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.