1. Alexander Ollerton
Purification of the NS1 nuclease domain of the Human Parvovirus B19
Horton Lab
Research Paper

2. Abstract
The human parvovirus B19 replicates in the human erythroid progenitor
cells. This causes a variety of unsettling diseases, which vary from a rash to death.
The B19 parvovirus genome encodes five proteins: two capsid proteins, two
unknown proteins and one NS1 protein. The NS1 protein assists with the replication
and cleavage of the viral DNA. Figuring out the functionality of the NS1 protein will
allow us to find the process of DNA cleavage and replication for the B19 viral
genome. Through a series of biochemical experiments we were able to find the
optimal condition for the cleavage and removal of the recombinant tags from the
nuclease domain via TEV-protease. Because of these biochemical experiments we
were able to get a pure protein and find out that the nuclease domain of the protein
is functional. Throughout this process the nuclease domain precipitated; the use of
10% glycerol aided in the solubilization of the nuclease domain. The nuclease
domain was successfully purified and functional DNA cleavage reactions revealed
specific and non-specific ssDNA cleavage of oligonucleotides Ori-Top 20, 24, and 67.
Introduction
Cossart et al. discovered the human parvovirus B19 in 1974 through a serum
sample in the 19th column of panel B (1). The B19 parvovirus was the only member
of the Parvoviridae known to be pathogenic to humans until the recent discovery of
human bocavirus and the human parvovirus 4(1, 2). The virus is highly
erythrotropic, which means that it replicates in human erythroid progenitor cells
(4).
The parvovirus B19 is widespread and when manifested in a healthy child,
the child can develop a rash illness and even erythema infectiosum (1).
The parvovirus B19 is also known to infect adults and can be fetal to pregnant
women causing fetal death in utero or hydrops fetalis (1). Figure 1 demonstrates the
schematic life cycle of the parvovirus B19 where the virus attaches itself to the cell
(1) and creates an entryway into the cell in order for replication to occur (8). NS1 is
a necessary component responsible for viral genomic replication.
Figure 1: Schematic structure of the parvovirus B19
The virus is categorized as a parvovirus due to the fact that it is among one of
the smallest viruses to contain a single-stranded DNA that is able to infect mammals
(1). The parvovirus B19 has genome of 5,596 nucleotides where the terminal

3. sequences are palindromic (1). This palindromic structure is important because it
acts as a duplex configuration, which serves as primers for the synthesis of the
complementary strand (1). The virus’s genome is relatively simple in structure and
is composed of two large open reading frames with several proteins (1).
Since the genome is coded of five proteins and a single-stranded DNA, it is
not a very complex genome (1). Two of the proteins are capsid proteins (VP1 and
VP2) and one is a nonstructural (NS1) protein; the other two proteins are 7.5 kDa
and 11 kDa with unknown functions within the parvovirus (1, 4). The capsid
proteins form icosahedral structure, which have sixty capsomers (figure 2).
The VP2 is the larger of the capsid proteins and is about 96% of total of the
two-capsid proteins (1). VP2 can self-assemble even when there is no viral DNA and
can produce viral-like particles (1). VP1 is similar in structure of VP2 with a
difference of 227 amino acids (1).
The NS1 protein is a major nonstructural protein and may have site-specific
DNA-binding, ATPase and helicase activity (1). The NS1 protein is essential for the
replication of the viral DNA and also plays a role for the virus’s gene expression (3).
It is known that the NS1 protein aids in gene expression directly for the viral genes
(1). The NS1 protein functions through two methods: 1) it assembles the cellular
location for replication and 2) it introduces a strand-specific and site-specific nick at
the terminal site allowing a 3’ to replicate (4). The helicase activity of the NS1 assists
in the unfolding of the double-stranded regions (palindromic regions) through ATP
hydrolysis (5). In order to focus on the strand and site-specific nick at the terminal
site by NS1, we are focusing on the nuclease domain. With the nuclease domain, we
are trying to determine its binding locations as well as its ability to cleave viral DNA.
This will be studied through a series of biochemical experiments where the nuclease
domain will be combined with oligonucleotide sequences that represent the DNA of
the B19 virus. The purpose of focusing on this domain is to see how it affects DNA
and how it functions in the overall B19 parvovirus.
Figure 2: Viral Particles of the Human Parvovirus B19

4. Methods
Inoculation and growing cells in Escherichia Coli (E. coli) BL21 (DE3)
A single colony of E. coli BL21(DE3) cells harboring the nuclease domain of the NS1
protein in the pMAL-c4e vector were inoculated in a 5mL culture tubes with
50ug/mL of ampicillin and Luria Broth Media and left in a 37℃ incubator overnight
while shaking at 250 RPM. The cells are induced with 0.5 mM of Isopropyl β-D-1-
thiogalactopyranoside when an optical density of 0.6 is reached. The cells are grown
out overnight at 37℃ and harvested through centrifugation at 5000 rpm at 4℃.
Purification
The cells are re-suspended in a lysis buffer (50mM NaH2PO4, 800mM NaCl, 1mM
Beta-Mercaptoethanol pH 8.0). A sonicator is used to lyse the cells until a constant
level of absorption at 260nm is observed. The lysate is centrifuged at 4℃ at 15000
RPM for fifty minutes. Once finished the supernatant is placed in talon resin at 4℃ at
120 RPM for forty minutes. After this the solution is poured in 10mL syringes, that
have filters at the bottom, and the solution is washed with a wash buffer (50mM
NaH2PO4, 300mM NaCl, 1mM Beta-Mercaptoethanol pH 8.0), a high salt buffer
(50mM NaH2PO4, 2M NaCl, 1mM Beta-Mercaptoethanol pH 8.0), and an elution
buffer (50mM NaH2PO4, 300mM NaCl, 250mM imidazole, 1mM Beta
Mercaptoethanol pH 8.0). Once finished the elution buffer is placed in a dialysis bag
and left in 4℃ overnight, changing the dialysis buffer (50 mM Tris (pH 7.5), 150 mM
NaCl, 1 mM EDTA, 1 mM DTT) three times every four hours. Once this is finished the
concentration is measured with the nanodrop and the protein is cut overnight at
room temperature by adding TEV-protease. 1.2mg of the recombinant nuclease
domain was added to 0.3125 mg of TEV-protease. This combination of protein and
TEV-protease is left overnight. All proteins were visualized in 12% SDS-PAGE under
coommassie or silver staining. The TEV-protease and recombinant nuclease
reaction is bound to the amylose resin. The flow through and wash from the
amylose was combined and ran through the DEAE using Fast protein liquid
chromatography.

5. Nuclease(SGGG(Maltose.Binding((
Protein(Tag(
His8dine(
Tag(
TEV(cut(site(
Results
Figure 3: Schematic of the construct for the NS1 nuclease domain
Figure 3 demonstrates the construct of the NS1 protein’s nuclease domain. With two
recombinant tags (histidine and maltose-binding protein) and a three-glycine
addition in between the TEV cutting site and the nuclease domain. The idea is that
the TEV will cleave the two recombinant tags just so that the three-glycine residues
and the nuclease domain are left together. This would be an ideal situation to obtain
a pure protein. Once the TEV and nuclease domain are combined they are put in
with the amylose resin. The flow through and wash of the amylose resin are
combined and run over the DEAE column to remove any other protein impurities.
Figure 4: Two gels (coommassie (left) silver stain (right)) showing the process after
running the protein through talon resin. This is the initial purification after the
sonication of the protein.
In order to isolate the recombinant nuclease domain it must run through talon
resin. Figure 4 demonstrates the steps that were taken in order to get an ideal pure
nuclease domain. The flow through has the Histidine and MBP tag along with the
nuclease domain around 66 kDa, but there are still other contaminants so it must go
through a wash step where other contaminants are washed away. After the wash
process it goes through an elution process where the protein is eluted from the
resin. This nuclease domain was from 1.5 L of E. coli BL21 (DE3) cells. One of the
12% SDS-PAGE was viewed through coommassie and the other was viewed through

6. silver stain. Once this process of isolation occurs we must remove the histidine and
MBP tag from the nuclease domain.
In figure 5 the gel shows the cleaving of the recombinant tag from nuclease domain.
The cleavage occurs when the TEV-protease specifically recognizes a sequence
within the recombinant nuclease domain; this creates the cleavage of the two
recombinant tags and the nuclease domain.
The nuclease domain of the NS1 protein is located at 20kDa and the two
recombinant tags are located at 43.7kDa(maltose-binding protein tag and histidine
tag). This was a test trial in order to obtain the optimum conditions in order for
cleaving to occur. The optimum conditions obtained are 8.56:0.3125 ug of
recombinant protein to TEV protease. After cleaving the histidine and MBP tag the
solution is ran through amylose resin in order to remove the recombinant tag.
In figure 6 the gel demonstrates the results of running the nuclease domain through
the amylose resin. Through this gel we can see that the nuclease domain is being
cleaved and purified from the two recombinant tags via TEV-protease.There are
trace amounts of the recombinant tag and TEV-protease present in the flow through
and wash of the amylose resin. To remove these impurities from the nuclease
domain, the flow through and wash of the amylose resin are run through the DEAE
column. The DEAE column was successful in removing the impurities from the
nuclease domain (figure 7). Fractions 1-9 from figure 7 were combined and
concentrated. 30 ug of the concentrated samples were visualized by coomassie to
determine purity (figure 8). The coommassie gel only shows the nuclease domain
band, which concludes that there is a pure enough nuclease domain in order to use
to find the binding functionality of the nuclease domain.
Figure 5: Silver stain gel with the reaction condition of the TEV-protease and the
NS1 protein. The cleavage of the two recombinant tags and nuclease domain can be
observed by the sizes of the bands.

7. Figure 6: Silver stain gel. Shows the process of the protein that is being ran through
the amylose resin.
Figure 7: Silver stain gel from the fractions that were collected from the DEAE
column. The impurities can be seen in the flow through fraction while the nuclease
domain elute out from 1-9.

8. Figure 8: Coommassie stained gel. This demonstrates the combination of the 1-9
samples from the DEAE column that were ran through the FPLC. Here we see the
NS1 nuclease domain is the only band that is shown, thus demonstrating a pure
protein.
Discussion
There were several issues that came about when trying to obtain a pure nuclease
protein. One of these issues was the precipitation of the nuclease protein, which
caused there to be a very tedious process. There were several experiments that
were approached in order to create a pure enough nuclease protein and these steps
proved to be very rewarding. One of the steps that were taken to obtain a non-
precipitating nuclease protein was trying different additives (table 1). These
additives stopped the precipitation of the nuclease domain, which allowed us to
keep more protein. Another step that was taken was changing resins. We first used
amylose resin and talon resin, but noticed the talon resin bound too much to the
protein. We then removed the talon resin and added a DEAE column to the
purification process. This became helpful for us because we can use this pure
nuclease protein to analyze the binding functionality of the nuclease domain. The
nuclease domain can help determine what sequences are necessary for binding by
measuring the affinity of the nuclease domain to different DNA substrates. The
nuclease domain may also form oligomers in its active state. This can be assessed
through analytical ultracentrifugation experiments. Alongside these experiments,
initial crystal trials can be implemented to obtain the structure of the nuclease
domain, which could lead to the discovery of the structure of the NS1 protein. Also
the purification methods can be used to clone the other domains of the NS1 protein
in this plasmid in order to figure out their functionality and structures as well. With
all of these items in place, we can determine the mechanism behind the activity of
the nuclease domain alongside the other NS1 domains. The discovery of the
functionality of the NS1 protein could lead to an understanding behind the
mechanistic properties of the viral genome replication as well as a potential to
develop therapeutic treatments to ameliorate symptoms from the Human
Parvovirus B19.

9. Table 1: Table of additives that were added to the nuclease domain
References
1. Heegaard, E. D., and Brown, K. E. (2002) Human parvovirus B19, Clinical
Microbiology Reviews 15, 485-+.
2. Hickman, A. B., and Dyda, F. (2005) Binding and unwinding: SF3 viral helicases,
Current Opinion in Structural Biology 15, 77-85.
3. Lou, S., Luo, Y., Cheng, F., Huang, Q. F., Shen, W. R., Kleiboeker, S., Tisdale, J. F., Liu,
Z. W., and Qiu, J. M. (2012) Human Parvovirus B19 DNA Replication
Induces a DNA Damage Response That Is Dispensable for Cell Cycle Arrest
at Phase G(2)/M, Journal of Virology 86, 10748-10758.
4. Servant-Delmas, A., Lefrere, J. J., Morinet, F., and Pillet, S. (2010) Advances in
Human B19 Erythrovirus Biology, Journal of Virology 84, 9658-9665.
5. Zhi, N., Mills, I. P., Lu, J., Wong, S., Filippone, C., and Brown, K. E. (2006) Molecular
and functional analyses of a human parvovirus B19 infectious clone
demonstrates essential roles for NS1, VP1, and the 11-kilodalton protein
in virus replication and infectivity, Journal of Virology 80, 5941-5950.