Human Papillomaviruses and Polyomaviruses

1. Human Papillomaviruses and Polyomaviruses

2. Introduction: Features common to all papillomaviruses Circular double stranded DNA genome within an icosahedral capsid Limited genetic capacity in ~8kbp genome Encodes capsid proteins, and non-structural proteins that promote viral DNA replication, and inactivate important cell cycle checkpoint proteins retinoblastoma (RB) and p53 Causative agents of papillomas on cutaneous skin and mucosal surfaces (called warts when found on skin and chondylomas on mucosal surfaces and genitalia) Some serotypes are specifically associated with cervical dysplasia and carcinoma (types 16, 18, 31, 35)

3. Family name: Papillomaviridae Closely related morphologicallyto Polyomaviridae family with whom they were originally grouped and collectively called Papovaviridae, an acronym derived from the three main branches of the family papillomavirus, polyomavirus and simian vaculating agent (SV40) Polyomavirus and SV40 were studied extensively as paradigms of tumor viruses The existence of promoter/enhancer regions and of mRNA splicing was first recognized in these viruses There are two human polyomavirus BK and JC; SV40-like viruses have been isolated from human tumors Cryo-EM reconstruction of HPV

5. Classification: >120 human serotypes, with >80 fully sequenced Numbered in order of discovery Type designation indicates >10% sequence divergence in three genes: E6, E7 and L1; all other genes are stable

6. HPV genome - schematic representation 1 2 3 E7 E1 E5 L2 Open Reading Frames E6 E2 L1 URR E4 Poly - A Poly - A 0 1 2 3 4 5 6 7 8 Kilobase

7. Early region 6 E6 protein of oncogenic HPVs binds to the p53 tumor suppressor gene product and abrogates it activity by accelerating its degradation Noncoding Upstream Regulatory Region Contributes to the control of DNA replication and transcription of ORFs Early region 7 E7 protein of oncogenic HPVs bind to the tumor suppressor gene product Retinoblastoma protein (RB) and related proteins, thus inhibiting their function Late region 1 L1 protein is the major capsid protein URR E6 E7 Poly - A Late region 2 L2 protein is the minor capsid protein L1 E1 Early region 1 E1 protein involved in viral plasmid replication Early region 5 E5 protein is located in the cellular membrane, prevents acidification of endosomes, and can stimulate the transformating activity of epidermal growth factor receptor and contribute to oncogenicity Poly - A L2 E2 E4 / E5 / Early region 2 E2 protein is an important modulator of viral transcription and play a role in viral replication Early region 4 E4 proteins form filamentous cytoplasmic networks and share the same cellular distribution. They appear to play a role in viral replication HPV Genome Structure

8. Replication cycle: • The replication cycle of HPVs is intimately associated with the differentiation state of the epithelial cell • following introduction of the virus into the basal squamous epithelial cell following breaks in the skin or mucous membrane, early viral gene expression (E1, E2, E5, E6 and E7) and steady-state viral DNA replication occurs, ensuring that as the basal cell divides, both basal and supra-basal daughter cells both contain viral DNA in the form of extra-chromosomal episomes • as cells move upwards in the epithelium and differentiate (exemplified by keratin 1 and 10 expression), late gene expression (E4, L1, L2) and vegetative viral DNA replication begins • virion assembly occurs in the upper spinous layers, coincident with disintegration of nucleus and other cellular organelles, and virus is shed in the stratum corneum. • little or no host cell death and inflammation associated with the replication cycle

9. HPV infection and replication in cervical epithelial cells Columnar epithelial cell Infection Shedding L1 & L2 expression Squamous epithelial cell Transformation zone Basement membrane Basal epithelial cell E1, E2, & E4-E7 expression

10. Malignant transformation of the cervix E7 E7 E2F RB RB E2F E2F E6 E6 P53 P53 P53 Cervical intraepithelial neoplasia (CIN) HPV transformed epithelial cell Invasive cervical cancer

11. Clinical manifestations: • breaks in epithelium necessary to gain access to basal epithelial cells • incubation period may be from 2-6 months; epithelial dysplasias (common warts on keratinized epithelium) can be found on hands and feet; condylomas, on mucosal epithelium, in the larynx and oral cavity, as well as genitalia and anus • expression of E5, E6 and E7 can drive hyper-proliferation and dysplasia of cells above the basal layer • associated parakeratosis (persistence of the nuclei of keratinocytes as they rise into the stratum corneum)and hyperkeratosis (hypertrophy of the stratum corneum) which produces the papillomatous cytoarchitecture of warts or chondylomas • except for those serotypes associated with cervical carcinoma, dysplasias eventually regress

12. Cutaneous warts (plantar, common and plane warts) • single or clusters of slightly raised papules, ~1cm in diameter found on palms, fingers, soles, neck and face; • 50-90% spontaneously resolve within 1-5y • Epidermodysplasia verruciformis • autosomal recessive disease with flat crusty, wart appearance but covering areas of the face, torso and upper extremities; • usually appear in first decade of life, in ~1/3 of young adults, these undergo malignant transformation into invasive squamous cell carcinoma on sun-exposed areas

13. Anogenital warts • hyperkaratotic papules either sessile or attached to skin by short, broad peduncle • range in size from 1mm to several • centimeters in areas when join together • usually on the penile shaft of circumcised males and perianal in MSM • in women usually on posterior vaginal • orifice

14. Recurrent respiratory papillomatosis • most common laryngeal neoplasm in children and usually results from vertical transmission of human papillomavirus at birth • usually multiple, papillomas most commonly • occur in the vocal cords and ventricular bands • but can involve any part of the larynx • most common in children between two and • four years of age • usual presenting symptom is hoarseness, • but some patients have stridor and other • signs of laryngeal obstruction.

15. Epidemiology: • transmission by direct contact with lesions of infected individuals or by contact with environmental surfaces harboring virus • incidence of common warts is highest in older children and adults with 75% life time risk of infection with one or more strains; first acquisition of HPV occurs with sexual debut (~75% of those who will be infected, were infected by age 18) • approximately 75% of US population has experienced one or more HPV infections; 60% of population are HPV antibody positive but DNA and lesion negative; 10% of population is DNA positive but lesion negative, 4% of population are DNA positive and display sub-clinical changes, and 1% have genital warts • ~5 million new cases of genital infection per year, with ~10% presenting with symptomatic genital warts • increased incidence of genital warts in US predicted to result in increased incidence of cervical cancer 10-30 years in the future

16. Pathogenesis: • Low-grade intraepithelial lesions support productive viral replication; an unknown number of high-risk HPV infections progress to high-grade cervical intraepithelial neoplasia (HGCIN) • this progression of untreated lesions to microinvasive and invasive cancer is associated with the integration of the HPV genome into the host chromosomes with associated loss or disruption of E2, and subsequent upregulation of E6 and E7 oncogene expression • E6 – targets p53 cell cycle checkpoint protein for ubiquitylation and destruction in the proteosome, while E7 - binds Rb and promotes its proteosomal destruction • consequently, normal cell cycle checkpoints are lost and unregulated DNA replication can lead to chromosomal damage, suppression of apoptosis, and cell proliferation • Paradox—low risk HPV strains also encode E6 and E7 proteins that can function to degrade p53 and Rb; why is HPV16 “high risk”?

17. Pathogenesis, cont. • Cervical cancer – the most frequently diagnosed cancer of women, accounting for ¼ of all cancers in women; 90% of cervical carcinomas contain HPV DNA; squamous cell carcinoma represents the majority of cervical cancers and is associated with presence of “high risk” serotypes 16, 18, 31 and, 35; cervical carcinoma rarely found in virgins, and its incidence increases with the number of sexual partners • Head and neck papillomas – includes laryngeal, recurrent respiratory and nasal papillomas are caused by “low risk” genital-mucosal strains HPV-6 and -11 • Oral cavity papillomas – primarily with HPV-6, 11, and 57 • Oral cancers – 20% are HPV associated, risk factors for oral cancers include cigarette smoking and alcohol consumption; a subset appear to be attributable to HPV; mainly on tonsils, tonsillar fossa, base of the tongue, and soft palette; HPV16 most frequent type isolated, types 6, 11, and 57 less frequently

18. Diagnosis: • Pap smears identify cervical dysplasia; specific strains are identified by PCR • genital warts are diagnosed by visual inspection • cervical cell changes (early signs of cervical cancer) can be identified by routine Pap tests. The HPV DNA test (PCR) can identify high-risk HPV types on a woman’s cervix • Prevention: • Limiting number of sexual partners decreases incidence of infection, regular Pap smears main defense against invasive disease, followed by surgical intervention; • virus-like particle (VLP) vaccines • Gardasil: induces immune responses to L1 proteins of HPV 6, 11, and 16 and 18), which together cause 90% of genital warts and 90% of cervical cancer, respectively • Cervarix: only targets HPV 16 and 18 • Treatment: • surgery or cryotherapy for removal of warts, though recurrence is common

23. Human Polyomaviruses Introduction: JCV and BKV are members of the polyomaviridae family dsDNA genomes within icosahedral capsids consisting of 72 pentameric capsomers first isolated in 1971 (JCV-brain tissue of patient with progressive multifocal leukoencephalopathy (PML); BKV from urine of renal transplant patient) Epidemiology: BKV first acquired by age 3-4; JCV by age 10-14, ~60-80% of adults are seropositive for both in pre-HIV era PML was seen in older adults with hematologic malignancies now seen in ~5% of HIV/AIDS patients

24. Virus replication: BKV and JCV share 75% nucleotide sequence homology; 70% with SV40 smaller genome compared to papillomaviruses (~5 kbp v. ~8 kbp genomes divided into two regions, early and late, transcribed from opposite DNA strands Early proteins: LT, MT and sT antigens expressed from differentially spliced mRNAs Late, capsid proteins VP1, VP2 and VP3 also expressed from differentially spliced mRNAs

25. Pathogenesis: acquisition by inhalation or close contact primary site of replication in oropharynx, followed by viremia and seeding of kidneys where clinical latent infection is established DNA and capsid antigen detected in mononuclear cells of patients with PML or AIDS neuropathology of PML likely due to infection of oligodendrocytes (viremic spread of reactivated virus) leading to decreased myelin production and de-myelination

26. PML: focal neurological defects due to demyelination parieto-occipital brain involvement associated with muscle weakness, gait disturbance, speech defect, or visual blind spot cerebellum involvement associated with broadlegged gait and imbalance Diagnosis: CT or MRI lumbar puncture for JCV PCR Treatment: none, except for highly active antiretroviral therapy( HAART) in AIDS patients H&E staining of PML brain biopsy; black arrows point out some of the reactive astrocytes; blue arrow points toward an oligodendrocyte nucleus which has been markedly enlarged with viral particles, giving it a magenta color

27. BK nephropathy: no evidence that BKV causes disease in the immune competent population possible route of infection through contaminated food or water or respiratory spread remains latent in lymphocytes, urogenital tract, and brain but may be reactivated if the host becomes immunocompromised. especially associated with disease in renal transplant patients; thought to cause graft failure in 2-5% of this population treatment is to decrease immunosuppression as much as possible without causing rejection Cidofovir appears to reduce BKV-associated nephropathy viral inclusions and cellular changes in BK nephropathy are seen in tubular epithelium of a renal transplant case