The Role of Polyadenylation in Human Papillomavirus Type 16 Late Gene Expression

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 4

DANIEL ÖBERG

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“It is better to burn out than to fade away” The Kurgan, Highlander

"What remains?" cried Ivanhoe; "Glory, maiden, glory! which gilds our sep- ulchre and embalms our name." Ivanhoe to Rebecca, who questions the value of chivalry and has asked what remains for knights when death takes them. (Chapter XXIX)

"Chivalry!-why, maiden, she is the nurse of pure and high affection-the stay of the oppressed, the redresser of grievances, the curb of the power of the tyrant-Nobility were but an empty name without her, and liberty finds the best protection in her lance and her sword." Ivanhoe explains to Rebecca the virtues of chivalry. (Chapter XXIX)

For my mother, to whom I owe everything Members of the committee

Opponent Assistant Prof. Sheila Graham Institute of Virology University of Glasgow

Members of the committee Chairman, Prof. Göran Akusjärvi IMBIM Uppsala University

Prof. Gerhart Wagner ICM Uppsala University

Assistant Prof. Öjar Melefors MTC Karolinska Institutet Stockholm List of manuscripts

This thesis is based on the following manuscripts, which in the text are referred by their roman numerals

Paper I Collier, B., D. Oberg, X. Zhao, and S. Schwartz. 2002. Specific inactivation of inhibitory sequences in the 5' end of the human papillomavirus type 16 L1 open reading frame results in production of high levels of L1 protein in human epithelial cells. J Virol 76:2739-52.

Paper II Oberg, D., B. Collier, X. Zhao, and S. Schwartz. 2003. Mutational inactivation of two distinct negative RNA elements in the human papillomavirus type 16 L2 coding region induces production of high levels of L2 in human cells. J Virol 77:11674-84.

Paper III Oberg, D., J. Fay, H. Lambkin, S. Schwartz. 2005. A downstream polyadenylation element in human papillomavirus type 16 encodes multiple GGG-motifs and interacts with hnRNP H. J Virol Submitted.

Reprints were made with permission from the publishers Related manuscripts

Rollman E, Arnheim L, Collier B, Oberg D, Hall H, Klingstrom J, Dill- ner J, Pastrana DV, Buck CB, Hinkula J, Wahren B, Schwartz S. HPV- 16 L1 genes with inactivated negative RNA elements induce potent immune responses. Virology. 2004 Apr 25;322(1):182-9.

Zhao X, Oberg D, Rush M, Fay J, Lambkin H, and S. Schwartz. 2005. A 57-nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hFip1, CstF-64, hnRNP C1/C2 and PTB. J Virol, In press. Contents

1 Introduction...... 11 1.1 Virology ...... 11 1.2 History of papillomaviruses...... 11 1.3 Human papillomaviruses and cancer...... 12 1.4 Human papillomavirus structure ...... 13 1.5 Papillomavirus life cycle ...... 15 1.6 Human papillomaviruses and gene expression...... 16 1.7 Long control-, early- and late region genomic structure and function. 18 1.7.1 LCR ...... 18 1.7.2 The early region ...... 18 1.7.2.1 E2 and E1 ...... 19 1.7.2.2 E6 and E7 ...... 20 1.7.2.3 E5 function and immune defence ...... 22 1.7.2.4 E4...... 22 1.7.3 The late region...... 23 1.8 Gene expression...... 23 1.8.1 Capping ...... 24 1.8.2 Polyadenylation...... 24 1.8.3 Splicing...... 25 1.8.4 The exon definition model...... 28 1.8.5 Linking mRNA processing and transcription ...... 29 1.8.6 Nuclear export of mRNA...... 29 1.8.7 Translation...... 31 1.8.8 Regulation of mRNA polyadenylation...... 33 1.8.9 Papillomavirus and late polyadenylation ...... 36 1.8.10 Papillomavirus early polyadenylation...... 38 1.8.11 Negative elements...... 40 2 Introduction to papers...... 42 2.1 Paper I (45)...... 42 4.2 Paper II (222) ...... 44 2.3 Paper III (223)...... 45 3. Future perspectives ...... 47 4. Populärvetenskaplig sammanfattning (In Swedish) ...... 49 5 Acknowledgement ...... 51 6 References...... 53 Abbreviations

3’ss 3’splice site, splice acceptor 5’ss 5’splice site, splice donor BPV Bovine papillomavirus C/EBP CCAAT/Enhancer binding protein CBC Cap binding complex CDK Cyklin dependent kinase CF Cleavage factor CMV Cytomegalovirus CPSF Cleavage and polyadenylation specific factor CStF Cleavage stimulatory factor CTD C-terminal domain CTE Constitutive transport element CTL Cytotoxic lymphocyte DSE Downstream element E6AP E6-associated protein EGFR Epidermal growth factor receptor ESS Exonic splicing silencer HDAC Histone deacetylase HIV Human immunodeficiency virus HLA Human leukocyte antigen hnRNP heterogeneous ribonucleo protein HPV Human papillomavirus HSV Herpes simplex virus IgM-mem IgM secretory polyadenylation signal IgM-sec IgM membrane polyadenylation signal ISE Intronic splicing enhancer LCR Long control region MAR Matrix attachment region MHC Major histocompability complex NES Nuclear export signal NK Natural killer cell NMD Nonsense mediated decay NPC Nuclear pore complex NRE Negative regulatory element ORF Open reading frame PABP Poly(A) binding protein pAE Early polyadenylation signal pAL Late polyadenylation signal PAP Poly(A)polymerase POD Promyelocytic leukaemia oncogenic domain PPT Polypyrimidine tract pRB Retinoblastoma protein RRE Rev responsive element RT-PCR Real time PCR snRNP small nuclear ribonucleo protein SV40 Simian virus 40 SVLPA Simian virus 40 late polyadenylation signal USE Upstream element 1 Introduction

1.1 Virology The elucidation of viruses as infectious agents started in the late 19th cen- tury. When the microbial organisms bacteria and fungi were filtered away from fluid shown to transmit certain diseases, the filtered fluid was still found potent to cause the infection. The name “virus”, which is the Latin word for “slimy liquid” or “poison”, was assigned to this invisible agent (89). The 1930s revolutionised the field of virology with the invention of the electron microscope. Electron micrographs on tobacco mosaic virus (TMV) visualised a virus particle for the first time. Other important findings were bacteriophages and plaque assays, chemical dissections of purified viruses and the assembled knowledge on mutated TMV variants. These discoveries revealed viruses as particles consisting of proteins and nucleic acids containing genetic information. The advances in the field of virology have shown that viruses are strictly parasitic, meaning that they need a host in which to propagate. Soon it became apparent that much information could be gained about the host systems infected by viruses. In particular the field of gene regulation has benefited greatly from the study of viruses and their host interactions. Many well established cellular mechanisms have been elucidated from the study of certain viruses, for example, messenger RNA regulation (Adenovirus) (18), regulation of transcription and transcription factors (SV40) (78), polyadenylation (poxvirus) (149) and translation (poliovirus) (233). During the last two decades, papillomavirus (PV) has been used more frequently as a model system for gene regulation and virally induced transformation. This virus has the advantage of having a relatively small genome where all known mechanisms of mammalian gene regulation are utilised.

1.2 History of papillomaviruses Warts on humans have been recognised as long as any source can date back. Studies on different human PV (HPVs) have indicated that the virus has evolved and followed the migration and settlement of man throughout history. As man wandered out from Africa about 52,000 years ago (138) HPVs seem to have followed (36, 127, 227). Research on HPVs dates back to 1907

11 when Cuiffo transmitted human warts upon intradermal inoculation through cell-free extracts (55). The first model of papillomavirus carcinogenesis was demonstrated in 1933 by Shope and Hurst. They showed that transmission of warts from wild cottontail rabbits caused ulcerative carcinomas on domestic rabbits (265). The morphology of HPV particles was visualised in 1949 using electron microscopy (286) (Fig. 1). However, it was not until 30 years ago that researchers started to suggest that human PV (HPV) could have a role in cancer (351, 352, 357). The International Committee on the taxon- omy of Viruses (ICTV) recently recognized papillomaviridae as a separate virus family and also updated the classification system (66). There are now more than 100 HPV types identified (354). HPV has a strict tropism for epithelial cells. Depending on the type of epithelia HPVs infect, they are divided into cutaneous (for example, HPV-1, -2, -5) or mucosal types (for example, HPV-6, -11, -16, -18).

Figure 1. Human papillomavirus capsid structure. Icosahedral structure with formation of pentamers (left). Assembled virion (right).

1.3 Human papillomaviruses and cancer About one third of all HPVs are sexually transmitted. It has been estimated that two-thirds of the individuals who have sexual relations with an infected partner will become infected. However, most infections are asymptomatic and are commonly cleared within a year (129). The first HPV types to be isolated directly from cancer biopsies of the cervix were HPV-16 and HPV- 18 (21, 77). The genital types can be divided into low- (for example, HPV-6, 11) and high-risk types (for example, HPV-16, -18, -31) depending on their association with cancer. The low-risk types induce benign lesions with low risk for progression to malignancy, whereas high-risk HPV types are associated with the development of malignant lesions such as squamous cell carcinoma or, less commonly, adenocarcinoma of the cervix (353). In the majority of the HPV-associated cancers the viral DNA has been integrated into the genome of the host cell (262, 336). This not only allows the viral oncoproteins E6 and E7 to remain expressed (262) but also upregulates their expression due to loss of the viral transcriptional regulator gene E2 (139, 140). The

12 tropism of mucosal types is not limited to the genital tract, since about 25% of the oropharynx cancers contain DNA from high-risk HPV types (355). Persistent infection, co-infection with HIV, immunosuppression, bad nutri- tion, cigarette smoking and a large number of sexual partners in a lifetime are some risk-factors for HPV mediated tumour development (160). Cervical cancer is the second most common cause of cancer in women in the world and accounts for 6% of all malignancies in women, with the mean age of development being 52 years (229, 230). Annually, about 470,000 women around the world are diagnosed with cervical cancer, leading to 230,000 deaths per year (229, 230). In these cases, HPV DNA sequences have been found in approximately 99% of the cervical smears, suggesting them to be the causative agents of these malignancies (318). Cervical cancer is most prevalent in developing countries where both information and controls are scarce. Since the Pap smear was introduced as a diagnostic test in the United States the number of cervical cancer cases has declined by over 80% (270). The most common sexually transmitted HPV type is the high risk type HPV-16, present in more than 50% of the cases (356). Thus, for elucidation of HPV’s role in cancer it is extremely important to study HPV- 16 life infectious cycle and gene regulation.

P97 P670 pAL LCR

Late Early Region Region

3ʻeUTR / pAE Figure 2. Papillomavirus episome. The double-stranded DNA genome consists of the long control region (LCR), an early region, the 3’untranslated region (3’eUTR) and a late region. Origin of replication and early promoter (P97) are situated in LCR. The early polyadenylation site (pAE) is located in the 3’eUTR, between the early and the late region. The late promoter (P670) is located in the beginning of the early region and the late region is followed by the late polyadenylation site (pAL).

1.4 Human papillomavirus structure The HPV possesses an icosahedral capsid with a diameter of 55-60nm (Fig. 1). The complete virus is composed of the major, L1, and the minor structural protein, L2, in addition to the viral DNA genome coated by cellular histones (12). No other molecules have been found integrated in the infec-

13 tious virus. The genome is circular, double stranded and approximately 8,000 bases long. It can be divided into a long control region (LCR), an early region, an early 3’untranslated region (3’eUTR) and a late region (Fig. 2). The LCR contains the late 3’UTR (3’lateUTR) and most of the cis- responsive elements influencing viral transcription and replication. The 3’eUTR contains the differentiation dependent early polyadenylation site (pAE). The LCR and 3’eUTR are the only non-coding sequences in the genome. The early region encodes the early (E) proteins named E1, E2, E4, E5, E6 and E7 (Fig. 3A). These proteins are responsible for the establishment and maintenance of the viral infection. The late region encodes the structural proteins, L2 and L1 (Fig. 3A).

A) P97 P670 pAE pAL E2 L1 E6 E7 E1 E4 E5 L2

E4 E4 L1 3ʼss 5ʼss 3ʼss LCR Early Region Late Region 3ʻeUTR 5‘ss 3‘ss

B) Early An E6/E7 An E2 An

Late An L2/L1 An L1 An E1/E4 An Figure 3. The HPV-16 genome, shown linearised above (A), consists of an early region, containing the early genes E1, E2, E4, E5, E6 and E7, and a late region, containing the late genes L1 and L2. Replication is controlled by elements in the long control region, LCR. Transcription starts from the constitutively active early promoter, P97, and the differentiation dependent promoter, P670. Polyadenylation occurs at the early polyadenylation site in 3’eUTR, pAE, or the late polyadenylation site, pAL. (B) The full-length early and late transcripts, represented by thicker lines, are alternatively processed to generate polycistronic mRNA coding for the different viral proteins. Only a few examples of the many different possible mRNA species are shown.

The viral capsid consists of 360 L1 but only about 12 L2 molecules. L1 alone has been shown to be able to assemble into pentamers which mul- timerise to form procapsids, virus-like particles (VLPs). This process is much less efficient than when L2 is present. L2 is thought to be integrated at the assembly stage into the capsid at each of its 12 vertices (309). This

14 slightly reconfigures the L1 pentamers so the capsid is formed easier (Fig. 1).

1.5 Papillomavirus life cycle It is thought that papillomavirus infects through sites of wound- ing/microlesions where they gain entry to basal epithelial cells (Fig. 4.).

Terminally differentiated Late layers Stage Suprabasal layers Early Basal layer Stage

Basement membrane Figure 4. Epithelium and papillomavirus life cycle. When a basal cell divides, a daughter cell will migrate upwards in the cell layers and start to differentiate. The cell will continue migrate upwards until it is terminally differentiated whereby it is sloughed off. Papillomavirus infects the basal cells of the epithelium (inwards arrow) through an abrasion/microlesion. It establishes infection but no viruses will be produced (early stage). Late in cell differentiation (Late stage), the viral structural genes will be expressed and infectious virus assembled. Viruses are released as cells are sloughed off (outwards arrow).

In these cells, PV genomes establish themselves as nuclear episomes at an average of 50 copies per cell (174). The viral DNA is replicated in Cairns structures (theta mode) in synchrony with the cellular DNA replication (288). That is, the replication is reinitiated for every round of copying. The establishment and maintenance of PV genomes are associated with expression of early PV transcripts that encode the oncoproteins E5, E6 and E7, and the replication proteins E1 and E2. After cell division, daughter cells leave the basal layer, migrate towards the suprabasal regions and begin to differentiate. Infected cells, in contrast to those uninfected, do not exit the cell cycle and start to differentiate as soon as they detach from the basal membrane, but are induced to proliferate by the viral oncoproteins E6 and E7 (In low-risk HPV infection, this results in clonal expansion of the infected cells and thereby formation of a papilloma, a wart). This phase is called the early or non-productive stage of the viral life cycle because production of progeny virus does not occur. The presence of E7 protein leads to the characteristic

15 retention of nuclei throughout all layers of infected epithelia (40). E6 and E7 have also been shown to be necessary for the maintenance of extrachromo- somal forms of HPV in undifferentiated cells (305). Following the early stage, a subset of daughter cells will undergo terminal differentiation as they enter suprabasal layers (74). This results in amplification of the viral genomes to thousands of copies per cell (171). This amplification mode appears to occur through rolling circle replication, where one initiation event leads to the generation of multiple daughter molecules (91). This is followed by the synthesis of the E4 protein and the L2 and L1 capsid proteins, resulting in the assembly of infectious virions. As the upper layer of the epithelium is shed, virions are released into the environment to infect new individuals or new sites in the same host (170). The E4 protein is suggested to help counteract the effects of the E7 protein, facilitating a productive life cycle (37, 90) and also to facilitate viral egress (73). The oncogenicity of HPVs arises from persistence of the viral infection, which in turn depends on several factors, including the genetic back- ground of the host (25, 167), environmental cofactors (35) and the ability of the virus to avoid immune clearance (99). The increased persistence and higher incidence of neoplastic progression of HPV infections in hosts with cell-mediated immune deficiencies (122, 123) demonstrates the importance of the immune system in the clinical outcome of HPV disease. This suggests that the virus subverts the immune defence. HPVs affect the immune response indirectly via the nature of the virus life cycle (98), allowing the virus to evade detection by the immune system as the progeny production and highest gene expression occurs within the terminally differentiated layers where immune-surveillance is thought to be limited. They also affect the immune response by direct interference with the host antiviral immune mechanisms, including the IFN response and MHC class I antigen presentation to cytotoxic lymphocytes (CTLs), mainly by the viral E5 protein (9). The numerous ways of MHC class I interference developed by viruses in general demonstrates the importance of this mechanism in viral clearance (337). Despite different approaches, the outcome is failure of the infected cells to efficiently present viral peptides to effector CTLs, with avoidance of detection and a subsequent destruction of the infected cell as result.

1.6 Human papillomaviruses and gene expression There are two major promoters controlling viral transcription, the early [P97 in HPV-16 (310)] and the late [P670 in HPV-16 (112)]. The early promoter produces early transcripts polyadenylated at pAE potentially coding for E1, E2, E4, E5, E6 and E7. Transcription from the late promoter generates late transcripts that encode E1, E2, E4 and E5 but can also contain the two late open reading frames (ORFs), L2 and L1, due to downregulation of pAE (See

16 1.8.10 “Papillomavirus and early polyadenylation”) and loss of inhibition at the pAL (See 1.8.9 “Papillomavirus late polyadenylation”). The transcripts produced are alternatively spliced and polyadenylated, generating several different polycistronic (4) messenger RNA (mRNA) (Fig 3B). Translation of polycictronic mRNAs is thought to occur by leaky scanning. In this process, a suboptimal translational start site would sometimes allow the ribosome to continue down the transcript scanning for other ORFs. The early promoter is activated by tissue specific transcription factors (52, 220, 221) and is regulated by levels of the E2 protein, through E2 binding sites in the early region (282, 287). Low levels have an activating effect whereas high levels sterically block the binding of cellular transcription factors (282, 287). E2 expressed from the early promoter produces a negative feedback loop on its own transcription activity during early stages of infection. In the late stages of infection the late promoter is switched on resulting in increased expression of E2 and E1. This is due to the fact that E2 protein does not repress the late promoter (163). It has been suggested that differentiation-specific cellular factors control the late promoter due to the tight correlation of viral late gene expression and epithelial differentiation. Interestingly, Grassman et al (112) reported the presence of potential CCAAT/enhancer binding protein (C/EBP) binding sites around the late promoters of HPV-6, -11, -16 and –31. C/EBP is preferentially expressed in terminally differentiated cells of various tissues, including skin, and plays a role in activating differentiation-specific genes (141, 275, 311). In hyper- plastic epidermis and squamous papillomas, C/EBP is present in all suprabasal layers, whereas its expression seems to be reduced in squamous cell carcinomas (224). Recently it was shown that the E2 proteins of HPV-8, -16 and –18 cooperate with C/EBP in transcriptional activation and that this synergism was independent of an E2 binding site (116). This suggests that C/EBP might be at least one of the factors upregulating the late promoter in differentiated cells. In contrast, C/EBP has been shown to inhibit the early promoter in HPV-11 (319, 342) and –16 (269). Speculatively, some of the gene family members of C/EBP give rise to smaller species through alternative translation. These are without their transactivation domain, thereby acting as dominant negative inhibitors to the full size C/EBP (244). Perhaps amounts of the smaller inhibiting species are prevalent during early infection, keeping viral gene expression from the early promoter at a low level and at the same time helping to silence the late promoter. In addition, it has been shown that methylation of E2 binding sites inhibits E2 binding (304). Recently, the LCR of HPV-16 was shown to be selectively hypermethylated in poorly differentiated basal-like cells and, in contrast, hypomethylated in highly differentiated cell populations (155). This presents an additional way of ensuring a low copy number in undifferentiated cells, since both translation and transcription are dependent on E2 binding in the LCR.

17 The alternative usage of splice signals and polyadenylation sites is controlled in a differentiation dependent manner. An optimal ratio of the different viral proteins is important for a productive viral infection. The early polyadenylation site (pAE) is downregulated in the later stages of cellular differentiation allowing the presence of the late transcripts, probably coincident with a release of inhibition at the late polyadenylation site (pAL) (See section 1.8.9 and 1.8.10). The most abundant transcript in late infection is E1/E4. It is spliced to encode the first 5 amino acids from the E1 ORF (41, 76, 214). The full-length late RNA, encoding the L2 protein, needs to be spliced to generate the L1 RNA. The regulation of this splice reaction is extremely important for two reasons: to achieve the correct ratio between the L2 and L1 proteins for efficient viral assembly and the strong immunogenicity (281) of the late proteins. Processing to generate the L1 transcript includes splicing to remove the pAE. If this occurs prematurely, the production of L1 protein could activate an immune response lethal to the infected cell.

1.7 Long control-, early- and late region genomic structure and function

1.7.1 LCR The HPV LCR is about 850 bp (Fig. 3A) and it has been shown to contain numerous elements. The early promoter, P97 in HPV-16 (310), also called the E6 promoter, has epithelial-specific enhancers with binding sites for several transcriptional activators and repressors contributing to the tissue tropism (52, 220, 221). The origin of replication (OriR) contains E2 binding sites for replication of the genome. In addition, two matrix attachment regions (MARs) are located in the LCR (289, 298). MARs are DNA segments with high affinity for the nuclear matrix. This interaction is believed to be the reason for the spatial organisation of DNA (316). The MARs are often found close to transcriptional enhancers and promoters. Most enzymatic machineries handling DNA and RNA associate with insoluble nuclear structures. In this way, MARs seem to bring together cis-responsive elements, the nuclear matrix and its attached enzymatic machineries (158, 313, 323).

1.7.2 The early region This region is approximately 4 kb long. It consists entirely of the overlap- ping early ORFs of E1, E2, E4, E5, E6 and E7. The late promoter is located

18 inside the E7 gene, at position 670 in HPV-16, hence its name P670 (112) (Fig 3A).

1.7.2.1 E2 and E1 The E2 protein has been found to form homodimers that recognise sequences in the transcriptional control region of the early promoter in the LCR (118). Low levels have an activating effect whereas high levels sterically block the binding of cellular transcription factors (282, 287). This differential function also contributes to copy number control in undifferentiated cells since E2 has also been found to be essential for replication. It binds to sequences surrounding the OriR and pulls down E1 to that region (17, 307, 312). E1 then forms a di-hexameric complex required for replication of the viral genome through its helicase activity (263). E1 has been shown to interact with components of the Swi/Snf complex and also sub-units of DNA polymerase- alpha to activate replication (180, 206). During HPV infection E7 establishes an S-phase environment supporting viral genome replication in the mid/upper strata (90). Multiple cyclins and their corresponding cyclin/cdk complexes bind and phosphorylate E1 (195). E1 contains four phosphorylation sites, the mutation of which drastically reduces the replication activity of E1 (195). One–round replication is normally regulated by phosphorylation of important factors, down- regulating their accumulation or affecting their localisation. Cdc6, a loading factor for the cellular helicase MCM2-7, becomes phosphorylated shortly after beginning of S-phase. This exposes a nuclear export signal (NES), causing its export to the cytoplasm (67). In contrast, E2 is not regulated by nuclear export but is always present. E1 on the other hand is regulated by a NES but with the opposite effect. Phosphorylation (during S-phase) of this site inactivates its NES and E1 is retained in the nucleus, supporting multiple rounds of viral replication (69). The E1 NES is probably important in early stages of infection contributing to the levels of E1 in nucleus ensuring low copy number of the genome. HPV-8 E2 inactivates transcription of beta-4 integrin (226), one of the two subunits of the alfa-6/beta-4 integrin, the function of which is thought to attach basal cells to the basement membrane and the extracellular matrix (322). This function probably ensures that infected cells producing viral proteins would have detached and migrated towards regions where immune surveillance is lower. The attachment of cells to the basement membrane suppresses keratinocyte differentiation (322). E2 associates with C/EBP transcription factors (116). These regulate many promoters of genes involved in cellular differentiation (244) (See 1.6 “Human papillomaviruses and gene expression”). Some studies suggest that E2 mediates viral DNA segregation in di- viding cells (136) by binding to the metaphase chromosomes and in this way facilitates the viral genomes to segregate into daughter cells in approxi-

19 mately equal numbers (183, 273). In HPV-11 it has been shown that the E2 protein localises to distinct foci. E2 also attracted the E1 protein to these structures (293). The two proteins were seen localising with the single- stranded DNA binding protein replication protein A (RP-A). E2 also attracted plasmid DNA containing the HPV origin region. These results suggest that the virus has distinct DNA replication sites. Interestingly, bovine papillomavirus (BPV) type 1 E2 and L1 exhibit a diffuse nuclear localisation until L2 is added, which causes E2 and L1 to localise to promyelocytic leukaemia oncogenic domains (PML-oncogenic domains or PODs, PML bodies, ND10) structures. This localisation partially, or completely, overlaps with the site for HPV-11 replication (292). HPV-5 E2 has been shown to be associated with splicing factors in subnuclear foci (169). The PODs contain a number of cellular proteins important in several nuclear processes. For example, many transcription factors are transient or permanent components of PODs (284). Several regulatory proteins encoded by DNA viruses target PODs at very early times of infection. Early steps of transcription and replication of some DNA virus families (Ad5, HSV-1, SV40) take place at the peripheries of PODs (2, 3, 34, 207, 284, 294). The co-localisation of L1, L2 and the genome (via E2) suggests the site is also used for virus assembly. That genome replication takes place in the vicinity strengthens this suggestion. HPV-16 E2 has been shown to induce apoptosis and G1 arrest. These functions have been assigned to p53 dependent and independent pathways, but independent of E2’s ability to regulate gene expression (70, 97, 110, 134). p53 has been shown to inhibit HPV DNA replication. Massimi et al. (205) showed that HPV-16 E2 and p53 interact specifically and efficiently. Interestingly, p53 has been found in the viral replication centres of SV40, HSV, CMV and adenovirus (23, 95, 103, 165, 326, 345).

1.7.2.2 E6 and E7 The replicative phase of high-risk HPV is confined to differentiated cells that have already exited the cell-cycle and are thus non-permissive for DNA synthesis (74). However, the E7 protein has been shown to target a number of cell cycle regulatory proteins, among them the “pocket protein” family of Rb, including pRb, p107 and p130, thereby upregulating genes required for G1/S and G2/M transition (61, 79, 80). Rb becomes increasingly phosphorylated approaching S-phase in the cell cycle. In this form Rb binds E2F transcription factors, actively preventing transcription from promoters containing E2F sites (324), such as DNA polymerase- and thymidine kinase (168, 274). Rb is bound by E7 in its hypophosphorylated state and thereby prevented from sequestering E2F (38). E7 interacting with Rb has also been shown to target Rb for ubiquitin-mediated degradation (106). In addition, E7 has been found to displace Rb-histone deacetylase (HDAC) interactions and be able to sequester HDAC independent of Rb (24). HDACs remove acetyl

20 groups from histones in the DNA bound nucleosomes silencing gene expression. Rb-E2F-HDAC complexes are the most commonly found complexes on promoters of quiescent or differentiating cells. Disturbance of the complexes would allow for acetylation of the promoter and/or free E2F with subsequent activation of transcription (133). HDACs can also directly target E2F-factors and remove acetyl groups, leading to export of these factors from the nucleus (202). Another ability of E7 is to enhance the activity and raise the level of different cyclins and also bind and inhibit cyclin dependent kinase (CDK) inhibitors (186). The inactivation of the CDK inhibitor p21 was shown to be necessary to prevent cell cycle arrest (124). This allows phosphorylation of E1 supporting processive viral proliferation (195). Cells expressing E7 have been shown to contain increased steady state levels of the tumour suppressor p53 (68) due to protein stabilisation (143, 144, 218). p53 is a site-specific DNA binding protein that is activated in response to DNA-damaging agents or unscheduled DNA replication, as in viral infection, resulting in cell cycle arrest or apoptosis (121, 189, 333). In HPV infections, the E6 protein reduces the elevated levels of p53 through degradation by recruiting E6AP, an ubiquitin ligase (132). In the complex E6-E6AP-p53, p53 is ubiquitinated and thereby sentenced to be degraded by the proteosome (132). E6 also induce ubiquitination of E6AP itself resulting in its degradation (148). Since E6AP targets the src family of tyrosine kinases, interactors in a variety of signalling networks (96), it was suggested that this was a new area of interest (186). HPV E6 carries out several other suppressive actions on p53. E6 can interfere with binding of p53 to its DNA recognition site (178, 306) and has also been shown to repress p53 responsive promoters by interacting with its transcriptional coactivators p300/CBP (231, 349). Recent studies have suggested that the interaction of E6 with proteins other than p53 is essential for immortalisation of cells. E6 binds members of the PDZ family sentencing them to degradation (159). PDZ proteins probably act as molecular scaffolds in signal transduction (51). Ad- ditionally, E6 has been shown to activate telomerase (162). This enzyme replicates telomeric DNA at the ends of chromosomes. Without this activity telomeres are gradually shortened through successive cell divisions. It has been suggested that critically shortened telomeres initiate the natural pathway leading to senescence and cell death (5, 120, 184, 246). Activation of telomerase is a critical step of cellular transformation (83, 117). Most tumour cells maintain telomere length and telomerase activity (27, 49, 156). In HPV-16 infected cervical cells there is an increase in abundance of tRNA and 5S rRNA, probably advantageous to the virus through heightened translational capacity facilitating production of viral proteins (59). One explanation for this is that E6 and E7 neutralise p53 and Rb, and that this leads to activation of TFIIIB (283). TFIIIB is responsible for recruiting RNA- polIII to its promoter, activating synthesis of many important short non- translated RNAs (59). Recently, it was shown that the level of Brf1, a limit-

21 ing subunit of TFIIIB, was upregulated by E6/E7 in HPV infection, possibly independently of Rb and p53 interactions (59). The approach to induce RNA-polIII activity is used by many viruses, but on different subunits in this system (175, 204, 271, 320).

1.7.2.3 E5 function and immune defence HPV-16 E5 has transforming functions. A key characteristic is its ability to associate with the epidermal growth factor receptor (EGFR) (135) and stimulating this receptor’s transforming activity (54, 182, 239, 339). E5 binds to the vacuolar ATPase thereby inhibiting the acidification of the endosomes. This results in an enhanced turn-over of EGFR leading to a pro- longed signalling by the EGFR/EGF complex (285). Recent studies on the effect of E5 in the context of the complete genome demonstrated an important activity in regulating the amplification and late gene expression in differentiated suprabasal cells (86). This suggests that E5 acts in conjunction with E7 in the late phase of the viral life cycle to support cell cycle progression (87). The lack of acidification of the endosomes also negatively affects the maturation of the MHCII complex (338), and also interferes with the MHC class I (HLA I) export pathway (9, 33). E5 selectively retains HLA- A/B and not HLA-C/E (9). The same effect renders cells infected with other viruses capable of avoiding both CTL and NK cell mediated destruction (43). HPV-16 E5 inhibition of the HLA class I transport is reversible by IFN treatment. For HPV-16 E5, this is probably due to the IFN induced overexpression of the HLA I heavy chain (9). E6 and E7 inhibits the type I IFN pathway, releasing IFN blockage of E5 inhibition in HLA class I transport (14, 181). Another role for HPV-16 E7 protein in counteracting the immune response is repression of the MHC class I heavy chain promoter (105). In addition E7 binds to TAP, important for transport of the MHC I heavy chain (173).

1.7.2.4 E4 In contrast to the other HPV early genes, the expression of E4 occurs in late infection prior to the expression of the viral structural proteins (53, 74) and is also only expressed from the differentiation dependent promoter (75). It has a diffuse and filamentous distribution in the cytoplasm despite of the association with viral genome amplification, which takes place in the nucleus (74). The filamentous distribution is due to its association with the cytokeratin network, which E4 has been shown to induce collapse of in vitro (73, 249). HPV-16 E4 induces a relocation of mitochondria, a drop in their membrane potential and when expressed in high levels, an apoptotic state of the infected cell (243). Davy et al. (62) showed that E4 has a potent G2-arrest (prior to mitosis) function that does not depend on its ability to bind keratins. This suggests that it has a possible role in antagonizing E7-mediated cell

22 proliferation during productive stages of infection giving rise to increased virus production.

1.7.3 The late region The late region contains the late genes L2 and L1 encoding the minor and major capsid proteins, respectively. L2 and L1 form a protective shell around the viral genome of the virus (For viral assembly see 1.4 “human papillomavirus structure”, for late protein expression see 1.6 “human papillomavirus gene expression” and 1.8.9/1.8.10 for “papillomavirus early and late polyadenylation”). The L2 protein is required for infectious virions to form and has also been shown to interact with a cell surface receptor (150, 225). L2 antiserum neutralises papillomavirus without preventing virion attaching to the cell surface (250). Three different surface molecules have been proposed to bind to the major capsid protein L1 and function as the primary receptor for papillomavirus (58, 84, 145). This suggests that L1 mediates the initial binding of virions to the cell surface, while L2 provides later functions for infection. HPV-16 L2 interacts with beta-actin to efficiently transport virions across the cytoplasm during infection. Destroying the amino acids 25-45 of L2 do not inhibit uptake of virus but inhibits the transport to the perinuclear region along radial tracts (334). Both late proteins contain nuclear localisation signals (NLS) directing them to the nucleus after synthesis. HPV-16 and -31 L2 accumulates in distinct nuclear dots identified as PODs (64, 93, 111). HPV-31 and HPV-16 L2 also relocates the corresponding L1 into these structures (64, 93) (222).

1.8 Gene expression Newly made transcripts (pre-mRNA) by RNA-polII undergo a series of processing events in the nucleus to become fully mature messenger RNAs (mRNAs). These three events are capping, splicing and polyadenylation. During these processing steps mRNA are bundled up with different factors. The assembly of the correct mRNA protein complex (mRNP) is important for the transcripts future fate, concerning stability, nuclear export and translatability. In addition, the processing events can occur both co- and post- transcriptionally and they affect each other greatly (200, 215, 247). Also regulation of translation is an important stage in the regulation of gene expression. Unexpectedly, it was found that the human genome might only consist of approximately 32,000 genes (172). Many simple organisms have almost as many genes as humans, and in some cases more! This revealed the ex- treme importance of alternative processing of gene products. These mecha-

23 nisms contribute greatly to the difference between lower- and higher organisms. Through alternative processing of transcripts, gene regulation is tightly controlled and the diversity of proteins is amplified by magnitudes.

1.8.1 Capping The first mRNA processing event to occur is capping. When 20-25 nucleotides have been synthesised, the 5’end on the nascent transcript is modified. This does not only identify the transcriptional start site but has also been shown to have important effects on mRNA maturation, translation and stability. RNA 5’-triphosphatase (RT) activity converts the 5’ most NTP into NDP. Guanylyltransferase (GT) adds a GTP resulting in an unphosphory- lated G in the 5’end, which is further methylated by N7G-methyltransferase (MT) forming the mature Cap (266). The Cap is recognized and bound by the cap binding complex (CBC), important in the stability of the mRNA (15). The CBC also enhances splicing of the first intron and is involved in the mRNP nuclear export. Once in the cytoplasm, the CBC is replaced by the translation initiator factor eIF-4E (264). However, eIF-4e can also be found in the nucleus, where it co-localises with splicing factors, suggesting that some translation or reading frame recognition could occur inside the nucleus and possibly even influence pre-mRNA splicing patterns (327).

1.8.2 Polyadenylation A stretch of up to 200-300 adenosine residues can be found at the 3’end of almost all eukaryotic mRNAs. This poly(A) tail confers stability (15), promotes translational efficiency (258, 301) and plays a role in nuclear export of mRNA (81, 131). The signal sequence for polyadenylation has in 90% of cases been shown to be the hexanucleotide AAUAAA. The majority of the remaining 10% have a substitution of the second A to a U residue. The hexanucleotide sequence is usually found 10-30 bases upstream of the cleavage/polyadenylation site. The cleavage and polyadenylation specific factor complex (CPSF) binds to AAUAAA (20). This is a weak interaction but is stabilised by the cleavage stimulatory factor complex (CStF) (Fig. 5), which binds to G/U-rich sequences 10-40 downstream of the cleavage site. The CStF binding sequence is much more variable than the AAUAAA and consists of one or many motifs of penta-uridine (U) interspersed by single G residues (196, 295). These motifs can be found in about 70% of the mammalian pre-mRNA. The exact sequence of the CPSF- and the CStF motifs and their location in relation to each other specify the cleavage site and also the strength of the poly(A) signal (39, 196). Two other factors, cleavage factor I and II (CFI and CFII), are involved in the complete reaction. These are required for cleavage but not poly(A) addition (296). CFI has been shown to stabilize

24 CPSF on its substrate RNA, but not vice versa, pointing to specific binding of CFI to the RNA (255). The actual endonuclease activity seems to origi- nate from one of the CPSF subunits, namely CPSF-73 (257).

CStF CPSF I II AAUAAA CS G/U

PAP I CStF II

CPSF PAP AAUAAA A AAAAn Figure 5. Schematic diagram of a polyadenylation site. The cleavage stimulatory factor (CStF) complex binds to the G/U-rich region. CStF stabilises the cleavage and polyadenylation specific factor (CPSF) interaction with the AAUAAA sequence. This interaction attracts the cleavage factors (CFI and CFII). Intrinsic activities of the CPSF complex then induce cleavage of the RNA between the two RNA elements (cleavage site, CS). The poly(A)-polymerase interacts with CPSF and synthesises the poly-A tail.

After cleavage, poly(A)-polymerase (PAP) associates with the polyadenylation complex through interaction with CPSF. It has been shown that only CPSF and PAP are needed to start the synthesis of the poly(A) tail. The poly(A)-binding protein II (PABII) binds to the poly(A) tail when 11-14 A residues have been added (211). PABII binding stabilizes the CPSF/PAP/RNA complex supporting processive synthesis of the long tail in a single rapid step (19). The interaction between PAP and CPSF is disrupted at the length of 200-300 nucleotides. This is thought to be due to sterical hindrance (317). Keller et al. (152) has shown that successive binding of PABII to the poly(A)-tail will form a compact oligomeric particle, the size of which, at the length of 200-300 adenosines is critical for the continued association between PAP and CPSF.

1.8.3 Splicing Approximately 50 % of the human genome is transcribed. Only about 20% of this RNA is pre-mRNA destined to become mature protein coding mRNA. Ninety percent of the pre-RNA destined to become mRNA consists of non-coding sequences (introns) interspersed with coding sequences (exons). For a mature mRNA to form, introns must be removed and selected

25 exons joined together. This process is called splicing and is a nuclear event. It gives rise to protein variability through the alternative joining of exons, mediated by splicing enhancers and inhibitors. Alternative splicing is very important for efficient use of the small genomes of many viruses. HPVs are more of the rule than the exception. Almost all mature HPV mRNA are produced through such splicing events. For splicing to take place, a number of conserved cis-acting elements on the pre-RNA and a number of trans-acting factors are needed. The cis- elements are the 5’splice site (5’ss), the 3’splice site (3’ss), the invariable adenosine (the branch point) and the polypyrimidine tract (PPT) (Fig. 6). The conserved elements lie largely in the intron sequence. The average size of an intron is several thousands of nucleotides, whereas the average size for an exon is only about 300 nucleotides. At the 5’ss the bases GU must be present, indicating the start (5’end) of the intron to be removed. At the 3’ss, the bases AG must be present. This dinucleotide ends the intron on the 3’end. The branch point is a single adenosine residue that in mammals is surrounded by the conserved sequence YNYURAC, where A is the invariable adenosine, R is A or G, Y is C or U and N is any base. The branch point is generally found 18-40 nucleotides upstream of the 3’ splice site. The polypyrimidine tract is located between the branch point and the 3’ splice site. It is rich in C and U residues, hence its name, and is variable in length. In metazoans the polypyrimidine tract is essential for efficient branchpoint usage and 3’splice site recognition (248).Trans-acting factors complex and form the huge spliceosome. These factors are small nuclear ribo nucleo protein (snRNPs) and extrinsic (non-snRNPs) protein factors (187). snRNPs are made up of an uridine rich small nuclear RNA (U snRNA) that complexes with a common set of seven core proteins and a set of specific proteins for each individual snRNP. The assembly of the spliceosome starts with the recruitment of the U1 snRNP to the 5’ splice site and the U2 snRNP to the branch point sequence. The pre-formed U4/U6-U5 triple snRNP complex then associates to form the immature spliceosome. This undergoes rearrangements that result in the formation of the fully formed spliceosome. Extrinsic factors, such as members of the SR protein family, help to stabilise the interactions of the snRNPs and the RNA. Re- ports suggest that up to 300 or more different proteins might be associated with the spliceosome (245, 347). Two major events make up the splicing reaction. Initially, the 2’hydroxyl group of the branch point adenosine attacks the phosphodiester group at the 5’ splice site. This is accompanied by the formation of a phosphodiester linkage between the branch point adenosine and the most 5’ nucleotide of the intron, the guanoside. Secondly, the free 5’exon is ligated to the 3’exon, which releases the lariat intron (Fig. 6).

26 Spliceo- some U2AF SR

BP PPT Exon A 5ʻss 3ʻss Exon B

EJC 5ʻss

PPT BP 3ʻss Exon A Exon B Intron Joined exons lariat

Figure 6. Schematic drawing representing a splice reaction. U2AF binds to the polypyrimidine tract (PPT) and 3’splice site (3’ss). This is sometimes regulated by splicing regulatory (SR) proteins binding downstream of the 3’ss. U2AF and the 5’ss assembles the spliceosome complex which bridges the intron gap. The branch point adenosine (BP) then attacks the 5’splice site (5’ss). This results in linkage between BP and the 5’ss guanosine. The free 3’end of exon A is ligated to the 5’end of exon B, which releases the intron lariat, completing the splice reaction. As a con- sequence of splicing a complex is deposited just upstream of the exon junction termed the exon junction complex (EJC), which has a role in nonsense, mediated decay (NMD) and perhaps nuclear export of the mRNA (See 1.8.6 “Nuclear export of mRNA”).

Splicing can be either constitutive or alternative. In constitutive splicing all introns are removed and all exons are joined together consecutively. In alternative splicing a 5’ss can be spliced to a 3’ss further downstream than the nearest 3’ss. Flanking elements being either inhibitors or enhancers regulate the flexible usage of splice sites in alternative splicing. Most human gene transcripts are alternatively spliced generating several different mRNAs that increase protein diversity (172). The main classes of factors interacting with these regulatory elements are the SR- and the hnRNP proteins. These factors are themselves controlled during the cell cycle through relative levels, phosphorylation and alternative processing. This allows tight control over alternative splicing (28, 113, 276). In mammals alternative splicing is probably used in more than 75% of pre-mRNA (142, 212). The number of different mRNA that can be produced increases by magnitudes when alternative splicing is being employed. In drosophila, the gene Dscam may give rise to as many as 38,000 different proteins due to alternative splicing. In humans the pre-mRNA of dystrophin consists of 2 million bases where 99.5% is intronic RNA, contains more than 70 introns (199), and takes about 15 hrs to synthesise. The mature RNA is only 14,000 bases and would, without in-

27 tronic RNA, take less than 4 minutes to produce. These exemplify the ex- treme regulation that must be exercised to control the huge number of events possible for a pre-mRNA. This also makes the system sensitive to mutations due to the regulation that must occur. A single point mutation in a splice site may cause skipping/inclusion of exons (29) in some cases leading to cancers of different types (314). Other kinds of diseases that can be caused by aberrant splicing include growth hormone deficiency, Frasier syndrome, Parkin- son’s disease, cystic fibrosis, retinitis pigmentosa, spinal muscular atrophy and myotonic dystrophy (85, 104). Treatments of diseases caused by faulty splicing using homing oligonucleotides against mutated sequences are under development.

1.8.4 The exon definition model The capping, splicing and polyadenylation events are interconnected as proposed in the exon definition model (216, 217) and further shown in numerous papers. This model defines first, interior and terminal exons (Fig. 7).

pA Figure 7. The exon definition model. First: The proximal 5’splice site (5’ss) is enhanced by the Cap binding complex (CBC). Interior: the 3’splice site (3’ss) and the 5’ss are bridged, having an activating effect on splicing. Last: complexes at the last 3’ss and the polyadenylation signal connects. The strength of each site enhances also the other site.

The 3’ss and the 5’ss across an exon connect through the binding of different factors at each site, U2snRNP at the 3’ss and U1snRNP at the 5’ss. These 3’ss/5’ss complexes are further bridged by other factors, stimulating splicing (113). Definition of the first exon is carried out by interaction between the CBC on the Cap-structure and the first 5’ss. CBC greatly enhances the strength of the first 5’ss. In the terminal exon the last 3’ss and the polyadenylation signal (pA) interact. The strength of each site in the terminal exon positively affects the usage of both sites (47). In addition, Cap can ex- change for the terminal 3’ss and enhance usage of the pA in a similar way. Insertion of a 5’ss between the terminal 3’ss (or Cap) and the pA inhibits polyadenylation. Transient complexes form and interact on coupled splice and polyadenylation substrates (332). The polyadenylation complex and the last 3’ splice acceptor complex fuse to form a mature complex coupling polyadenylation and last-intron removal, well before splicing/polyadenylation products are detected (47). Unique polyadenylation

28 complexes rapidly form on substrates containing only the polyadenylation signal.

1.8.5 Linking mRNA processing and transcription That transcription and pre-mRNA processing are tightly linked has been widely accepted (reviews (16, 125, 200, 241, 247, 350)). Both pA and splicing factors have been shown to be associated with the promoter region and later on the active RNA-polII machinery. The general transcription factor (GTF) transcription factor II D (TFIID), which has a central role in promoter recognition, recruits CPSF to the promoter and transfers it to the C- terminal domain (CTD) of RNA-PolII during transcriptional initiation (60, 209). In addition, it has also been found that CstF binds to the CTD (209). Deletion of the CTD does not necessarily affect transcription initiation in vertebrate cells; rather it reduces the overall level of transcription. The pre- mRNA processing events of capping, splicing and polyadenylation are all inhibited by the deletion of CTD (208, 209). The CTD consists of 52 tandemly arranged heptad repeats. Position 2 and position 5 on each heptad are serine residues, called ser2 and ser5, respectively. Ser5 is phosphorylated during transcription initiation by the TFIIH associated kinase kin28. In this state the CTD recruits the capping enzymes and promotes their capping activity (126). As the elongating process continues ser5 is dephosphorylated, whereas phosphates are added to ser2 by the positive transcription elongation factor pTEFb. Different splicing factors, such as SR-proteins, associate with the CTD (16). The elongating rate of a traversing RNA-polII complex has been shown to affect the choice of alternative splice sites. Slower transcription reduces exon skipping by shifting the choice of alternative 3’ss in the favour of the proximal one (65, 130). This explains why different promoters can affect the choice of splice sites differently (50) since transcription factors can influence the efficiency of elongation (335). Vice versa, transcription is also affected by splicing. Both initiation and elongation can be positively affected by splice sites and splicing factors (94, 100). Addition- ally, some polyadenylation factors associates with the CTD already at the promoter and follows the CTD throughout the length of the gene. The ser2 phosphorylation status of CTD positively affects the association of polyadenylation proteins (350). In conclusion, CTD is a key player that connects all stages of RNA-polII transcription and RNA processing into an “mRNA factory” complex.

1.8.6 Nuclear export of mRNA The different processing steps all affect the quality of the mature mRNP complex. The different processes leave tags in the form of protein complexes containing export factors (Fig. 8). In Xenopus oocyte injection experiments,

29 splicing generated mRNAs are exported at higher rates than non-intron containing transcripts (190). Splicing deposits an exon-junction complex (EJC) 20-24 nucleotides upstream of the exon-exon junction (177). Many of its components have been shown to function in mRNA export (251, 290, 348). Recent evidence, however, have shown that components of the EJC only modestly affect mRNA export, whereas the export receptor TAP/NXF1 and the splicing factor UAP56, a RNA helicase involved in early spliceosome assembly, are essential for the export of both spliced and intron-less transcripts (review (252)). Also, the addition of an artificial poly-A tail or 3’end with insufficient polyadenylation signals on RNAs does not support export (81, 131). Improperly spliced or polyadenylated transcripts are retained within the nucleus, perhaps as part of quality control mechanisms ensuring that only fully mature mRNP complexes reach the cytoplasm (review, (291)).

Protein Cytoplasmic degradation (iv) through NMD or NSD Translation decay pathways Cytoplasm

Nucleus (iii) Retention and degradation at nuclear periphery Nuclear export (ii) Degradation close to transcription site due to abberant processing

mRNP C An

Capping (i) C Growing pre-mRNA Splicing Polyadenylation

polII Transcription Intron Exon pA Promoter Figure 8. mRNA nuclear export and quality control. (i) The mRNA is capped, spliced and polyadenylated. These processes leave proteins attached to the transcript forming an mRNA protein complex (mRNP). (ii) Improperly assembled mRNP does not have correct transport signals whereby it is retained in nuclear dots close to the transcription site and subsequently degraded by the nuclear exosome. (iii) Nuclear pore complex (NPC) associated proteins monitor the mRNP for nuclear export signals. Lack of such retains the mRNP in the nucleus. (iv) mRNA undergoes exonu- clease-mediated degradation by nonsense-mediated decay (NMD), due to premature stop codons, and non-stop decay (NSD) pathways, as in normal degradation or induced by specific instability elements.

30 Indeed, a complex of 3’ to 5’ exonucleases, called the exosome, exists in the nucleus and has been implicated in the degradation of unprocessed pre- RNAs (22). Improperly assembled mRNP complexes are retained within nuclear dots at or close to the site of transcription. The faulty mRNP is released from the nuclear foci and “normal" mRNA levels restored if a nucleus specific exosome factor is deleted, suggesting retention and elimination of defective mRNPs by an exosome dependent quality control (review (252)). Furthermore, exosome recruitment to active genes by elongation factors (7) suggests exosome monitoring of mRNPs at an early step of transcription. Growing evidence shows that nuclear pore complex (NPC) associated proteins monitor mRNP quality at the nuclear periphery and retains unprocessed or malformed mRNP complexes within the nucleus (review (252)). Finally, transport factors and NPC are subject to regulation during different phases, such as in the cell cycle, development and differentiation (30, 88, 198).

1.8.7 Translation After transport out to the cytoplasm the mRNP can undergo a series of different fates. It can be stored (151), degraded (328) or translated. Translation is the process done by a multi-protein complex called the ribosome to deci- pher the aminoacid sequence of an mRNA and through that synthesise a protein (109). Briefly, the Cap structure is recognized by the 43S pre- initiation complex through the help of a number of different initiation factors. 43S then scans the mRNA until it finds the start AUG codon whereby it associates with the 60S subunit forming the complete 80S ribosome. Initia- tion of translation is synergistically enhanced by the mRNA Cap and poly-A tail bound PABP (166). Translation efficiency is not only affected by availability of circulating initiation factors, but also of such phosphorylation status. Next phase is elongation, where codon specified successive loading of aminoacyl-tRNA by the ribosome synthesizes the protein chain. Transla- tion termination ensues when a stop codon is reached, resulting in release of the newly synthesized polypeptide. The PABP bound poly-A tail is thought to interact with the Cap-complex and make the translation more effective. This loop structure promotes re-initiation due to shunting of the ribosome onto another round of translation. An additional level of control against faulty mRNA is nonsense- mediated decay (NMD). EJC complexes promotes NMD during the first round of translation on transcripts containing premature termination codons (PTCs) upstream a EJC complex (157, 176, 193, 194). NMD occurs in the cytoplasm and in addition, a wide accumulation of evidence points to NMD occurring also in the nucleus (327). It has been proposed that an initial round of translation or at least a NMD scanning event could occur in the nucleus, mediated by several translation factors found to be co-localised with different mRNA processing factors in nuclear foci (327).

31 +

pA i) 5ʻss 3ʻss -

ii) 5ʻss pA 3ʻss

Weak Strong iii) pA1 pA2 -/+

iv) +pA

v) CBC pA Figure 9. Alternative polyadenylation. (i) A highly active 3’splice site (3’ss) can form a complex with the downstream polyadenylation (pA) signal greatly enhancing the polyadenylation reaction. (ii) Competition between a pA and splicing reaction. A strong 5’ss inhibits a pA site in itself. (iii) Alternative use of tandemly arranged pA signals due to changes in concentration of common cellular pA factors. High concentration favours the proximal weak pA signal, whereas low concentration favours the distal strong signal. (iv) The usage of a pA signal can be directly or indirectly affected by factors other than common pA factors or a proximal upstream 3’- or 5’ss. (v) Proximity of a Cap-binding complex (CBC) have the same effect as proximity of an active 3’ss (see above(i)). These single events are often part of a more complex situation involving a mix of above examples.

Structural elements throughout the whole length of the mRNA have been shown to affect translation and through that regulate gene expression. In the 5’UTR of ferritin there is a region called iron responsive element (IRE). The interaction between the 43S pre-initiation complex and the Cap-structure is thought to be disturbed upon iron-deficiency, due to that the Cap-proximal IRE interacts with an IRE-binding protein (161). Other 5’UTR elements are upstream start/stop codons and stretches of GC-richness (63). Some 3’UTR elements have been suggested to inhibit 80S ribosome assembly (228). Inter- estingly, elements in both coding regions and 3’UTR inhibiting translation has been identified in HPV genes (154, 280, 299). Due to the degeneracy of the genetic code it is possible to build the same protein from different codons. Codon usage can vary significantly between genes and between species. The expression of certain genes is affected by tRNA availability relative to codon usage (147). This mechanism has been proposed for the alternative expression of the late genes of papillomaviruses, BPV-1 L1 (346) and HPV-16 L1 (179). However, it has since been

32 shown that this is rather due to intragenic regulatory elements on the mRNA (45, 222, 223, 344).

1.8.8 Regulation of mRNA polyadenylation There have been many reports about regulation of polyadenylation signals. Among others, these include calcitonin/calcitonin gene related peptide (CT/CGRP) exon 4 (188), retroviruses (HTLV-1 and HIV-1 (137)), as well as immunoglobulin type M (IgM) and papillomaviruses polyadenylation signals, both discussed below. One of the most extensively studied sites is the simian virus 40 late polyadenylation signal (SVLPA), discussed below. In the above examples, the regulated polyadenylation region with its sequences is highly variable but the factors found to be involved are often the same. By no means have all the regulators been found. In general, the binding strength of regulatory factors and their relative positioning to the polyadenylation site can have diverse effects on polyadenylation in different systems. The same factor may enhance in one and inhibit in another. Motifs regulating a polyadenylation site are called upstream elements (USEs) or downstream elements (DSEs) depending on where they reside relative to the pA. Regulation occurs most often in tandemly arranged polyadenylation signals or when a polyadenylation event competes with a splicing event in a mutually exclusive situation (Fig.9). Downregulation of polyadenylation to make transcripts more unstable also occurs. During B-cell differentiation there is a shift from production of the membrane to the secretory form of immunoglobulin (Ig) mRNA and protein (6, 253). The shift in Ig has been suggested to occur because of changes in CstF-64 levels (297). When resting primary human splenic B-cells or a fi- broblast cell line leave the G0 and enter S-phase, CstF-64 levels increase (203). The strong downstream polyadenylation site, pA-mem, would be preferred for cleavage if the CstF-64 levels were low, whereas if CstF-64 levels increase the weak proximal polyadenylation site, pA-sec, would be used (82). Many viruses rely on the differential use of polyadenylation sites. In expressing L1, during adenovirus infection, a shift occurs to the L3 mRNA. The L1 message utilises a weak CstF binding site for polyadenylation, while L3 uses a strong downstream signal (201). Variations in the levels of CstF subunits cannot account for all the polyadenylation regulation observed. CstF levels do not consistently differ between B-cells and plasma-cells (82). The large change in concentration of such a general polyadenylation factor as CstF would have too great an im- pact on gene expression in the cell. A smaller change in a few more specific factors involved in a particular reaction would seem like the better alternative to regulate polyadenylation efficiency. Indeed, in the usage of alternative polyadenylation sites on IgM RNA the factor hnRNP F inhibits the binding of CstF-64 at the polyadenylation site for secretory heavy chain

33 (315) and it is a higher ratio of hnRNP F to hnRNP H that is important. The U1A protein, normally found associated with U1snRNP but also existing in non-U1snRNP complexes (219), binds between the two DSEs in pA-sec and thereby inhibits CStF binding (238). A 29 kDa protein binds to and influences the pA-sec and the level of this factor varies in cellular differentiation (237). One could speculate that this 29 kDa protein, not having been identified and was actually exchanged for a 55 KDa factor in binding to pA-sec (237), could be the 2H9 member of the hnRNPH/H’/F family and the 55 KDa could be the now found pA-sec binding factor hnRNP F, since the hnRNP F/H-ratio affects the pA-sec (315) and the different members have similar RNA binding domains (128). It has also been shown that sequences 50-200 nucleotides downstream of pA-sec induce pausing/stalling of the passing RNA-polII (234). This would give pA-sec more time to assemble the necessary complex for polyadenylation to commence. Introduction of pA-sec stalling region downstream of a heterologous pA conferred the pausing effect (234) indicating that pausing can be a general trait for polyadenylation signal. It has also been proposed that the splicing environment can influence the choice between pA-sec and pA-mem. When RNA-polII has synthesised the downstream 3’ss, splicing out of the pA-sec, to produce the IgM-mem mRNA, would initiate. The reports differ in this aspect. The most recent, by Bruce et al (26), shows that artificially altering the concentration of some splicing factors changed the IgM-sec/IgM-mem ratio. They further show that the natural splicing environment is different between B-cells and plasma cells, suggesting a model in which changes in both cleavage-polyadenylation and splice activities ensure proper regulation in alternative IgM mRNA processing. SVLPA has been studied extensively. It contains four USEs, one binding U1snRNP (321) inhibiting polyadenylation (48) and three sharing a common sequence providing efficiency to the pA (32, 261). Three DSEs have also been found, a G/U-rich element interacting with CstF (46) followed by a G-rich element interacting with hnRNP H (11, 242) followed by another G/U-rich element (259, 260). The latter was shown to interact with hnRNP C (329, 330). All three elements contribute to the efficiency of SVLPA. It has earlier been reported that binding of the cellular factor hnRNP H to the G-rich motif stimulates polyadenylation (10). It has also been shown that the G-rich hnRNP H binding site significantly influences the overall secondary structure of the core elements in SVLPA (119). When Arhin et al (8) screened a database they found that 34% of all mammalian polyadenylation sites contain G-rich downstream tracts. They investigated many of these and showed that they bind hnRNP H at different affinities and that this stimulates polyadenylation both in vivo and in vitro. They suggest that polyadenylation regions containing G-rich elements could fold into suboptimal conformations. hnRNP H binding to the elements would alter its structure and present the core elements to the general polyadenylation fac-

34 tors in a productive fashion. Alternatively, hnRNP H interacts directly with the polyadenylation signal stimulating CStF binding and complex assembly. The formation of secondary structures around polyadenylation signals has been shown to be a common functional feature. The importance of secondary structure has been seen in several pA, including those of the bovine growth hormone gene (108), the human CD59 gene (308), the adenovirus L4 gene (272), equine infectious anaemia virus (114) and the murine IgM-sec gene (237). In human T-cell leukaemia virus type 1 (HTLV-1) a large stem- loop secondary structure brings the more than 200 nucleotide distant AAUAAA into optimal proximity to the polyadenylation enhancing DSE and the cleavage site (1, 13). A USE in HIV-1 appears to be involved in secondary structure formation aiding the recruitment of CPSF (107). Hans and Alwine (119) have shown that the downstream region of SVLPA can form into extensive secondary structures and that this ability correlates with cleavage efficiency. Recently, it was speculated that a marginally stable secondary structure may be more efficient and easily affected by regulatory trans-acting factors. The SVLPA was shown to fold into three competing structures (332). This “could-go-either-way” state could flick into the favour of one secondary structure with the help of some auxiliary factor. It is interesting to speculate about factors affecting the outcome of folding of transient secondary structures being themselves regulated in a differentiation dependent manner, virally or cellularly induced. Indeed, many of the factors discussed above have been shown to exist in different abundance and localisation between cell types and differentiation stages. A con- crete example, U1A has been shown to inhibit the usage of pA-sec of IgM in B-cells in favour of IgM-mem (238), mentioned above. The total amount of U1A, relative to other snRNP proteins, was significantly reduced during differentiation into plasma-cells suggesting a redistribution between U1snRNP and mRNA pools. It has also been found that the total level of U1A and the proportion that is not snRNP bound is raised in undifferentiated cells (235). Genes that follow the same pattern of regulation have also been shown to contain binding sites for U1A (236). As mentioned above, the ratio between hnRNP F/H changes during B- to plasma-cell differentiation, in favour of hnRNP F, inhibiting pA-sec (315). Retrospectively, the diverse sequences between different polyadenylation signals mirror the need for the transcripts to be differentially expressed, but do not necessarily have to result in this. A certain sequence might have evolved to do the same job as another. Two very different sequences can still form almost identical RNA structures due to complemen- tarities in their own sequence. Factors might have an effect on stability of the secondary structure and/or directly interact with core polyadenylation factors in a productive or inhibitory fashion.

35 B) pAE

E4 3ʻss

Act. E4 5ʼss A) pAL E5 +

L1 3ʻeUTR hFip1 4*U1snRNP hnRNP C1/C2 USE 3ʻlateUTR + PTB - CStF-64 CStF-64 - HuR pAE G/U U2AF(+ASF/SF2) U1A - + pAL + + L2

6*hnRNP H CStF-64 DSE

Figure 10. Regulation of human papillomavirus type 16 (A) late and (B) early polyadenylation site, pAL and pAE, respectively. pAL is inhibited in the early stages of infection by factors binding to upstream sequences. The most inhibitory effect comes from a region containing four U1snRNP binding sites. As cells differentiate, the levels and phosphorylation status of ASF/SF2 are upregulated, in correlation with an increased activity of pAL and that this factor is associated with the pAL complex at this stage. (B) The pAE contains upstream (USE) and downstream sequences (DSE) that affect its efficiency positively. In correlation with a downregulation of pAE in late differentiation the USE and DSE interacting CstF-64 and the DSE interacting hnRNP H has been found to be downregulated. In addition, a splice site suppressor has been found close to the E4 5’ss. Activation of this site, as in terminally differentiated cells, seems to disturb the last exon definition between E4 3’ss and pAE, negatively affecting polyadenylation at pAE.

1.8.9 Papillomavirus and late polyadenylation Regarding the regulation of papillomavirus polyadenylation, the first reports were on bovine papillomavirus type 1 (BPV-1). It was shown that polyadenylation was inhibited by U1snRNP binding to a 5’ss located upstream of the pAL site (101, 102) via direct interaction between PAP and the 70K U1snRNP (115). The presence of negative regulatory elements (NREs) in the late 3’UTR seems to be a common characteristic of papillomaviruses, including BPV-1 (101), HPV-1 (299), HPV-16 (153) and HPV-31 (57). The NRE in HPV is called ARE and consists of two AUUUA and three UUUUU motifs (280, 299). These motifs reduce the mRNA half-life (280) and inter-

36 acts with several cellular proteins (340, 341) also binding to the c-fos ARE (280). Two of these proteins were identified as HuR and hnRNP C (277, 280). The shuttling protein HuR binds to both types of motifs (277) whereas hnRNP C exclusively bound the penta-U motif (278). HuR localising in the nucleus correlates with inhibition of late gene expression, whereas inhibition is lifted when HuR localises to the cytoplasm (31). The HPV-1 ARE mediated negative effect is dependent on nuclear experience (325). It was suggested that the HPV-1 ARE acts similarly as c-fos ARE in retaining the transcript in the nucleus resulting in degradation of the RNA when HuR is nuclear. Interestingly, the presence of the transport elements HIV RRE (with REV) and SRV-1 CTE overcomes the inhibitory effect (300). The 79- nucleotide NRE in HPV-16 contains four weak 5’ss in its 5’part and its 3’region is G/U-rich (153). The HPV-16 NRE interacts with many different factors: CstF-64, HuR (164) and U2AF65 (71) interact via the 3’G/U region, while a U1snRNP-containing complex interacts with the 5’region (56). Re- cently, phosphorylated ASF/SF2 was found to be indirectly associated with NRE, in a complex with U2AF65 (210). The 5’part of NRE contains the greater inhibitory activity, showing that the factors involved can bind to the separate parts independently. Both the 5- and the 3’part though is needed for full repression of pAL (56) (Fig. 10A). The U1A component of the U1snRNP-like complex binds to the 3’-G/U rich sequence. It would be interesting to see whether U2AF65 and U1A can compete for the binding of this sequence. A model could be proposed in undifferentiated cells, in which U1snRNP binding to the 5’part is strong, stabilised by U1A interacting with the 3’part and/or stabilised from U2AF65 also interacting with the 3’part. This would inhibit polyadenylation at pAL. In differentiated cells, ASF/SF2 levels and also its phosphorylation status is upregulated (210). This protein was found to be associated with U2AF65 in the NRE (210). Perhaps its addition to the NRE protein-complex in differentiated cells would alter the NRE from having a 5’ss function to a 3’ss function according to the terminal exon definition model (see 1.8.5 “The exon definition model”) and thereby NRE would play an activating role in late polyadenylation. The upregulation of ASF/SF2, in both quantity and activity, is of interest also when considering the L1 splice acceptor. This site is regulated by both splice inhibitors (344) and splice enhancers ((223) and Zhao et al, unpublished observation). The SR-protein ASF/SF2 has been shown to be a forefront splicing enhancing factor, stabilising the U2AF complex binding to a 3’ss (113). In the light of the regulation of ASF/SF2, it would be interesting to see, as was suggested by Zhao et al. (344), whether hnRNP A1 interacting with the splice inhibitor of HPV-16 late 3’ss could also be alternatively regulated during the different stages of HPV infection. A concomitant regulation of both inhibitory and enhancing splicing and polyadenylation factors is bound to occur during epithelial differentiation. In addition to the

37 normal pattern of regulation, viral factors can also contribute to the changing of splicing and polyadenylation environment. Both adenovirus and HIV infection affect the expression and activity of ASF/SF2 (213, 240). In HPV-16 infected epithelial cells, a 4 to 8-fold increase of ASF/SF2 levels was seen (210). At least one of the effectors is the viral E2 protein, a known transcriptional regulator, since cells transformed with this factor showed almost as high an increase of ASF/SF2 levels. In the late stages of infection E2 levels are increased (197), in correlation with above.

1.8.10 Papillomavirus early polyadenylation Papillomavirus mRNA contains tandemly arranged polyadenylation signals. Late transcripts encoding the capsid genes L1 and L2 must ignore the pAE and use the pAL (Fig. 2 and 3). Terhune et al (303) have shown that HPV-31 has three weak CstF sites around its pAE and that CstF-64 binds to them in vitro. They also show that pAL has a strong CstF binding site. When inducing keratinocyte differentiation a 40% drop was seen in CstF-64 cellular concentration. This would correlate with a switch in the usage of polyadenylation sites during differentiation. The same group also reported that the 5’region of HPV-31 L2 is the primary determinant of the usage of the pAE (302) and that as much as 800 nts of the 5’ region of L2 were needed for pAE to be fully functional. We have recently found and characterised USE elements in HPV-16 pAE (343) (Fig. 10B). These interacted with the core polyadenylation factors CstF-64 and the newly identified hFip-1, as well as PTB and hnRNP C1/C2. The deletion of USE marginally lowered pAE polyadenylation efficiency (343) in favour of the L2 and L1 mRNA. Deletion of the pAE site resulted in increased late gene expression. However, the effect was reduced by the activation of upstream cryptic polyadenylation sites (343). This points to the existence of strong activating elements in the downstream region of pAE directing cleavage and polyadenylation to this region. In support of this, the same effect was seen for HPV-31 (302). Speculatively, possibly the weak sequences in the USE only guide, “put into place”, the strong DSE in order to only affect the pAE site. Interestingly, SVLPA DSE-U2 interacts with hnRNP C and is suggested to couple pA and splicing (48). Perhaps the small enhancing effect on pAE by HPV-16 USE is conferred in the same way. SVLPA USEs also interact with U1A (191), which facilitates either polyadenylation complex formation or is involved in coupling pA and splicing (192). Recently, our lab found that the efficiency of the HPV-16 E4 3’ss at nt 3358 correlated with the efficiency of pAE and that unmasking the E4 5’ss at nt 3652, by deletion of its upstream inhibitor or optimisation, ren- dered the pAE less effective (256) (Fig. 10B). This is in keeping with the last intron definition (see 1.8.5 “The exon definition model”) and the reported findings by Cooke et al (48) about the disruptive effect a putative 5’ss has

38 when inserted in between the last 3’ss and the pA. This demonstrates that there is cross-talk between the 3’ss and the pAE and that it can be inter- rupted by a stronger 5’ss. Thus one can argue for a differentiation dependent de-masking of the wild-type 5’ss, inhibiting pAE and allowing the presence of L2 and L1 transcripts. We found that the presence of the USE had an inhibitory effect on the RNA levels of the early transcripts (343), in line with previously published data (140). This suggested yet another level of controlling early gene expression and copy number in undifferentiated cells (See 1.6 “Papillomavirus gene expression”). In paper III in this thesis we have studied the downstream region of HPV-16 pAE and its structure. Consecutively destroying hnRNP H binding motifs 150-250 nucleotides downstream of pAE correlated with both inhibition of polyadenylation and a decrease in bound hnRNP H (223) (Fig. 10B), suggesting that hnRNP H mediates polyadenylation efficiency to pAE by associating with its downstream region. Interestingly, the hnRNP H levels vary between different tissues and also in response to cellular and pathologi- cal changes (146). We performed immunohistochemical staining of cervical epithelia against hnRNP H and could see that the hnRNP H protein levels declined in differentiated cell layers (223). The G-richness of the region seems to be phylogenetically conserved but not identical (223). This is interesting considering that late gene expression for different HPV types is activated at different levels of epithelial differentiation (232). Perhaps the same factors interact but with different affinities depending on sequence variation between the types. In a database search of different mammalian polyadenylation sites, 34% of the downstream regions were found to contain short tracts of G-residues (8). This suggests that HPVs utilise a polyadenylation enhancing mechanism in the regulation of their pAE that is used naturally in the infected cells. Since HPVs have adapted to the differentiation pattern of epithelia there might be host genes with similar differentiation-dependent polyadenylation elements. The pAE will not be totally shut off during HPV infection. In fact, in late infection E1/E4 mRNAs polyadenylated at pAE are the most abundant viral transcripts (41). This reveals that the pAE must only be partly downregulated in the differentiation process. Most interestingly, we found that at least 400 nucleotides were needed for full restoration of the polyadenylation effect at HPV-16 pAE (223). This is supported by the finding that HPV-31 needs at least 800 nucleotides of the pAE downstream region for full efficiency (302). pAE does not appear to be a strong polyadenylation signal. Destroying an exonic splicing silencer (ESS) in the L1 region, L1 mRNA was detected (223), showing that transcription must have continued at least 1500 nucleotides downstream of pAE (nt 4215) and splicing from the E4 5’ss at nt 3652 to the L1 3’ss at nt 5637 occurred before polyadenylation reaction ensued. The fact that polyadenylation at pAE and splicing to L1 3’ss are mutually exclusive, suggests that HPV-16 pAE is regulated by a post-

39 transcriptional mechanism. Interestingly, data from a luciferase reporter assay on HPV-31 pAE showed that significant read-through occurred in undifferentiated cells with only a 50% increase upon differentiation (303). Surprisingly, making the pAE strong in HPV-31 recombinant genomes reduced replication, showing that a weak pAE is important for a productive viral life cycle (302). A stronger pAE is expected to enhance the expression of the early genes leading to a more productive replication. This suggests that the splicing pattern of early transcripts has been affected in such a way that replication is inhibited, since the strength of a polyadenylation site enhances the activity of the last 3’ss. One explanation would be that with a strong pAE, E4 3’ss is strengthened and dominates over the alternative 3’ss responsible for the splicing to produce transcripts encoding the viral replication factor E1 and the viral transcription factor E2. In addition, a regulated weak pAE allowing read-through would suggest a control mechanism of early HPV RNA level due to instability the longer RNAs.

1.8.11 Negative elements It has been reported that many cellular and viral mRNAs contain inhibitory sequences that are important determinants in gene regulation. Inhibitory sequences can be located in both the coding regions and in 3’ untranslated regions (UTR) of mRNA. For example, negative sequences reside in both coding region and in the 3’ UTR of c-fos (267) and c-myc (331) mRNAs, acting by reducing mRNA half-life. Interestingly, papillomavirus late mRNAs also contain inhibitory sequences in both coding region and 3’UTR (101, 153, 154, 280, 299). These reduce mRNA stability (154, 280, 299) and inhibit translation (154, 280, 299) and polyadenylation (102). The relevant sequences interact with numerous factors (340, 341, 344). These factors have been shown to be involved generally in polyadenylation, splicing and shuttling between nucleus and cytoplasm. The expression levels of transient or stably transfected genes from subcloned complementary DNA (cDNA (reverse transcribed mRNA)) are dramatically decreased, compared to levels when they are expressed from their natural context, in mammalian cells (268). This could be due to the presence of intragenic splicing and polyadenylation elements. For many mRNAs certain events must have occurred for it to be loaded with the ap- propriate factors, such as those mediating nuclear export or further processing. This complex has been shown to be important for signalling that the RNA is processed and ready for export. If these important events have been bypassed, as in a cDNA or an isolated gene, regulatory elements surrounding splice sites or polyadenylation sites present on the transcript will bind factor complexes, now without function. This situation will signal that the mRNA is defective, its export from the nucleus will be heavily inhibited and subsequently it will be degraded. Alternatively, located in its new context a cryp-

40 tic splice/polyadenylation site can be activated resulting in additional processing of the transcript. Ultimately, the changed transcript would either just be degraded or, at best, be able to produce a truncated version of the protein. For a deeper understanding and explanation of the reader is referred to section 1.8.6, “Nuclear export of mRNA”. It is not unlikely that many cDNAs that have been “codon optimised” in order to increase protein production, including early HPV genes (42, 72, 185), have contained various regulatory RNA elements that have been de- stroyed by the mutations. This is true for the HPV late mRNA where we have shown that a mutant L1 and L2 cDNA with changed nucleotide sequence but retained amino acid sequence expressed under the control of the CMV-promoter can produce the late RNA and proteins whereas no late RNA and proteins were produced from the wild type late cDNAs (45, 222). Further, we have used the mutant late genes in vaccination trials and there detected strong immune activation against the HPV proteins (254). Thus, negative elements on RNA expressed from isolated late genes have most probably been the cause for the inability to develop viable HPV- vaccines.

41 2 Introduction to papers

We are interested in the regulation of HPV-16 late gene expression. The activation of late gene expression depends on terminal differentiation of the infected epithelia. This has until recently prevented the production of virus in tissue culture system due to unavailability of a convenient differentiation system (288). In our studies to unravel the absence of L1 and L2 production in proliferating cells, we have found that their mRNA contain inhibitory sequences (44, 279, 299, 300). Previously we have shown that there is an inhibitory sequence present in the 5’end of the HPV-16 L2 coding region. This acts by reducing the stability of the L2 mRNA (44, 279). Another element was found in the 3’ part of L2, which acted by reducing L2 protein production (279). It was shown that the RNA binding protein hnRNP K and poly(rC) binding proteins (PCBP-1 and –2) interact with the 3’end of the mRNA coding region and cause reduced translation (44). In previous work on HPV-16 L1 we demonstrated the presence of cis-acting negative elements in the L1 coding region. These elements were shown to reduce L1 mRNA levels (300). We have speculated that the role of some of these elements in the viral life cycle is to regulate HPV-16 polyadenylation and/or splicing. Our lab has recently shown that a negative element in the L1 region interacts with hnRNP A1 and acts as a splicing silencer (344). In paper III in this thesis, we show that the region conferring inhibition on HPV-16 L2 transcripts expressed from the genes in an isolated form, are important for polyadenylation. For further information see section 1.8.11 “Negative Elements”.

2.1 Paper I (45) Having established the presence of inhibitory sequences in both HPV-16 L2 and L1 mRNA we constructed a system for mapping and characterising them. An expression plasmid was created consisting of the strong viral CMV promoter, a polylinker (for easy insertion of genes of interest) and the HPV- 16 late polyadenylation signal. Any gene expressed from this plasmid would have the same 5’ leader sequence on the resulting RNA produced in this system, and therefore would be detectable in a Northern blot assay using a probe for this region. In this system we showed that a variety of genes were readily expressed and detectable whereas L2 and L1 were undetectable. Hy-

42 brids were made between the highly expressed equine infectious anaemia virus (EIAV) p55gag gene and L1 to map the location of the inhibitory elements. The negative region on L1 was found to be in the first 367 nucleotides. The inhibitory effect of the region was not position dependent. We mutated the first 514 nucleotides in L1 by site directed mutagene- sis, changing the nucleotide sequence without altering the amino acid sequence. The L1 mutant (L1mut) produced high levels of both mRNA and protein when analysed by Northern- and Western blotting. Immunofluores- cense showed no production from the wild type L1 (L1wt) gene. In contrast, L1mut gene produced L1 with the localisation of the protein in the nucleus. To further map the elements, hybrids were made of the 514 region between wild type and mutant sequence. The region was divided into three segments. Northern blot analysis showed that the first 188 nucleotides contained a strong negative element. It appeared that another weaker element was located between 188 and 356 and that there could be an element between 356 and 514. Even further mapping, again using EIAV/L1wt hybrids, showed that the major negative element was situated within the first 129 nucleotides. This was confirmed in a CAT reporter assay. To determine where the elements were active, L1wt and L1mut mRNA were synthesised in vitro and electroporated directly into the cytoplasm of cells. The two different mRNAs were shown by Western blot to produce similar levels of L1 protein. This shows that the inhibitory elements are not active in the cytoplasm, at least not in the absence of a nuclear experience of the mRNA. We next showed by in vitro translation that L1wt and L1mut mRNA are as efficiently translated in a coupled rabbit reticulocyte lysate system. This is in contrast to bovine papillomavirus -1 (BPV) L1, which has been shown to be translated inefficiently in the same system. The effect could be overcome by replacing rare codons in the BPV-1 L1 gene sequence with those preferred in humans (346). The L1 genes of a number of distantly related HPV types were cloned and tested for the presence of inhibitory elements. The inhibitory effect was found in the majority of the HPV types tested since L1 mRNA was undetectable in most transfections. In this study we report the presence of regulatory elements located in the beginning of HPV-16 L1 coding region. We show that the negative effect is abolished by destroying regulatory elements and not by exchanging rare codons. The elements lie in the context of splicing and have since been shown by Zhao et al (344) to contain a hnRNP A1 dependent splicing silencer.

43 4.2 Paper II (222) In this paper we aimed to characterise the negative elements in the L2 coding region further. The experimental approach was similar to Paper I. When hybrids were made between L2 and EIAV we found that the first 361 nucleotides contained inhibitory sequences, named inhibitory region I. We also found that nucleotides 358-1422 contained inhibitory sequences, which were named inhibitory region II. The first 533 nucleotides were mutated by changing the nucleotide sequence without altering the amino acid sequence. By making cross-hybrids of both wild type (wt) and mutant L2 and L1 sequences we confirmed the presence of inhibitory sequences in the 5’ and 3’ part of L2. Mutating the first 533 nucleotides inactivated the inhibitory effect of this region. This mapped the 5’ boundary of inhibitory region II to lie between nt 367 and 533. We continued by mutating the remainder of the L2 gene. The complete mutation replaced 85 % of all the codons. The full L2 mutant produced high levels of mRNA and protein when analysed by Western and Northern blotting. Fractionation of transfected cells and immunofluorescence revealed that the L2 was found in the nucleus. We also saw that L2 had a punctuate pattern and that L2 could induce relocation of L1 into the same region, dem- onstrating that the L1 and L2 sequences interacted in the cellular nuclei. This interaction has earlier been shown in BPV–1 and HPV-33 (64, 92). We went further by making hybrids of the L2wt and L2mut. The en- tire region could be divided up into 10 segments numbered 1 to 10, each roughly 150 nts long. The segments 1-2 and segments 5-6 were shown to have inhibitory effects. The two regions together reduced the mRNA to undetectable levels. The 1-2 segments alone had almost 100 % inhibition on mRNA. The 5-6 segments only mildly affected the mRNA levels but inhibited protein production very effectively. We concluded that segments 1-2 contained the inhibitory region I, and that segments 5-6 contained the inhibitory region II. The remaining sequence was absent of strong negative elements. We next showed that the L1 gene mutated in only the first 514 nt produced mRNA and protein levels as high as a full-length mutant of L1. This demonstrated that specific inactivation of negative RNA elements alleviates inhibition and results in high expression of L1, dispelling the hypothesis of codon usage as the negative conveyer on late mRNA levels. As previously shown in our lab, the human immunodeficiency virus type 1 (HIV-1) Rev and RRE and simian retrovirus type 1 CTE induces HPV-16 L1 production. However, when these were employed by L2 they did not overcome the inhibition exerted by the negative elements. This indicates that the elements in L1 and L2 act by different mechanisms.

44 In this study we report the presence of two inhibitory elements in HPV-16 L2 coding region. These affect the mRNA level and mRNA utilisa- tion negatively. We show that the negative effect is abolished by destroying regulatory elements and not by exchanging rare codons. We also show that HPV-16 L2 protein localises in a punctuated pattern in the nucleus and that L2 attracts L1 to these regions.

2.3 Paper III (223) In this paper we have investigated the downstream region of HPV-16 early polyadenylation site (pAE) and its contribution to the efficiency of polyadenylation. The complete 3’early UTR together with the full-length HPV- 16 L2 gene were fused downstream of a cytomegalovirus (CMV) promoter driven chloramphenichol acetyl transferase gene, generating the construct pCATL2. A parallel construct was generated where the full-length L2 gene was replaced by a codon-optimised version of L2, producing pCATL2M. In codon-optimisation of the HPV-16 L2 gene it was earlier shown that RNA elements inside the L2 ORF, negative on the RNA and protein levels (222, 279), had been deactivated (222). pAE in pCATL2 was fully operational, whereas no activity was detected at all in pCATL2M. This demonstrated that sequences contained in the L2 region had activating effects on pAE and that these had been deactivated by codon-optimisation of the sequence. By making hybrids between wild-type and mutant sequences it was found that more than 410 nucleotides downstream of pAE, located in the L2 gene, were needed for full efficiency of pAE. Deletions in the first 299 nucleotides showed that the 160-240 nucleotides downstream of pAE contained a major enhancing polyadenylation element. Interestingly, deletion of the intervening region between the pAE and the major enhancing region did not affect polyadenylation efficiency. These results suggests that a major part of the downstream region could be involved in the formation of a complex secondary structure important in presenting certain regions to the pAE. Closer inspec- tion of the major enhancing region revealed the presence of multiple triple-G motifs and a hexa-U stretch. Consecutively mutating the motifs gradually lowered the polyadenylation efficiency of pAE, whereas mutating the hexa- U had no effect. The negative effect from the triple-G mutations was repro- duced when introduced into the subgenomic pBEX constructs. In correlation with the earlier suggestion, deactivation of L1 splicing inhibitory sequences in pBEX, generating pBEXM, allowed the L1 3’ss to compete with the utili- sation of the pAE. This again showed that a large downstream region of pAE was involved in the polyadenylation reaction. Apparently, in some cases, there is time for both RNA-polII to synthesise the full late region and the assembly of the mature splicing machinery to have its action before polyadenylation at pAE occur. In addition, exchanging the L2 gene for the full-

45 length L2 mutant version in the pBEX and pBEXM plasmid demonstrated the importance of some L2 sequences, distinct from common necessary splicing elements, for the L1 3’ss to be used. In screening for protein interactions with the pAE downstream region, we found that the major enhancing region bound a 55 kDa protein and that binding strength coincided with number of triple-G present in the sequence studied. Searching the literature allowed a candidate 55 kDa poly-G binding protein to be proposed, hnRNP H. By immunoprecipitation it was further shown that hnRNP H was indeed the factor that strongly interacted with the HPV-16 major polyadenylation region. The core polyadenylation factor CstF-64 was also shown to have specificity to the major enhancing region. An immunohistochemical detection of hnRNP H revealed that hnRNP H was expressed in the lower- and suprabasal layers in cervical squamous epithelium, but not in the superficial layers consisting of highly differentiated cells. This correlates with a differentiation hnRNP H- dependency of HPV-16 pAE.

46 3. Future perspectives

The knowledge in the field of papillomavirus is rapidly expanding. The win- dows to how the virus regulates its life cycle are quickly opening as methods like raft culturing has been established and widely used. This enables one to closely study papillomavirus in a natural environment but still in vitro. Many other new techniques would be interesting to apply in unveiling the intrica- cies in the virus-host relationship. These could be whole genome microarrays, confocal microscopy, RNAi, new services and others. The fast growing knowledge of the transcriptosome together with the development and reliability of splicing arrays has now come so far that they are being employed in evaluating patterns of gene expression in different sicknesses. Microarrays has been employed in genome-wide steady-state, degradation and translation levels of mRNA. The integration of viral infections in these approaches would vastly expand the amount of knowledge. Huge overlaps of information would probably be gotten between the studies strengthen- ing/demolishing/surfacing many biological models. It is now also possible to follow the path of individual RNAs in living cells. In addition, small inter- fering RNA directed against important viral transcripts could be applied either directly or their expression could be induced through site directed targeting with special carriers. Importantly, it is now possible for a relatively small amount of money and in a couple of weeks to get totally synthetic genes that earlier would have taken many months to synthesise and cost ten times as much. Interestingly, the quickly growing amount of antibodies against all kinds of regulatory proteins opens up the possibility to also apply antibody-arrays. A combination between conventional micro-arrays, antibody-arrays and RT-PCR would be a powerful tool in elucidating, for example, the cell environment at certain stages in the viral life cycle or the oncogenicity of individually expressed viral proteins. In a more technical and detailed perspective, it would be interesting to see the expression pattern of hnRNP H and other factors possibly involved in late gene expression in infected epithelia and epithelia in malignant state. Since both HPV E2 and E4 have been shown to have late gene expression or differentiation inducing capabilities, it would be of interest to co-express these factors and evaluate a possible effect on the pAE. Alternatively, a direct induction or knocking down of specific factors could be performed, either through anti-sense expression or RNA interference. In addition, there are many different cell lines available showing different expression patterns

47 for certain regulatory factors. pAE could be tested in a B-cell line compared and a plasma-cell line, between which hnRNP H/F levels and ratios have been shown to vary, and in that having regulatory roles in polyadenylation. Both HPV-16 and HPV-31 pAE need a large part of their downstream region for full polyadenylation efficiency. Perhaps this is because a complex secondary structure needs to be formed. Perhaps it has loose structures with some being stabilised by multiple factors, such as hnRNP H, for the polyadenylation mechanism to function. A thorough evaluation of the downstream region in both a computational and experimental approach would be valuable. Are the negative elements in L2 and L1 coding regions only artefacts because the genes are expressed from isolated forms? Or do the elements have those functions also in the certain stages of the viral life cycle? A further evaluation of the splicing promoting effect on the L1 3’ss from the L2 region is required (See Paper III and summary thereof (section 2.3)). Both exact sequence and eventual interacting proteins should be determined. Perhaps it is only structure of the upstream region of the L1 3’ss that is important, mirrored in the A/U to G/C content. Results from isolated regions of the genome must eventually be evalu- ated in the context of the whole genome, where individual events are highly influenced by upstream and/or downstream events. Alternatively, the effect of small genomic alterations in a whole genome approach might not be detectable, hence, each signal/site should be analysed individually to evaluate its full sensitivity. In the process to elucidate the complex interactions between HPV and humans not only do we battle the oncogenicity of the virus but we also learn a lot about normal cellular processes. Many important findings have been done in papillomaviruses. The materialization of systems biology where individual biological processes are put together as whole systems will shed light and truly explain many individual and strange phenomena concerning viral infection and diseases. I believe the scientific future for papillomavirus to be bright and in the near time problems surrounding its oncogenicity to be completely revealed and solved.

48 4. Populärvetenskaplig sammanfattning (In Swedish)

Virologin föddes i slutet av 1800-talet då man påvisade att en vätska där bakterier och svamp var bortfiltrerade fortfarande kunde överföra smittsamma sjukdomar. Detta fenomen gavs det latinska ordet “Virus”, för “slemmig vätska” eller “gift”. Det var i början av 1900-talet som det för första gången vetenskapligt visades att vårtor var smittsamma. Vanliga hand- och fotvårtor orsakas av humant papillomvirus (HPV). Till denna virusfamilj finns också de som orsakar könsrelaterade vårtor såsom kondylom och många andra mer obskyra hud- och slemhinneinfekterande typer. Denna virusfamilj innehåller över 100 medlemmar, vilka kan delas upp i olika grupper beroende vilken typ av epitel (hud/slemhinnor) de infekterar. Det kom att dröja fram till för 30 år sedan innan man började förstå att HPV kunde orsaka cancer. Nu är det allmänt vedertaget att vissa typer ur denna virusfamilj, främst de sexuellt överförbara/slemhinneinfekterande, kan ge upphov till olika cancerformer bl.a. livmodershalstappscancer. Oftast så ger infektionen inga symptom och i de allra flesta av fallen så är infektionen bekämpad inom ett år. Kända riskfaktorer för HPV-medierad tumöruppkomst är främst förlängd infektion, som i sin tur påverkas av att vara HIV-positiv, svagt immunförsvar, dålig diet, rökning samt många sexpartners under sin livstid. Livmoderscancer är den näst vanligaste cancerformen hos kvinnor världen över, med en medelålder för uppkomst på 52 år. I 99% av cancerfallen så har man funnit HPV närvarande. Sedan man införde Cellskrap i USA så har antalet uppkomna livmoderscancerfall minskat med 80%. Dock så kommer denna metod inte att bekämpa HPV som så, samt att många utvecklingsländer ej har råd till ett nationellt program. Utveckling av ett billigt vaccin skulle kunna vara lösningen. Fram till nyligen så har produktionen av virus protein för vaccinering varit förhindrad på grund av komplexiteten i virusets gener. Vi och andra har visat att genom att förändra virusets genstruktur så kan viralt protein produceras och vaccinering utföras med positivt resultat. Den vanligaste sexuellt överförbara HPV typen är den cancerassocierade HPV-16. Denna typ återfinns i över 50% av alla livmoderscancrar. Detta varför vi valde att studera just HPV-16. HPV infekterar de basala epitelcellerna, vilka är de innersta lagrena där cellerna fortfarande är delningskompetenta. När celldelning sker och en

49 cell puttas utåt i lagrena så kommer den att ej att dela sig igen utan att börja åldras. När så cellen till slut når det sista lagret så kommer den att dö och ramla bort. HPV infekterade celler kommer inte att sluta dela sig efter att ha lämnat de basala lagrena utan fortsätter dela sig ett tag. På detta sätt så kommer det att finnas många fler infekterade celler, med följden av högre virus produktion. Det är dessutom denna effekt som ger upphov till den karaktäristiska vårtformationen. I ett tidigt stadium, i basala celler och nära detta lager, så kommer bara de mest nödvändiga virala proteinerna kunna detekteras och detta endast i mycket små mängder. Såsom cellerna ytterst i ”vårtan” börjar åldras så kommer virusets gener att uttryckas annorlunda. Det är först där som virusets strukturella proteiner börjar produceras. En hög produktion av dessa samt expandering av antalet viruskromosomer leder till formationen av enorma mängder HPV som slutligen avsöndras till omgivningen redo för att infektera nya värdar. Vi är intresserade av åldringsprocessen av cellen och vad som händer för att virusets strukturella proteiner skall börja produceras. Vi studerar virusets gener i både isolerade former och dess naturliga plats på viruskromosomen. Genom att punktvis eller med större förändringar i virusets DNA så har vi funnit ett flertal cellulära proteiner som möjligen interagerar och reglerar de strukturella generna. Initiala experiment visade på att dessa gener innehöll delar som gjorde så att genprodukterna blev både instabila samt proteinproduktionen från dem inhiberades. Mer fortgående resultat visar att dessa negativa delar, i alla fall delvis, istället inducerar ett alternativt genuttryck hos viruset beroende på åldringsstadium hos den infekterade cellen. Ett av proteinerna som verkar spela en roll heter hnRNP H. Det binder till ett flertal ställen på en av de två strukturella generna, medförande en kumulativ ned-reglerande effekt. När vi märkte in oinfekterat livmodersepitel med antikroppar specifika mot detta protein så fann vi en mycket god korrelation med detta proteins ned-reglering och strukturella proteiners upp-reglering. I fortsättningen så kommer vi vilja studera hur andra faktorer är reglerade i åldringsprocessen samt vad som händer med dessa i infekterade celler och celler som har utvecklats till cancerceller. Ett mål är att kartlägga regleringen av de virala generna för att finna ställen optimala för alternativa vaccineringsmetoder.

50 5 Acknowledgement

As always, one will forget to thank a lot of people. I sincerely apologise to all I have not mentioned below.

First of all I would like to thank my supervisor, Stefan, for giving me the opportunity to do a thesis under your tutelage. During the five years we have worked together I have learnt a lot from you, of which I am looking forward to use in future work. Present and past lab members: Marcus, Brian, Lisa, Annette, Karin, Anna, Margaret and Xiaomin. Thanks for all the help. It was great getting to know you and I hope you all the very best. “Thank you” a million times would not be enough to Margaret, for all the help and support I have gotten from you. It has been invaluable. Thanks also for all the Irish lessons! Good luck with your upcoming writing! Special thanks to Brian for introducing me into the lab and for great gym sessions. Also to Xiaomin, if I say it myself I have become a great singer in Chinese, bao xie xie! Mutated rabbits? Some would say: Zhen Qi Guai… What is the name in English for the African animal with a long neck? Well, you ask Max and he knows! Thanks mate! I could easily fill a page. All the co-authors for past, present and, hopefully, future collaborations. I would like to thank Dr. Sheila Graham who agreed to be my opponent. I am looking forward to an interesting discussion and wish you great fortune in your life and science. All the IMBIM people: Good luck with everything you all do in the future. Especially to group leaders Göran Akusjärvi, Catharina Svensson and Göran Magnusson. All present and past people of the virology corridor: For many good advice and nice and fun company. All the administrative people have been invaluable during the time. Tack så mycket! Ylva, tack för alla snälla ord under alla dessa år. Norrlänningar är bäst! Jag hoppas innerligen att du blir friskare och så förblir. Lena, alltid uppmuntrande och go. Minns fortfarande frukten för 7 år sedan. Min andra mamma… Må ditt liv förbli friskt och lyckligt! “The Deep Vikings”, my brothers with whom I have slain dragons and saved princesses a countless number of times. What a grey and boring world it would have been without our time together. Thanks a lot! May the dice always roll your way… In Erik’s case “our way”, no offence!

51 Rocío, I am the luckiest guy. !Te quiero! My family, I love you all! Thanks for all the encouragement, belief and help during the years. Mum, I owe it all to you.

52 6 References

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