1. PAPILLOMA VIRUSES

2. Papilloma Viruses Characteristics –dsDNA viruses (circular) –Genone 8 kbp –Genome associated with cellular histones –Naked capsid Widespread in humans and other animals –They cause ‘warts’ –Some strains 16,18 and 31 associated with high risk of cervical cancer –Over 100 types of HVP found so far

4. Papilloma Viruses Different types infect different anatomical sites –HPV-1 target feet –HPV-2, 4, 7 hands –Virus targets mucosa (oral, nasal, genital) OR skin Transmission of genital tract HPV thru sexual contact Transmission of skin HPV thru touching, or contaminated surface HPV that infect genital tract mucosal can cause –cervical warts referred to as ‘condylomas’ (benign rarely progress to cancer) –Strains 16,18 and 31 associated with high risk of cervical cancer such as Squamous Cell Carcinoma Referred to as Oncogenic HPVs Genital tract infections are transient in many cases cause no symptoms Lack of symptoms allows for easier spreading

5. HPV 16 HPV 16 is of interest due to oncogenic properties 6 Early genes (E1, E2, E4, E5, E6 and E7) 2 Late genes (L1 and L2) Genome has multiple ORFs Variable Splicing of mRNA transcripts yields variety of mRNAs with multiple ORFs –Virus targets mucosa (oral, nasal, genital) OR skin Transmission of genital tract HPV thru sexual contact Transmission of skin HPV thru touching, or contaminated surface

6. Infectious Cycle Infection requires entry to NON-DIFFERENTIATED basal cells Virus binds to heparin and  6 -integrin Virions are taken in by endocytosis Genome ends up inside nucleus –Stays as circular DNA –Does not incorporate itself into host genome Replication of genome occurs generating 50-100 copies –Every cell division genome is duplicated and split equally between parent and daughter cell –This type of division is referred to as ‘plasmid replication’ When Basal Cells become Keratinocytes burst of viral replication occurs –This burst is called vegetative replication –During this same period L1 and L2 genes are expressed producing capsids –Release of assembled virions occurs during cell death

7. HPV 16, 31 Genome Structure  In undifferentiated basal cells P early transcribes all six E genes  Transcript terminates at Poly(A)early  Poly(A)early immediately after E genes resulting in no expression of Late genes  mRNAs generated have multiple ORFs, unclear how ribosomes translate them

8. HPV 16, 31 Genome Structure  Late promoter (P late ) active in Keratinocytes  When E1 and E2 levels increase a shift occurs towards vegetative DNA replication  Transcripts from P late terminate to Poly(A) late Giving rise to L1 and L2  Alternative splicing is responsible for the shift in E1 and E2 and ultimately increase in L1 and L2  L1 and L2 encode for capsid proteins which leads to an increase in virion production  Splicing of P late -Poly(A)late transcript results in removal of E genes and expression of only L1 and L2 genes

9. Functions of HPV Proteins  E1 is a DNA helicase, binds replication origin and initiates DNA replication. By unwinding genome cellular DNA polymerases can replicate viral genome  E2 enhances binding of E1 onto replication origin  E6 enhances degradation of p53 protein which is a major cell cycle control protein  E7 binds Rb protein increasing cell cycling  L1 major capsid protein  Splicing of P late -Poly(A)late transcript results in removal of E genes and expression of only L1 and L2 genes  Some cellular transcription factors known to bind regulatory sequences of PV genome are: NF-1, SP1 and AP1

10. Viral Replication Surges in Keratinocytes  Replication surge is unusual given the fact that keratinocytes are terminally differentiated and do not carry any significant DNA replication  E7 is responsible for this peculiarity, binds Rb resulting in release of E2F transcription factor  E2F then participates in expression of a variety of genes involved in cell cycle  E7 binds Rb protein increasing cell cycling, essentially convincing keratinocytes to divide  Rb is a major tumor suppressor protein. Many retinoblastoma cancers are due to mutated Rb

11.  p53 normally induces apoptosis of cells that should not be dividing such as Keratinocytes  p53 neutralization is achieved by ubiquitination which leads to proteasome degradation  similar to Rb, p53 mutations are observed in a variety of cancers E6 Eliminates p53

12.  p53 normally is in low levels in the cytosol and inactive  In stress conditions: increase in phosphorylation occurs making it more stable Increase in p53 expression also occurs  p53 activation can stop cell cycling at G1 for repair  OR cause apoptosis  In many cancers p53 is mutated, in HPV is NORMAL!!  No reason to mutate, simply eliminate by ubiquitination p53 Function

13.  Surprisingly do not produce HPV virions!  In these cell lines genome is inserted into host genome  Normal HPV infected cells do not incorporate HPV genome into their own genome  E6 and E7 are the only genes being expressed in HPV related cancers  No surprise E6 and E7 target p53 and Rb respectively  Viral integration into host cellular genome has no advantage  Expression of E7 allows neutralization of Rb, release of E2F and expression of cell cycling genes  Normally p53 would eliminate them but p53 itself is eliminated through E6 Cervical Cancer Cell Lines Induced by HPV

14.  Pap test has been the main diagnostic tool for cervical cancer for decades.  The discoverer is George Papanicolaou  Significantly reduces mortality rates of cervical cancer  Cervical warts are pre-malignant tumors and often removed surgically or freeze killed with liquid nitrogen  HVP vaccines are now available that raise immunity against L1 protein of HPV strains 16, 18 (Guardasil includes L1 from stains 6, 11, 16 and 18)  L2 is considered in vaccination trials, could provide immunity against wider range of HPV types  Vaccination is recommended at young age before encounter with virus i.e ages 11-12 Cervical Cancer Diagnosis and Prevention