CA García Sepúlveda MD PhD

1. Retrotransposons & Retrovirus CA García Sepúlveda MD PhD Laboratorio de Genómica Viral y HumanaFacultad de Medicina, Universidad Autónoma de San Luis Potosí

2. Session #27 RetroposonsIntroduction • Retroposon or Retro-transposons are transposons that mobilize via an RNA form. • The DNA element is transcribed into RNA and then reverse-transcribed into DNA, which is inserted at a new site in the genome. • Transposition that involves an obligatory intermediate of RNA is unique to eukaryotes. • Some eukaryotic transposons are related to retroviral proviruses in their general organization. • Retroviruses insert DNA copies (proviruses) of an RNA viral genome into the chromosomes of a host cell.

3. Session #27 RetroposonsIntroduction • The life cycle of retroviruses and retroposons involve alternation of reverse transcription from RNA to DNA with transcription from DNA to RNA. • Only retroviruses can generate infectious particles. • Retroposons are confined to an intracellular cycle. • Retroposons & retrovirus share features with other transposons • short direct repeats of target DNA at the site of an insertion.

4. Session #27 RetroposonsRetroviruses dsRNA ssRNA+ dsDNA ssRNA- RNAVirus RT DNAVirus ssDNA Virus

5. Session #27 RetroposonsRetrovirus • Belong to the Retroviridae family of single-stranded RNA viruses that Reverse Transcribe (ssRNA-RT). • Enveloped viruses possessing an RNA genome. AKA Pararetrovirus dsDNA-RT ssRNA-RT

6. Session #27 RetroposonsRetrovirus • Belong to the Retroviridae family of single-stranded RNA viruses that Reverse Transcribe (ssRNA-RT). • Enveloped viruses possessing an RNA genome. • Its members include: ssRNA-RT

7. Session #27 RetroposonsRetrovirus • Belong to the Retroviridae family of single-stranded RNA viruses that Reverse Transcribe (ssRNA-RT). • Enveloped viruses possessing an RNA genome. • Its members include: ssRNA-RT • Avian leukosis virus

8. Session #27 RetroposonsRetrovirus • Belong to the Retroviridae family of single-stranded RNA viruses that Reverse Transcribe (ssRNA-RT). • Enveloped viruses possessing an RNA genome. • Its members include: ssRNA-RT • Mouse mammary tumour virus

9. Session #27 RetroposonsRetrovirus • Belong to the Retroviridae family of single-stranded RNA viruses that Reverse Transcribe (ssRNA-RT). • Enveloped viruses possessing an RNA genome. • Its members include: ssRNA-RT • Murine & feline leukemia virus

10. Session #27 RetroposonsRetrovirus • Belong to the Retroviridae family of single-stranded RNA viruses that Reverse Transcribe (ssRNA-RT). • Enveloped viruses possessing an RNA genome. • Its members include: ssRNA-RT • Human T-lymphotropic virus

11. Session #27 RetroposonsRetrovirus • Belong to the Retroviridae family of single-stranded RNA viruses that Reverse Transcribe (ssRNA-RT). • Enveloped viruses possessing an RNA genome. • Its members include: ssRNA-RT • Walleye dermal sarcoma virus

12. Session #27 RetroposonsRetrovirus • Belong to the Retroviridae family of single-stranded RNA viruses that Reverse Transcribe (ssRNA-RT). • Enveloped viruses possessing an RNA genome. • Its members include: ssRNA-RT • Human immunodeficiency virus 1

13. Session #27 RetroposonsRetrovirus • Belong to the Retroviridae family of single-stranded RNA viruses that Reverse Transcribe (ssRNA-RT). • Enveloped viruses possessing an RNA genome. • Its members include: ssRNA-RT • Chimpanzee foamy virus

14. Session #27 RetroposonsRetrovirus • Rely on reverse transcriptase to perform the reverse transcription of its genome from RNA into DNA. • cDNA is integrated into the host's genome with the help of an integrase. The virus then replicates as part of the cell's DNA, which means: mRNA Positive sense 5'-Capped 3'-Poly-A

15. Session #27 RetroposonsRetrovirus • These retrovirus are exogenous. • A retroviral sequence that is integrated in the germline remains in the cellular genome as an endogenous PROVIRUS. • Like a lysogenic bacteriophage, a provirus behaves as part of the genetic material of the organism. • They are inherited to the progeny. ssRNA-RT

16. Session #27 RetroposonsRetrovirus • Retrovirus that have been incorporated to the genome are called ENDOGENOUS (ERV). • They are derived from ancient infections of germ cells in humans, mammals & other vertebrates. • ERVs make up 5-8% of the human genome (98,000 elements). • Most insertions have no known function (junk DNA) but some play important roles in host biology: • Control of gene transcription. • Control of cell fusion during placental development. • Resistance to exogenous retroviral infection. • Immunosuppresion

17. Session #27 RetroposonsRetrovirus • ERVs are activated during pregnancy in viviparous mammals (not Monotremes) and particularly during the implantation of the embryo. They act as immunosupresors protecting the embryo from its mother's immune system. Viral fusion proteins involved in the formation of placental syncytium limits cell migration (something an epithelium will not do well, as certain blood cells are able to diapedize).

18. Session #27 RetroposonsRetrovirus • ERV's are similar to HIV (which causes AIDS in humans). • The immunodepressive action was the initial normal behavior of the virus, as in HIV. • The fusion proteins were a way to spread the infection to other cells by merging them with the infected one, as in HIV. • It is believed that the ancestors of modern vivipary mammals evolved after an infection by this virus, enabling the fetus to survive the immune system of the mother.

19. Session #27 RetroposonsRetrovirus • 24 ERV families identified by Human Genome Project (HGP). • Broadly classified into 3 classes, on the basis of relatedness to exogenous genera: • Class I are similar to the gammaretroviruses • Class II are similar to the betaretroviruses & alpharetroviruses • Class III are similar to the spumaviruses

20. Session #27 RetroposonsRetrovirus • Most ERVs appear to be defective, containing nonsense mutations or major deletions which prevent them from producing infectious virus particles. • However, there is one family of viruses that have been active since the divergence of humans and chimpanzees [ hERV-K(HML2)]. • Make up less than 1% of hERV elements but is one of the most studied. • There are indications it has been active in the past few hundred thousand years, as some humans carry more copies of the virus than others.

21. Session #27 RetroposonsRetrovirus • Human ERVs (hERVs) are suspected of involvement in some autoimmune diseases (multiple sclerosis). • Especially human endogenous retrovirus W known (MSRV). • Also a possible hERV involvement in the HELLP (Hemolytic anemia, Elevated Liver enzymes & Low Platelet count) syndrome & pre-eclampsia. • hERVs very likely associated with some types of schizophrenia.

22. Session #27 RetroposonsRetrovirus • Retroviruses have ssRNA genomes.

23. Session #27 RetroposonsRetrovirus • Retroviruses have ssRNA genomes. • Genomes are replicated through a dsDNA intermediate.

24. Session #27 RetroposonsRetrovirus • Retroviruses have ssRNA genomes. • Genomes are replicated through a dsDNA intermediate. • The life cycle involves an obligatory stage of integration in which the proviral dsDNA is inserted into the host genome by a transposition-like event that generates short direct repeats of target DNA.

25. Session #27 RetroposonsRetrovirus • The enzyme responsible for generating the initial DNA copy of the RNA is reverse transcriptase. • Converts the RNA into a linear (or circular) duplex of DNA in the cytoplasm of the infected cell.

26. Session #27 RetroposonsRetrovirus • The enzyme responsible for generating the initial DNA copy of the RNA is reverse transcriptase. • Converts the RNA into a linear (or circular) duplex of DNA in the cytoplasm of the infected cell. • The enzyme responsible for integrating the DNA copy is integrase.

27. Session #27 RetroposonsRetrovirus • Implications of proviral DNA integration: • A retroviral sequence that is integrated in the germline remains in the cellular genome as an endogenous provirus (behaves as part of the genetic material of the organism). • Cellular sequences may be transposed with proviral DNA and inserted into the genome as duplex sequences in new locations. • Retrovirally transposed cellular sequences may change the properties of the cell that becomes infected with the virus.

28. Session #27 RetroposonsHIV as an example • Tropism • Fusion • RT • Integration • Transcription • Assembly • Budding • Release • Maturation

29. Session #27 RetroposonsHIV as an example • Tropism • Fusion • RT • Integration • Transcription • Assembly • Budding • Release • Maturation Integration mediated by integrase. Can occur at one or more sites.

30. Session #27 RetroposonsHIV as an example • Tropism • Fusion • RT • Integration • Transcription • Assembly • Budding • Release • Maturation Proviral DNA is transcribed by the host machinery to produce viral RNAs: - mRNAs - genomic RNA.

31. Session #27 RetroposonsHIV as an example • Tropism • Fusion • RT • Integration • Transcription • Assembly • Budding • Release • Maturation The enzymes reverse transcriptase and integrase are carried with the genome in the viral particle.

32. Session #27 RetroposonsHIV as an example Two copies of the RNA genome are packaged into each virion (diploid virus). When a cell is infected by two different viruses, it is possible to generate heterozygous virus particles carrying one genome of each type.

33. Session #27 RetroposonsRetroviral translation & processing A typical retroviral sequence contains 3 or 4 "genes". Gag-Pol-Env Coding regions give rise to multiple proteins by processing reactions. Retroviral mRNA has a conventional structure; it is capped at the 5′ end and polyadenylated at the 3′ end.

34. Session #27 RetroposonsRetroviral translation & processing Retroviral mRNAis represented by two subgenomic species. 1. Gag-Pol-Env (Full length) 2. Env

35. Session #27 RetroposonsRetroviral translation & processing (Gag) The Gag product is translated by reading from the initiation codon to the first termination codon. Gag derived products p6, p7, p17 & p24 are generated by post-translational processing. Gag derived products have structural functions.

36. Session #27 RetroposonsRetroviral translation & processing (Pol) To translate the Pol product, the first termination codon must be bypassed. Depends on the “reading frame correspondence” between Gag and Pol. If Gag and Pol are in the same reading frame... Termination codon usage is suppressed by a glutamyl-tRNA, generating a Gag-Pol peptide.

37. Session #27 RetroposonsRetroviral translation & processing (Pol) Gag-Pol peptide is then processed to yield: 1- Gag derived products (p6, p7, p17 & p24). 2- Pol derived products (p10, p32, p51, p66). Pol derived products have enzymatic functions.

38. Session #27 RetroposonsRetroviral translation & processing (Pol) When Gag & Pol are in different reading frames, a ribosomal frameshift occurs to generate a single protein. Readthrough is ~5% efficient, so Gag protein outnumbers Gag-Pol protein by 20x. ←20x ←1x

39. Session #27 RetroposonsRetroviral translation & processing (env) The Env polyprotein is expressed by post-transcriptional splicing generating a shorter subgenomic messenger that is translated into the Env product. Env products gp120 & gp41 are envelope proteins.

40. Session #27 RetroposonsRetroviral translation & processing