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The 3' region of Human Papillomavirus type 16 early mRNAs decrease expression
© Vinther et al; licensee BioMed Central Ltd. 2005
Received: 17 June 2005
Accepted: 14 October 2005
Published: 14 October 2005
High risk human papillomavirus (HR-HPV) infects mucosal surfaces and HR-HPV infection is required for development of cervical cancer. Accordingly, enforced expression of the early HR-HPV proteins can induce immortalisation of human cells. In most cervical cancers and cervical cancer cell lines the HR-HPV double stranded DNA genome has been integrated into the host cell genome.
We have used a retroviral GUS reporter system to generate pools of stably transfected HaCaT and SiHa cells. The HPV-16 early sequences that are deleted upon integration of the HPV-16 genome was inserted into the 3' UTR of the reporter mRNA. Pools containing thousands of independent integrations were tested for the steady state levels of the reporter mRNA by Real Time PCR and reporter protein by a GUS enzymatic activity assays. In addition, we tested the cellular distribution and half lives of the reporter mRNAs. The integrity of the reporter mRNAs were tested by northern blotting.
We show that the 3' region of the HPV-16 early mRNAs (HPV-16 nucleotide (nt.) 2582–4214) act in cis to decrease both mRNA and protein levels. This region seems to affect transcription from the exogenous minimal CMV promoter or processing of the reporter mRNA. The observed repression was most pronounced at the protein level, suggesting that this sequence may also affect translation. For the HPV types: 2, 6, 11, 13, 18, 30, 31, and 35 we have investigated the regulatory effect of the regions corresponding to the HPV-16 nt. 3358–4214. For all types, except HPV-18, the region was found to repress expression by posttranscriptional mechanisms.
We find that the 3' region of HPV-16 early mRNAs interfere with gene expression. It is therefore possible that the deletion of the 3' part of early HPV-16 mRNAs occurring during cervical oncogenesis could contribute to transformation of cells through deregulation of the viral oncogene synthesis. Moreover, we find that the corresponding region from several other HPV types also repress expression, suggesting that the repression by this region may be a general feature of the HPV life cycle.
All HPV-16 mRNAs that encode E6 and E7 contain the sequence from the splice acceptor located at nt. 3358 to the early polyadenylation signal located at nt. 4215 and this sequence may be involved in regulation of the HPV-16 E6 and E7 expression in the normal HPV life cycle. Furthermore, the large majority of chimeric mRNAs created by integration of the HPV-16 genome in cervical cancer cells are spliced from the 880 splice donor located shortly downstream of the E7 reading frame to a cellular splice acceptor (figure 1) . Thus, the elimination of the HPV-16 sequence from nt. 3358–4214 may also be important for regulation of E6 and E7 during the normal HPV-16 lifecycle and could contribute to selection of cells containing integrated HPV-16 genomes during cervical oncogenesis. Here, we demonstrate that the HPV sequence downstream of the HPV-16 nt. 2582 and 3358 splicesites decrease mRNA steady state level and may also affect mRNA translation. Homologous sequences from other HPV types were also found to decrease gene expression.
Cloning of reporter construct
The HRSp-GUS retroviral reporter vector [26, 27] was generously provided by professor Helen Blau, Stanford University School of Medicine, California. HPV sequences were PCR-amplified from plasmid templates (pBR322-HPV-2, pSV010-HPV-6, pBR322-HPV-11, pBR322-HPV-13, pBR322-HPV-16, pBR322-HPV-18, pBR322-HPV-30, pBR322-HPV-31 and pBR322-HPV-35 (short BamHI fragment)) with pfu polymerase (Promega) and primers from MWG Biotech, Ebersberg, Germany. The following regions were amplified : HPV16: 2582–4214, 3358–4214, 4063–4214, 4063–4310, HPV-18: 3378–4234, HPV-31: 3295–4137, HPV-6: 3325–4377, HPV-11: 3325–4370, HPV-35: 3301–4158, HPV-2: 3299–4387, HPV-13: 3332–4331, HPV-30: 3206–4216. Primers included restriction sites for cloning into Apa I / PinA I digested pHRSp-GUS or into Xho I / PinA I digested pHRSp-GUS (HPV-16 nt. 4063–4310). Plasmid DNA for transfection was prepared with Qiagen kits and all inserts were sequenced.
Cell culture and retroviral infections
The SiHa cell line was obtained from American Type Culture Collection (ATCC number HTB-35) and professor Norbert Fusenig, DKFZ, Heidelberg, kindly provided the HaCaT cell line. The PG13 amphotropic packaging cell line was a gift from PhD Lene Pedersen, Aarhus University, Denmark. 8,6 cm dishes with PG13 cells (grown in 10% neonatal calf serum) were transfected with pHRSp-GUS reporter constructs using the LipofectaminePLUS reagent (Invitrogen). 72 hours post transfection Puromycin (Sigma P7255) was added to a final concentration of 2,5 μg/ml for selection of stably transfected cells. The cells were transferred to T175 flasks that were allowed to grow to confluence. Viral supernatants were harvested by changing the media to 10 % Foetal calf serum (Invitrogen) and incubating the cells for 24 hours at 32°C, 5% CO2. The supernatants were filtered through a 0,45 μm syringe filter (Satorius) and Polybrene was added to a final concentration of 8 μg/ml. For each construct three 8.6 cm dishes of HaCaT or SiHa were each infected 4 hours with 4 ml of viral supernatant. 72 hours after transduction the infected cells were selected by adding 2,5 μg/ml Puromycin (Sigma P7255) to the cell media. The number of colonies was evaluated for each infection (pool), so that at least 1000 different colonies were present in each pool. For each construct three independent infections were performed.
Quantification of mRNA levels by Real time RT-PCR
1 × 106 HaCaT or SiHa cells from the different cell pools (three for each construct) were plated in 8.6 cm cell culture dishes and after 48 hours total RNA was isolated with the Qiagen RNeasy kit according to the instructions of the manufacturer. 10 μg RNA were treated with Amplification grade RNase free DNase I (Invitrogen). Real Time PCR reactions were run on a MJ Research DNA Engine Opticon™. Primers pairs specific for the GUS (5'-GTGATGATAATCGGCTGATG-3' and 5'-CCTGCGTCAATGTAATGTTC-3') and PURO (5'-AGGGCAAGGGTCTGGGCA-3' and 5'-TCGGCGGTGACGGTGAAG-3') coding sequences were designed. The GUS primer set was used with an annealing temperature of 58°C and the PURO primer set with an annealing temperature at 63°C. The QuantiTect SYBR Green RT-PCR Kit from Qiagen was used to assemble 25 μl RT-PCR/PCR reactions in triplicate. The following PCR profile was used: 30 min. at 50°C, 15 min. at 95°C, (15 s at 94°C, 30 s at 58 or 63°C, 1 min at 72°C) × 40 followed by recording of a melting curve. The threshold cycle Ct for individual reactions was determined with the Opticon software. All templates were checked for DNA contamination by omitting the reverse transcriptase in control reactions. The GUS mRNA level relative to the PURO mRNA level for each construct was calculated with the 2-ΔΔCt method and expressed relative to the empty vector . The GUS mRNA levels were normalized to the PURO mRNA levels to ensure that differences between constructs in the efficiency of retroviral infection does not influence the results . ΔCt values for individual pools were calculated and the ΔCt average for the empty vector was treated as an arbitrary constant and used to calculate ΔΔCt values for all pools. The mRNA levels for individual pools were calculated by evaluating the 2-ΔΔCt term. The results for the three independent pools for each construct were averaged and the standard error of the mean was calculated. For mRNA half-life experiments Actinomycin D (Sigma A9415) was added to the medium to a concentration of 2 μg/ml and total RNA was harvested from the cells at t = 0, 1, 3, 6, 9, 15 hrs. Aliquots containing equal amounts of total RNA were used as templates for Real Time PCR analysis with the GUS specific primerset. The GUS mRNA levels for each timepoint were calculated from the Ct values, averaged and expressed relative to the mRNA level at the time of Actinomycin D addition. For examination of nuclear/cytoplasmic mRNA ratios SiHa cells were trypsinised and 2 × 106 cells were used for preparation of cytoplasmic RNA with the RNeasy kit from Qiagen. The nuclear pellets were washed three times in RLN buffer (Qiagen) and used for isolation of nuclear RNA with the Qiagen RNeasy procedure for total RNA. The mRNA levels were determined with Real time PCR. The GAPDH mRNA was detected with the following primer set (5'-GAAGGTGAAGGTCGGAGTC-3') and (5'-GAAGATGGTGATGGGATTTC-3').
Northern blot analysis
Total RNA (app. 7,5 μg) from the SiHa cell pools stably transfected with the different constructs was separated on a MOPS 1% agarose gel, transferred to a BrightStar Membrane (Ambion) and crosslinked with the autocrosslink setting in a Stratalinker (Stratagene) according to the recommendations in the NorthernMax kit from Ambion. Nt. 6–131 and nt. 538–1037 of the GUS reporter mRNA were cloned into pBluescript II SK (+) and used for preparation of radioactively labelled probes by in vitro transcription with Radioactive labelled UTP [α-32P, 800 Ci/mmol] and the T7 Maxiscript kit (Ambion) according to the recommendations of supplier. The crosslinked membrane was hybridised over night with 1 × 106 cpm probe per ml of ULTRAhyp solution at 72°C and washed the following day according to the instructions in the NorthernMax protocol. Finally, the membrane was exposed to Biomax MR film (Kodax) for 72 hours at 4°C.
Quantification of total protein and GUS protein
1 × 106 HaCaT or SiHa cells from the different cell pools (three for each construct) were plated in 8.6 cm cell culture dishes. After 48 hours cells were scraped in lysis buffer: 100 mM potassiumphosphate (pH 7.8) and 0.2 % Triton X-100 with DTT added to 0,5 mM immediately before use. Membrane debris was pelleted by centrifugation (15000 g, 4°C) for 3 min. and the supernatant was collected and stored at -80°C. The Coomassie®Plus Reagent (PIERCE) was used to determine the total protein concentration of the extracts. The level of GUS protein in the extracts was determined with the FluorAceTM β-glucuronidase Reporter Assay kit from BIORAD. All cell extracts were diluted 1:10, 1:100, 1:1000 or 1:10000 to ensure that the measurements were within the linear range of the assay. Fluorescence was counted on a Wallac VICTOR2 Multilabel Counter. The GUS protein levels were normalized with the total protein levels and expressed relative to the HRSp-GUS vector. For each construct the three independent pools were averaged and the standard error of the mean was calculated. Translational was evaluated for the different constructs by normalizing the GUS protein levels with the total levels of the GUS mRNA (not normalized to PURO) as determined by Real Time RT-PCR.
It has previously been demonstrated that the HPV-31 early polyadenylation signal functions inefficiently  and we therefore made a construct with the HPV-16 early polyadenylation region included instead of the bovine Growth Hormone (bGH) polyA region present in the HRSp-GUS vector (figure 2a). In our experiments usage of the HPV-16 early polyadenylation signal (HPV-16 nt. 4063–4310) does not significantly reduce the steady state levels of the reporter mRNA compared to the similar construct containing the bGH polyadenylation region (HPV-16 nt. 4063–4214) (figure 2b). This indicates that in SiHa and HaCaT keratinocytes the HPV-16 early polyadenylation signal functions reasonably efficiently.
Although it is quite possible that the expression of the HPV-16 E6 and E7 proteins is regulated at the protein level by the sequences downstream of the reading frames, this has to our knowledge never been investigated in any detail. We determined the steady state levels of the GUS reporter protein normalized to total protein levels for the different constructs. The normalized protein levels reflect the full regulatory influence of the inserted sequences, including effects on mRNA processing, mRNA transport, mRNA stability and translation. To assess the influence that the inserted HPV sequences have on the amount of protein produced per mRNA we calculated the ratio between the steady state levels of GUS protein (normalized to total protein) and the steady state levels of the GUS mRNA (normalized to total RNA)(figure 2d). This normalization reveals that in neither cell line the HPV-16 nt. 4063–4214 region changes the protein/mRNA ratio, whereas the HPV-16 nt. 2582–4214 region greatly reduces the protein/mRNA ratio. The HPV-16 nt. 3358–4214 region significantly reduces the protein/mRNA ratio in the SiHa cell line, but not in HaCaT cells. Inclusion of the HPV-16 polyadenylation region instead of the bGH polyadenylation region in the HPV-16 4063–4310 construct should give mRNAs with native HPV-16 3' ends. Compared with the similar mRNA that contains the bGH polyadenylation region (HPV-16 nt. 4063–4214), this mRNA produces less protein in the HaCaT cell line, whereas no significant difference is present in SiHa cells (figure 2d). Only the cytoplasmic pool of mRNA is available for translation and it is therefore possible that the reduction in protein/mRNA ratio that we observe is caused by an altered distribution of the reporter mRNAs. We therefore isolated cytoplasmic and nuclear mRNA fractions and determined the mRNA concentrations by real time PCR. None of the inserted HPV-16 sequences affect the distribution of the GUS reporter mRNA or the PURO and GAPDH controls (figure 2e).
In this study we have investigated if the HPV-16 early genes are regulated at the posttranscriptional level. We find that the HPV-16 nt. 2582–4214 region very efficiently reduce the steady state level of a reporter mRNA and the amount of protein that is produced from the mRNA when compared to the empty reporter vector HRSp-GUS vector (see figure 2). Moreover, a truncated construct containing the HPV-16 nt. 3358–4214 region also decreased the steady state level of reporter mRNA and in one of the two cell lines tested reduced the protein/mRNA ratio. In contrast the HPV-16 nt. 4063–4214 region encompassing the short U rich HPV-16 early 3' UTR only, did not affect expression. The reporter assay that we have used in our experiments is based on observation of steady state levels of the different reporter mRNAs and of reporter protein. It is possible that splice acceptors present in the inserted sequences could stimulate splicing from cryptic splice donors in the reporter sequence and this would make our results very difficult to evaluate. For that reason we used Northern Blot analysis to confirm that all the reporter mRNAs have the expected unspliced structure. All constructs, except the HPV-18 construct, produced only the expected full length unspliced reporter mRNA (figure 2b and 4b). The 2582–4214 construct did not express sufficient amounts of reporter mRNA (full length or spliced) to be detected by Northern Blot. Thus, the lowered steady state levels of reporter mRNA that we observe could reflect a decrease in reporter mRNA half-life, a decreased rate of transcription or inhibition of export of mRNA. Surprisingly, we found that none of our reporter mRNAs containing HPV-16 sequence had a decreased half-life (see figure 3). This indicates that the inserted HPV-16 sequence influence the transcription from the minimal CMV promoter included in the pHRSp-GUS vector or affect the nuclear export of the mRNAs. It is known that HPV-16 E5 region contain a strong matrix attachment region [23, 29] and it is possible that this region or other unidentified enhancer elements influence the CMV promoter. Moreover, it is well established that the late mRNAs from several different papillomavirus types contain sequences that interfere with mRNA export [30–33]. Often these sequences bind factors involved in splicing such as U2AF65  and the U1 snRNA . The HPV-16 nt. 3358–4214 and 2582–4214 regions both contain a splice donor located at HPV-16 nt. 3632 and the HPV-16 nt. 2582–4214 region also contain the splice acceptor at HPV-16 nt. 3358. Both these splice sequences diverge from the consensus sequences, but are used during the viral lifecycle and could therefore potentially contribute to the repression that we observe by inhibiting mRNA export and mediation of rapid degradation within the nucleus. Still, we investigated the distribution of the different reporter mRNA (figure 2e) and found that the HPV-16 sequences does not change the distribution of the reporter mRNAs. This suggests that the decrease in steady state mRNA levels is caused by inhibition of the minimal CMV promoter. Alternatively ineffective mRNA processing of the reporter constructs may lead to very rapid degradation (cotranscriptional) of the reporter mRNA that cannot be detected in our RNA half-life experiments.
Jeon and Lambert have previously reported that the very A/U rich 4005–4213 region from HPV-16 very efficiently decreases the stability of a reporter mRNA . The 3' UTRs from short-lived mRNAs often contain destabilizing A/U-rich elements (ARE) , but the HPV-16 A/U rich sequence does not contain any of the canonical ARE sequences (AUUUA). AREs and a few other RNA sequences have been found to be highly overrepresented in mRNAs with high turnover rate, but they are not strong predictors of the mRNA turnover rate , This emphasizes that the ability of a RNA sequences to destabilize mRNAs is highly context dependent and requires cooperative binding of several factors. In our experiments the HPV-16 sequence from nt. 4063–4214 does not influence the steady state level or decrease the half-life of the reporter mRNA (figure 2a and 3). Schwartz and coworkers did not find any repressive effect of the HPV-16 nt. 4005–4213 region on protein expression in their reporter system  and weak inhibitory effect (1.6 fold inhibition) on the mRNA level . It is possible that the upstream nt. 4005–4063 sequence is necessary for the repression observed by Jeon and Lambert and Schwartz and coworkers. Another possibility is that the reporter systems and cell lines used in the different studies influence the regulatory properties of this HPV-16 region.
It has been reported that the early polyadenylation region from HPV-31 is app. 20 fold less efficient than the SV-40 late polyadenylation region and that this difference could be reduced to a 5 fold difference by introduction of an efficient binding site for the cleavage stimulatory factor CstF-64 to the HPV-31 polyadenylation region . Our experiments indicate that the efficiency of the HPV-16 early polyadenylation region from nt. 4215 to 4310 is comparable with the efficiency of the bGH polyadenylation region present in the HRSp-GUS vector (compare 4063–4214 with 4063–4310, figure 2c). Clearly, these findings may reflect a difference in the efficiency of the HPV-16 and HPV-31 early polyadenylation regions or the efficiency of the bGH and SV40 polyadenylation regions. Interestingly, Schwartz and coworkers have recently shown that the U-rich sequence present in the HPV-16 3' UTR enhances polyadenylation from the early HPV-16 polyadenylation signal . The HPV-31 early 3' UTR is less U-rich than the HPV-16 early 3' UTR and may therefore not enhance polyadenylation in the same way. Still, different cells and reporter systems were used in the different studies.
In addition to decreased reporter mRNA steady state levels we also observe that the mRNAs containing the HPV-16 nt. 2582–4214 and nt. 3358–4214 sequences produce less protein per mRNA than the control mRNA. Only cytoplasmic mRNA is available for translation and since we have measured total mRNA concentration, inhibition of mRNA export and accumulation of reporter mRNAs in the nucleus would result in a decreased protein/mRNA ratio in our experiments. We find that the ratio of nuclear to cytoplasmic mRNA is unaltered by the HPV-16 sequences (figure 2e), which indicates that the inserted HPV sequences may influence translation.
A productive HR-HPV infection relies on carefully regulated expression of the HPV gene products. The expression is coupled to the differentiation of the infected cells, so that the viral proteins are expressed at low levels in the basal and suprabasal cell layers, but are expressed at higher levels in the more superficial cells that have exited the cell cycle [37, 38]. This strategy most likely helps to keep the HPV infection hidden for the immune system. The repression that we observe by the HPV-16 nt. 3358–4214 and nt. 2582–4214 regions shows that HPV-16 exploits posttranscriptional mechanisms probably to keep the expression of the E6 and E7 proteins at a suitably low level. This is analogous to the repression of the late structural proteins of HPV-16, which also partly occur on the level of mRNA processing, export, stability and translation [30–32, 39, 40].
We next wondered if posttranscriptional repression could be a general feature of early mRNAs from different HR-HPV types and possibly all HPV types. The HPV types tested include the high risk types HPV-16, HPV-18, HPV-31, and HPV-35 and also the low risk types HPV-6, HPV-11 and HPV-13, which frequently infect the genital tract and cause condylomata acuminata, but are practically absent in high grade lesions and in cervical cancers. In addition, we have tested HPV-2, a benign HPV type with a preference for cutaneous tissue and HPV-30, which is a rather rare HPV type occasionally present in genital infections. We found that the region from the E2 splice acceptor to the early polyadenylation signal from all the tested types except HPV-18 repress expression of the reporter by decreasing mRNA steady state levels or reducing the amount of protein produced or both (figure 4). Like the HPV-16 sequence these HPV sequences therefore contain elements that interfere with transcription and/or processing of the mRNAs to reduce the steady state level of the reporter mRNAs. Moreover, most of them also reduce translation and it is therefore very likely that repression by this region of the HPV genomes in a general feature of the HPV life cycle. The HPV-18 sequence seems to increase expression of the reporter through increased mRNA steady state level and enhanced protein production, but since we find that the HPV-18 construct in addition to the expected full length reporter mRNA also express a shorter mRNA this may be the reason for the increased expression. The finding that the 3' end of most HPV E6 and E7 mRNAs repress expression by posttranscriptional mechanisms is interesting, since it suggests that posttranscriptional repression of the early HPV genes may be a general feature of many HPV types and possibly has evolved to facilitate evasion of the host immune system. Still all the HPV types tested are part of the A supergroup and more distantly related types could be regulated very differently.
In conclusion, we demonstrate that the HPV-16 nt. 2582–4063 region represses expression at the mRNA and possibly also the protein level. This regulation could be involved in the regulation of the HPV-16 E6 and E7 oncogenes during the normal HPV lifecycle. Also for the HPV types 2, 6, 11, 13, 16, 30, 31, 32 and 35 the region from the E2 splice acceptor to the early polyadenylation signal represses expression of the reporter construct. This indicates that regions downstream of these splicesites function in the HPV lifecycle to repress the expression of the early HPV proteins including the E6 and E7 oncoproteins. For the HR-HPV types deletion of this region through genomic integration occurring in the E1 or E2 open reading frames could contribute to enhanced expression of E6 and E7 and increase the oncogenic potential of the infected cells during cervical oncogenesis.
We thank Professor Helen Blau, Stanford University School of Medicine, California, for the generous gift of the pHRSp-GUS vector. Also, we are grateful for plasmids containing the different HPV genomes that we obtained from professors Harald zur Hausen, DKFZ, Heidelberg, (HPV-2, HPV-11, HPV-16, HPV-18), Wayne Lancaster, Wayne State University School of Medicine, Detroit, (HPV-31), Gérard Orth, Institut Pasteur, Paris, (HPV-13, HPV-33), Attila Lorincz, Digene Corporation, (HPV-35), Tom Broker, University of Alabama, Birmingham (HPV-6). The HaCaT cell line was very kindly provided by professor Norbert Fusenig, DKFZ, Heidelberg. Furthermore we thank Solvej Jensen for excellent technical assistance.
Our Real Time PCR machine is funded by: The Carlsberg Foundation, Dir. Ib Henriksens Foundation, Fonden Til Lægevidenskabens Fremme, Frænkels Mindefond, Fonden af 17.12.1981 and Sven Hansen og hustru Ina Hansens Fond. In addition this work was supported by Fabrikant Einar Willumsens Fond, The Novo Nordisk Foundation, Oda og Hans Svenningsens Foundation and Gerda og Aage Haensch's Foundation. KK was supported by a scholarship from The Danish Cancer Society and JV by The Danish Medical Research Council.
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