Papillomaviridae Classification Essay

1. Introduction

Papillomaviruses have been discovered in a wide array of vertebrates. More than 300 papillomaviruses have been identified and completely sequenced, including over 200 human papillomaviruses (PaVE: Papillomavirus Episteme [1]). One of the most distinctive characteristics of the papillomavirus group is their genotype-specific host-restriction, and the preference of particular papillomavirus types for distinct anatomical sites, where they cause lesions with distinctive clinical pathologies [2]. These include benign hyper-proliferative lesions such as warts, as well as unapparent or asymptomatic precursor lesions, that can in some instances progress to high-grade neoplasia and invasive malignant cancer. The association of “high-risk” human papillomavirus (HPV) types (see legend to Figure 1) with cervical cancer is now well established, and provides a rationale for the introduction of HPV DNA testing in cervical screening, as well as the development of prophylactic vaccines against HPV16 and 18 which are the major papillomavirus types responsible for cervical cancer. Such typing studies have also revealed a plethora of HPV types, both high and low-risk, in oral mouthwash samples despite the absence of apparent clinical disease [3], as well as in skin swabs and plucked hairs taken from immunocompetent individuals [4,5,6,7,8,9]. The predominant asymptomatic skin HPV types come primarily from the genera Betapapillomavirus and Gammapapillomavirus, and at a population level, members of these genera are very successful, infecting children at a young age to produce persistent subclinical infections [4]. Changes in the epithelial micro-environment, as can occur following immunosuppression or in individuals suffering from epidermodysplasia verruciformis (EV), can allow these HPV types to produce visible papillomas, and in some situations can facilitate the development of cancers [10]. Certain Beta HPV types are a significant cause of non-Melanoma skin cancer in susceptible individuals, although the molecular mechanism by which they facilitate cancer progression appears to be somewhat different from what has been worked out for the Alphapapillomavirus types [11]. When considered together, it appears that different papillomavirus types have evolved distinct life-cycle strategies, which allow them to thrive and produce viral progeny at different epithelial sites. At least part of this variation reflects the different ability of papillomaviruses to interact with the immune system, and to produce productive infections that are visible at the macroscopic level. The complex immune evasion strategies that underlie the ability to produce such lesions are a particular characteristic of the Genus Alphapapillomavirus, with low-risk Alphapapillomavirus types in particular (Figure 1), often being responsible for recalcitrant warts even in immunocompetent hosts [12]. By contrast, most members of the genera β- and Gammapapillomavirus only cause such visible lesions when the normal immune response of the host is compromised [13,14,15,16]. The clinical importance of papillomaviruses, and their relatively small genomic size, has led to many full genomic sequences becoming available in recent years. The availability of such extensive sequence information, combined with a developing clinical and biochemical understanding of disease-biology, means that papillomaviruses are an ideal model system to understand how evolution can influence viral tropisms, pathogenicity, and the underlying molecular processes that govern disease outcome [17,18,19,20]. As discussed below, this “natural experiment” in infection is also providing us with insight into epithelial cell biology and immunology.

2. Papillomavirus Diversity at the Level of Genotype, Epithelial Tropism and Pathogenicity

Papillomaviruses comprise a diverse group of viruses that infect both humans and animals. Their origin is linked to changes in the epithelium of their ancestral host that occurred at least 350 million years ago. Since then, they have co-evolved as their different host species have evolved, with little cross-transfer between species [17,18]. They are now found in birds, reptiles, marsupials and other mammals, pointing to an earlier evolutionary appearance than was initially suspected. Recent phylogenetic analysis suggests, however, that the “generalist” ancestral papillomavirus may not have followed an identical evolutionary path to that of their hosts, but paralleled the evolution of host resources or attributes, such as the presence or absence of fur or the evolution of sweat glands. These host-adaptations are thought to have created new ecological niches for papillomavirus to colonise, which in turn drove viral diversity followed by co-speciation with their hosts [17]. Through this route, papillomaviruses have developed their remarkable species specificity as well as a great diversity of epithelial tropisms. With over 240 distinct papillomavirus types classified into 37 genera, papillomavirus may perhaps be considered as one of the most successful families of vertebrate viruses [17,21,22].

The classification of papillomaviruses is based on nucleotide sequence comparison rather than on serology, with individual HPVs being referred to as genotypes [22]. Data from vaccine trials has shown that there is limited antibody cross-reactivity, except between closely related genotypes [23], with the major coat protein of the virus (L1) containing hypervariable loops that are exposed on the virion surface [24,25]. The L1 gene was chosen early on as the standard for PV classification, and for some papillomavirus types, there is little sequence information outside of this region. To be classified as distinct types, individual papillomaviruses must be at least 10% divergent from each other in their L1 nucleotide sequence. These papillomavirus “types” are grouped into larger phylogenetic groupings or genera, which are categorised with a Greek letter followed by a number that indicates the species (Figure 1) [22].

Thus the species Alphapapillomavirus 9 includes HPV types 16, 31, 35, 33, 52, 58 and 67. Ideally, papillomavirus (PV) classification should integrate phylogeny, genome organization, biology and pathogenicity as a single property, rather than being based simply on genomic sequence analysis, or the analysis of genomic fragments [21]. This phylogenetic species concept, although potentially useful, is complex to implement, however, and for many papillomavirus types, detailed information regarding their biology is only poorly defined.

Figure 1. Evolutionary Relationship between Human Papillomaviruses. The human papillomaviruses types found in humans fall into five genera, with the Alpha-, Beta- (blue) and Gammapapillomavirus (green) representing the largest groups; Human papillomaviruses types from the Alphapapillomavirus genus are often classified as low-risk cutaneous (light brown); low-risk mucosal (yellow); or high-risk (pink) according to their association with the development of cancer. The high-risk types highlighted with red text are confirmed as “human carcinogens” on the basis of epidemiological data. The remaining high-risk types are “probable” or “possible” carcinogens. Although the predominant tissue associations of each genus are listed as either cutaneous or mucosal, these designations do not necessarily hold true for every member of the genus. The evolutionary tree is based on alignment of the E1, E2, L1, and L2 genes [26]. HPV sequence data was be obtained from PaVE [1].

Figure 1. Evolutionary Relationship between Human Papillomaviruses. The human papillomaviruses types found in humans fall into five genera, with the Alpha-, Beta- (blue) and Gammapapillomavirus (green) representing the largest groups; Human papillomaviruses types from the Alphapapillomavirus genus are often classified as low-risk cutaneous (light brown); low-risk mucosal (yellow); or high-risk (pink) according to their association with the development of cancer. The high-risk types highlighted with red text are confirmed as “human carcinogens” on the basis of epidemiological data. The remaining high-risk types are “probable” or “possible” carcinogens. Although the predominant tissue associations of each genus are listed as either cutaneous or mucosal, these designations do not necessarily hold true for every member of the genus. The evolutionary tree is based on alignment of the E1, E2, L1, and L2 genes [26]. HPV sequence data was be obtained from PaVE [1].

In general, sequence-based phylogeny does provide some useful insight into disease association, although closely related types can in some instances show distinct pathologies. HPV4, 65 and 95 are encompassed in the Gammapapillomavirus 1 species, for instance, but while HPV4 and 65 induce indistinguishable pigmented wart-like lesions mainly on the palmoplantar or lateral surface of hands and feet [27,28,29], HPV95 induces less obvious unpigmented papules primarily on plantar epithelial surfaces [30]. HPV6 and 11 are similarly contained within a common species-grouping (Alphapapillomavirus 10), and although both cause papillomas of similar appearance, HPV6 shows a marked predilection for genital sites, when compared to HPV11, which is the most prominent type at oral sites [31,32,33,34]. A third example comes from the Alphapapillomavirus 8 species which includes HPV40, which causes mucosal lesions, and HPV7, which is the cause of “butchers” warts that develop at cutaneous sites, particularly the hands [35]. Explaining such subtle tropism differences is beyond our current understanding of virus biology, and is not of obvious medical importance. It is however likely that such tropism differences reflect differences in viral gene function, patterns of gene expression and epithelial regulation at different body sites. Perhaps of greater importance are the differences in cancer risk associated with the high-risk types. HPV16, 31 and 35 are all contained within the Alphapapillomavirus 9 species group and are classified as “carcinogenic to humans” by The International Agency for the Research on Cancer (IARC) [36]. The association between HPV16 and cervical cancer is more than 10 times stronger than that between either HPV31 or HPV35 however [37], and of these three types, HPV16 is uniquely associated with tumours of the oropharyngeal region [38]. To explain this will require a dissection of virus-specific gene expression at this particular epithelial site, and an understanding of virus protein function and the extent to which proteins encoded by different HPV types affect common molecular pathways.

From the above, it is apparent that viral pathogenicity depends on multiple factors, including the virus genotype, the nature of the cell infected (tropism) and the status of host immunity. Our current thinking suggest that tropism is controlled primarily at the level of viral gene expression rather than at the level of viral entry into the cell [39], and that regulatory elements within the long control region (LCR) are of key importance in determining the tissue range of different HPV types [40,41]. Differences in infectivity may however be a contributing factor, and it has been suggested that this may correlate with differences in surface charge distribution between cutaneous and mucosal virions [42]. Although the diseases caused by specific HPV types sometimes occur at non-typical sites, this is uncommon, with such lesions often exhibiting non-typical morphology and pathology [43]. The idea that papillomaviruses have epithelial sites where they have evolved to complete their productive life-cycle, and epithelial sites where they do this less-well or not at all, makes good sense when we consider the small number of instances where cross-species infection has been documented. Bovine Papillomavirus type 1 infection of cattle leads to the development of benign cutaneous lesions that may regress, whereas infection of horses typically results in the development of locally aggressive, non-regressing equine sarcoids that are non-permissive for virus production [44,45,46,47]. In this case, it appears that the BPV E5 gene, which is important for the production of fibropapillomas in cattle, can stimulate aberrant cell proliferation in the dermal layers in the horse [47]. Although cottontail rabbit papillomavirus (CRPV) is not a fibropapillomavirus like BPV, a similar concept explains its ability to generate non-productive lesions in domestic rabbits, that have a tendency to develop over time into neoplasia and cancer [48,49]. In this case, it is the E6 and E7 proteins, along with the viral transcription factor E2, that are ultimately important for the development of the cancer phenotype [50,51]. Historically, these ideas are not new to the field of tumour virus biology, with a large body of experimental work using animal models to understand how members of the polyoma and adenoviruses families can cause tumours [52,53]. In the case of adenoviruses, it is the E1A and E1B proteins, and with polyomaviruses it is the T antigens, that when deregulated can cause tumours in experimental systems [54,55], and it is now well known that these proteins share functional similarity with the E6 and E7 proteins of the high-risk papillomaviruses [56,57]. In fact, our current knowledge suggests that it is the deregulated expression of the high-risk E6 and E7 proteins that leads to the development of neoplasia at specific epithelial sites, and that these infections should be regarded as “non-productive or abortive infections” rather than “ordered” productive infections, where the level and pattern of viral gene expression is properly controlled [58,59]. It has been reported that several factors such as the type of cell, hormone as well as inflammatory cytokines control viral gene expression [41,60,61,62]. With this in mind, it is clearly important to understand how epithelial environment, cell type and host immunity act together to drive neoplasia, and we suspect that this will be an important topic of future research. Although this line of thinking fits well with our understanding of how high-risk HPV types cause neoplasia and cancer at specific sites, including the cervix, anus and oropharynx, the same broad principles also explain the elevated risk of non-melanoma skin cancer posed by Betapapillomaviruses, even though the molecular detail of how these viruses cause cancer is different [11]. Betapapillomavirus types are prevalent as asymptomatic infections in normal skin and mucosa in the general population [3,63,64], with infection occurring soon after birth [9]. In immunocompromised individuals, including those suffering from epidermodysplasia verruciformis, it appears that normal patterns of viral gene expression can become deregulated, allowing a level of viral gene expression that would not be tolerated in immune competent individuals (Figure 2) [11].

A Viral Biorealm page on the family Papillomaviridae

Baltimore Classification

Higher order taxa

Viruses; dsDNA viruses, no RNA stage; Papillomaviridae

Genera

Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Deltapapillomavirus, Epsilonpapillomavirus, Zetapapillomavirus, Etapapillomavirus, Thetapapillomavirus, Iotapapillomavirus, Kappapapillomavirus, Lambdapapillomavirus, Mupapillomavirus, Nupapillomavirus, Xipapillomavirus, Omicronpapillomavirus, Pipapillomavirus

Description and Significance:

Papillomaviruses are a class of viruses that infect many vertebrates and can cause benign epithelial growths, known as papillomas, such as skin and genital warts.

Human Papilloma Virus (HPV) is a very common virus that causes the growth of abnormal tissue or cells on body skin. HPV can cause abnormal tissue changes on the vocal cords, mouth, hands, feet and genital organs. Each type of HPV infects certain parts of the body and over 60 such types have already been identified.

HPV is of extreme significance because some types of this virus lead to the growth of abnormal tissues that can lead to cancer of the female organs. Cancer can be prevented by finding and treating HPV-related tissue changes.

Genome Structure

The genome of papillomavirus is not segmented and contains a single molecule of circular, superoiled, double-stranded DNA. The complete genome is 5300-8000 nucleotides long. The genome has a guanine + cytosine content of 40-50%. (source: ICTVdB Descriptions)

Virion Structure of a Papillomavirus

The virions of papillomavirus consist of a non-enveloped capsid that is round with icosahedral symmetry. The isometric capsid has a diameter of 40-55 nm. The capsids appear round and the capsomer arrangement is clearly visible. The capsid consists of 72 capsomers in skew arrangement. The surface appears rough and the surface projections are small. (source: ICTVdB Descriptions)

Reproductive Cycle of a Papillomavirus in a Host Cell

The individual isolates for papillomavirus are very species-specific. All of these isolates are tropic for squamous epithelial cells but the receptors are unknown. The virus infects the basal cells of the dermal layer, and early gene expression can be detected in these cells (in situ hybridization). However, late gene expression, expression of structural proteins and vegetative DNA synthesis is restricted to terminally differentiated cells of the epidermis which implies a link between cellular differentiation and viral gene expression.

The functions of each Gene are given below:

E1-- Replication and replication repression
E2-- Activates transcription in HPV types 6, 11 and 16, represses transcription and binds to long control region.
E3-- No known product or function
E4-- Cytoplasmic protein in HPV-1-induced warts.
E5-- Transformation in HPV-6.
E6-- Transformation in cooperation with E7 in HPV-16 and HPV-18.
E7-- Transformation in cooperation with E6.
E8-- No known product or function.
L1-- Major capsid protein.
L2-- Minor capsid protein.

The expression of the Papillomavirus genome is complex because there are multiple promoters, alternative splicing patterns, and a link between differentiation and gene expression. Only one strand of the genome is transcribed, producing two classes of proteins: the non-structural regulatory proteins, known as early proteins, and the structural proteins L1 and L2, known as late proteins.

The transformation process is very complex and depends largely on the early gene products. The transferring proteins appear to vary from one virus type to another and the function and mechanism of these transforming proteins are still not clear. The general principle appears to be that two or more early proteins co-operate to give a transforming phenotype.The most confusing thing is that in most cases, all or part of the papilloma genome including the putative "transforming genes" is maintained in the tumour cells, whereas in other cases, the virus DNA may be lost after transformation. BPV-4 is an example of one such "hit-and-run" mechanism.

In case of Human Papillomaviruses (HPVs), E2 binds to the early promoter and decreases expression of E6/E7. The loss of E2 is thus the first stage of transformation. E6 then binds to p53 via a cellular protein, p100, and targets it for degradation via the ubiquitin pathway. E7 binds pRB and prevents phosphorylation. This would normally result in apoptosis but both E6 ad E7 interact with a number of cellular proteins which influence the outcome of infection.

The HPV E7 proteins are small (HPV16 E7 comprising 98 amino acids), zinc binding phosphoproteins which are localised in the nucleus. They are structurally and functionally similar to the E1A protein of subgenus C adenoviruses. The first 16 amino-terminal amino acids of HPV16 E7 contain a region homologous to a segment of the conserved region 1 (CR1) of the E1A protein of subgenus C adenoviruses. The next domain, up to amino acid 37, is homologous to the entire region 2 (CR2) of E1A. Genetic studies have established that these domains are required for cell transformation in vitro, suggesting similarities in the mechanism of transformation by these viruses. The CR2 homology region contains the LXCXE motif (residues 22-26) involved in binding to the tumour suppressor protein pRb. This sequence is also present in SV40 and polyoma large T antigens. The high risk HPV E7 proteins (of, for example, types 16 and 18) have an approximately ten-fold higher affinity for pRb protein than the low risk HPV E7 proteins (of, for example, type 6). Association of the E7 protein with pRb promotes cell proliferation by the same mechanism as the E1A proteins of adenoviruses and SV40 large T antigen. Recent studies have shown that E7 promotes degradation of Rb family proteins rather than simply inhibiting their function by complex formation. The CR2 region also contains the casein kinase II phosphorylation site (residues 31 and 32). The remaining 61 amino acids of E7 protein have very little similarity to E1A, however a sequence CXXC involved in zinc binding is present in both proteins. The E7 protein contains two of these motifs which mediate dimerisation of the protein. Mutation in one of the two Zn binding motifs destroys transforming activity, although this mutant is able to associate with Rb protein. Therefore dimerisation may be important for the transforming activity of E7.

The HPV E6 are small basic proteins (HPV16 E6 comprising 151 amino acids) which are localised to the nuclear matrix and non-nuclear membrane fraction. They contain four cysteine motifs which are thought to be involved in zinc binding. E6 encoded by high risk HPVs associates with the wild type p53 tumour suppressor protein. For association with p53, the E6 protein requires a cellular protein of 100 kDa, termed E6-associated protein (E6-AP). Like SV40 large T antigen and Ad5 E1B 58 kDa, E6 proteins of high risk HPVs abrogate the ability of wild type p53 to activate transcription. However, the mechanism of E6 action is different than that of SV40 large T and the E1B protein since it involves degradation of p53. It has been shown that E6-dependent degradation of p53 occurs through the cellular ubiquitin proteolysis pathway.

Genome Replication: The genome is replicated as a multicopy nuclear plasmid (episome). There are two mechanisms involved in genome replication:

Plasmid Replication involves the E1 protein and occurs in cells in the lower levels of the dermis. The virus DNA is amplified to 50-400 diploid genomes and then it replicates once per cell division, with the copy number/cell remaining constant.
Vegetative Replication occurs in terminally differentiated cells in the epidermis. Here, the control over the number of copies seems to be lost and the DNA is amplified to hundreds of copies/cell. The virus is shed from epidermal cells when these are sloughed off and is transmitted by both direct and indirect contact.

The differentiation of the host cell determines the productive infection by HPV. A low level of gene expression occurs when HPV genomes are established as autonomous replicating extrachromosomal elements following entry into basal epithelial cells. After the differentiation of infected cells, the synthesis of progeny virions is made possible because of the induction of productive replication and expression of caspid genes. Certain HPV types can also exist in a latent state, as exemplified by evidences from immunosupressed patients as well as individuals with recurring laryngeal papillomatis.While HPV DNA may be present, differentiation-dependent synthesis of virions is not possible in latently infected cells. The effectiveness of therapeutic methods for treatment of infections could be determined by the presence of a latent state for HPVs.

Viral Ecology & Pathology

HPV can grow on the cervix, vagina, vulva, urethra and the anus. HPV causes condyloma (warts) and dysplasia (pre-cancer), two kinds of abnormal tissues. There is very little information available as to how or when people become infected with HPV. A lot of medical research is in progress to answer these questions and understand the exact nature of HPVs. It is only known now that HPV is mainly spread through sexual contact. The symtoms for HPV are not very explicit and both men and women infected with the virus may not know about it, nor develop dysplasia or genital warts for many years. Interestingly, smoking ciggarettes is also closely associated to HPV with smokers having a much higher chance of developing dysplasia than non-smokers.

References

Karl R. Beutner, MD, PhD, Stephen Tyring, MD, PhD; "Human Papillomavirus and Human Disease"; The American Journal of Medicine; Vol 102, May 5, 1997

ICTVdB Descriptions

Virus Alpha Structure; Electron Micrograph Images: Papillomavirus

The Big Picture Book of Viruses: Papovaviridae

Department of Obstetrics and Gynecology: Human Papillomavirus

Papillomavirus genome structure, expression, and post-transcriptional regulation

Human papillomavirus life cycle: active and latent phases

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