Inhibition of Translation by UAUUUAU and UAUUUUUAU Motifs of the AU-rich RNA Instability Element in the HPV-1 Late 3′ Untranslated Region

Abstract

The human papillomavirus type 1 (HPV-1) late mRNAs contain a 57-nucleotide adenosine- and uridine-rich RNA instability element termed h1ARE in their late 3′ untranslated regions. Here we show that five sequence motifs in the h1ARE (named I–V) affect the mRNA half-life in an additive manner. The minimal inhibitory sequence in motifs I and II was mapped to UAUUUAU, and the minimal inhibitory sequence in motifs III–V was mapped to UAUUUUUAU. We also provide evidence that the same motifs in the AU-RNA instability element inhibit mRNA translation, an effect that was entirely dependent on the presence of a poly(A) tail on the mRNA. Additional experiments demonstrated that the h1ARE interacted directly with the poly(A)-binding protein, suggesting that the h1ARE inhibits translation by interfering with the function of the poly(A)-binding protein. The human papillomavirus type 1 (HPV-1) late mRNAs contain a 57-nucleotide adenosine- and uridine-rich RNA instability element termed h1ARE in their late 3′ untranslated regions. Here we show that five sequence motifs in the h1ARE (named I–V) affect the mRNA half-life in an additive manner. The minimal inhibitory sequence in motifs I and II was mapped to UAUUUAU, and the minimal inhibitory sequence in motifs III–V was mapped to UAUUUUUAU. We also provide evidence that the same motifs in the AU-RNA instability element inhibit mRNA translation, an effect that was entirely dependent on the presence of a poly(A) tail on the mRNA. Additional experiments demonstrated that the h1ARE interacted directly with the poly(A)-binding protein, suggesting that the h1ARE inhibits translation by interfering with the function of the poly(A)-binding protein. Human papillomaviruses (HPVs) 1The abbreviations used are: HPV(s), human papillomavirus(es); UTR, untranslated region; ARE, AU-rich element; nt, nucleotides; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; hGH, human growth hormone; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S-transferase; PABP, poly(A)-binding protein. are a group of non-enveloped, double-stranded DNA tumor viruses with tropism for epithelial cells (1Howley P.M. Fields B.N. Knipe D.M. Howley P.M. 3rd Ed. Fields Virology. 2. Lippincott-Raven Publishers, Philadelphia, PA1996: 2045-2076Google Scholar, 2zur Hausen H. Biochim. Biophys. Acta. 1996; 1288: 55-78Google Scholar). Expression of the late mRNAs is restricted to the terminally differentiated cells in the upper layers of the epithelium and at least four papillomaviruses (bovine papillomavirus type 1 (BPV-1), HPV-1, -16, and -31) have been shown to contain cis-acting inhibitory RNA elements located in the late 3′ UTR (reviewed in Refs. 3Baker C.C. Billakanti S.R. Calef C.E. Farmer A.D. Malpern A.L. Myers G.L. Human Papillomaviruses: a Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos National Laboratory, Los Alamos, NM1997: 11-16Google Scholar, 4Schwartz S. Semin. Virol. 1998; 8: 291-300Google Scholar, 5Schwartz S. Sokolowski M. Collier B. Carlsson A. Goobar-Larsson L. Recent Res. Dev. Virol. 1999; 1: 53-74Google Scholar, 6Schwartz S. Upsala J. Med. Sci. 2000; 105: 171-192Google Scholar). In addition, negative RNA elements have been identified in the HPV-16 L1 and L2 open reading frames (4Schwartz S. Semin. Virol. 1998; 8: 291-300Google Scholar, 7Tan W. Felber B.K. Zolotukhin A.S. Pavlakis G.N. Schwartz S. J. Virol. 1995; 69: 5607-5620Google Scholar, 8Sokolowski M. Tan W. Jellne M. Schwartz S. J. Virol. 1998; 72: 1504-1515Google Scholar). We have previously identified and characterized an inhibitory AU-rich element (ARE) located in the HPV-1 late 3′ UTR region named h1ARE (4Schwartz S. Semin. Virol. 1998; 8: 291-300Google Scholar, 5Schwartz S. Sokolowski M. Collier B. Carlsson A. Goobar-Larsson L. Recent Res. Dev. Virol. 1999; 1: 53-74Google Scholar, 6Schwartz S. Upsala J. Med. Sci. 2000; 105: 171-192Google Scholar, 9Tan W. Schwartz S. J. Virol. 1995; 69: 2932-2945Google Scholar) (Fig. 1). Using actinomycin D we showed that the presence of the h1ARE reduced the mRNA half-life (10Sokolowski M. Zhao C. Tan W. Schwartz S. Oncogene. 1997; 15: 2303-2319Google Scholar). The minimal inhibitory sequence termed XB spans 57 nucleotides (nt) and contains 93% A and U. The element contains two AUUUA- and the three UUUUU-containing sequences (9Tan W. Schwartz S. J. Virol. 1995; 69: 2932-2945Google Scholar, 10Sokolowski M. Zhao C. Tan W. Schwartz S. Oncogene. 1997; 15: 2303-2319Google Scholar). Replacing two uridines (U) with cytidines (C) in each motif inactivated the h1ARE (10Sokolowski M. Zhao C. Tan W. Schwartz S. Oncogene. 1997; 15: 2303-2319Google Scholar). The h1ARE interacts with cellular factors (11Zhao C. Tan W. Sokolowski M. Schwartz S. J. Virol. 1996; 70: 3659-3667Google Scholar, 12Zhao C. Sokolowski M. Tan W. Schwartz S. Virus Res. 1998; 55: 1-13Google Scholar), which bind to the c-fosARE (10Sokolowski M. Zhao C. Tan W. Schwartz S. Oncogene. 1997; 15: 2303-2319Google Scholar). Two of the h1ARE binding factors interacted with the wild type h1ARE but not with a functionally inactive mutant of the h1ARE (10Sokolowski M. Zhao C. Tan W. Schwartz S. Oncogene. 1997; 15: 2303-2319Google Scholar). These proteins were identified as HuR and hnRNP C (10Sokolowski M. Zhao C. Tan W. Schwartz S. Oncogene. 1997; 15: 2303-2319Google Scholar, 13Sokolowski M. Furneaux H. Schwartz S. J. Virol. 1999; 73: 1080-1091Google Scholar), and we later showed that binding of the HuR protein correlates with inhibitory activity of a panel of h1ARE mutants (13Sokolowski M. Furneaux H. Schwartz S. J. Virol. 1999; 73: 1080-1091Google Scholar). While HuR binds to both AUUUA- and UUUUU-motifs (13Sokolowski M. Furneaux H. Schwartz S. J. Virol. 1999; 73: 1080-1091Google Scholar), hnRNP C binds exclusively to the UUUUU-motifs (14Sokolowski M. Schwartz S. Virus Res. 2001; 73: 163-175Google Scholar). The role of hnRNP C in HPV-1 late gene expression is unclear. The HuR protein shuttles between the nucleus and the cytoplasm (15Keene J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5-7Google Scholar), and we observed that there was an inverse correlation between the levels of HuR in the cell cytoplasm and the inhibitory activity of the h1ARE (16Carlsson A. Schwartz S. Arch. Virol. 2000; 145: 491-503Google Scholar), suggesting that the presence of high levels of HuR in the cytoplasm antagonizes the inhibitory effect of the h1ARE, whereas a primarily nuclear association of HuR is associated with inhibition of HPV-1 late gene expression. Interestingly, the HIV Rev and RRE, and the SRV-1 CTE can overcome the inhibition (9Tan W. Schwartz S. J. Virol. 1995; 69: 2932-2945Google Scholar), suggesting that the h1ARE traps the HPV-1 late mRNAs in the nucleus and that this may lead to rapid mRNA degradation. Interestingly, the inhibitory effect of the h1ARE was greater at the protein level than at the mRNA level, suggesting that the h1ARE also inhibited the utilization of the mRNA. Here we present results of a mutational analysis of the h1ARE, and we provide evidence that the h1ARE inhibits mRNA translation. All the eukaryotic expression plasmids containing the h1ARE mutants are derived from pΔKX (11Zhao C. Tan W. Sokolowski M. Schwartz S. J. Virol. 1996; 70: 3659-3667Google Scholar) (Fig. 1 B). Oligonucleotides were annealed and cloned into pΔKX digested with KpnI and XbaI, which resulted in the insertion in pΔΚX of the wild type and mutant h1ARE sequences displayed in the figures. All mutants were sequenced. pCMVlacZ was constructed by replacing the chloramphenicol acetyltransferase (CAT) gene in pΔKX by the lacZ gene. To generate pCCΔ that lacks the h1ARE and its derivatives, a fragment containing the first 75 nts that are transcribed from the CMV promoter, the CAT gene and HPV-1 late 3′ UTR sequences spanning nt 7184–7447 was first amplified from pΔKX using oligonucleotides HCMV-S (5′-CGAGCTCTCAGATCGCCTGGAGACGCC-3′) and XhoIpA (9Tan W. Schwartz S. J. Virol. 1995; 69: 2932-2945Google Scholar), introducing unique 5′-SacI and 3′-XhoI sites. The PCR fragment was ligated to pCR2.1 (Invitrogen), downstream of the T7 RNA polymerase promoter, generating pΔ. pΔ was digested withApaI and EcoRV, filled in with T4 DNA polymerase, and religated to remove polylinker sequences between the T7 promoter and the cloned PCR fragment, generating pCCΔ. pCCΔ also lacks the downstream polylinker sequences between KpnI andXhoI, which are replaced by a unique NsiI site. To generate pCC, a PCR fragment was first amplified from pCCKH1 (11Zhao C. Tan W. Sokolowski M. Schwartz S. J. Virol. 1996; 70: 3659-3667Google Scholar) (Fig. 1 B) by using oligonucleotides HCMV-S (see above) andXhoIpA (9Tan W. Schwartz S. J. Virol. 1995; 69: 2932-2945Google Scholar) followed by insertion into pCR2.1 (Invitrogen). This step was followed by transfer of a SacI-XhoI fragment from the pCR2.1-based intermediate to pCCΔ, resulting in pCC. To generate pCC(A), which contains the h1ARE in the antisense orientation, a PCR fragment amplified from pCCKH1 by using oligonucleotides H1KPNI-A (5′-GGTACCGAACACTACTGTAGAATATGTG-3′) and H1XBA-S (5′-TCTAGAGCTACTAGTTCCACCACAAAGCGC-3′) was inserted intoEcoRV-digested pBlueScript (Stratagene) generating pKS-H1XK. This was followed by transfer of aKpnI-XbaI fragment from pKS-H1XK to pCC, resulting in pCC(A). Plasmids pCCXB, pCCAUM/UM, pCCAUM, pCCUM, pCCB2, and pCCC1 were generated by transfer of KpnI-XbaI fragments from the previously described plasmids pKSXB, pKSAUM/UM, pKSAUM, pKSUM, pKSB2, and pKSC1 (10Sokolowski M. Zhao C. Tan W. Schwartz S. Oncogene. 1997; 15: 2303-2319Google Scholar), respectively, to pCC digested with KpnI and XbaI. pCMVhGH has been described previously (17Ramirez-Solis R. Resendez-Perez D. Alvidrez-Quihui L. Rincon-Limas D. Varela-Martinez R. Martinez-Rodriguez H. Barrera-Saldana H. Gene. 1990; 87: 291-294Google Scholar). Radiolabeled RNAs for UV cross-inking were produced from pKSXB, pKSAUM/UM, pKSB2, and pKSC1 (10Sokolowski M. Zhao C. Tan W. Schwartz S. Oncogene. 1997; 15: 2303-2319Google Scholar). DNA transfections were performed with FuGENE (Roche Molecular Biochemicals) as described previously (10Sokolowski M. Zhao C. Tan W. Schwartz S. Oncogene. 1997; 15: 2303-2319Google Scholar). Transfections were performed in triplicates, and mean values and standard deviations are displayed in the figures. For RNA analysis triplicate samples were pooled and analyzed by Northern blot. Each plasmid was analyzed in at least three independent transfection experiments. RNA synthesis and transfections were performed as described previously (18Wiklund L. Spångberg K. Goobar-Larsson L. Schwartz S. J. Hum. Virol. 2001; 4: 74-84Google Scholar). To generate RNAs with a poly(A) tail of fixed length, two PCR fragments were first amplified with the following primer pairs: T7CATS (5′-GTAATACGACTCACTATAGGGTACTGCGATGAGTGGCAGGG-3′) and HPV1ANTIPA (5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCACACTTGTGTATAATGCACCGG-3′) (which encodes a A60 tail) or T7CATS and HPV1ALW (5′-CACACTTGTGTATAATGCACCGG-3′). The PCR fragments that were generated from plasmid pΔKX or pΔKX containing the 57-nt h1ARE fragment using the two primer pairs described above were gel-purified and used for synthesis of capped RNAs with and without the A60tail. Plasmids or RNA encoding CAT, human growth hormone (hGH), or β-galactosidase were in transfections to the transfection To CAT protein cells were as described previously M. Tan W. Jellne M. Schwartz S. J. Virol. 1998; 72: 1504-1515Google Scholar) at For DNA cells were at The levels of CAT, hGH, and β-galactosidase proteins were using CAT, and β-galactosidase (Roche Molecular All CAT were to the protein of the cell as by the RNA and primer were performed as described previously M. Tan W. Jellne M. Schwartz S. J. Virol. 1998; 72: 1504-1515Google Scholar). To Northern RNA was at as previously described (18Wiklund L. Spångberg K. Goobar-Larsson L. Schwartz S. J. Hum. Virol. 2001; 4: 74-84Google Scholar). Northern analysis was performed as described (10Sokolowski M. Zhao C. Tan W. Schwartz S. Oncogene. 1997; 15: 2303-2319Google Scholar). of or RNA was on containing followed by transfer to a and of the DNA was performed using a to the UV and synthesis of RNA were performed as previously described (11Zhao C. Tan W. Sokolowski M. Schwartz S. J. Virol. 1996; 70: 3659-3667Google Scholar, K. L. Schwartz S. Virology. 2000; Scholar). and were on the To the HPV-1 late 3′ UTR element named h1ARE the 57-nt minimal element (Fig. 1 B) or a functionally inactive mutant (Fig. 1 B) was inserted between the CAT by the human promoter, and the late HPV-1 poly(A) resulting in and B). we used pΔKX (which lacks the of the late 3′ pCCKH1 (which contains the late 3′ and (which contains the region of the late 3′ UTR containing the XB sequence in antisense cells were in the CAT was in each and mean values and standard deviations are displayed in the figures. pCMVlacZ was as an in and the was than between the samples in the For RNA from the were pooled and to RNA and Northern Analysis of CAT in transfections of cells and of between and that the between and were and demonstrated that the 57-nt XB contains the inhibitory sequence not The mRNAs containing the 57-nt XB fragment have a half-life In with the presence of the h1ARE on the mRNA also results in a of mRNA not We that and used for of the AU-rich The XB sequence contains two motifs and three motifs that were (Fig. These motifs were in (Fig. To motifs were for the motifs were by (Fig. there was a in CAT RNA and protein levels (Fig. for each To a on the of each were in the motifs (Fig. The results that expression levels were as motifs were (Fig. and that each motif to in four of the motifs as high expression levels as in five motifs (Fig. and To the motif to was in (Fig. resulting in The results that RNA and protein levels than These levels were to produced from (Fig. and that also the motif to Interestingly, the effect was greater at the protein level than at the RNA for analyzed mutants (Fig. We that five motifs to inhibition in an additive HPV-1 sequences inserted into the plasmid are The sequence motifs in XB and the plasmids were into cells in and protein was for CAT followed by of the three samples and RNA plasmid was in CAT levels were by CAT ELISA, and the levels are displayed as of CAT produced from values and standard deviations are the RNA samples were to Northern followed by The RNA levels are displayed as of RNA with RNAs from triplicate experiments are To the minimal inhibitory sequence of the were in motifs I and II in the the or the the were not (Fig. and in the the not affect the inhibitory activity of motifs (Fig. and that the inhibitory motif was The two were by a sequence (Fig. this sequence with four not affect the inhibitory activity of the h1ARE (Fig. and that this sequence not to the inhibitory To the minimal inhibitory sequence of the the nucleotides the were The results that in both the the and the the resulted in CAT protein and RNA expression levels (Fig. and two in the sequence with two the whereas in the the the effect on the inhibitory activity (Fig. and the minimal motif was UAUUUUUAU. The h1ARE may into the region with the two motifs and the region with the three motifs The inhibitory than the XB as inhibited CAT and reduced mRNA levels to the same as the region (Fig. and Two or two were as inhibitory as the XB (Fig. that type of motif or for the two motifs were to two motifs by insertion of two in each resulting in or the three motifs were to contain three as in the resulting inhibitory elements were as inhibitory as the wild type h1ARE (Fig. and by mRNA levels and protein was also greater at the protein level than at the mRNA the results demonstrated that both motifs in a HPV-1 sequences inserted into the plasmid are The sequence motifs in XB and the plasmids were into cells in and protein was for CAT followed by of the three samples and RNA plasmid was in CAT levels were in CAT ELISA, and the levels are displayed as of CAT produced from values and standard deviations are the RNA samples were to Northern followed by The RNA levels are displayed as of RNA with RNAs from triplicate experiments are For mutants that inhibitory we observed that the inhibitory effect was greater at the protein level than at the mRNA level (see and and and In the mRNAs that the h1ARE, or mutants were by the translation than mRNAs the h1ARE or mRNAs containing functionally inactive To the effect on protein and the effect on the mRNA XB sequences were inserted into the plasmid resulting in and and the inhibition at the protein and mRNA levels was the of on the mRNA. can there is a in CAT protein and mRNA levels for inserted of the XB fragment (Fig. and and the inhibition was greater at the protein level than at the RNA level and of the in a a of the inhibitory activity of XB sequence at protein and RNA levels (Fig. the of plasmid The results demonstrated that XB a greater effect at the protein level than at the RNA level (Fig. from we that CAT protein levels were reduced XB and RNA levels XB in the of the mRNAs with (Fig. We that in to the effect on mRNA mRNAs the h1ARE are for translation. J. 1995; Scholar, Sci. 1995; Scholar) the inhibition of translation observed To the poly(A) tail of the mRNAs shown in were to with the shown in A followed by Northern using a located downstream of the The results that mRNAs poly(A) of that inhibition of translation was not a of To the effect of the h1ARE on translation we replaced the CMV promoter with the T7 promoter and the and with the in the and pCCKH1 (Fig. 1 B) used previously to the These resulted in and pCC that were and used as for in synthesis of capped and and mRNAs as described These mRNAs the same sequences as the mRNAs produced by the pCCKH1 and plasmids in the of cells (Fig. 1 B). and and mRNAs were in into cells by and the CAT levels produced at were by using a CAT The results that mRNAs that contain the h1ARE in produced CAT protein levels than the which contain the h1ARE in antisense (Fig. mRNAs in samples as an produced protein levels (Fig. of cells were at and the levels of CAT protein were and (Fig. B). Two of the results shown in were the mRNAs with the HPV-1 late 3′ UTR were CAT protein or the mRNAs were not for of translation as a of a inhibition of translation, by factors binding to the To the of the mRNAs were reduced by the presence of the HPV-1 late 3′ UTR, and mRNA levels at and were by primer (Fig. and mRNAs at as at and (Fig. also of and mRNA at not The mRNA were to for mRNAs and for A of experiments were performed using mRNA. These experiments that RNAs were not to the of the cell and that the of the mRNAs are by the translation not We that the inhibitory effect on the by the HPV-1 late 3′ UTR, was not a of reduced mRNA half-life in the cytoplasm and that the HPV-1 late 3′ UTR by mRNA translation. In addition, the previously observed effect on the mRNA half-life was dependent on a nuclear of the mRNA and was not in mRNAs were into To that the 57-nt minimal XB sequence containing the two and the three motifs inhibited translation in the RNA transfection and mRNAs were into cells in The mRNAs produced levels of CAT than the mRNAs (Fig. B). protein levels produced from the mRNAs were in both samples (Fig. B). capped and and mRNAs were into and the levels of CAT protein produced at and were mRNAs were in samples as an C that mRNAs produced CAT protein levels than the mRNAs as whereas and mRNAs showed intermediate inhibition of CAT protein with and mRNAs (Fig. protein in the cell was in samples at each (Fig. The CAT protein at the for four which the CAT protein levels This is the the mRNAs have The mRNA in the cells with and mRNA were by primer on RNA from the D that the levels of and mRNAs were at 1 and and with a to that observed for and mRNAs D The results that the h1ARE primarily by mRNA translation in the RNA transfection experiments performed and demonstrated that both the two motifs and the three motifs inhibit translation. The poly(A) tail is for mRNA translation A. 1997; Scholar). We to the HPV-1 AU-rich element with the function of the poly(A) We and capped and mRNAs and the levels of CAT protein in the cells at A that the which contains the HPV-1 AU-rich element in orientation, produced CAT protein levels than which contains the HPV-1 AU-rich element in antisense In the presence of the h1ARE on the mRNA not inhibit CAT (Fig. The effect of the to the capped which contains the h1ARE, was with to the mRNA. of mRNA was in samples (Fig. results were in a using the XB sequence and the inactive mutant (Fig. a between the 57-nt XB sequence and the poly(A) the results demonstrated that inhibition of CAT by the h1ARE was dependent on a the results showed that the effect on translation by the tail on translation was by the the inhibitory effect of the AU-rich RNA element on translation To the h1ARE of the in mRNAs that were into the mRNAs with a poly(A) tail of fixed were into the cells and the of the poly(A) tail was at 1 or by Northern mRNAs with and without the h1ARE or poly(A) tail were The results that the mRNAs were not at 1 (Fig. In addition, analysis of the mRNAs containing the h1ARE at showed that the poly(A) (Fig. inhibition of translation was not by that the h1ARE inhibited the function of the poly(A) was to that the h1ARE, or h1ARE binding directly with the poly(A)-binding protein We bind directly to the XB was UV to XB RNA or UV of that to the XB RNA but to the RNA (Fig. not bind to an RNA derived from the L1 region in HPV-16 (Fig. The protein was used as and interacted with as (13Sokolowski M. Furneaux H. Schwartz S. J. Virol. 1999; 73: 1080-1091Google Scholar), and not bind to of the RNA sequences To that the binding of to the XB sequence was sequence a was The XB RNA with the RNA for binding to whereas the HPV-16 RNA not that the with XB was Analysis of the mutants and that were shown to inhibit translation to a also interacted with the (Fig. binding of to the mutants and with their inhibitory effect on translation. The mutational analysis that the HPV-1 AU-rich element of the two and the three on the to the that the motif was for mRNA but not and that were for Scholar). This is in with the results on a mutational analysis and sequence that the minimal motif may In on the a analysis of the to the that the and not the was the motif 1995; 15: Scholar). These also that mRNAs containing of the are than mRNAs with Using the HPV-1 ARE, we also that of the motif were inhibitory than in of the not affect its to mRNA In the of the HPV-1 ARE, the was the motif with inhibitory that the sequence may affect the of an AU-rich In to the HPV-1 AU-rich element that contains two and three the element on the mRNA contains in three of motifs the same effect as the element S. C. J. Scholar). The HPV-1 AU-rich element contains five and we show that five to inhibition in an additive manner. in three motifs in the resulted in mRNA Scholar). that of the motifs are for function of the AU-rich RNA We have previously shown that the mRNA half-life is reduced by the presence of the h1ARE using DNA transfections in which the mRNAs are in the cell (9Tan W. Schwartz S. J. Virol. 1995; 69: 2932-2945Google Scholar, 10Sokolowski M. Zhao C. Tan W. Schwartz S. Oncogene. 1997; 15: 2303-2319Google Scholar). In the same mRNAs were directly into the cytoplasm as described the there was effect on the mRNA half-life by the These results that a nuclear of the mRNAs is for rapid mRNA suggesting that the mRNAs are in the or with nuclear factors that mRNA degradation. Recent results on the mRNA showed that HuR nuclear of mRNAs 2001; Scholar). HuR also the mRNA suggesting that nuclear and mRNA are and that mRNA to degradation. In a we that the mRNA Rev in with or the SRV-1 CTE overcome the inhibitory effect of the h1ARE (9Tan W. Schwartz S. J. Virol. 1995; 69: 2932-2945Google Scholar), that of the mRNAs an inhibition and results in high expression. These results also demonstrated that the h1ARE has an inhibitory function in the in to its inhibitory effect of translation in the cytoplasm described The h1ARE binds and may inhibit the between and of the mRNA and the of on the mRNA. This is not without was shown that the mRNA protein interacts with and that with the for the M. J. D. J. 1998; Scholar). of the or lead to to the poly(A) tail and by the poly(A) resulting in C. M. S. 2001; Scholar). was not observed has also been that the proteins are in mRNA translation directly Sci. 2001; Scholar, J. Dev. 1999; Scholar). of HuR protein from the nucleus to the cytoplasm is associated with protein from mRNAs containing with HuR binding A. Proc. Natl. Acad. Sci. U. S. A. 2000; Scholar, K. K. K. H. 1999; 4: Scholar). HuR may to and of Rev protein that the inhibitory effect of the h1ARE in cells also of mRNAs D.M. Felber B.K. Pavlakis G.N. Scholar, Dev. Scholar). proteins as HuR lead mRNAs a that nuclear and The role of the h1ARE, and the in the HPV-1 to We are to H. Furneaux for J. for H. for and A. for reading the