MicrobiologyBytes: Virology: Papillomaviruses Updated: April 8, 2009 Search

Papillomaviruses

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The viral nature of human warts was first identified in 1907; the first Papillomavirus was isolated from rabbits by Richard Shope in 1933. In spite of this early start, human Papillomaviruses (HPV) remained largely unstudied until the advent of molecular virology (cloning) in the 1970s. This is because to date, no HPV replicates in vitro. Much of our knowledge comes from Bovine Papillomavirus (BPV) for which animal host systems exist.

Morphology:

Papillomaviruses are small, non-enveloped icosahedral particles ~52-55nm diameter. There are 72 capsomers (60 hexameric + 12 pentameric) arranged on a T = 7 lattice. Apart from the larger size, these appear very similar to Polyomaviruses particles (N.B. no sequence relatedness). There are 2 capsid proteins, 1 major (encoded by the L1 gene) and 1 minor (L2):
Negatively-stained electron micrograph of papillomavirus particles

Genome:

The papillomavirus genome consists of circular, d/s DNA ~8kbp in size, associated with cellular histones to form a chromatin-like substance. At least 12 different HPV genomes have been sequenced, and the genetic organization of all is similar.
HPV genome

Replication:

Individual isolates are highly species specific. All are tropic for squamous epithelial cells (receptors 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.
HPV gene expression

Gene: Function:
E1 DNA-dependent ATPase, ATP dependent helicase: allow unwinding of the viral genome and act as an elongation factor for DNA replication.
E2 Responsible for recognition and binding of origin of replication. Exists in two forms: full length (transcriptional transactivator) and truncated (transcriptional repressor). The ratio of these found in the heterotrimeric complex formed before complexing with E1 regulates transcription of viral genome.
E3 ???
E4 Late Expression: C terminal binds intermediate filament, allowing release of virus-like particles. Also involved in transformation of host cell by deregulation of host cell mitogenic signalling pathway.
E5 Obstruction of growth suppression mechanisms: e.g EGF receptor; activation of mitogenic signalling pathways via transcription factors: c-Jun and c-Fos (important in ubiquitin pathway degradation of p53 complex by E6). Inactivation of p21 (p53 induced expression halts cell cycle until DNA is proof-read for mutations).
E6 E6 Transformation of host cell by binding p53 tumour suppressor protein.
E7 Transforming protein, binds to pRB/p107.
E8 ???
L1 Major capsid protein: can form virus-like particles.
L2 Minor capsid protein: possible DNA packaging protein.

Expression of the Papillomavirus genome is complex because there are:

Transcription has been studied in detail by transfection of cloned Papillomavirus DNA into cells. Only one strand of the genome is transcribed. Two classes of proteins are produced:
Early Proteins: Non-structural regulatory proteins, including trans-acting transcriptional regulators (E2, E7).
Late Proteins: The structural proteins L1 and L2.
Transformation: Is complex! Depends on the early gene products. The transforming proteins appear to vary from one virus type to another. There is still some confusion about the function/mechanism of the transforming proteins. The general principle appears to be that two (or more) early proteins co-operate to give a transforming phenotype. Although some viruses can transform cells on their own (e.g. BPV-1), others also appear to require co-operation with an activated cellular oncogene (e.g. HPV-16/ras). More confusingly, in most cases, all or part of the Papillomavirus genome including the putative "transforming genes" is maintained in the tumour cells, whereas in other cases (e.g BPV-4), the virus DNA may be lost after transformation - a "hit-and-run" mechanism.

In HPVs:

HPV and p53

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.

O'Brien PM, Campo MS. Papillomaviruses: a correlation between immune evasion and oncogenicity? Trends in Microbiol. 2003 11: 300-305.

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

  1. Plasmid Replication - occurs in cells in the lower levels of the dermis. Initially, the virus DNA is amplified to 50-400 copies/diploid genome. After this, it replicates once per cell division, the copy number/cell remaining constant. The E1 protein is involved in this phase of replication.
  2. Vegetative Replication - occurs in terminally differentiated cells in the epidermis. In terminally differentiated cells (or growth-arrested cells in culture) control of copy number appears to be lost and the DNA is amplified up to very high copy numbers (000's copies/cell).
  3. Virus is shed from epidermal cells when these are sloughed off and is transmitted by direct contact (esp. genital warts) and indirect contact.

 

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Pathogenesis:

These viruses are widespread in nature and infect birds and mammals. The usual outcome of infection is the formation of a benign outgrowth of cells, a wart or papilloma. These may occur almost anywhere in or on the body. Skin warts are divided into flat warts (superficial) and plantar warts (verrucae, on soles of feet or toes). Genital warts (condylomas) occur in the genital tract and are transmitted by sexual intercourse. Warts can be treated by topical application of caustic substances or freezing, but surgical removal is more reliable, and is required for internal warts e.g. laryngeal. Warts may persist for many years, but may regress spontaneously due to a CTL response. There may be some enhanced risk of skin warts exposed to U.V. light developing into invasive squamous cell carcinoma (very rare).
Medscape: Therapeutic Options for External Genital Warts

Papillomaviruses are associated with tumours in humans and animals, including >95% of cervical cancers and approximately 20% of head and neck cancers. At least 58 different HPV have been identified using molecular techniques. In the last few years, a number of types have been suggested to be associated with particular tumours:

Cancer: Predominant types: Co-factors:
Skin carcinomas HPV-5, 8 U.V., genetic?
Lower genital tract cancers HPV-16, 18, 31, 33 ???
Malignant transformation of respiratory papillomas HPV-6, 11 X-rays

500,000 new cases of cervical neoplasia are diagnosed every year, making this one of the three most common causes of cancer death in woman globally (Peto, R, 1986). About half the women afflicted will die. The total prevalence of cervical cancer is estimated to be 1.4 million cases worldwide. HPV is a primary cause of cervical cancer, 93% of all cervical cancers test positive for one or more high risk type HPV (Schiffman MH, et al. 1993). There is a genetic link to cervical tumours, partly dependent on the virus and, in part, on the genotype of the patient.

Human papillomaviruses and cancer

Over one in four U.S. women between the ages of 14 and 59 years is infected with human papillomavirus – a sexually transmitted virus that can cause genital warts and cervical cancer. Around a quarter of teenage girls and half of women in their early 20s carry the virus. More than 2% of the women tested positive for HPV 16, HPV 18, or both, strains of the virus known to cause cervical cancer. High-risk strains of HPV are found in 99% of women with cervical cancer (Prevalence of HPV Infection Among Females in the United States. JAMA 2007 297: 813-819).

Vaccines

MicrobiologyBytes Podcast: Should we cure cancer?

Cost-Effectiveness of a Potential Vaccine for Human papillomavirus: "A vaccine with a 75% probability of immunity against high-risk HPV infection could result in a life-expectancy gain of 2.8 days or 4.0 quality-adjusted life days at a cost of $246 relative to current practice. If all 12-year-old girls currently living in the United States were vaccinated, >1,300 deaths from cervical cancer would be averted during their lifetimes. Vaccination of girls against high-risk HPV is relatively cost effective even when vaccine efficacy is low. Although gains in life expectancy may be modest at the individual level, population benefits are substantial."


© MicrobiologyBytes 2007.