Progress Towards Understanding Cytomegalovirus UL133/8
Locus Through Chemical Biology
Louis Chavez,1 Eric Lau,1 Michael Rak,2 Felicia Goodrum,2 and Eli Chapman1
A Thesis Submitted to the Department of Chemistry and Biochemistry in Partial Fulfillment of
the Bachelors of Science Degree
Department of Chemistry and Biochemistry1
Department of Immunobiology2
The University of Arizona
May, 2015
Approved By:
Dr. Eli Chapman Dr. Nancy Horton
Faculty Thesis Advisor Biochemistry Undergraduate Advisor
Department of Pharmacology and Toxicology Department of Chemistry and Biochemistry
Abstract
Cytomegaloviruses (CMV) are DNA viruses belonging to the Herpesviridae family. Human
cytomegalovirus (HCMV) is estimated to persist in >60% of the population. The virus goes
through stages of lysogeny and latency. Persistent seroprevalence and latency of HCMV pose a
challenge to ameliorating HCMV infection and preventing further spread of the virus. Although
often innocuous in healthy individuals, HCMV has severe implications on morbidity and mortality
for immuno-compromised individuals, including newborns. HCMV also poses a risk for
otherwise healthy elderly individuals, exacerbating age related pathologies such as
atherosclerosis, restenosis, immune senescence, and frailty. The recently discovered ULb’
locus in the HCMV genome encodes four viral proteins: UL133, UL135, UL136, and UL138. The
ULb’ region is postulated to be important because the proteins encoded in this region regulate
latency and reactivation of the herpesvirus. Several of these viral proteins are thought to interact
with each other and it is proposed that these protein-protein interactions are involved in latency
or reactivation of the viral cycle. To date, many questions remain regarding the interactions the
proteins have with one another and with other proteins, the mechanism that these proteins
employ to regulate latency or reactivation of HCMV, and how the viral proteins are modulated.
From the experiments outlined in our study, the groundwork has been set to develop tools to
study these viral proteins. Future experiments center around the use of functional and
biochemical procedures to identify small molecule binders of UL133, UL135, UL136, and UL138
and to use these modulators to probe the function of these proteins in cellular infectivity models.
This will provide insights into their utility as drug targets to either send the virus into a latent
state or reactivate the virus so anti-virals will be affective.
Introduction
Cytomegaloviruses (CMV) are part of a broad category comprised of large, complex
DNA viruses belonging to the Herpesviridae family. More specifically, human cytomegalovirus
(HCMV) is one of the eight human herpesviruses that, once infected, persist indefinitely in
infected individuals via latent infection (1). The latency of this virus is the basis of its high
seroprevalence worldwide (6, 9), affecting >60% of the world’s population (2). One major
contributor to the prevalence of new infections is congenital infection. Congenital infection
occurs in 1 in 50 children in the United States and can lead to a number of complications
including birth defects, which makes HCMV the leading cause of infectious disease-related birth
defects (8). HCMV infection also has a pronounced impact on immuno-compromised individuals
with weakened T-cell immunity. In these individuals, such as AIDS patients, cancer patients, or
transplant patients (2), HCMV often exacerbates morbidity and mortality rates (1). In healthy
individuals, an increased risk of age related pathologies may persist, such as atherosclerosis,
restenosis, immune senescence, and frailty (1, 5).
The elusive nature of this virus, like many other viruses, can be attributed to its latency.
During latency, the viral genome persists in the host without assembly of infectious virions (4).
When the appropriate, undescribed, stimuli is present, there is still a potential for reactivation of
the virus (7). The HCMV genome is 235 kbp, encoding the largest genome of any virus known
to infect humans (7). The ULb’ region of the viral genome was first described over a decade ago
(1). Studies of the UL133-UL138 (UL133/8) locus indicate the four viral proteins within the
UL133/8 locus are all associated with the Golgi apparatus as integral membrane proteins, with
large C-terminal cytosolic domains (1,2). Preliminary studies of the UL133/8 proteins’
biochemical function suggest that the locus influences progression of the viral lytic cycle (1, 7).
However, these studies do not clarify how each viral protein promotes reactivation or latency of
the virus. One novel finding of the locus demonstrates protein-protein interactions, specifically
between UL133 and UL138, that likely promote latency and active infection (5). The two
demonstrate a robust interaction, and although the functional significance has not yet been
elucidated, UL138 has been shown to regulate and promote latency of the virus. Disruption of
UL138 in the ULb’ region results in a virus with an increased efficiency when compared to WT
(1). This demonstrates that UL138 plays a role in promoting latency of the virus. Contrastingly,
deletion of the UL135 sequence in the region results in defective viruses that are incapable of
viral genome amplification and reactivation (8). The UL135 and UL138 viral proteins have
recently been thought to comprise a molecular switch. The proposed function of this molecular
switch is to modulate replication through UL135 and latency through UL138. In other words, this
molecular switch is responsible for balancing between states of latency and reactivation. In
addition to this UL135/UL138 molecular switch, UL136 may be involved in the viral
latency/activation cycle. There are five separate isoforms of UL136: 33 kDa, 26 kDa, 25 kDa, 23
kDa and 19 kDa (9). Some UL136 isoforms promote viral genome latency, while others promote
amplification. For example, disrupting the 23 kDa and 29 kDa isoforms causes an increase in
replication of HCMV (9), which is similar to the outcome of disrupting UL138. Furthermore, the
UL136 26 kDa and 33 kDa isoforms do not associate with the membrane to the same degree as
the other three isoforms. In fact, the 26 kDa and 33 kDa isoforms are more prominent in the
cytoplasm, which indicates that these proteins dissociate more from the Golgi apparatus than
the other isoforms and the other viral proteins (9).
From past studies conducted on the UL133/8 locus, three of the four viral proteins and
their roles in regulating reactivation or latency of HCMV have been postulated. However, none
of the current research regarding the UL133/8 locus has described the mechanism by which
these viral proteins promote reactivation or latency (1-9). Therefore, the research outlined here
aims to generate recombinant versions of the four viral proteins. Once soluble proteins have
been purified, we can utilize functional chromatography (10, 11) to probe for modulators of
these proteins. These modulators will become probes in understanding the localization and
interaction of the viral proteins. From these experiments, we will have more knowledge
regarding the latency and reactivation states of HCMV and how this may be used in a
therapeutic setting.
Materials and Methods
Cloning. Forward and reverse primers were designed for UL133, UL135, UL136 and UL138
viral proteins (Table 1). The template DNA for each gene was acquired from our collaborator,
and these sequences were cross-referenced with sequences published in the NCBI. Only the
cytoplasmic portion of the genes was amplified. The protein size, amino acid length, and
cytoplasmic amino acid residues to be derived from these clones are shown in Table 2. To
amplify the viral proteins, the polymerase chain reaction (PCR) was employed. The PCR
amplification was performed as a 50 μL reaction in a specialized PCR eppendorf tube with 10x
Buffer, 50 mM MgSO4, 1 μL pGEM-T-ULb’, 50 mM forward primer, 50 mM reverse primer, 1 μL
Pfx DNA Polymerase, and ddH2O. The reaction was added to a thermo-cycler (Techne),
programmed with the following cycle: 94°C for 2 minutes, 94°C for 30 seconds, 50°C for 30
seconds, 68°C for 1 minute, 68°C for 10 minutes, 4°C until retrieved; the 94°C for 30 seconds,
50°C for 30 seconds and 68°C for 1 minute periods were cycled through 30 times. Once viral
DNA had been amplified, the PCR product was purified via a DNA agarose gel. First, the DNA
agarose gel solution was made by adding 1 g of high melting temperature Agar to 100mL of 1x
TAE buffer (40 mM Tris, 20 mM acetic acid and 1 mM EDTA) and heating the solution until all
agar was dissolved. The resulting solution was then poured into a DNA electrophoresis well
plate, and left at room temperature to polymerize. Once polymerized, the gel was placed into
the electrophoresis unit with 1x TAE buffer. Next, 6x DNA Gel Loading Dye was added directly
to the 50 μL of amplified PCR product. The 60 μL of dyed PCR product was loaded into three
wells. 2 μL DNA ladder with 6x loading dye was loaded in a separate well. The electrophoresis
gel was set to run at 80 V for 60 minutes. Then, the gel was stained with ethidium bromide for
10 minutes. After staining, the gel was rinsed with water and placed on a UV lamp for
visualization. The band that corresponded to the desired viral protein gene was excised, placed
in a 1.7 mL eppendorf tube, and frozen overnight. The next step of DNA recovery was to
process the excised DNA gel bands with a ZymogenTM Gel DNA Recovery Kit. The gel fragment
with the gene of interest was transferred to a 2 mL eppendorf tube and three volumes of
agarose dissolving buffer were added. This was heated for 10 minutes at 60°C. Once the gel
had completely dissolved in solution, 500 μL of the solution containing DNA was loaded onto a
Zymo-SpinTM Column. The column was spun at 7000xg for 60 seconds, and the flow through
was discarded. After all of the DNA had been captured on the column, 200 μL of DNA Wash
Buffer was loaded onto the column, centrifuged at 7000xg for 60 seconds and flow through was
discarded; this step was repeated twice. The column was then transferred to a clean 1.7 mL
eppendorf tube and 30 μL of DNA Elution Buffer was added directly onto the column. The
column was centrifuged at 7000•g for two minutes and the purified DNA was collected. A 50 μL
reaction was employed to digest the viral DNA, which contained CutSmart 10x Buffer, 44 μL
viral DNA, 50 mM forward restriction enzyme, 50 mM reverse restriction enzyme. A similar
digest was performed to cut the pGex-6P-1 vector, which had CutSmart 10x buffer, 30 μL pGex-
6p-1, 50 Mm forward restriction enzyme, 50 mM reverse restriction enzyme, and ddH2O. The
specific cut sites for each viral protein and the pGex-6P-1 vector are shown in Table 3. The
tubes were then placed in a 37°C water bath for one hour. After an hour, the tube containing
pGex 6P-1 was removed from the water bath and treated with 1 μL Calf-intestinal alkaline
phosphatase (CIP). All tubes were placed in a 37°C water bath for another hour. The digested
products were purified using the DNA-concentrating kit. Once purified, concentrations were
determined using a Nanodrop Spectrophotometer. The concentrations were then used in the
calculations for the gene to vector ratio for optimal ligation. The following equation was used:
.
The gene and the vector with the corresponding cut sites were ligated in a 20 μL reaction, which
contained 10x T4 DNA Ligase Buffer, 1 μL digested pGex-6P-1, 1 μL digested viral gene, and 1
μL T4 DNA Ligase. Once all reagents were added, the reaction proceeded overnight for 14
hours at 16°C. 10 μL of ligated product was then added to 200 μL DH5-α cells. The reaction
was left on ice for 30 minutes, transferred to a 42°C water bath for 45 seconds, and then put
back on ice again for two minutes. 800 μL of Super Optimal Broth (SOC) medium was added to
the cells. The 1 mL reaction was set in a 37°C incubator shaker, and all 1 mL of the incubated
product was plated on an ampicillin-resistant agar plate. The plate was set in a 37°C incubator
overnight. A 15 mL culture tube was filled with 3 mL of Terrific Broth (TB) and ampicillin was
added to a final concentration of 100 μL/mL. One colony was picked from each plate and mixed
in the media. The mini preps were put in the 37°C incubator and incubated overnight. Each mini
prep was centrifuged at 3,000•g for 10 minutes. The supernatant was decanted and discarded.
The pellet was resuspended with 250 μL P1 Buffer from the QIAprep Spin Miniprep Kit and
transferred to a 1.7 mL eppendorf tube. After resuspsension, 250 μL P2 Buffer was added, and
mixed thoroughly. 350 μL of N3 Buffer was then added to neutralize the solution, the solution
was mixed and then centrifuged at 13,000•g for 10 minutes. The supernatant from this solution
was then run through the QIAprep Spin Miniprep Columns and flow through was discarded. The
column was then washed with 750 μL Buffer PE. The column was transferred to a new, labeled
1.7 mL tube, and 50 μL Elution Buffer was loaded onto the column. The Elution Buffer was left
on the column for 1 minute before spinning the column at 13,000•g for two minutes. The
minipreps for each viral protein were stored in the freezer. The miniprep for each viral protein
was then mapped by cutting with their respective restriction enzymes and ran on an agarose gel
to verify that the genes had been properly inserted into the vector. Three different conditions
were tested for each viral protein: no restriction enzyme added, one restriction enzyme added,
and both restriction enzymes added. The restriction enzyme map was performed in a 10 μL
reaction, and the reagents added can be seen in Table 4. Once the reagents had been added,
the tube was incubated in a 37°C water bath for two hours. All 10 μL was added and ran on the
DNA gel. The gel was observed over UV light.
Small Scale Expression. Three different expression strains of E. coli cells were used: BL21
(DE3), BL21 (AI), and BL21. Thawed cells (50 μL) and DNA (1 μL) were mixed in a tube. They
were then put on ice for 30 minutes, immediately transferred to a 42°C water bath for 45
seconds, put back on ice for 2 minutes, and recovered by adding 450 μL of SOC media to the
cells. The cells were placed in the 37°C shaking incubator for one hour. Half of the cells were
plated on an ampicillin resistant agar plate, and put in the 37°C incubator overnight. The next
day, in 15 mL culture tubes, 2 mL Luria-Bertani Broth (LB) medium was mixed with 100 μg/mL
ampicillin. Roughly the same amount of colony was removed from the transformed plates,
added to the culture tubes, and mixed thoroughly. The bacteria were grown at 16°C or 37°C.
When the bacteria were grown at 16°C, they were grown to an Optical Density 600 nm (OD600)
of 0.4 - 0.5. Bacterial cells (500 μL) then were transferred into a new tube, and induced with 800
μM isopropyl β-D-1-thiogalactopyranoside (IPTG). Both samples were placed in a 16°C shaker
and left to grow for 16 hours. The next day, 300 μL of bacteria was aliquoted into a 1.7 mL
eppendorf tube and centrifuged at 13,000xg for 10 minutes. The supernatant was then aspirated.
When the bacteria were grown in 37°C, they were grown to an OD600 of 0.8. Once the bacteria
reached an OD600 of 0.8, an aliquot of bacteria (500 μL) was induced with 800 μM IPTG. The
induced and uninduced cell culture tubes were both left in the shaker for three hours. After three
hours of induction, the bacterial cells were harvested by centrifugation as above. Laemmli Buffer
(2x) was added to the uninduced and induced pellets. Once the pellets were resuspended, they
were heated at 90°C for 10 minutes. The samples were then loaded onto a 12% SDS
polyacrylamide gel, Running Buffer (1x) (25 mM Tris, 192 mM glycine, 0.1% SDS) was added to
the gel electrophoresis unit, and the gel was run at 15 milliamps until the Staining Buffer dye ran
off the gel. The gel was transferred to staining solution (0.025% Coomassie R-250, 45%
methanol, 10% acetic acid, 45% water) for two hours. The gel was then soaked in destaining
solution (30% methanol, 10% acetic acid, 60% water) overnight and the following day, the gel
was visualized on a transilluminator to see the expression of the various viral protein.
Protein Expression and Purification. Once the optimal cell line and induction temperatures for
protein expression were determined, the appropriate bacterial cells were prepared for a larger
scale of expression. In a 2 L baffled flask, 1 L of autoclaved LB media was mixed with 100
μg/mL ampicillin. Using a pipette controller, 10 mL of media was used to transfer the entire agar
plate of bacteria that had been transformed the day before. The culture was grown in a 37°C
shaker until reaching an OD of 0.8. At an OD of 0.8, a 300 μL sample was taken for the
uninduced control. The culture was then induced with 800 μM IPTG final concentration, and left
in the 37°C shaker for three hours. After three hours, the bacteria was transferred into large
centrifuge tubes and centrifuged at 7,000xg for 10 minutes. The supernatant was discarded,
and the pellet was resuspended in 10 mL Lysis Buffer. Then, the resuspended cells were lysed
by pumping through a LM10 Microfluidizer (microfluidics). The lysate was transferred to ultra-
centrifuge tubes and centrifuged at 4°C for 60 minutes at 136,000xg. A sample of the
supernatant and pellet was collected. The supernatant was run through an equilibrated
Glutathione column, and a sample of the flow-through was collected. The column was washed
two times with 10 column volumes of Wash Buffer, and a sample was collected of each wash.
To elute the protein, 2 column volumes of Elution Buffer was run through the column three times,
and fractions of each elution was collected. The recipe for the buffers are summarized in Table
5. In order to discern if any protein was present in any given fraction collected from the resin, a
SDS-PAGE gel was ran.
Results
Initial attempts to purify the His-tagged viral proteins were not successful (Figure 1).
There are many different proteins of varying sizes apparent in the pellet lane. The band in the
pellet lane between 10 and 17 kDa corresponds to a protein size that indicates the likely
presence of the viral protein. However, in subsequent purification steps, such as lysate, load,
wash and elution, this band is lost.
In order to ensure that the viral DNA had been amplified successfully, a 1% agarose gel
was run (Figure 2). The lanes in the gel were loaded as follows: DNA ladder, UL133, UL135,
UL136, and UL138. From comparing the bands from the gel to the bands of the DNA ladder, it
can be determined that the four viral DNA strands that were amplified are all between 500 and
1000 base pairs. Specific restriction endonuclease sites were introduced to both the viral DNA
and vector. In the first restriction enzyme digest experiment, the four viral proteins were cleaved
using the specific restriction enzymes corresponding to the recognition sites. This was repeated
for the pGex6P-1 vector using the two restriction enzymes that corresponded to each viral
protein. In Figure 3, the digested UL133, UL135, UL136, and UL138 inserts can be seen. The
band size for the digested viral proteins traversed the gel to the same extent as their undigested
counterparts, between 500 and 1000 base pairs. The digested pGex6P-1 vectors with restriction
enzymes corresponding to UL133, UL135, UL136, and UL138 can be observed in Figure 4. The
bands for the digested vectors are between 4000 and 5000 base pairs.
After restriction enzyme digestion, the products were ligated, DH5a E. coli cells were
transformed and colonies were picked for minipreps. Five minipreps were chosen for UL133. To
ensure that each construct had the viral gene incorporated into the vector, a restriction enzyme
digest map was employed. BamHI and XhoI were the two restriction enzymes used to cut the
UL133 viral gene out of the vector. Once the gene had been removed, a DNA gel was run to
verify the presence of the viral gene and vector in each of the minipreps. In Figure 5, the band
between the 4000 and 5000 base pair maker corresponds to the pGex-6P-1 vector and the
band between the 500 and 750 base pair marker corresponds to the UL135 viral DNA.
A small-scale experiment was run in order to see if the viral DNA could express the viral
protein. The UL135 viral protein was grown in 2 mL LB and induced with 800 μM IPTG. In
Figure 6, the sample uninduced by IPTG can be compared to its IPTG-induced counterpart
(uninduced denoted by a “-” and induced denoted by a “+”). In the induced lanes, there are
arrows that point to thick bands that correspond to the viral protein, which is discernible through
molecular weight and thought to be the correct protein.
Discussion
Experiments to express and purify the viral proteins in a His-tagged vector (pSpeedET)
were unsuccessful, which can be observed in Figure 1. Although there was protein present, the
protein that was synthesized was insoluble. This is apparent by the 17 kDa sized protein
present in the gel. This band corresponds to the size of the UL136 viral protein. However, this
band is only present in the pellet, not in any subsequent purification fractions. Since solubility
was the issue and not expression, we postulated that our protein could be solubilized in a
different vector with an N-terminal fusion. This will also aid in purification The vector that we
chose to subclone our DNA into was pGex6P-1. This vector has an N-terminal GST tag, and
the size and stability of this tag are known to help induce folding of the molecule upon
purification. To create this GST-tagged viral protein, primers for the viral protein inserts had to
be designed, which were subsequently cut with restriction enzymes and ligated into the
pGex6P-1 vector.
The size of each PCR amplified viral gene was verified through agarose gel
electrophoresis. The polymerization of the agarose forms a matrix that makes it more difficult for
larger DNA to traverse. Thus, the viral DNA in Lane 3 from Figure 2 is the largest of the four
proteins ran, which corroborates data from previous studies that implicate UL135 to be the
largest viral protein (849 base pairs). The band in Lane 2 travels further than the band in Lane 3,
which implies that the DNA in Lane 2 (UL133, 579 base pairs) is the second largest. The bands
in Lane 4 and Lane 5 travel about the same distance, and also travel the furthest out of all four
viral DNA bands. These lanes correspond to UL136 (459 base pairs) and UL138 (429 base
pairs), respectively. The restriction enzyme digest in Figure 3 shows that the viral proteins are
still the appropriate size. The restriction enzyme digest in Figure 4 shows that the pGex-6P-1
vectors are also between the 4000 and 5000 base pair bands (the uncut pGex6P-1 vector is
4984 base pairs).
The colonies from the transformation of the ligated products of UL133 were prepared as
minipreps, and a gel was ran to discern if the viral gene had been correctly incorporated into the
vector. There are two visible bands in each lane in Figure 5. The band between the 4000 and
5000 base pair marker indicates the presence of the vector. The second set of bands, between
the 500 and 750 base pair markers, correspond to the UL133 viral DNA. Thus, it can be
deduced that the transformation of the ligated products was successfully performed.
The SDS-PAGE gel that was performed showed expression of each of the viral proteins
in uninduced and IPTG-induced samples. With the GST tags, the viral proteins increase in
molecular weight by about 26 kDa. This was taken into account when calculating where each
viral protein’s band should be on the gel. Each band in the (+) lane with a red arrow in Figure 6
corresponds to correctly sized GST-tagged viral protein. This indicates that the GST-fusion
proteins were successfully expressed.
Conclusion
The insolubility of the proteins is a challenging obstacle. However, there are experiments
that can be performed to overcome this unexpected outcome. In a paper published by
Petrucelli et al. (5), the interactions that all four viral proteins have with each other are proposed.
The strongest interaction is between UL138 and UL133. Two moderately strong interactions are
proposed between UL138 and UL136 and UL133 and UL136. Thus, a co-purification
experiment that takes advantage of these proteins’ abilities to interact, stabilize, and solubilize
could be a potential experiment to address protein solubility. Another factor to consider is the
media used to grow the bacteria. All large-scale expressions were performed with LB as the
media. While this media is effective in expressing the protein, perhaps other media options such
as Yeast Extract Tryptone (2XYT) or Terrific Broth (TB) could be explored to see if these have
any effect on solubility. Lastly, since the viral protein is present in the pellet (Figure 1), a
denatured preparation experiment could be employed. This experiment would be useful
because it would denature the protein and keep it completely unfolded and insoluble until the
very last step of purification via dialysis, at which time it is hoped the proteins will refold.
Although we are currently working on overcoming this obstacle to come up with the
UL133, UL135, UL136, and UL138 viral proteins, we have taken a large step forward from
where current research stands. Once soluble protein is purified, we will be enabled to test the
viral protein modulators with functional chromatography. From these experiments, we will
hopefully be able to discover modulators of UL133/8 function to gain new insights into protein
physiologic, pathologic, and biochemical functions. This end-goal will ultimately enable
researchers to find inhibitors that can up- and down-regulate the viral proteins as necessary,
controlling the latency and reactivation and ultimately eradicating HCMV from its host.
Table 1. Viral proteins and primers with cut sites highlighted
Viral Protein Sequence 5’ --> 3’
UL133 Forward
UL133 Reverse
GGA ATT G|GA TCC ATG CGT TAC CGG GAA TTC TTC AAG
GGC ATT C|TC GAG TTA CGT TCC GGT CTG ATG CTG CTG
UL135 Forward
UL135 Reverse
GGC ATT G|GA TCC ATG ACC CAG CGC CGA GGC CGC AAG
GGC ATT C|TC GAG TTA GGT CAT CTG CAT TGA CTC GGC
UL136 Forward
UL 136 Reverse
GGC ATT G|GA TCC ATG TTA AGA TAT TAC CAC CAG GAC
GGC ATT G|AA TTC TTA CGT AGC GGG AGA TAC GGC GTT
UL138 Forward
UL138 Reverse
GGC ATT G|AA TTC ATG GCT TAC CAT TGG CAC GAC ACC
GGC ATT C|TC GAG TTA CGT GTA TTC TTG ATG ATA ATG
Table 2. UL viral protein size and characteristics
Viral Protein Number of Base Pairs Number of Amino
Acid Residues
Cytoplasmic Amino
Acid Residues
UL133
579
258
68-258
UL135 849 328 47-328
UL136 459 240 88-240
UL138 429 169 28-169
Table 3. Viral proteins and restriction enzymes utilized (highlighted) for digest
Protein Cut Sites
UL133
Forward Primer: EcoRI, HpaII, MspI, NlaIII, BamHI, DpnII, MboI, NdeII
Reverse Primer: TaqI, AvaI, TliI, XhoI
UL135 Forward Primer: HaeIII, PhoI, NlaIII, BamHI, DpnII, MboI, NdeII
Reverse Primer: TaqI, AvaI, TliI, XhoI
UL136 Forward Primer: MseI, NlaIII, BamHI, DpnII, MboI, NdeII
Reverse Primer: SnaBI, EcoRI
UL138 Forward Primer: NlaIII, EcoRI
Reverse Primer: TaqI, AvaI, TliI, XhoI
pGex-6P-1 BamHI, EcoRI, SmaI, SalI, XhoI, NotI
Table 4. Restriction enzyme map reactions
Reagent μL added, No
Restriction Enzyme
Added
μL added, One
Restriction Enzyme
Added
μL added, Both
Restriction Enzymes
Added
ddH2O
5
4.8
4.6
DNA 4 4 4
Cutsmart 10X 1 1 1
Restriction Enzyme 1 0 0.2 0.2
Restriction Enzyme 2 0 0 0.2
Table 5. Reagents in protein purification buffers
Buffer Reagents (final concentrations)
Lysis Buffer
50 mM NaH2PO4, 150 mM NaCl, 1 mM EDTA,
1 % Triton, 1 mM DTT, 1 mM PMSF
Wash Buffer 50 mM NaH2PO4, 150 mM NaCl, 1 mM EDTA
1 mM DTT,
Elution Buffer 50 mM pH 8 Tris Buffer, 0.4 M NaCl, 0.1 %
Triton, 1 mM DTT, 50 mM reduced GSH
Figure 1. Large Scale expression and purification of BL21(DE3) [pSpeedET-UL136]
Figure 2. Viral DNA PCR amplification of UL133, UL135, UL136, and UL138 inserts
Figure 3. Restriction enzyme digest of UL133, UL135, UL136, and UL138
Figure 4. Restriction enzyme digest of pGex-6P-1 vectors
Figure 5. Restriction enzyme map of UL133 minipreps 1-5
Figure 6. Small scale expression of viral proteins
References
1. Umashankar M, Petrucelli A, Cicchini L, Caposio P, Kreklywich CN, Rak M, Bughio F,
Goldman DC, Hamlin KL, Nelson JA, Fleming WH, Streblow DN, Goodrum F. A novel
human cytomegalovirus locus modulates cell type-specific outcomes of infection. PLOS
Pathogens.2011;7:e1002444. doi: 10.1371/journal.ppat.1002444.
2. Alex Petrucelli, Michael Rak, Lora Grainger, Felicia Goodrum. Characterization of a
novel golgi apparatus-localized latency determinant encoded by human cytomegalovirus.
J Virol. 2009 June; 83(11): 5615–5629. Published online 2009 March
18. doi: 10.1128/JVI.01989-08
3. Lora Grainger, Louis Cicchini, Michael Rak, Alex Petrucelli, Kerry D. Fitzgerald, Bert L.
Semler, Felicia Goodrum. Stress-inducible alternative translation initiation of human
cytomegalovirus latency protein pUL138. J Virol. 2010 September; 84(18): 9472–
9486. Published online 2010 June 30. doi: 10.1128/JVI.00855-10
4. Siok-Keen Tey, Felicia Goodrum, Rajiv Khanna. CD8+ T-cell recognition of human
cytomegalovirus latency-associated determinant pUL138. J Gen Virol. 2010
August; 91(Pt 8): 2040–2048. doi: 10.1099/vir.0.020982-0
5. Petrucelli A, Umashankar M, Zagallo P, Rak M, Goodrum F. Interactions between
proteins encoded within the human cytomegalovirus UL133-UL138 Locus. J Virol. 2012
Aug;86(16):8653-62. doi: 10.1128/JVI.00465-12. Epub 2012 Jun 6.
6. Farah Bughio, David A. Elliott, Felicia Goodrum. An endothelial cell-specific requirement
for the UL133-UL138 locus of human cytomegalovirus for efficient virus maturation. J
Virol. 2013 March; 87(6): 3062–3075. doi: 10.1128/JVI.02510-12
7. Gang Li, Michael Rak, Christopher C. Nguyen, Mahadevaiah Umashankar, Felicia D. An
epistatic relationship between the viral protein kinase UL97 and the UL133-UL138
latency locus during the human cytomegalovirus lytic cycle. Goodrum, Jeremy P. Kamil J
Virol. 2014 June; 88(11): 6047–6060. doi: 10.1128/JVI.00447-14
8. Mahadevaiah Umashankar, Michael Rak, Farah Bughio, Patricia Zagallo, Katie Caviness,
Felicia D. Goodrum. Antagonistic determinants controlling replicative and latent states
of human cytomegalovirus infection. J Virol. 2014 June; 88(11): 5987–
6002. doi: 10.1128/JVI.03506-13
9. Caviness K, Cicchini L, Rak M, Umashankar M, Goodrum F. Complex Expression of the
UL136 gene of human cytomegalovirus results in multiple protein isoforms with unique
roles in replication. J Virol. 2014 Dec;88(24):14412-25. doi: 10.1128/JVI.02711-14. Epub
2014 Oct 8.
10. Lau EC, Mason DJ, Eichhorst N, Engelder P, Mesa C, Kithsiri Wijeratne EM,
Gunaherath GM, Gunatilaka AA, La Clair JJ, Chapman E. Functional chromatographic
technique for natural product isolation. Org biomol Chem. 2015 Feb 28;13(8):2255-9. doi:
10.1039/c4ob02292k.
11. Kang MJ, Wu T, Wijeratne EM, Lau EC, Mason DJ, Mesa C, Tillotson J, Zhang DD,
Gunatilaka AA, La Clair JJ, Chapman E. Functional chromatography reveals three
natural products that target the same protein with distinct mechanisms of action.
Chembiochem. 2014 Sep 22;15(14):2125-31. doi: 10.1002/cbic.201402258. Epub 2014
Aug 14.