Amplifying the Human Papillomavirus (HPV)-18 genome in HEK 293TT

cells via heterologous expression of the E1/E2 viral replication proteins

Min Jung Kim, Caleb McKinney, Dan Chen, and Alison McBride
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda MD

Currently, the HPV field uses four ways to study the early phases of the HPV replication cycle from viral entry to early
stage replication. Transfection is used to introduce the viral genome to cells to study viral gene expression and genome
replication mechanism. Raft-derived virus introduces genomes to keratinocytes from viruses isolated from terminallydifferentiated HPV-infected keratinocytes grown in 3D organotypic raft cultures. While virus produced by this method is
ideal for studying de novo infection, raft cultures are challenging and virus yield is low. Pseudoviruses are recombinant
HPV capsids that usually contain non-viral expression vectors to study various aspects of HPV entry. Quasiviruses
capitalize on pseudo-virus biology by encapsidating HPV genomes, and they are what our lab uses to study the onset of
viral gene expression programs after infection. Quasivirus production yields high infectious titer viral preps which makes it
more feasible to conduct higher throughput studies to dissect mechanisms of initial establishment such as particle
attachment, intracellular trafficking and particle neutralization.
We produce quasiviruses by co-transfecting a HPV capsid expression vector along with the HPV genome into 293TT.
Capsid proteins are produced to high levels and self-assemble into capsids but because the viral genome does not
replicate at all, this cell line ends up with 100 to 1000 more capsids than viral genome. This leads the capsids to take up
random 8 kb chunks of the non-viral genome, and results in a heterogeneous virus prep. To have higher virus yield with
the desired genome, there is a need for a way to replicate the HPV genome in the 293TT cell line. We hypothesized that
the reason why the viral genome cannot be replicated in 293TT is because the two conserved DNA replication proteins,
E1 and E2, are not expressed. Since E1 is a replicative helicase and E2 works cooperatively with E1 as a helicase loader
and allows a more specific binding to the DNA (see cartoon, below), we also hypothesized that by ensuring that E1 and
E2 are expressed in trans, the viral genome will replicate. Remarkably, I found that heterologous co-expression of the
HPV18 E1 and E2 replication proteins from cadmium sulfate-responsive promoters, was sufficient to induce replication of
HPV18 genomes in 293TT cells. Furthermore, I was able to amplify two variations of HPV18 genome in the 293TT cell
line: the wild type and the recombinant marker viral genome expressing genes for GFP and Zeocin resistance. In future
studies, we hope to see a higher yield of HPV18-encapsidated viruses in our virus stocks if the E1/E2 replication proteins
are co-expressed with capsid proteins in HPV18 genome-transfected 293TT packaging cells. The marker genome would
also be a desirable genome to amplify for a quasivirus prep since it will allow cells infected with quasivirus to be more
efficiently screened and/or selected.

DNA extraction: Total DNA was isolated from cell pellets with DNeasy total DNA isolation kit (Qiagen).
DNA digestion for qPCR or Southern Blot analysis: 0.5 g of total DNA was digested with EcoRI (Roche) , which
cuts the viral genome only once, and one of the following: DpnI (Roche) to digest unreplicated DNA or MboI (New
England BioLabs) to digest replicated DNA.
qRT-PCR for viral amplification: Real-time qRT-PCR was performed with the ABI Prism 7900HT Sequence Detector
(Applied Biosystems) and SYBR green PCR master mix (Roche).
Southern Blot: After digestion, samples were separated on 0.8% Tris-acetate-EDTA (TAE) agarose gels. DNA was
visualized with 0.5 mg/ml ethidium bromide and transferred onto nylon membranes with a TurboBlotter downward
transfer system (Whatman). Membranes were UV cross-linked, dried, incubated with prehybridization blocking buffer,
and then incubated overnight with [32P]dCTP-labeled HPV18 DNA in hybridization buffer (0.75 SSC [1 SSC is 0.15
M NaCl plus 0.015 M sodium citrate], 2% SDS, 5 Denhardts solution, 0.2 mg/ml sonicated salmon sperm DNA, 25
ng [32P]dCTP-labeled HPV18 DNA). Radiolabeled probe was generated from 50 ng of 2 gel-purified linear HPV18
DNA with the Random Prime DNA labeling kit (Roche). Hybridized DNA was visualized and quantitated by
phosphorimaging on a Typhoon Scanner (GE Bioscience).
SDS PAGE and Western Blotting: Cells were lysed in SDS extraction buffer (50 mM Tris-HCl [pH 6.8], 5% [wt/vol]
SDS, 10% [vol/vol] glycerol). Protein concentration was determined with a BCA protein assay kit (Thermo-Pierce), and
10 g total protein was separated on a 4 to 12% bis-Tris polyacrylamide gel (Invitrogen). Proteins were transferred
overnight onto PVDF membrane (Millipore) and subsequently immunoblotted. The antibodies used were rabbit antiflag for E2 (Sigma, 1:10,000), chicken anti-EE for E1 (Bethyl lab 1:1000), and mouse anti-Tubulin (Sigma, 1:10,000).

Not Replicated

60

Replicated

50
40
30
20
10

70
60

Total HPV18 GFP ZEO

Replicated + Not Replicated

50
40
30
20
10
0

HPV18 HPV18 - E2 HPV18 HPV18 - E2 HPV18 - HPV18 - E2 - HPV18 - HPV18 - E2 pMEP4 PMEP+ 4/9 - PEMP
- 9 - PEMP+4 - E1 E1 - CAD
+ 4/9 - 9 - PEMP +4 - E1 - E1 - CAD
PEMP
4
4 pMEP4 PMEP
CAD 4 HRS CAD 4 HRS CAD 4 HRS
HRS
HRS
pMEP9 CAD 4+ HRS CAD 4
+ HRS - CAD -4 HRS
pMEP9
+
+
E1-pMEP9

E1-pMEP9

E2-pMEP4

E2-pMEP4

Figure 5: A) As expected, the quantity of non replicated (transfected) HPV18-GFP-ZEO was similar among all of the
conditions 24 hours post transfection and 4 hours of incubation with CdSO4. Co-expression of E1/E2 increased Dpnsensitive HPV18 genome levels about 80 fold. B) The sum of replicated (Dpn-sensitive) and non-replicated (MboIsensitive) viral genome was confirmed to be similar to the total amount of total viral genome (EcoRI only).

Not Replicated

Replicated

80

Verification of E1 and E2 expression

HPV18 E1
Figure 2: Analysis of protein
lysates confirmed expression of
viral replication proteins E1 and E2
only in cells transfected with the
corresponding recombinant pMEP
vectors. The antibody used for
HPV18 E1 did not result in strong
signals and there were no
detectable levels of E1 expression
in cells that were collected after
four hours of CdSO4 incubation.

HPV18 E2
Tubulin

70
60
50
40
30
20
10

pMEP4

pMEP9

E1-pMEP9

E2-pMEP4

CdSO4
incubation

24

24

24

24

Time Post
Transfection

28

28

28

28

48

48

48

48

48

48

48

48

0
pMEP4 HPV18WT
+
+
+
- - HPV18WT
- - HPV18WT
pMEP4/9 - NO pMEP4 - E1 - pMEP4/9 pMEP9
+
+ NO -CAD - CAD +
CAD
4 HRS
E1-pMEP9
+
+
-

- HPV18WT
+
+
+
+
- - HPV18WT
- - HPV18WT
- - HPV18WT
pMEP4 - E1 - pMEP4/9 - NO pMEP4 - E1 - pMEP4/9 + CAD 4- HRS - CAD+O/N +NO CAD
- O/N - CAD 4+ HRS
O/N
+
+
+
+
-

- HPV18WT
+
- pMEP4 - E1 + CAD 4- HRS +
+

E2-pMEP4

CdSO4
incubation

Time Post
Transfection

24

24

24

24

28

28

28

28

48

48

48

48

48

48

48

48

Analyzing Viral Genome Replication: Southern Blot

Figure 6: Substantial amounts of replicated genome only appeared with transfection of both recombinant plasmids. As a
result of co-expression of the E1 and E2 viral replication proteins, the total viral genome abundance increased 3 to 8 fold,
whether or not cells were treated with CdSO4 to induce E1 and E2 expression. This indicates that leaky, undetectable
amounts of E1 and E2 are sufficient to induce viral genome replication in 293TTs. This is clear from the modest difference
in the abundance in replicated genome with and without CdSO4 incubation from cells collected 48 hours post transfection.

Genome
Maintenance and
Partitioning
Figure 3: HPV18 WT was used as a
probe for Southern blots. Since the
recombinant pMEP plasmids contained
segments of HPV18 WT for viral
replication proteins, we predicted that the
probe may detect viral gene-containing
pMEP vectors, which are 0.7 kbp bigger
than HPV18 WT.

Comparing DpnI/MboI-Resistant HPV18 WT to Total EcoRI

Digested Genome Abundance
100
Total HPV18 WT

90

HPV18 WT (picograms)

Initial Viral Replication

70

90

E2
helicase
loader

HPV18 GFP ZEO Genome Replication

in HEK293TT Cells

Comparing DpnI/MboI-Resistant HPV18

GFP ZEO to Total EcoRI Digested
Genome Abundance

HPV18 WT Genome Replication in HEK293TT Cells

E1
replicative
helicase

HPV18 WT (picograms)

Papillomavirus (PV) is an ancient group of non-enveloped, double stranded, circular DNA virus that infects keratinocytes
of the skin or mucous membranes. Viral DNA replication is highly attuned to host cell differentiation, and human
papillomavirus (HPV) life cycle has three main components. Initially, the virus infects a basal keratinocyte and its genome
replicates just enough to establish a small number of viral genomes. As the infected basal cells divide, the viral genome
replicates with the host DNA and partitions to daughter cells. This mode of replication continues as the basal cells move
upward through the epithelium and differentiate, at which point productive infection is activated, viral genomes are
amplified and packaged into viral capsids.

Transfection of Human Embryonic Kidney (HEK) 293TT cells:

HEK293TT cells were transfected with
one of four different plasmid mixtures. All mixtures contained wild type HPV18 or HPV18 GFP/Zeo. In addition to the
viral genome, the control plasmid mixture contained equimolar amounts of empty pMEP4 and empty pMEP9 vectors.
The second mixture contained pMEP4, harboring the HPV18 Flag-tagged E2 gene (pMEP4-E2) and empty pMEP9.
The third mixture contained pMEP9, harboring the HPV18 EE-tagged E1 gene (pMEP9-E1), with empty pMEP4. The
last mixture contained pMEP4-E2 and pMEP9-E1. The plasmid mixtures were incubated with Lipofectamine 2000
transfection reagent (Life Technologies) for 20 minutes in Optimem (Life Technologies). The mixture was added to
cells and allowed to incubate overnight.

Quantification of Viral Genome Replication: RT-qPCR

HPV18 GFP ZEO (picograms)

6.25x105

HPV18 GFP ZEO (picograms)

The overall focus of this project is to determine the role of cellular factors and processes that regulate efficient
gene expression and genome replication programs of Human Papilloma Viruses (HPVs) at early stages of
infection. We have utilized an HPV18 quasivirus infection system to study initial infectivity of primary
keratinocytes. We produce recombinant HPV18 quasiviruses in Human Embryonic Kidney (HEK) 293TT
packaging cells by co-expressing HPV18 genome with the L1 and L2 capsid proteins. It is a promising system to
study the early viral replication program when low levels of de novo-introduced viral genome would require
optimal efficiency of viral-mediated processes. The major drawback to the quasivirus system is that 293TT cells
do not replicate the viral genome, likely due to silencing of HPV18 replication protein expression in these cells.
Since the cell line expresses the capsid protein at high levels, the viral capsids take up non-viral segments of
cellular DNA resulting in a virus prep where only 0.1% to 1% of a typical virus stock contains encapsidated
HPV18 genome. In this study, we determined that heterologous co-expression of the HPV18 E1 and E2
replication proteins was sufficient to induce replication of HPV18 genomes in 293TT cells. Cells expressing both
replication proteins contained about 10 times more HPV18 genome than those with HPV genome transfected
alone. This suggests that the HPV genome can be amplified in HEK293TT. Future studies will be done to assess
whether the viral amplification achieved with heterologous expression of viral replication proteins lead to an
increase in HPV18 genome-containing capsids in virus preps.

Replicated + Not Replicated

80
70
60
50
40
30
20
10

pMEP4

+
+
+
+
+
+
+
+
HPV18WT HPV18WT HPV18WT HPV18WT HPV18WT HPV18WT HPV18WT HPV18WT pMEP9 pMEP4/9
+ - NO + pMEP4- - E1 - -pMEP4/9
+ - CAD+ pMEP4- - E1 - - pMEP4/9
+ - NO + pMEP4- - E1 - -pMEP4/9
+ - CAD+ pMEP4- - E1 - CAD
NO CAD
4 HRS
CAD 4 HRS
CAD O/N
NO CAD O/N
4 HRS O/N
CAD 4 HRS
E1-pMEP9
+
+
+
+
+
+
+
+

Kb

E2-pMEP4

CdSO4
incubation

Time Post
Transfection

24

24

24

24

28

28

28

28

48

48

48

48

48

48

48

48

Figure 7: The sum of replicated (Dpn-sensitive) and non-replicated (MboI-sensitive) viral genome was confirmed to be
similar to the total amount of total viral genome (EcoRI only).

Figure 1: Human Embryonic Kidney (HEK) 293TT cells were seeded onto four six well plates. The next day, cells in each
well were transfected with 2.4 g of DNA from one of four plasmid mixtures (1). After 24 hours, cells from one plate were
collected and centrifuged into a pellet (2). CdSO4 was added to two of the three remaining plates for a final concentration
of 3 M CdSO4 to induce E1 and E2 expression. The third plate was given fresh media without CdSO4. After four hours of
incubation, the CdSO4 media was exchanged for fresh media for one plate and the cells in the other plate incubated with
CdSO4 were collected and spun down (3). After 24 hours, the cells in the two remaining plates were collected (4, 5). The
cell pellets DNA was extracted from resuspended cells, and the extracted DNA was analyzed using RT-qPCR and
Southern blots for HPV18 genome amplification. Western blots were used to analyze the HPV18 E1 and E2 protein
expression levels.

HPV18 Genome re-ligation: HPV genomes were cloned into plasmids and thus can be rapidly amplified in bacteria.
To obtain transfection-ready genome, 10 G of pUC18-HPV18 WT and pRB322-HPV18 GFP/ZEO genome-containing
plasmids were digested with restriction endonucleases, NcoI and EcoRI, respectively, to remove the genomes from
their respective plasmids. Linearized genomes were then intramolecularly religated O/N with 0.4 U/l of T4 DNA
Ligase. Religated genomes were precipitated O/N with NaCl/isoproponal and resuspended in TE buffer.

Figure 4: DNA was extracted from cells that were transfected with HPV18-GFP-ZEO and incubated with CdSO4 for 4
hours. Lanes 7 10 show that there are equal amounts of linearized HPV18 -GFP-ZEO, but lane 11 shows that the cells
transfected with vectors containing E2 and E1 had more detectable Dpn-resistant HPV18. In lanes 12 15, there were
similar amounts of digested, non-replicated DNA, and lanes 17 20 also show similar amounts of non digested, non
replicated DNA. Note that lane 15 and 20, corresponding to E1/E2 co-expression were the only lanes with replicated
HPV18 DNA, evidenced by Dpn-resistant HPV18 in lane 15 and MboI digestion products in lane 20. This indicates that
the co-transfection of both viral replication proteins are necessary for viral genome amplification. Slower migrating
species were detected in lanes 8, 10, 18, and 20. As described in Figure 3, we suspect that these bands correspond to
pMEP-E2 detected by the HPV18 probe.

HPV18 WT and HPV18 GFP ZEO can be amplified in the HEK293TT cell line by co-expressing the E1 and E2
viral replication proteins.

Further studies will be done to determine if co-transfection of E1 and E2 leads to a higher yield of HPV18
genome-containing capsids in quasivirus stocks. If so, additional studies can be done to determine if the
increase in packaging is merely due to an increase in total abundance of viral genome or if replicated genome
has increased accessibility to viral capsid proteins.

I would like to thank everyone in the DNA Tumor Virus Section of the Lab of Viral Diseases. I especially want to thank Dr.
Caleb McKinney for his invaluable mentorship and guidance, Dr. Alison McBride for all of her encouragement and
support, and Dan Chen for helping me with the Southern Blots. I would also like to thank the OITE staff and NIAID for
providing this incredible opportunity. Funding was provided by the Amgen Foundation.