Papillomaviruses Chris Buck Email: buckc@nih.gov National Cancer Institute, Bethesda, Maryland Papillomaviruses National Cancer Institute*, Bethesda, Maryland *The views expressed in this talk are my own and do not necessarily represent the views of the NCI *I receive royalties from inventions related to HPV vaccines Baltimore Classification Scheme Baltimore Classification Scheme No integration into host cell DNA. Viral genome only exists as an “episome” Papillomavirus Virion •Non-enveloped icosahedral shell formed by 72 pentamers of a single protein, L1 (basis of current HPV vaccine) •60 nanometer diameter •Many features in common with polyomaviridae (e.g., SV40). Most notably: the viral genome is a small circular dsDNA molecule decorated with host cell histones (chromatin) Human papillomavirus type 16 Taxonomy •“Crown Group” hypothesis: the last common ancestor of placental mammals already had several distinct types of papillomavirus. Each ancestral virus has co-speciated with its host animal. Little or no evidence of papillomaviruses transmission between distantly related animals Taxonomy Genus: Mu Species: 1 Type: HPV1 causes warts on the soles of the feet Taxonomy Genus: Alpha Species: 4 Type: HPV2 causes warts on the palms of the hand Taxonomy Genus: Gamma Species: 1 Type: HPV4 Caused a wart on the palm of my hand. Grrr! Taxonomy Genus: Alpha Species: 10 Type: HPV6 causes genital warts. Does NOT cause cervical cancer Taxonomy Genus: Alpha Species: 9 Type: HPV16 Generally asymptomatic, but causes cervical cancer in a small minority of infected individuals Genome •8 kilobase circular dsDNA. Persists as episome (doesn’t integrate into cellular DNA) L1 E6 E7 7904/1 1000 7000 2000 6000 5000 L2 3000 4000 E5 E4 E2 E1 Genome •8 kilobase circular dsDNA. Persists as episome (doesn’t integrate into cellular DNA) L1 E6 7904/1 1000 7000 2000 6000 5000 L2 •One coding strand. Genome is divided into Late and Early regions E7 3000 4000 E5 E4 E2 E1 Capsid Genes •virion formation •cell association L1 7904/1 1000 7000 2000 6000 5000 L2 E7 E6 3000 4000 E5 E4 E2 E1 Capsid Genes E7 E6 7904/1 L1 1000 7000 2000 6000 5000 L2 •genome encapsidation •membrane penetration •post-entry trafficking 3000 4000 E5 E4 E2 E1 Capsid Genes E7 E6 7904/1 L1 1000 7000 2000 6000 5000 L2 •genome encapsidation •membrane penetration •post-entry trafficking 3000 4000 E5 E4 E2 E1 Oncogenes •Together the viral oncogenes immortalize the cell and prime it for viral DNA replication by disrupting the cell cycle. L2 E6 E7 7904/1 L1 1000 7000 2000 6000 5000 3000 4000 E4 E2 E5 E1 Oncogenes •Together the viral oncogenes immortalize the cell and prime it for viral DNA replication by disrupting the cell cycle. L2 E6 E7 7904/1 L1 1000 7000 2000 6000 5000 E1 3000 4000 E4 Small genome! No E2 room for fancy stuff like DNA polymerase, E5 DNA primase, ribonucleotide reductase, etc. The host cell must be forced to provide these factors. Cell Cycle Disruption Adenovirus E1B SV40 T Ag Adenovirus E1A •The E6 and E7 genes of cancer-causing HPVs mediate destruction of the tumor-supressor genes p53 and pRB (respectively) •Other types of DNA virus also disrupt p53 and pRB. This promotes entry into the S phase of the cell cycle, allowing replication of the viral DNA E6 & E7 - Other Functions E6: Activates telomerase. Interferes with about a dozen other known cellular targets, resulting in p53-independent effects. E7: Triggers chromosomal instability Interferes with about a dozen other known cellular targets, resulting in pRB-independent effects. Ongoing E6 and E7 expression is crucial for maintenance of HPV-transformed cells. For example, the famous HeLa cell line dies if E6 and/or E7 expression is knocked down. DNA Handling Genes LCR/ Ori E6 E7 7904/1 L1 1000 7000 2000 6000 5000 L2 •Helicase, recruits cellular DNA polymerase E1 3000 4000 E4 E2 E5 •Transcriptional regulation •recruit E1 to Ori, •tethers viral DNA to chromosomes during cell division Viral Lifestyles •“Live fast, die young” - high-level viremia. Virus attempts to outrun the adaptive immune response. Examples: Ebola, influenza •“Tiptoe past the graveyard” - virus can linger and lie low in a safehouse cell type (neurons, stem cells). Latently infected cells express few or no viral proteins. Example: HPVs, polyomaviruses, herpesviruses. Immunosuppression can lead to emergence from latency and high level virion production •Second strategy is somewhat more common among DNA viruses (RNA is hard to preserve in a latent state?) Life Cycle - Initial Infection (Skin) micro-trauma virions Epidermis (Keratinocytes) Basement Membrane Basal Cells Dermis Slide courtesy M. Kast Gene Expression (Wart) Progeny virions Viral Proteins Capsid & Early Epidermis Early only Basement Membrane Dermis None Immune Evasion Epidermis Langerhans cells Basement Membrane Dermis Dendritic cells Draining Lymphatics Immune Evasion Epidermis Langerhans cells Basement Membrane Dermis Dendritic cells In traditional cell culture monolayers, the virus thinks it’s in a basal keratinocyte, where hiding from the immune system is top priority. The only way to trick the virus into late phase Draining Lymphatics(virion production) in vitro is through the use of keratinocyte raft cultures HPV Reporter Vectors (Pseudoviruses) GFP codon-modified L1+L2 (helper plasmid) GFP reporter (8 kb) SV40 Ori transfect transduce T Ag “293TT” HPV Reporter Vectors (Pseudoviruses) GFP codon-modified L1+L2 (helper plasmid) GFP reporter (8 kb) Neutralizing antibody SV40 Ori transfect transduce T Ag “293TT” HPV-Based Gene Transfer Vectors •L1 and L2 can package non-viral DNA and deliver packaged DNA to cells with high efficiency. HPVderived DNA sequences are not required for efficient packaging (though some types of non-viral DNAs are packaged better than others) •HPV vectors may become useful for genetic delivery of vaccine immunogens (e.g., vaccines capable of eliciting cytotoxic T cells against HIV or other viruses) Cryo Therapy, Wounding Epidermis Langerhans cells Basement Membrane Dermis Dendritic cells Draining Lymphatics DC & LC Activation = HPV Specific Immunity Vaccination Epidermis Langerhans cells Basement Membrane Dermis Draining Lymphatics Dendritic cells DC Activation = HPV-specific Response Progression to Cancer is Accompanied by Deregulation of Viral Gene Expression CIN 1 CIN 2 CIN 3 Doorbar, J Clin Virol 32:7-15, 2005 Common molecular events: •Viral genome integration into cellular DNA •Loss of E2 leads to increased E6/E7 expression •Loss of L1, L2 expression. Therefore, current vaccine can’t clear pre-cancerous lesions. Therapeutic Vaccines CIN 1 CIN 2 CIN 3 Doorbar, J Clin Virol 32:7-15, 2005 •Goal: recruit T cells against HPV E6 and/or E6 •Goal: eradicate transformed cells (including precancerous lesions) Gene Expression (Wart) Progeny virions Viral Proteins Capsid & Early Epidermis Early only Basement Membrane Dermis Bad news: stably infected keratinocyte stem cells don’t produce L1. Therefore, the L1 vaccine can’t possibly clear existing infections. This is why you must vaccinate kids before they’re exposed. None Therapeutic Vaccines Expert Opinion on Emerging Drugs, November 2012 •Good news: cervical cancer cells depend on ongoing E6/E7 expression •Bad news: effective T cell immunity is a lot more difficult to elicit than effective antibody responses History and Medical Significance •1894-1924: Ciufo, Variot and others show that genital and skin warts can be transmitted between individuals by a filterable infectious agent History and Medical Significance •1894-1924: Ciufo, Variot and others show that genital and skin warts can be transmitted between individuals by a filterable infectious agent •1933 (Shope) rabbit papillomas have viral etiology •1935 (Rous) papillomas can progress to carcinoma •Papillomaviruses found to cause skin warts and other tumors in vertebrates ranging from birds to people Infection with Shope cottontail rabbit papillomavirus History and Medical Significance •1842: Rigoni-Stern reports that prostitutes have much higher incidence of cervical cancer than nuns History and Medical Significance •1842: Rigoni-Stern reports that prostitutes have much higher incidence of cervical cancer than nuns •1928: Georgious Papanicolaou develops the “Pap smear” technique for microscopic detection of cervical cancer and pre-cancer History and Medical Significance •1842: Rigoni-Stern reports that prostitutes have much higher incidence of cervical cancer than nuns •1928: Georgious Papanicolaou develops the “Pap smear” technique for microscopic detection of cervical cancer and pre-cancer •1951: George Otto Gey establishes in vitro culture of HeLa (Henrietta Lacks) cells derived from a lethal cervical cancer History and Medical Significance •1842: Rigoni-Stern reports that prostitutes have much higher incidence of cervical cancer than nuns •1928: Georgious Papanicolaou develops the “Pap smear” technique for microscopic detection of cervical cancer and pre-cancer •1951: George Otto Gey establishes in vitro culture of HeLa (Henrietta Lacks) cells derived from a lethal cervical cancer •1983: Harald zur Hausen discovers new HPV types (types 16 and 18) lurking in HeLa cells and other cervical cancer cells. •2008: Harald zur Hausen wins Nobel Prize for his work establishing a causal link between HPVs and cervical cancer Harald zur Hausen HPV Vaccine: What Is It? •Recombinant virus-like particles based on HPV capsid protein L1 •Produced in yeast (Merck) or baculovirus (GSK) •Elicits HPV-neutralizing antibody responses that protect against initial infection HPV Vaccine: Not Just for Girls Anymore •Summer 2006: FDA and CDC recommend new HPV vaccine “Gardasil” (Merck) for girls and women age 9 26 •October 2009: A second vaccine, “Cervarix” (GlaxoSmithKline) approved by FDA October 2009: Gardasil approved for use in boys and young men HPV Vaccine: Highly Effective Common Cancers in Women “Pap” smear Adapted from Parkin et al, Eur J Cancer 37:S4, 2001 Worldwide Incidence of Cancers Attributable to HPV Estimates from IARC HPV and Non-Cervical Cancers •About half of cancers of the upper throat (for example, tonsils) are caused by HPV infection. A great majority of HPV-associated throat cancers contain HPV type 16 (covered by the vaccine) •Work by Maura Gillison and others has shown that oral sex, particularly with multiple partners correlates with risk of throat cancer (Michael Douglas: not crazy! actually kind of heroic!) •Most cases of anal cancer are caused by HPV. As with throat cancer, HPV16 accounts for a great majority of cases. No clear connection between anal sex and anal cancer, though overall history of multiple sex partners is a clear risk factor. HPV Vaccine: Not Just for Girls Anymore % of cervical cancers Distribution of HPV Types in Cervical Cancer by Geographical Region 100 90 80 70 60 50 40 30 20 10 0 HPV 45 HPV 31 HPV 18 HPV 16 Africa Bosch et al, JNCI, 1995 Latin South-East Europe America Asia North America HPV Vaccine Some Degree of Cross-Protection? HPV31? “high risk” types partial HPV16 HPV18 HPV45 no yes (weak?), Cervarix may offer greater cross-protection? Harper (2006) Lancet 367:1247 HPV Vaccine HPV Vaccine HPV Vaccine HPV Vaccine HPV Vaccine HPV Vaccine - Theory HPV Vaccine - Theory HPV Vaccine - Theory •Other virologists should be trying to figure out how to apply this magic to their favorite virus. We’re looking at you, HIV biologists! Current HPV Vaccine - What It Will Do •Protect against two HPV types that cause 70% of all cervical cancer. Given the amazingly strong antibody responses, protection will probably be life-long •Gardasil (but not Cervarix) will protect against two HPV types that cause 90% of all genital warts •Protect against other HPV-induced cancers, such as throat cancer and anal cancer Current HPV Vaccine - What It Won’t Do •Won’t be affordable for women in developing countries •Won’t protect against all of the dozen or so HPV types that can cause cervical cancer •Won’t protect against non-carcinogenic HPV types that may still cause abnormal Pap smear •Won’t protect women who are already infected (more on why this is so coming up later in the lecture) Life Cycle: Tissue Tropism Life Cycle: Entry Infectious Entry Pathway Step 1: Attachment + + + - + - - cell membrane heparan sulfate proteoglycans (=surface proteins decorated with sulfated polysaccharide side-chains) Infectious Entry Pathway Heparin, carrageenan - - - Step 1: Attachment Topical Microbicides •Several brands of sex lube happen to use carrageenan as the principal gelling agent Topical Microbicides •Recently-announced clinical trial results suggest that a carrageenan gel protects women from HPV infection when applied before sex Infectious Entry Pathway conformational shift? Step 2: L2 exposure Infectious Entry Pathway Step 3: Furin cuts L2 furin protease Infectious Entry Pathway Step 3: Furin cuts L2 furin protease Note: many viruses use furin during assembly. •Some toxins (anthrax toxin) use furin during entry Infectious Entry Pathway conformational shift? Unidentified second receptor? Infectious Entry Pathway conformational shift? Receptor = Annexin-A2? Infectious Entry Pathway conformational shift? nAb Many hours Receptor? Recent Developments •Infection of mouse female genital tract reveals that the virus initially attaches to heparan sulfate on the basement membrane furin Day et al (2009) PNAS 106:20458 L2-Neutralization - Chop the Whole Tree? Immunogen: BPV1 L2 aa 1-88 cutaneous, mostly asymptomatic foot warts CRPV BPV1 neutralization HPV16, 31 hand warts genital mucosa, cancerassociated HPV18 HPV6, 11 genital warts Richard Roden Diana Pastrana (2005) Virol 337:365 Infectious Entry Pathway Step 5: Endocytosis PO4? clathrin 2 to 8 hours? Infectious Entry Pathway Step 6: Endosome H+ H+ protease(s)? disulfide reduction? H+ + H+ H Infectious Entry Pathway Step 7: Membrane disruption L2 carboxy-terminus Syntaxin-18? Sortin Nexin 17? ~8 to 20 hours? Infectious Entry Pathway Step 8: L2 drags viral genome to nucleus DNA L2 Tubulin Actin? nuclear pore Infectious Entry Pathway Step 8: L2 drags viral genome to ND-10 24 to 72 hours mRNA ND-10 Future Directions: L2-Based Vaccines •Antibody responses to certain portions of L2 can be broadly cross-neutralizing. For example, antibody response to HPV16 L2 can neutralize other genital and skin-tropic HPV types. Maybe also true for conserved portions of L1? •Single immunogen might protect against all HPV types. Would eliminate the need for expensive Pap testing •Added bonus: protection against skin warts might facilitate implementation of childhood vaccination Future Directions: Therapeutic Vaccines •T cell responses to E6 and E7 might cure existing cervical cancer and pre-cancerous lesions •The fact that E6 and E7 are likely required for maintenance of transformed phenotype in cervical cancers makes them excellent cancer vaccine targets Practicalities •Unvaccinated college-age adults should consider getting the HPV vaccine. This advice now applies to both men and women •In contrast to other STIs, condoms provide limited (possibly no) protection against HPV transmission. Large areas of genital surfaces can be productively infected •Papanicolaou (“Pap”) testing is highly effective for detecting cervical pre-cancer •Newer HPV DNA testing is also a highly effective screening method •Topical microbicides are in trials. Carrageenan, which is already on store shelves, appears to be effective for protecting women against HPV infection in an early clinical trial the end