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MOLECULAR AND CELLULAR BIOLOGY, Oct. 2005, p. 8643–8655
0270-7306/05/$08.00⫹0 doi:10.1128/MCB.25.19.8643–8655.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 19
Gene Codon Composition Determines Differentiation-Dependent
Expression of a Viral Capsid Gene in Keratinocytes In Vitro
and In Vivo
Centre for Immunology and Cancer Research, The University of Queensland, Research Extension, Building 1,
Princess Alexandra Hospital, Ipswich Road, Woolloongabba, Queensland 4102, Australia
Received 18 March 2005/Returned for modification 28 April 2005/Accepted 11 July 2005
By establishing mouse primary keratinocytes (KCs) in culture, we were able, for the first time, to express
papillomavirus major capsid (L1) proteins by transient transfection of authentic or codon-modified L1 gene
expression plasmids. We demonstrate in vitro and in vivo that gene codon composition is in part responsible
for differentiation-dependent expression of L1 protein in KCs. L1 mRNA was present in similar amounts in
differentiated and undifferentiated KCs transfected with authentic or codon-modified L1 genes and had a
similar half-life, demonstrating that L1 protein production is posttranscriptionally regulated. We demonstrate
further that KCs substantially change their tRNA profiles upon differentiation. Aminoacyl-tRNAs from differentiated KCs but not undifferentiated KCs enhanced the translation of authentic L1 mRNA, suggesting that
differentiation-associated change to tRNA profiles enhances L1 expression in differentiated KCs. Thus, our
data reveal a novel mechanism for regulation of gene expression utilized by a virus to direct viral capsid protein
expression to the site of virion assembly in mature KCs. Analysis of two structural proteins of KCs, involucrin
and keratin 14, suggests that translation of their mRNAs is also regulated, in association with KC differentiation in vitro, by a similar mechanism.
modification utilizing mammalian preferred codons without
changing the protein sequence (36, 41, 42, 69), but it remains
unclear whether codon modification assists L1 protein synthesis by removal of sequences inhibitory to mRNA translation or
destabilizing of mRNA or by some other mechanism. Mechanisms postulated to determine instability of L1 mRNA in undifferentiated cells (32, 54, 62) have not been demonstrated to
distinguish between undifferentiated and differentiated KCs,
the host cells of PV infection.
Terminally differentiated KCs flatten and develop a cornified envelope, which provides the barrier function of epithelia
(1). The proliferation and differentiation capacity of cultured
epidermal cells makes KCs ideal candidates for gene targeting
and drug therapy (35). It is therefore desirable to develop models
for the study of regulation of gene expression in KCs as they
develop. In this study, we examined expression of the PV major
capsid (L1) proteins from authentic or codon-modified (Mod) L1
gene expression plasmids in KC culture in vitro and in mouse skin
in vivo. We demonstrate that gene codon composition determines
the timing of PV L1 capsid protein expression in KC culture in
vitro upon differentiation and the differential expression of the L1
gene between the superficial and basal epithelium of the skin in
vivo. Substantial differences were demonstrated in the tRNA
pools of differentiated and undifferentiated KCs. A change in the
aminoacyl-tRNA (aa-tRNA) pool upon KC differentiation enhances translation of authentic but not Mod PV L1 mRNA, likely
reflecting a better match in differentiated KCs between available
aa-tRNAs and the codons present in the PV L1 gene but rarely
used in most mammalian genes.
Direction of gene expression to undifferentiated or differentiated cells is classically determined by altered promoter methylation or by production of specific transcription factors or posttranscriptionally by interaction of regulatory mRNA sequences
with translational regulators (38). Papillomaviruses (PVs) are a
family of double-stranded DNA viruses which replicate exclusively in epithelium, promote cell growth, and affect cellular differentiation, giving rise to benign tumors with, for some virus
types, potential for malignancy. mRNA encoding the PV major
capsid L1 protein can be transcribed from L1 gene expression
plasmids in many types of mammalian cells (20). However, translation of the transcribed mRNA to L1 protein is limited in vivo to
differentiated keratinocytes (KCs) (5, 53) and to yeast cells (50,
67). Although inhibitory mechanisms have been proposed to explain the blockage of PV L1 gene translation in undifferentiated
cells (10, 11), no inhibitory factors have been identified as specific
for epithelial cells in vitro or in vivo. Thus, the mechanism for the
tight differentiation-specific translation of the PV L1 gene in KCs
remains to be determined.
PVs, like many mammalian DNA viruses, use relatively few
“mammalian consensus” codons to encode their capsid genes,
manifesting a high A⫹T genome content due to third-nucleotide bias to A⫹T (68). In humans, codon-mediated translational controls may play an important role in the differentiation
and regulation of tissue-specific gene products (47). Blockage
to translation of PV L1 mRNAs has been overcome by codon
* Corresponding author. Mailing address: Centre for Immunology
and Cancer Research, The University of Queensland, Research Extension, Building 1, Princess Alexandra Hospital, Ipswich Road, Woolloongabba, Queensland 4102, Australia. Phone: 07 3240 5282. Fax: 07
3240 5946. E-mail for Kong-Nan Zhao: [email protected]. Email for Ian H. Frazar: [email protected].
MATERIALS AND METHODS
Construction of native (Nat) and Mod PV L1 genes. The plasmids used in the
experiments described here (pCDNA3HPV6b Nat L1, pCDNA3HPV6b Mod L1,
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Kong-Nan Zhao,* WenYi Gu, Ning Xia Fang, Nicholas A. Saunders, and Ian H. Frazer*
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monoclonal antibody against PV1 L1 protein (67), followed by Cy3-conjugated
anti-mouse IgG (Sigma). L1-labeled KCs were further blocked with 5% skim
milk–PBS and probed with fluorescein isothiocyanate-conjugated monoclonal
antibody against keratin 14 (K14; Covance). Fixed KCs were probed with antibody against involucrin protein (Covance), followed by fluorescein isothiocyanate-conjugated secondary antibody. Nuclei were counterstained by 4⬘,6⬘-diamidino-2-phenylindole (DAPI). KCs were examined by immunofluorescence
microscopy. Fixed skin section samples were similarly stained for PV L1 protein.
Isolation of differentiated and undifferentiated KCs from mouse and cow skin.
Differentiated and undifferentiated KCs were isolated from mouse and cow skin
as previously described (48).
Preparation of aa-tRNAs. Total tRNAs were extracted and purified from
undifferentiated and differentiated KCs using a QIAGEN kit (QIAGEN). aatRNAs were produced as previously described (69).
High-pressure liquid chromatography (HPLC) analysis of tRNAs. HPLC
analysis was carried out on a Waters liquid chromatography system equipped
with an LC-100 column oven, a spectrophotometric detector with a 254-nm filter,
and a Waters Chromatopac (Waters). tRNA (40 to 60 ␮g) treated with 20%
trifluoroacetic acid (TFA) and formic acid for 1 h was injected into a Luna 5-␮m
C18 column prefitted with a 7-mm guard column (Phenomene). Elution was
achieved using a 0% to 30% linear gradient of 1 M ammonium acetate–0.1%
TFA, pH 3.2, and 50% acetonitrile–0.025 M potassium orthophosphate–0.1%
TFA, pH 5.0, over 120 min. The chromatographic run was carried out at 37°C at
a flow rate of 0.4 ml/min.
tRNA dot blot hybridization. One hundred nanograms of tRNA, after denaturation in 1 M deionized glyoxal–20 mM NaPO4 (pH 7.0) at 50°C for 1 h, was
applied to a Nytran blot using a 24-well slot blot apparatus. The blot, after
incubation in 20 mM Tris-HCl (pH 8.0) at 100°C for about 10 min, was then air
dried and cross linked by 254-nm irradiation. The cross-linked blot was prehybridized with hybridization buffer containing 6⫻ SSC (1⫻ SSC is 0.15 M NaCl
plus 0.015 M sodium citrate), 10⫻ Denhardt solution, 0.2% SDS, and 1 mM
EDTA at 37°C for at least 4 h. The blot was then hybridized with DNA oligonucleotide probes complementary to tRNAMet(initiator), tRNAAla(CGA),
tRNAArg(CGA), tRNAAsp(GAC), tRNAAsn(AAC), and tRNAAsn(AAG) in 4⫻
SET buffer (1⫻ SET is 0.15 M NaCl, 0.03 M Tris HCl, and 2 mM Na2EDTA, pH
8.0) at 37°C overnight. The DNA oligonucleotide probe complementary to mammalian tRNAMet(initiator) is 5⬘-TAGCAGAGGATGGTTTC-3⬘, and that complementary to tRNAAsn(AAT) is 5⬘-CGTCCCTGGGTGGGCTC-3⬘. DNA oligonucleotide probes complementary to mouse and bovine tRNAAla(GCA) are
5⬘-TAAGGACTGTAAGACTT-3⬘ (mouse) and 5⬘-TAAGGATTGCAAGACT
A-3⬘ (bovine), those complementary to mouse and bovine tRNAArg(CGA) are
5⬘-CGAGCCAGCCAGGAGTC-3⬘ (mouse) and 5⬘-TTGGTAATTATGAATT
A-3⬘ (bovine), those complementary to mouse and bovine tRNAAsp(GAC) are
5⬘-TAAGATATATAGATTAT-3⬘ (mouse) and 5⬘-TGAGGTGTACAGGACT
T-3⬘ (bovine), and those complementary to mouse and bovine tRNAAsn(AAC)
are 5⬘-CTAGATTGGCAGGAATT-3⬘ (mouse) and 5⬘-CTAGACTGGTGGGC
TCC-3⬘ (bovine). The DNA oligomers were labeled with T4 polynucleotide
kinase (Amersham) and [␥-32P]ATP (3,000 Ci/mmol; Amersham) at the first 5⬘
end. Specific activities of 108 to 109 cpm/␮g were generally reached. Approximately 107 cpm of oligomers was used per blot in hybridization reactions. Blots
were washed with 1⫻ SET buffer at 37°C and autoradiographed.
Cell-free in vitro translation assay. For in vitro translation, L1 plasmid (1 ␮g)
was added to 20 ␮Ci of [35S]methionine (Amersham), and 40 ␮l of T7 DNA
polymerase-coupled rabbit reticulocyte lysates (Promega), with or without additional aa-tRNAs as indicated. Translation was performed at 30°C and stopped by
adding SDS loading buffer. The L1 proteins were separated by SDS-polyacrylamide gel electrophoresis on a 10% gel and blotted onto polyvinylidene difluoride membrane. The blots were imaged by phosphor screen and quantified by
densitometric analysis using the ImageQuant program (Molecular Dynamics).
Codon usage analysis of mouse K14, involucrin, and HPV6 L1 genes. DNAcoding sequences for the K14, involucrin, and HPV6 L1 genes were downloaded
from the website of the National Center for Biotechnology Information (http:
//www.ncbi.nlm.nih.gov/mapview/). The codon usage pattern of the three genes
was analyzed by a computer program [CodonFrequency(GCG)] at the website of
the ANGIS Bioinformatic Forum (http://www.angis.org.au/html/index.html).
The frequency of use of 64 codons for each gene was tabulated (available on
request).
RESULTS
Codon usage determines differentiation-dependent expression of PV L1 genes in KC cultures in vitro. We prepared
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pCDNA3BPV1 Nat L1, and pCDNA3BPV1 Mod L1) have previously been described (69). Briefly, both the bovine PV 1 (BPV1) and human PV 6b (HPV6b)
wild-type L1 open reading frames (ORFs) are about 1.5 kb in length, encoding
⬃500 amino acids. The PV wild-type L1 genes show a strong codon usage bias,
among degenerately encoded amino acids, toward 18 codons mainly with T at the
third position that are rarely used by mammalian genes (68, 69). We artificially
modified BPV1 and HPV6b L1 genes in which the L1 ORFs are substituted with
codons preferentially used in the mammalian genome. We made about 250 base
substitutions in 250 codons rarely used in mammalian cells to produce unmodified L1 proteins encoded from the L1 ORFs with consensus codon usage (69).
All the Nat and Mod PV L1 sequences were sequenced and found to be error
free; they were then cloned into the mammalian expression vector pCDNA3
containing simian virus 40 ori (Invitrogen), giving four expression plasmids,
pCDNA3HPV6b Nat L1, pCDNA3HPV6b Mod L1, pCDNA3BPV1 Nat L1, and
pCDNA3BPV1 Mod L1.
Cell culture and DNA transfection. KCs were isolated from newborn mouse
skin as previously described (51). Isolated KCs were grown as adherent cultures
in a freshly prepared medium (365 ml DMEM medium, 2 mM glutamine, 100
U/ml penicillin, 100 U/ml streptomycin, 125 ml Ham’s F12 medium, 50 ml fetal
bovine serum, 2.5 mg transferrin, 2.5 mg insulin, 4.2 mg cholera toxin, 0.12 mg
hydrocortisone, 17 mg adenine, 10 mg gentamicin) for 1 day and then cultured
in KC-SFM medium with low calcium (GIBCO) for 7 days to induce cell differentiation. KCs cultured for 1 or 7 days were transfected with PV L1 gene
expression constructs (pCDNA3 Nat HPV6b L1, pCDNA3 Mod HPV6b L1,
pCDNA3 Nat BPV1 L1, and pCDNA3 Mod BPV1 L1) using Lipofectamine
(Invitrogen) according to the manufacturer’s protocol. After transfection, DNAtransfected KCs continued to grow in KC-SFM medium for 42 h before collection for RNA and protein preparation.
RNA Northern blot analysis. Total RNA was extracted from L1 DNA-transfected KCs using a NucleoSpin RNAII Kit (Mackery-Nagel). For cytoplasmic
RNA purification, buffer RLN (50 mM Tris [pH 8.0], 140 mM NaCl, 1.5 mM
MgCl2, 0.5% Nonidet P-40) was directly added to monolayer cells, and cells were
lysed at 4°C for 5 min. After the nuclei were removed by centrifugation, cytoplasmic RNAs were purified by a QIAGEN kit (QIAGEN). Following DNase I
treatment, 10- or 15-␮g RNA samples were electrophoresed in 1.2% denatured
agarose gels and blotted onto a Nylon N⫹ membrane (Amersham). The Northern blots were probed with an equal mixture of 32P-labeled Nat and Mod PV L1
gene probes. To visualize internal controls, the Northern blots were stripped and
reprobed with a 32P-labeled glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) gene probe.
Reverse transcriptase PCR (RT-PCR) and quantitative RT-PCR. A 0.5-␮g
sample of RNA purified from cultured KCs transfected with different PV L1
gene expression constructs was converted to cDNA using random primers and
PowerScript RT (Clontech) according to the manufacturer’s protocol. We used
20 ng of cDNA from each RNA sample in a 20-␮l RT-PCR mixture using the
FastStart DNA Master SYBR Green I kit (Roche) supplemented with 3 mM
MgCl2 and Platinum Taq polymerase (Invitrogen). Quantitative RT-PCR was
undertaken using the TaqMan system (Applied Biosystems). The efficiency of
amplification for each pair of primers was determined using a standard curve that
was generated using serially diluted plasmid DNA. Transcription of each investigated L1 gene, mouse K14, and involucrin was compared to ␥-actin (46).
Western blot analysis. DNA-transfected KCs were collected for protein preparation 42 h posttransfection. Cell pellets were resuspended in phosphate-buffered 0.15 M sodium chloride (PBS), pH 7.4, containing 2 mM phenylmethylsulfonyl fluoride and sonicated for 40 s. Fifty-microgram total protein samples were
separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis
and blotted onto polyvinylidene difluoride membrane. The blots were first
probed by monoclonal antibodies against PV L1 protein and ␤-tubulin. Blots
were then probed with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG; Sigma) and visualized using a chemiluminescence system
(Amersham).
Use of the gene gun. Flank and belly skin of BALB/c mice 6 weeks old was shot
by particle bombardment with DNA-coated gold beads coated with Nat and Mod
human and bovine PV L1 gene plasmids (2 ␮g DNA per dose) using the
helium-powered Helios gene gun delivery system at a pressure setting of 480
lb/in2 based on the expression of a Mod gfp gene (Bio-Rad Laboratories, Richmond, CA). Eight mice were used for the gene gun delivery of each L1 plasmid
DNA. Skin was collected 42 h after particle bombardment, fixed using 10%
neutral buffered formalin, and embedded in paraffin for sectioning.
Immunofluorescence labeling of L1, K14, and involucrin protein in vitro and
in vivo. KCs were grown on eight-well chamber slides, transfected with the
different plasmids, fixed, and permeabilized with 85% ethanol 42 h posttransfection. Fixed KCs were blocked with 5% skim milk–PBS and probed with
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primary KC cultures from newborn mouse skin and induced
differentiation in vitro. Progressive differentiation of the KCs
cultured from day 1 to day 7 was confirmed by microscopic
appearance, increased Papanicolaou staining for cytokeratins
(43), progressive loss of expression of the basal cell keratin K14
(4, 19, 44), terminally differentiation-dependent expression of
involucrin (13, 40), and progressive nuclear condensation (Fig.
1A to F). KCs cultured for 1 or 7 days were transiently transfected with the Nat L1 gene of two PVs (HPV6b, BPV1). After
42 h, L1 mRNA was detected in undifferentiated and differentiated KCs, whereas L1 protein was detected only in cultures
of differentiated KCs, as determined by immunofluorescence
in situ (Fig. 2A and B) or by immunoblotting of cell extracts
(Fig. 2C and D). In further experiments, KCs were transfected
after a period of 0 to 7 days in culture, during which time
progressive KC differentiation was observed. Expression of L1
protein over the 24 h following transfection increased progressively, when cells were allowed to differentiate more prior to
transfection (data not shown). These results confirm that for
adherent primary KC cultures, as for suspension cultures (49),
expression of PV L1 proteins is differentiation dependent.
PV L1 and L2 genes demonstrate a strong bias toward the
usage of 18 codons with T at the third position that mammalian
genes rarely include (68, 69). To test the hypothesis that codon
usage bias might determine the differentiation-dependent
translation of the L1 genes in cultured KCs, we examined the
expression of two heavily codon-substituted L1 genes in differentiating KCs, as these genes, encoding unmodified L1 proteins, have been shown to be expressed well in a wide range of
cells in vitro (24, 41, 69). The Mod L1 genes were each transcribed in both undifferentiated and differentiated KCs (Fig.
2C and D); L1 protein, however, was expressed in D1-transfected (undifferentiated) KCs but not in in vitro D7-transfected (differentiated) KCs (Fig. 2A and B). Thus, these data
suggest that the synonymous codon use of the Nat L1 genes is
adapted to expression of their protein products in KCs according to the differentiation stage of the cells.
Codon usage determines how L1 protein expression is targeted to different sites of the mouse skin in vivo. To confirm
our in vitro findings in vivo, expression of the Nat and Mod L1
genes in mouse skin was investigated following gene delivery
by DNA particle bombardment. A gfp gene encoding green
fluorescent protein modified for expression in mammalian
cells, and known to be expressed well in all layers of skin, was
used as a control for DNA particle bombardment (Fig. 3B).
Using the same helium pressure setting, both Nat and Mod L1
genes were delivered to mouse skin. RNA in situ hybridization
revealed that the L1 genes, whether Nat or Mod, were each
transcribed in both basal and superficial epithelial cells (data
not shown). However, in keeping with the in vitro findings, L1
protein was expressed from the Nat sequence L1 genes only in
the most superficial epithelial cells (Fig. 3C and D), including
superficial differentiated KCs in deeper hair follicles (Fig. 3C
and D). In contrast, the Mod L1 genes were expressed more
extensively throughout the epidermis and dermis, except for
the most superficial KC layers (Fig. 3E and F). Thus, the
results indicate that the codon usage of the PV L1 gene determines the site of expression of L1 protein within the mouse
epidermis in vivo.
Codon modification does not affect the stability of PV L1
mRNA in undifferentiated and differentiated KCs. The above
results indicate that the site of expression of L1 protein from
either Nat or Mod PV L1 genes is determined by a differentiation-linked process apparently acting at a posttranscriptional
level. Regulation of mRNA stability is an important element in
the posttranscriptional control of gene expression (37). Accordingly, we examined the stability of the Nat and Mod PV L1
mRNAs in undifferentiated and differentiated KCs. KCs cul-
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FIG. 1. Cultured primary KCs differentiate between day 1 (D1) and day 7 (D7) in vitro. Primary in vitro murine KC cultures were exposed to
differentiation medium for 7 days. At days 1 and 7, cells were compared for morphology (A) and for cellular differentiation using Papanicolaou
stain (B). Expression of K14, an early differentiation marker detected by indirect immunofluorescence, is shown in green (C), and nuclei from the
same field were counterstained blue with DAPI (D). Expression of involucrin, a late differentiation marker detected by indirect immunofluorescence, is shown in green (E), and nuclei from the same field were counterstained blue with DAPI (F).
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FIG. 2. Codon usage determines the timing of expression of the PV L1 capsid gene as KCs differentiate in vitro. KCs cultured for 1 day (D1)
or 7 days (D7) were transfected with an expression construct for a Nat or Mod PV L1 capsid gene as indicated. (A and B) At 42 h after transfection,
cells were stained by indirect immunofluorescence (A, 1 day; B, 7 days) for human PV L1 protein (red) and for K14 (green) and nuclei
counterstained with DAPI (blue). Overlaid panels combine all three images. Magnification of all images, ⫻400. (C and D) For 1- and 7-day
cultured KCs similarly transfected with Nat or Mod human (C) and bovine (D) PV L1 genes, L1 mRNA was assessed by RT-PCR using cellular
actin (Act) as an internal standard and by quantitative RT-PCR. Results are shown as the mean (⫾ the standard error of the mean) of four
individual assays from two independent experiments. L1 protein levels were assessed by immunoblotting using cellular tubulin (Tub) as an internal
standard.
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tured for 1 or 7 days were transiently transfected with the Nat
or Mod L1 genes of two PVs (HPV6b, BPV1). At 18 h posttransfection, the KCs, treated with actinomycin D (Act D) to
prevent further gene transcription for 0, 2, 5, or 8 h, were
harvested for total RNA preparation. L1 mRNA was detected
by Northern blot analysis and compared with GAPDH mRNA
(Fig. 4A and B, insets). As shown by quantitative RT-PCR
(Fig. 2C and D), the steady-state levels of L1 mRNA transcribed from the Mod PV L1 genes were several times higher
than the levels expressed from Nat PV L1 genes (Fig. 4A and
B, insets). The half-lives of both Nat and Mod L1 mRNAs were
then calculated as the time period necessary to reduce the
amount of L1 mRNA in Act D-treated KCs to 50% of the
original L1 mRNA abundance at time point zero (0 h) (Fig. 4A
and B). In D1-transfected undifferentiated KCs, the half-lives
of the Nat L1 mRNAs were 5.3 ⫾ 0.8 h for HPV6b and 5.2 ⫾
0.7 h for BPV1, whereas those of the Mod L1 mRNAs were
nonsignificantly shorter at 4.7 ⫾ 0.8 h for HPV6b and 4.8 ⫾
0.8 h for BPV1 (Fig. 4A and B). In D7-transfected differentiated KCs, the half-lives of the Nat L1 mRNAs were 4.3 ⫾ 0.4 h
for HPV6b and 4.4 ⫾ 0.6 h for BPV1 and the half-lives of the
Mod L1 mRNAs were nonsignificantly longer at 5.0 ⫾ 0.7 h for
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FIG. 3. Codon usage determines the spatial expression of the PV L1 capsid gene in skin in vivo. Expression constructs for Nat (C and D) or
Mod (E and F) HPV6 and BPV1 L1 genes or for humanized (hm) green fluorescent protein (B) were delivered on gold beads (2 ␮g/dose) to the
flank skin of BALB/c mice using a biolistic delivery system (Helios; Bio-Rad Laboratories, Richmond, CA). Control skin (A) was left untreated.
After 42 h, skin was fixed in 10% neutral buffered formalin, sectioned, and stained as indicated for L1 protein by indirect immunofluorescence (red)
or for nuclei using DAPI (blue). Epidermis (E), dermis (D), and hair follicles (H) of the skin section are indicated in panel A. Magnification of
all sections, ⫻400.
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HPV6b and 4.6 ⫾ 0.4 h for BPV1 (Fig. 4A and B). Thus, codon
modification does not alter the stability of PV L1 mRNAs in
cultured KCs sufficiently to explain the observed differences in
steady-state L1 protein levels.
As the transport of mRNAs to the cytoplasm is delayed until
RNA splicing is completed (30, 56), we repeated, using cytoplasmic mRNA, the L1 mRNA half-life studies in differentiated KCs and quantitated mRNA by Northern blotting hybridization (Fig. 5A and B) and quantitative RT-PCR (data not
shown). L1 mRNAs, whether transcribed from Nat or Mod PV
L1 genes, were efficiently exported from the nucleus to the
cytoplasm of KCs (Fig. 5A and B). The half-lives of the cytoplasmic Nat L1 mRNAs were 3.9 ⫾ 0.7 h for HPV6b and 5.2 ⫾
0.7 h for BPV1, whereas those of the Mod L1 mRNAs were
nonsignificantly different from the Nat mRNAs at 4.3 ⫾ 0.5 h
for HPV6b and 5.5 ⫾ 0.8 h for BPV1 (Fig. 5A and B). Thus,
differences in the cytoplasmic pools of mRNA are unlikely to
account for the observed differences in L1 production from Nat
and Mod L1 genes in differentiated cells.
tRNA profiles differ between undifferentiated and differentiated KCs. To explain the observation that codon modification of the L1 gene could alter the expression of L1 protein in
differentiating KCs without altering mRNA levels or stability,
we hypothesized that observed translational differences likely
reflected different availability of aa-tRNAs between differen-
tiated and undifferentiated KCs. We therefore extracted
tRNAs from murine and bovine epidermal cells, sorted by size
and buoyant density into smaller undifferentiated cells and
larger differentiated cells, and confirmed the differentiation
state of the cells by K14 and involucrin staining and morphology (Fig. 6). tRNA species were then profiled for each cell
population by HPLC separation (Fig. 6). tRNA profiles of
murine and bovine epithelial cells were similar (Fig. 6A, B, C,
and D), and the profiles of differentiated cells were in each
case distinct from undifferentiated cells (Fig. 6), showing, as
for studies with tumor cells and cells of parent tissues (34), that
cell differentiation can alter the aa-tRNA pool. As no validated
technology is available for identification and quantitation of
individual tRNA species in cells by HPLC or otherwise, we
arbitrarily chose several tRNA oligonucleotide probes complementary to specific regions of six tRNAs, tRNAMet(Initiator),
tRNAAla(CGA), tRNAArg(CGA), tRNAAsp(GAC), tRNAAsn
(AAC), and tRNAAsn(AAT), to examine whether reactivity
with these probes differed between undifferentiated and differentiated KCs. Reactivity with the tRNAMet(Initiator) probe
was similar between undifferentiated and differentiated KCs
(Fig. 6E). Reactivity with the probes corresponding to four
tRNAs, tRNAAla(GCA), tRNAArg(CGA), tRNAAsp(GAC),
and tRNAAsn(AAC), was consistently stronger in undifferentiated KCs than in differentiated KCs (Fig. 6E), and reactivity
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FIG. 4. Half-lives of L1 mRNAs transcribed from the Nat or Mod PV L1 capsid gene in cultured KCs. KCs cultured for 1 day (D1) or 7 days
(D7) were transfected with an expression construct for a Nat or Mod PV L1 capsid gene as indicated. At 18 h after transfection, cells were treated
with Act D (500 ng/ml) and total RNAs were harvested at 0, 2, 5, and 8 h for Northern blot hybridization and mRNA stability analysis. Fifteen
micrograms of total RNA from Nat PV L1-transfected KCs (10 ␮g of total RNA from Mod PV L1-transfected KCs) treated with DNase I was
electrophoresed on a 1.2% denatured agarose gel and blotted onto a nylon membrane. The Northern blots were probed with an equal mixture of
32
P-labeled Nat and Mod PV L1 gene probes. To visualize the internal controls, the Northern blots were stripped and reprobed with a 32P-labeled
GAPDH gene probe. (A, B) Densitometric quantification of Nat and Mod HPV6b and BPV1 L1 mRNA levels following normalization to GAPDH.
The half-lives of Nat and Mod HPV6b and BPV1 L1 mRNAs, calculated as the times required for a given transcript to decrease to 50% of its initial
abundance (horizontal dotted lines), are shown in parentheses. The data shown are the means (⫾ the standard error of the mean) of four individual
blotting assays from two independent experiments. Insets are representative Northern blot analyses (n ⫽ 4) of Nat and Mod HPV6b and BPV1
L1 and GAPDH RNA expression in day 1 and day 7 L1-transfected KCs. Statistical analysis of the results from the experiments was conducted
using one-way analysis of variance. No significant difference between Nat and Mod L1 half-lives was obtained (P ⬎ 0.05).
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with tRNAAsn(AAT) was consistently weaker (Fig. 6E). Thus,
HPLC tRNA profile differences between differentiated and
undifferentiated KCs are reflected in differences in hybridization-determined tRNA reactivity.
Translation of Nat PV L1 genes is preferentially enhanced
by aa-tRNAs from differentiated KCs. We have previously
shown that altering the available pool of aa-tRNAs alters the
expression of a Nat L1 gene in cell-free in vitro translational
studies (69). To show directly that the aa-tRNA populations of
differentiated KCs favor translation of Nat L1, we firstly confirmed that translation of L1 protein from Nat L1 genes was
much slower than that from the Mod L1 genes (Fig. 7A) using
a rabbit reticulocyte lysate cell-free in vitro translation system
that has fixed pools of aa-tRNAs and amino acids. We then
examined whether addition of aa-tRNAs from undifferentiated
or differentiated murine KCs could enhance the translation of
the Nat and Mod L1 genes (Fig. 7B). Introduction of exogenous tRNAs from differentiated KCs but not undifferentiated
KCs enhanced the translation of Nat L1 genes in a dosedependent manner, with optimum efficiency at 10⫺7 M (Fig.
7B). aa-tRNA from differentiated KCs at 10⫺7 M significantly
enhanced the translation of L1 3.1-fold ⫾ 0.7-fold (standard
error of the mean) for HPV6b and 2.5-fold ⫾ 0.9-fold for
BPV1 at 12 min, whereas 10⫺7 M aa-tRNA from undifferentiated KCs gave less-significant enhancement (1.7-fold ⫾ 0.5fold increase for HPV6b and 1.4-fold ⫾ 0.3-fold for BPV)
compared to control reaction mixtures without added aatRNAs (Fig. 7C). Supplementation of the aa-tRNAs from undifferentiated or differentiated murine KCs had no significant
effect on the translation of the Mod L1 genes (data not shown).
We thus conclude that aa-tRNA availability changes in KCs
with cellular differentiation, and the aa-tRNA pool in differentiated KCs enables more-efficient translation of the “rarely
used” codons in the Nat L1 gene than the aa-tRNA pool in
undifferentiated KCs.
Codon usage may also regulate the expression of some
mammalian proteins associated with KC differentiation. To
investigate whether differentiation-dependent expression of
mammalian proteins might in part be determined by their
codon composition and by tRNA abundance differences associated with cellular differentiation, we examined the expression
of the K14 and involucrin genes in day 1 and day 7 cultured
KCs by RT-PCR and immunoblotting (Fig. 8A and B). As
shown by RT-PCR, the mRNA transcripts of both K14 and
involucrin were at a steady-state level in day 1 and day 7
cultured KCs (Fig. 8A and B). However, immunoblotting of
day 7 cultured KCs revealed that expression of K14 protein was
down-regulated, in contrast to the significantly up-regulated
expression of involucrin (Fig. 8A and B), consistent with the
observations of immunofluorescence in situ (Fig. 1C and E).
We examined the codon usage of mouse K14 and involucrin
genes in comparison with the HPV6 Nat L1 gene by tabulating
the percentage of codons with G/C or A/T at the third position
for each gene (Table 1). The K14 gene has a significantly
greater usage of third-position G/C residues than the involucrin gene (Table 1), while the HPV6 L1 Nat gene has the least
use of third-position G/C residues (Table 1). The involucrin
protein includes several glutamine residues, 102 of which are
encoded by a Glu(GAG) codon. If these glutamine residues
are excluded, the involucrin gene has as high a percentage of
AT-ending codons (56/100) as the HPV6 L1 Nat gene. In view
of this finding and tRNA dot blot hybridization data showing
that undifferentiated KCs are apparently rich in tRNAAsp(GAC) and tRNA-Asn(AAC) (Fig. 6E), we compared the
codon usage of the three genes for the amino acids Asp and
Asn (Table 2). The K14 gene, in contrast to the involucrin and
HPV6 Nat L1 genes, has a high percentage of GAC codons for
amino acid Asp, supporting our finding that tRNA-Asp(GAC)
abundance correlates with high-level expression of K14 protein
in undifferentiated KCs (Fig. 6E and 8A). Thus, at least two
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FIG. 5. Stability of cytoplasmic L1 mRNAs transcribed from the Nat or Mod PV L1 capsid genes in differentiated KCs. KCs cultured for 7 days
(D7) were transfected with an expression construct for a Nat or Mod PV L1 capsid gene as indicated. At 18 h after transfection, KCs were treated
with Act D (500 ng/ml) and cytoplasmic RNAs were prepared at 0, 2, 5, and 8 h for Northern blot hybridization and mRNA stability analysis. Ten
micrograms of cytoplasmic RNA treated with DNase I was electrophoresed on a 1.2% denatured agarose gel and blotted onto a nylon membrane.
The Northern blots were probed with an equal mixture of 32P-labeled Nat and Mod PV L1 gene probes. As internal controls, the Northern blots
were stripped and reprobed with a 32P-labeled GAPDH gene probe. (A and B, left) Representative Northern blot analysis (n ⫽ 2) of Nat and Mod
HPV6b and BPV1 cytoplasmic L1 and GAPDH RNA expression in day 7 L1-transfected KCs. (Right) Half-lives of Nat and Mod HPV6b and BPV1
cytoplasmic L1 mRNAs assessed by densitometric quantification following normalization to GAPDH. Data shown in parentheses are the means
(⫾ the standard error of the mean) of duplicate blot assays.
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FIG. 6. tRNA profiles and dot blot-examined tRNA species differ between differentiated and undifferentiated KCs. (A to D) Total tRNA was
extracted, using a kit (QIAGEN), from single-cell suspensions derived by collagenase and trypsin digestion from murine flank skin (A and B) and
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PAPILLOMAVIRUS CAPSID GENE EXPRESSION IN KERATINOCYTES
8651
bovine ear skin (C and D) and separated into basal (undifferentiated) cells (A and C, insets) and squamous (differentiated) cells (B and D, insets)
by density flotation on a discontinuous 30% to 70% Percoll gradient. The differentiation state of the isolated undifferentiated and differentiated
cells was confirmed by K14 and involucrin (Invol.) staining (A and B). Nucl., nucleus. Extracted tRNAs were dissolved in water, and HPLC
retention profiles were determined using a Luna 5-␮m C18 column (Phenomene) and a linear elution gradient of acetonitrile up to 30% over 2 h
at 37°C. (E) tRNA dot blot hybridization of tRNA samples from undifferentiated (Undi.) and differentiated (Diff.) KCs. The details are described
in Materials and Methods.
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FIG. 7. Translation of PV L1 capsid genes is preferentially enhanced by aa-tRNA from differentiated KCs. L1 genes from HPV6 and BPV1
were translated in a cell-free system using rabbit reticulocyte lysate (in the presence of [35S] methionine. Production of 35S-labeled L1 was assessed
by autoradiography of polyacrylamide gel electrophoresis-separated proteins. (A). Production over time of L1 protein from Nat L1 genes (HPV6b
and BPV1) is less rapid than from Mod L1 genes. (B). Supplementation of the in vitro translation reaction mixture with aa-tRNA from
differentiated (Diff.) or undifferentiated (Undi.) KCs enhances L1 production from Nat L1 genes in a dose-dependent manner from 10⫺8 to 10⫺6
M; inhibition of translation is evident at the highest concentrations of aa-tRNA. (C) Addition of aa-tRNAs prepared from differentiated KCs (10⫺7
M) increased the rate of translation of Nat L1 genes more effectively than aa-tRNAs from undifferentiated KCs over a time course of 8 to 16 min.
(D) Means (⫾ the standard error of the mean) of L1 production from three independent experiments quantitated by scanning densitometry of
autoradiographs. Statistical analysis of the results from individual time points from these experiments was conducted using analysis of variance
(P ⬍ 0.05 and P ⬍ 0.01 compared with no addition of aa-tRNAs as shown).
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TABLE 2. Usage of codons corresponding to abundant aa-tRNAs
in KCsa
mammalian genes differentially expressed between differentiated and undifferentiated KCs may use a subset of favored
codons matching the abundance of the isoacceptor tRNAs to
regulate expression of the corresponding proteins during KC
differentiation.
DISCUSSION
We demonstrate here that KCs differentiated in vitro and
displaying KC differentiation markers produce PV L1 protein
more efficiently from the L1 gene than undifferentiated KCs.
Further, mRNA quantitation demonstrates that the difference
in L1 protein expression between differentiated and undifferentiated KCs is largely determined posttranscriptionally. Generalized substitution, within the L1 gene, of isoencoding
codons with a G or a C at redundant positions within the codon
triplet enhances expression of L1 protein in undifferentiated
KCs and impairs expression in differentiated KCs. In vivo observations in epidermis transduced with Nat or Mod L1 are
consistent with our in vitro data and with the specific expression of PV L1 protein in differentiated KCs observed in the
course of natural PV infection.
Several posttranscriptional mechanisms for regulation of expression of broad subsets of genes on cell differentiation are
recognized, including changes in the assembly of the eukaryotic translation-initiation factor complex and expression of
specific RNA binding proteins targeting cis-regulatory sequences associated with genes expressed on differentiation (re-
TABLE 1. Comparison of codon usage between K14, involucrin,
and HPV6 L1a
n (%), P value
Codon usage
K14
Total
Involucrin
511 (100) 467 (100)
GC ending 337 (66)
AT ending 174 (34)
HPV6 L1
500 (100)
268 (57)
174 (35), 0.0000 (K14-L1)
199 (43), 0.0045 (K14-Inv) 326 (65), 0.0000 (Inv-L1)
a
Codon usage for the whole gene sequence: the number of codons, thirdposition GC codons (GC ending), and AT codons (AT ending) (n), and their
frequencies (%) are shown. P values reflect whether or not two genes differ in
their expected usage of GC-ending codons and AT-ending codons (Fisher exact
test).
Codon
Asp
GAT
GAC
Asn
AAT
AAC
K14
Involucrin
7 (33) 10 (77),0.0323 (K14-Inv)
14 (67) 3 (23)
8 (53)
7 (47)
0 (0), 0.4706 (K14-Inv)
2 (100)
HPV6 L1
20 (77), 0.0036 (K14-L1)
6 (23), 1.0000 (Inv-L1)
14 (54), 0.9999 (K14-L1)
12 (46), 0.4815 (Inv-L1)
a
For each codon, we report the total number in each gene (n) and the relative
frequency (%). The P values reflect whether the two compared genes differ in
their expected encoding of the amino acids (Fisher exact test).
viewed by Calkhoven et al. [6]) (47). However, we show here
that provision of aa-tRNAs from differentiated KCs but not
undifferentiated KCs enhances the translational efficiency of
the Nat L1 gene in a cell-free system and has no effect on
translation using the same expression vector of a Mod L1 gene
which, as a result of codon modification, is well expressed in
undifferentiated KCs. This observation is difficult to explain as
a consequence of the generally active posttranslational mechanisms for regulation of gene expression discussed by Calkhoven et al. as associated with cell differentiation and more
strongly supports a hypothesis that aa-tRNA changes associated with cell differentiation may, in association with selective
codon usage, regulate the translation of some genes within a
cell lineage.
Replacement of less-preferred codons within a prokaryotic
gene with synonyms more commonly observed in mammalian
genes can greatly increase gene expression in eukaryotic cells,
with improvement in translational efficiency attributed to correction of a mismatch between eukaryotic cell tRNA pools and
preferred prokaryotic gene codon bias (26, 39, 61, 70). Plotkin
et al., using in silico analysis of codon usage in eukaryotic genes
differentially expressed in uterus and testis tissues and in brain
and liver tissues (47), have recently hypothesized that posttranscriptional controls based on gene codon usage may also play
an important role in the regulation of tissue-specific gene expression in mammals. Further, impaired viral protein expression in mammalian host cells, attributed to codon usage divergence of viral and human genes, is observed for the latent
genes of Epstein-Barr virus (31), the env gene of human immunodeficiency virus type 1 (25), and the E7 (9), L1, and L2
genes (69) of PV. Thus, the codon composition of PV L1 genes
might be expected to influence L1 expression according to the
availability of aa-tRNA within the cell. The correlation observed in the present study between differences in aa-tRNA
species and differential L1 gene expression for differentiated
and undifferentiated KCs may have broad significance for selective gene expression within multicellular eukaryotic organisms. In Escherichia coli and in yeasts, highly expressed genes
use a subset of codons corresponding to the highly expressed
isoacceptor tRNAs (3, 23) and the synthesis of a number of
colicins is linked to the difference in tRNA availability for the
various codons used by the relevant genes (60). More generally, the pool of available aa-tRNAs is held to be rate limiting
for accuracy and efficiency of gene translation (18, 27, 28, 59,
66). However, in multicellular eukaryotes, there are very few
experimental data on tRNA abundance (15), though a rela-
Downloaded from http://mcb.asm.org/ on October 26, 2015 by University of Queensland Library
FIG. 8. mRNA translation efficiency of KC structural genes differs
with KC differentiation. KCs cultured for 1 day (D1) or 7 days (D7)
were harvested for RNA and protein preparation. K14 and involucrin
(Invol) mRNAs were assessed by RT-PCR and proteins by immunoblotting using actin as an internal standard. The results are representative of two independent experiments (n ⫽ 4) in day 1 and day 7
cultured KCs.
n (%), P value
Amino
acid
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PAPILLOMAVIRUS CAPSID GENE EXPRESSION IN KERATINOCYTES
stability in differentiated and undifferentiated KCs, as are
mRNAs from Mod L1. Thus, it is unlikely that L1 mRNA
stability is a major determinant of the differentiation-determined selective posttranscriptional block to L1 capsid protein
synthesis we observed for Nat and Mod PV L1 transcripts in
cultured KCs. Further, the half-life of the less efficiently translated Nat and Mod PV L1 transcripts in KCs appears paradoxically to be somewhat longer than for the more efficiently
translated L1 mRNAs. The stability of some mRNAs is affected by translation (52, 57, 64). Based on the present observations, the stability of PV L1 transcripts, rather than being a
major determinant of efficient L1 translation, may be regulated
in part by L1 mRNA translation efficiency, determined in KCs
by a differentiation-determined match between L1 codon usage and the availability of aa-tRNAs.
In the present study, HPLC analysis has revealed that tRNA
profiles of differentiated cells were in each case distinct from
undifferentiated cells in both murine and bovine epithelium.
What mechanism may drive the difference in tRNA profiles
between undifferentiated and differentiated KCs? Epidermal
KCs are highly specialized epithelial cells designed to perform
a very specific function, separation of the organism from its
environment. During the KC differentiation process, numerous
genes are turned on and off at specific stages (16, 17). One
possibility is therefore that the observed changes in the available aa-tRNA pool between differentiated KCs and undifferentiated KCs reflects regulated tRNA production or aminoacylation of tRNAs with differentiation. Alternatively, the pool
of free aa-tRNAs might be determined by the extent to which
particular aa-tRNAs are needed by the cell for protein production, as the majority of charged tRNAs are associated with
nascent protein production in the ribosome in metabolically
active cells (29). Further elucidation of the mechanism varying
the pool of aa-tRNA with epithelial differentiation must await
better methods of quantitating specific aa-tRNA species
change in real time.
ACKNOWLEDGMENTS
This work was funded in part by the Queensland Cancer Fund (Q68)
and by a National Health and Medical Research Council of Australia
Industry research fellowship (301256) to K.N.Z.
We thank Kelly Minto for assistance with the animal work.
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