«1. Introduction The human papillomavirus (HPV) is one of the most common causes of sexually transmitted disease in both men and women around the ...»
Biology and Pathogenesis
José Veríssimo Fernandes1 and
Thales Allyrio Araújo de Medeiros Fernandes2
1Federal University of Rio Grande do Norte
2University of Rio Grande do Norte State
The human papillomavirus (HPV) is one of the most common causes of sexually transmitted
disease in both men and women around the world, especially in developing countries,
where the prevalence of asymptomatic infection varies from 2 to 44%, depending on the population and studied region (Sanjosé et al., 2007). Most HPV infection is transient and some studies show that the majority of sexually active individuals are exposed to and acquire infection from this virus at some phase in their lives (Baseman and Koutsky, 2005;
Trottier and Franco, 2006). HPV infection is more prevalent in young adults, at the beginning of their sexual activity, with a subsequent decline in the prevalence rate with increasing age, likely as a result of development of an immune response against the virus and reduction of sexual activity (Castle et al., 2005; Fernandes et al., 2009; Chan et al., 2010).
HPV can infect basal epithelial cells of the skin or inner-lining tissues and are categorized as cutaneous types or mucosal types. Cutaneous types are epidermotropic and infect the keratinized surface of the skin, targeting the skin of the hands and feet. Mucosal types infect the lining of the mouth, throat, respiratory, or anogenital tract epithelium (Burd, 2003).
Some HPVs are associated with warts while others have been well established as the main risk factor of invasive cervical cancers and their associated pre-cancerous lesions (Clifford et al., 2005; Zekri et al., 2006; Muñoz et al., 2006). However, only few HPV-infected individuals progress to invasive cervical cancer (Burd, 2003). Most infected individuals eliminate the virus without developing recognized clinical manifestation. (Bosch et al., 2008).
Today, more than 150 different HPV types have been cataloged and about 40 can infect the epithelial lining of the anogenital tract and other mucosal areas of the human body. Based on their association with cervical cancer and precursor lesions, HPVs can also be classified as high-risk (HR-HPV) and low-risk (LR-HPV) oncogenic types. LR-HPV types, such as HPV 6 and 11, can cause common genital warts or benign hyperproliferative lesions with very limited tendency to malignant progression, while infection with HR-HPV types, highlighting HPV 16 and 18, is associated with the occurrence of pre-malignant and malignant cervical lesions (Muñoz et al., 2003; Bosch et al., 2002; Bosch et al., 2008). HR-HPV types are also associated with many penile, vulvar, anal, and head and neck carcinomas, and contribute to over 40% of oral cancers (Stanley, 2010).
Persistent infection with HR-HPV is unequivocally established as a necessary cause of cervival cancer (Trottier & Franco, 2006). The critical molecules for initiation and progression of this cancer are the oncoproteins E5, E6, and E7, that act largely by overcoming negative growth regulation by host cell proteins and by inducing genomic instability, a hallmark of HPVassociated cancers (Munger et al., 2004; Moody & Laimins, 2010).
Once HPV transmission to the genital tract occurs through sexual contact, the risk factors for the infection and cervical lesions, including cervical cancer, are the same classic risk factors for other sexually transmitted diseases. The number of sexual partners is the risk factor more consistently associated with genital HPV infection and therefore with cervical cancer.
In addition, other indicators of sexual behavior and reproductive activities, heredity, immune and nutritional status, and smoking can contribute in some way to the development of cervical cancer (Tarkowski et al., 2004; Muñoz, 2006; Fernandes et al., 2010).
In this chapter we will discuss the biology and pathogenesis of human papillomavirus, analyzing some specific aspects of their interactions with the infected host and specific host cell components.
2. Biologic properties of HPV
2.1 Structure of viral particle and regulation of gene expression The human papillomavirus (HPV) is a relatively small non-enveloped virus that contains a double-stranded closed circular DNA genome, associated with histone-like proteins and protected by a capsid formed by two late proteins, L1 and L2. Each capsid is composed of 72 capsomeres, each of which is composed of five monomeric of 55kDa units that join to form a pentamer corresponding to the major protein capsid, L1. The L1 pentamers are distributed forming a network of intra- and interpentameric disulfide interactions which serve to stabilize the capsid (Sapp et al., 1995). In addition to L1, minor capsid proteins with approximately 75kDa exist within the virion and are called the L2 protein. To assemble the viral capsid, the pentamers join to copies of L2 that occludes the center of each pentavalent capsomere. (Jo & Kim 2005; Buck et al., 2008; Conway & Meyers, 2009). Thus, each virion contains 72 copies of the L1, the major component of the capsid, and a variable number of copies of L2, a secondary component of the viral capsid, forming a particle with icosahedra symmetry and approximately 50 to 60 nm in diameter ( Burd, 2003; Longworth & Laimins, 2004; zur Hausen, 2009).
Fig. 1. The structure of HPV. (Adapted from Swiss Institute of Bioinformatics, Viral Zone. Available in http://viralzone.expasy.org/all_by_species/5.html )
The viral genome of the HPV consists of a single molecule of double-stranded and circular DNA, containing approximately 8000 base pairs and harboring an average of 8 open reading frames (ORFs) (Jo & Kim 2005; Zheng & Baker, 2006). In a functional point of view, the HPV genome is divided into three regions. The first is a noncoding upstream regulatory region (URR) or long control region (LCR) that has regulatory function of the transcription of the E6 and E7 viral genes; The second is an early region (E), consisting of six ORFs: E1, E2, E4, E5, E6, and E7, which encodes no structural proteins involved in viral replication and oncogenesis. The third is a late (L) region that encodes the L1 and L2 structural proteins. The LCR region of the anogenital HPVs ranges in size between 800-900 pb, representing about 10% of the genome, and varies substantially in nucleotide composition between individual HPV types (Fehrmann & Laimins, 2003; Jo & Kim, 2005).
Only one strand of the double-stranded DNA serves as the template for viral gene expression, coding for a number of polycistronic mRNA transcripts. (Stanley et al., 2007).
The regulation of viral gene expression is complex and controlled by cellular and viral transcription factors. Most of these regulations occur within the LCR region, which contains cis-active element transcription regulators. These sequences are bound by a number of cellular factors as well as the viral E2 product (zur Hausen, 1996). A large number of cellular transcription factors have been identified and the dysfunction of some of them appears to play a significant role in papillomavirus-linked carcinogenesis (Thierry et al., 1992; Hamid & Gaston, 2009).
The transcription start sites of viral promoters differ depending on the virus type, but, in all types, promoter usage is keratinocyte differentiation-dependent (Smith et al., 2007). The replication origin and many transcriptional regulatory elements are found in the upstream LCR region. The virus early promoter, differentiation-dependent late promoter, and two polyadenylation signals define three general groups of viral genes that are coordinately regulated during host cell differentiation. The E6 and E7 genes maintain replication competence. E1 E2, E4, E5, and E8 are involved in virus DNA replication, transcriptional control, beyond other late functions and L1 and L2, responsible for the assembly of viral particles (Bodily & Laimins, 2011).
The regulation of expression of the late genes in genital HPVs is not well understood.
However, it has been shown that the second, or later, promoter is initiated in a differentiation-dependent manner, and thus is activated only when cells are grown in the host’s stratifying/differentiating tissue. Once activated, the later promoter directs transcription from a heterogeneous set of start sites and will serve to produce a set of transcripts that facilitate the translation of L1 and L2 proteins (Smith et al., 2007; Conway & Meyers, 2009). Activation of the later promoter is accompanied by acceleration of viral DNA replication and by high levels of viral protein expression. As a result, virus copy-number amplifies from 50 copies to several thousands of copies per cell. So when a late promoter is activated, the expression of genes will occur, encoding the structural proteins L1 and L2, which join to assemble the capsids and to form virions (Stanley et al., 2007).
2.2 Functions of viral proteins E1 Protein The E1 protein represents one of the the most conserved proteins among different HPV types. It has DNA-binding functions and a binding site in the origin of replication localized
in the LCR region. It assembles into a hexameric complex, supported by the E2 protein, and the resultant complex has helicase activity and initiates DNA bidirectional unwinding, constituting a prerequisite for viral DNA replication (Wilson et al., 2002; Frattini & Laimins, 1994). The carboxyl terminal domain of E1 has an ATPase/helicase activity and is necessary subunit p70 of DNA polymerase α, but is not sufficient to support replication (Amin et al., and sufficient for oligomerization. This domain also interacts with the E2 protein and 2000). A segment of approximately 160 amino acid residues upstream of the ATPase/helicase domain is the DNA-binding domain (Titolo et al., 2003). A stretch of about 50 amino acids within the amino terminus of E1 acts as a localization regulatory region (LCR) and contains a dominant nuclear export sequence (NES) and a nuclear localization signal (NSL), which are regulated by phosphorylation (Deng et al., 2004).
E2 protein The E2 open reading frame of HPV gives rise to multiple gene products by alternative RNA splicing. The proteins derived from the E2 gene are involved in the control of viral transcription, DNA replication, and segregation of viral genomes (McPhillips et al., 2006;
Kadaja et al., 2009). These different E2 types represent the major intragenomic regulators (Bouvard et al., 1994).
The E2 protein can bind to factors on mitotic chromatin and join the virus genome to host cell chromosomes during mitosis; it contributes to coordinating the HPV DNA replication with host cell chromosome duplication, allowing the viral genomes to be distributed to the daughter cell. This constitutes an important requirement for the persistence of virus DNA in undifferentiated basal cells (McPhillips et al., 2006). Furthermore, the E2 protein interacts with E1 and stimulates viral DNA replication, favoring the binding of E1 to the origin of replication ( Seo et al., 1993; Chow et al., 1994).
In lesions containing HPV episomes, the E2 protein directly represses the expression of early genes as a mechanism to regulate the copy number. In addition, it has been reported that HPV E2 proteins are able to repress telomerase promoter activity mediated by the HPV E6 protein (Hamid et al., 2009). Integration of the HPV genome in the host cell chromosome usually disrupts E2 expression, causing a deregulated expression of early viral genes, including E6 and E7, and this event can favor the transformation of human cells and the transition into a malignant state (Romanczuk & Howley, 1992) In addition to the full-length E2 protein, the infected cells can express an E8^E2C transcript, in which the small E8 domain is fused to the C-terminal domain of E2 (E2C). The full-length E2 protein forms heterodimers with repressor forms of E2, and these E2 heterodimers serve as activators of transcription and replication during the viral cycle. The single-chain E2 heterodimer in the HPV 18 genome initiates genome replication, but is not sufficient for long-term replication of the HPV 18 genome. This is due to the capacity of HPV18 in encoding the repressor E8/E2, which acts as a negative regulator of HPV18 genome replication (Kurg et al., 2010). Moreover, it has been shown that inactivation of E2 in the HPV16 genome increases E6/E7 transcription (Soeda et al., 2006), and that mutation of E8^E2C in the HPV31 or HPV16 genome increases the genome copy number and the E6/E7 transcription, suggesting that the transcriptional repressing by E8^E2C has an important role in viral replication (Lace et al., 2008). It was also noted that the E2C domain not only mediates specific DNA binding but has also an additional role in transcriptional repression www.intechopen.com 7 Human Papillomavirus: Biology and Pathogenesis by recruitment of co-repressors, such as the CHD6 protein. This suggests that repression of the E6/E7 promoter by E2 and E8^E2C involves multiple interactions with host cell proteins through different protein domains (Fertey et al., 2010).
E4 protein Despite being considered an early protein, E4 is exclusively located in the differentiated layers of the infected epithelium (zur Hausen, 1996). Although its expression occurs in highly differentiated cells that express the capsid genes and synthesize new progeny virions, and coincides with the onset of vegetative viral DNA replication, E4 is not found in virion particles. The role of this protein in the virus life cycle has not yet been determined, but E4 is not required for transformation or episomal persistence of viral DNA, but interacts with the keratin networks and causes their collapse (Doorbar et al., 1991).
It has been suggested that E4 may have an important role in favoring and supporting the HPV genome amplification, besides regulating the expression of late genes, controlling the virus maturation, and facilitating the release of virions (Londgworth & Laimins 2004). E4 also interacts with and disrupts the organization of intermediate filaments. The role of E4 in providing the release of virus is supported by the association of E4 with the cornified cell envelope (CCE), a highly resistant structure under the plasmatic membrane of differentiated keratinocytes in the stratum corneum. Furthermore, E4 may play role in regulating gene expression and has been shown to induce G2 arrest in a variety of cell types (Londgworth & Laimins 2004).