
component of the National Institutes of Health (NIH). We
would especially like to thank Ana Betancourt for DNA
sequencing.
ACKNOWLEDGEMENTS
Cloning and expression of the Nodamura virus RNA-
dependent RNA polymerase
Poster presentation at Society for the Advancement of
Chicanos and Native Americans in Science (SACNAS)
National Conference, October 2012, Seatltle, WA