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Mcpyv Presentation

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Mcpyv Presentation

  1. 1. The role of Merkel cell polyomavirus in human cancer Qualifying Examination Dominik Satory Department of Molecular and Human Genetics Baylor College of Medicine, Houston, TX Tuesday, September 9, 1:30 P.M. – 3:30 P.M. Room T934
  2. 2. Polyomaviruses <ul><li>small, non-enveloped </li></ul><ul><li>DNA based </li></ul><ul><li>capable of inducing tumorigenesis </li></ul><ul><li>pathogens of birds and mammals including human </li></ul><ul><li>best-characterized polyomavirus is SV40 </li></ul>
  3. 3. Polyomavirus genome <ul><li>circular </li></ul><ul><li>~ 5000 base pairs </li></ul><ul><li>regulatory region </li></ul><ul><li>early unit </li></ul><ul><ul><li>large T-antigen </li></ul></ul><ul><ul><li>small T-antigen </li></ul></ul><ul><li>late unit </li></ul><ul><ul><li>VP1, 2 and 3 </li></ul></ul>Poulin and DeCaprio, 2006
  4. 4. Polyomavirus life cycle <ul><li>infection of </li></ul><ul><ul><li>permissive cells </li></ul></ul><ul><ul><li>nonpermissive cells </li></ul></ul><ul><li>viral replication, cell lysis and release of progeny (lytic phase) </li></ul><ul><ul><li>permissive cells </li></ul></ul><ul><li>transformation (lysogenic phase) </li></ul><ul><ul><li>nonpermissive cells </li></ul></ul><ul><ul><li>permissive via loss of lytic potential </li></ul></ul><ul><ul><li>rare event – requires integration </li></ul></ul>permissive cell nonpermissive cell viral infection cell death transformed cell
  5. 5. Large T-antigen <ul><li>functional polyomavirus protein </li></ul><ul><li>binds and degrades/sequesters cellular tumor suppressors – promotes S-phase entry </li></ul><ul><li>binds viral replication origin, regulatory elements and cellular replication machinery – perpetuates synthesis of large number of progeny and induces cell lysis </li></ul>Hsc70-binding motif 0 100 200 300 400 500 600 700 817 DnaJ Rb Ori binding Helicase Rb-binding motif C N p53-binding sites
  6. 6. Human polyomaviruses <ul><li>Previously characterized: </li></ul><ul><li>BK </li></ul><ul><li>JC </li></ul><ul><li>Novel: </li></ul><ul><li>WU </li></ul><ul><li>KI </li></ul><ul><li>MC </li></ul>(Feng et al., 2008)
  7. 7. Merkel cell carcinoma (MCC) <ul><li>the deadliest form of skin cancer </li></ul><ul><li>rapidly increasing number of cases </li></ul><ul><li>unknown origin </li></ul><ul><li>lack of effective treatment </li></ul><ul><li>frequent metastases </li></ul><ul><li>prevalent in immunosuppressed patients </li></ul>
  8. 8. Merkel cell polyomavirus (MCPyV) <ul><li>low copy number polyomavirus-like transcripts found in 80% of MCC tumors </li></ul><ul><li>clonal DNA integration </li></ul><ul><li>closely related to MPyV and HaPyV </li></ul><ul><li>point mutations in TAg-encoding gene are predicted to result in synthesis of truncated protein </li></ul>C 0 100 200 300 400 500 600 700 817 DnaJ Rb Ori binding Helicase MCV350 MCV339 N
  9. 9. Hypothesis <ul><li>1. MCPyV infects permissive human cells, replicates and causes their lysis, which releases the infectious progeny. </li></ul><ul><li>2. Mutated MCPyV infects a cell and integrates into its genome, which may result in tumor transformation. </li></ul><ul><li>The transformed cell initiates tumors, which can lead to further metastases. </li></ul>
  10. 10. Hypothesis <ul><li>1. MCPyV infects permissive human cells, replicates and causes their lysis, which releases the infectious progeny. </li></ul><ul><li>2. Mutated MCPyV infects a cell and integrates into its genome, which may result in tumor transformation. </li></ul><ul><li>The transformed cell initiates tumors, which can lead to further metastases. </li></ul>
  11. 11. Hypothesis <ul><li>1. MCPyV infects permissive human cells, replicates and causes their lysis, which releases the infectious progeny. </li></ul><ul><li>2. Mutated MCPyV infects a cell and integrates into its genome, which may result in tumor transformation. </li></ul><ul><li>The transformed cell initiates tumors, which can lead to further metastases. </li></ul>
  12. 12. Aims of the project Aim 2: Lytic cycle Aim 1: Transformation and tumorigenesis Aim 3: Expression and interactions of viral proteins
  13. 13. Aim 1 : The transforming and oncogenic potentials of MCPyV tumor trans- formation genomic integration tumor growth, accumulation of mutations
  14. 14. Aim 1 : The transforming and oncogenic potentials of MCPyV <ul><li>Does MCPyV transform human cells in vitro ? </li></ul><ul><li>Can MCPyV cause tumors in laboratory animals? </li></ul>
  15. 15. Transforming potential of MCPyV T-antigen <ul><li>SV40 TAg immortalizes mammalian cells in vitro </li></ul><ul><li>primary cells divide several times and senesce </li></ul><ul><li>transformed cells proliferate indefinitely </li></ul><ul><li>if MCPyV TAg causes cellular transformation, primary cells should become immortalized </li></ul>
  16. 16. Transforming potential of MCPyV T-antigen <ul><li>MCPyV and SV40 TAgs will be delivered using retroviruses </li></ul><ul><li>transformed cells should continue dividing </li></ul>
  17. 17. Aim 1 : The transforming and oncogenic potentials of MCPyV Does MCPyV T-antigen immortalize human cells? YES NO <ul><ul><li>MCPyV may cause cellular transformation. </li></ul></ul><ul><ul><li>p53-binding sites may be required. WT TAg will be used. </li></ul></ul>
  18. 18. MCPyV T-antigen-induced oncogenesis <ul><li>murine PyV causes tumors in mice </li></ul><ul><li>SV40, JC and BK viruses induce brain tumors in hamsters and rats </li></ul><ul><li>is MCPyV also a tumor-causing agent? </li></ul>
  19. 19. MCPyV T-antigen-induced oncogenesis inject newborn hamsters with the virus examine tumors in hamster’s body polyomavirus induces tumorigenesis presence of virus suggests its oncogenic potential detect viral proteins by IHC and viral DNA by PCR
  20. 20. Aim 1 : The transforming and oncogenic potentials of MCPyV Does MCPyV cause tumors in laboratory animals? YES NO <ul><ul><li>MCPyV may cause tumors in humans. </li></ul></ul><ul><ul><li>Mutations in VP genes? </li></ul></ul><ul><ul><li>SV40/JC/BK/MC recombinants to answer this question. </li></ul></ul>
  21. 21. Aims of the project Aim 2: Lytic cycle Aim 1: Transformation and tumorigenesis Aim 3: Expression and interactions of viral proteins
  22. 22. Aim 2 : Identification of WT MCPyV and its potential infectivity infection of permissive cell cell lysis, release of virions virus replication
  23. 23. Aim 2 : Identification of WT MCPyV and its potential infectivity <ul><li>Is WT MCPyV present in non-tumor tissues of MCC patients? </li></ul><ul><li>Is WT MCPyV capable of lytic growth? </li></ul>
  24. 24. Identification of WT MCPyV <ul><li>truncated TAg is incapable of lytic phase </li></ul><ul><li>two distinct truncating mutations identified </li></ul><ul><li>only two MCPyV genomes sequenced </li></ul>
  25. 25. Identification of WT MCPyV <ul><li>test for MCPyV sequences in: </li></ul><ul><ul><li>non-tumor tissues of MCC patients </li></ul></ul><ul><ul><li>environment of MCC patients (i.e. air, dust, parasites) </li></ul></ul><ul><li>PCR-amplify TAg- and VP-coding genes </li></ul><ul><li>sequence the viral DNA </li></ul>MCPyV
  26. 26. Aim 2 : Identification WT MCPyV and test its infectivity Does WT MCPyV exist in humans and the environment? YES NO <ul><ul><li>WT MCPyV may infect humans and cause tumors upon loss of lytic potential. </li></ul></ul><ul><ul><li>Pathogen may be cleared from the organism by immune system. Make recombinant WT MCPyV. </li></ul></ul>
  27. 27. Lytic potential of MCPyV <ul><li>polyomaviruses cause lytic death of permissive cells </li></ul><ul><li>WT (isolate or recombinant) MCPyV is expected to be capable of lytic growth </li></ul><ul><li>lytic growth results in abundance of virions and allows infectivity </li></ul>
  28. 28. Lytic potential of MCPyV <ul><li>plaque assay - figure </li></ul>
  29. 29. Aim 2 : Identification WT MCPyV and test its infectivity Is WT MCPyV capable of lytic growth? YES NO <ul><ul><li>WT MCPyV may infect humans and cause tumors upon loss of lytic potential. </li></ul></ul><ul><ul><li>Recombinant WT may contain additional unknown mutations depriving such a virus from lytic potential. </li></ul></ul>
  30. 30. Aims of the project Aim 2: Lytic cycle Aim 1: Transformation and tumorigenesis Aim 3: Expression and interactions of viral proteins
  31. 31. Aim 3 : Mechanism of MCPyV-induced transformation viral gene expression synthesis of viral proteins interactions with cellular proteins
  32. 32. <ul><li>Are MCPyV proteins present in MCC? </li></ul><ul><li>Do MCPyV proteins interact with cellular partners? </li></ul><ul><li>What is the mechanism of MCPyV TAg-induced cellular transformation? </li></ul>Aim 3 : Mechanism of MCPyV-induced transformation
  33. 33. Expression and interactions of MCPyV proteins <ul><li>T-antigen is critical for PyV-induced transformation </li></ul><ul><li>interacts with: </li></ul><ul><ul><li>pRb </li></ul></ul><ul><ul><li>p53 </li></ul></ul><ul><li>deregulates cell cycle </li></ul><ul><li>prevents apoptosis </li></ul>✗
  34. 34. Stability of mRNA and expression of MCPyV proteins <ul><li>proteins – Western hybridization </li></ul><ul><li>mRNA – quantitative rtPCR, DTS </li></ul>TAg (94 kDa) VP1 (44 kDa) actin tumor SV40 MCC antibody SV40 SV40 MCPyV MCPyV TAg TAg TAg TAg VP1 VP1 VP1 VP1
  35. 35. Aim 3 : Mechanism of MCPyV-induced transformation Are MCPyV proteins present in MCC tumors? YES NO <ul><ul><li>MCPyV proteins may interact with cellular partners and lead to transformation. </li></ul></ul><ul><ul><li>MCC caused by MCPyV integration: </li></ul></ul><ul><ul><li>transactivation of oncogenes </li></ul></ul><ul><ul><li>disruption of tumor suppressors </li></ul></ul>
  36. 36. Interactions of MCPyV proteins <ul><li>co-immunoprecipitation </li></ul><ul><ul><li>antibodies against TAg, VP and cellular proteins </li></ul></ul><ul><li>yeast two-hybrid </li></ul><ul><ul><li>test for interactions between TAg and cellular tumor suppressors </li></ul></ul><ul><ul><li>identify new interactions using expression library </li></ul></ul><ul><li>BiFC </li></ul>
  37. 37. Aim 3 : Mechanism of MCPyV-induced transformation Do MCPyV proteins interact with cellular partners? YES NO <ul><ul><li>MCC can be caused by functional MCPyV proteins. </li></ul></ul><ul><ul><li>MCC caused by MCPyV integration: </li></ul></ul><ul><ul><li>transactivation of oncogenes </li></ul></ul><ul><ul><li>disruption of tumor suppressors </li></ul></ul>
  38. 38. Mutation analysis of TAg TAg transformation 0 100 200 300 400 500 600 700 817 DnaJ Rb Ori binding Helicase MCV350 MCV339 N DnaJ Rb Ori binding N C C Ori binding Helicase C N Helicase C DnaJ Rb N N C
  39. 39. Aim 3 : Mechanism of MCPyV-induced transformation Are p53-binding sites necessary for transformation? YES NO <ul><ul><li>Translation re-initiation allows synthesis of C-terminus. </li></ul></ul><ul><ul><li>Truncating mutations occurred after transformation. </li></ul></ul><ul><ul><li>TAg-dependent degradation of pRb is sufficient for tumor transformation. </li></ul></ul>
  40. 40. Epistasis analysis of TAg <ul><li>identification of the mechanism of MCPyV TAg-mediated transformation </li></ul><ul><li>introduction of TAg to pRb and p53 depleted cells and animals </li></ul>
  41. 41. Aim 3 : Mechanism of MCPyV-induced transformation Is transformation in presence of TAg epistatic with loss of pRb/p53? YES NO <ul><ul><li>TAg transforms cells by targeting pRb/p53. </li></ul></ul><ul><ul><li>Transformation occurs via disruption/transactivation. </li></ul></ul><ul><ul><li>Unknown tumor suppressors/oncogenes involved. </li></ul></ul>
  42. 42. Aim 3 : Mechanism of MCPyV-induced transformation Is stability of MCV339 and MCV350 mRNA affected by mutations? YES NO <ul><ul><li>Lower amount of potential proteins as compared to WT. </li></ul></ul><ul><ul><li>MCV339 and MCV350 proteins are synthesized on WT level. </li></ul></ul>
  43. 43. Summary and Significance <ul><li>MCC is a deadly skin cancer with unknown etiology and lacking effective treatment. </li></ul><ul><li>Evidence of a novel polyomavirus capable of causing cancer may allow development of MCC treatment. </li></ul><ul><li>MCPyV may provide association of human cancer and infectious polyomaviruses. </li></ul>
  44. 44. Future directions <ul><li>Test for presence of MCPyV in other tumors. </li></ul><ul><li>Explore the mechanism by which MCPyV spreads. </li></ul><ul><li>Test whether TAg truncation is a common feature. </li></ul><ul><li>Estimate seropositivity among human population. </li></ul><ul><li>Develop effective treatment. </li></ul>

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