1. Retroviruses and Trans(retro)posons

2. DNA fragments (elements) with the capacity to move in the genome Mobile DNA (elements) in the genome Jumping genes Function: structural ~ 50% genomic in mammals. Clasification: DNA transposons Retro (RNA) transposons Transposons

3. 3 IS elements (insertion sequence)_bacterias > 20 types Frequency of movements (1/100.000 a 1/10.000.000 per generation) Gene inactivation F plasmids Mechanism: Cut and paste Structure: Inverted repeats Target repeat Sequence coding region-Enzyme “Transposase” DNA transposons

4. 4 ADN transposones Elementos IS (insertion sequence) bacterianos (eucarioticos) Más de 20 tipos distintos Movimiento poco frecuente (1/100.000 a 1/10.000.000 por generación) Pueden inactivar genes Pueden transferirse a otras bacterias mediante su transposición a plásmidos o a viruses Estructura de los elementos IS ･ Repeticiones invertidas en los extremos Region codificante central: Transposasa

6. 6 Retro (RNA) transposons Mostly in Eukaryotes Yeast Drosophila Mammals (humans y ~8%) Mechanism: ~ retroviruses (copy and paste) Types: ---Retroviruses like (LTR) ---No related to retroviruses Structure: Inverted repeats LTR (Long Terminal Repeats) (250-600 pb) Sequence coding region-Enzymes Reverse Transcriptase (RT), Integrase (IN) and other proteins.

7. Types: ---Retroviruses like (LTR)

8. 8 Figure 22.12 Absence of LTRs Mostly in Eukaryotes Two types: LINES (Long Interspersed Elements) ~6 Kb SINES (Short Interspersed Elements) ~300 bp Structure: Target-site direct repeats A/T rich regions Sequence coding region- RNA binding proteins (ORF 1) and Enzymes ~Reverse Transcriptase (RT) and endonuclease (ORF 2) Types: ---No related to retroviruses

9. 9

10. 10 Function: Alterations in the patterns of gene expressions DNA fragments (elements) with the capacity to move in the genome Mobile DNA (elements) in the genome Jumping genes Transposons Function: structural

11. 11 Introduction Figure 22.1 --Retrovirus (two copies of RNA packaged in a capsid) attaches to cell membrane (Env) and fuses (Env), allowing entry of RNA into cell. --RNA is reverse transcribed into dsDNA and integrated into the host genome as a provirus. --The host transcribes the provirus, making all the proteins necessary for assembly of the virus which buds out of the cell as the complete retrovirus. Retrovirus life cycle

12. 12 The Retrovirus Life Cycle Involves Transposition-Like Events 1-A retrovirus has two copies of its genome of single-stranded RNA. 2-An integrated provirus is a double-stranded DNA sequence. 3-Polyproteins are initially produced and then processed (proteases) to give all the viral protein products necessary for retrovirus reproduction.

13. 13 A retrovirus generates a provirus by reverse transcription of the retroviral genome. Figure 22.2 ---Retroviral RNA (plus strand) is reverse transcribed (Pol – reverse transcriptase) into ssDNA (minus strand). ---The complementary strand of DNA (plus strand) which adds LTRs (long terminal repeats) to the 5’ and 3’ end, the linear dsDNA is inserted into the host genome (Pol-integrase). ---Transcription of the provirus (integrated retrovirus sequence) into RNA (plus strand) by the host RNA polymerase. v vm

14. 14 Retroviral Genes Codes for Polyproteins A typical retrovirus has three genes: gag, pol, and env. Figure 22.3 vmRNA U5U3 Gag and Pol requires either stop codon suppression or a frame-shift. This reduces the amount of Pol produced. Env is translated from a separate viral mRNA that is generated by splicing. Each of the three protein products are processed by proteases to give multiple Proteins. Full length viral mRNA is packaged as the viral genome. The terminal region of the viral mRNA contains specific sequence (U and R segments) mRNA is capped 5’ and polyadenylated 3’

15. 15 vmRNA U5U3 Gag - structural proteins in matrix, capsid, and nucleocapsid. Pol – protease, reverse transcriptase, and integrase. Env – surface protein, transmembrane protein for recognition and fusion Retrovirus encoded polyproteins that must be processed

16. 16 Figure 17.4 What is the source of the viral membrane?

17. 17 Influenza Virus Must “Uncoat” in Cytoplasm H5N1 hemagglutinin (HA) type 5 and neuraminidase type 1hemagglutininneuraminidase

18. 18 Viral DNA Is Generated by Reverse Transcription A short sequence (R) is repeated at each end of the viral RNA. Figure 22.5 1-Reverse Transcription 2-Integration 3-Transcription U5 – unique sequence at 5’ end (80-100 bp) U3 – unique sequence at 3’ end (170-1350 bp). A short sequence (R): is repeated at each end of the viral RNA, so the 5’ and 3’ ends are R-U5 and U3-R. Virus RNA structure

19. 19 Reverse transcriptase starts synthesis when a tRNA primer binds to a site 100 to 200 bases from the 5′ end. –(Synthesis= 5’ to 3’ direction and requires a double stranded region to initiate) –( tRNA is a “viral transcriptional RNA “?”) When the enzyme reaches the end, the 5′-terminal bases of RNA are degraded. –This exposes the 3′ end of the DNA product. The exposed 3′ end base pairs with the 3′ terminus of another RNA genome –Strand switching event Synthesis continues, generating a product in which the 5′ and 3′ regions are repeated. –This gives each end the structure U3-R-U5. Figure 22.6 U3 R U5 Viral DNA is generated by reverse transcription -RT converts RNA to DNA -RT shows DNA polymerase activity -RT has RNAase H activity Quality and Quantity---Control

20. 20 Similar strand switching events occur when reverse transcriptase uses the DNA product to generate a complementary strand. Figure 22.7 -Synthesis continues, generating a product in which the 5’ and 3’ regions are repeated, giving each end the structure U3-R-U5 (LTR) (Long Term Repeat) -Similar strand switching events occur when reverse transcriptase uses the DNA product to generate a complementary strand. -Strong stop plus strand DNA jumps to 3’ end of cDNA. This adds U3 to 5’ end of dsDNA and completes the formation of the LTR. U3 R U5 Quality and Quantity---Control

21. 21 Strand switching is an example of the copy choice mechanism of recombination. Figure 22.8

22. 22 Viral DNA Integrates into the Chromosome The organization of proviral DNA in a chromosome is the same as a transposon. –The provirus is flanked by short direct repeats of a sequence at the target site. Linear DNA is inserted directly into the host chromosome by the retroviral integrase enzyme. Two base pairs of DNA are lost from each end of the retroviral sequence during the integration reaction.

23. 23 Figure 22.9 RNA has direct (inverted) repeats, linear DNA form of virus has Long Terminal Repeats (LTR) and the integrated DNA form (provirus) has LTRs that are shortened by 2 bp And a 4-6 bp repeat of host DNA. Integrase, a viral product, acts on both viral DNA and target-host DNA. -It targets random sites by making staggered cuts at the selected-host DNA sites. -it targets end of the viral DNA converting the blunt ends into the recessed ends by removing bases beyond the CA dinucleotide sequence. -it needs sticky ends -1 to 10 copies can be introduced into host cells. (amplification and rearrangement of DNA) U3 region of each LTR carries a promoter and enhancer ( initiates transcription)

24. http://www.whfreeman.com/catalog/static/whf/kuby/content/anm/kb03an01.htm Non-germline vs germline?

25. 25 Figure 22.10 Transducing retro-viruses: a virus variant containing cellular RNA. Replication-defective: a virus variant in which a viral sequence has been replaced. Helper virus: a retro-virus that provides the missing viral function to a replication- defective virus. How are the Replication-defective viruses generated? U3 U5 U3U5

26. 26 Retroviruses May Transduce Cellular Sequences Transforming retroviruses are generated by a recombination event: –A cellular RNA sequence replaces part of the retroviral RNA. Figure 22.11 Model for the formation of the transforming viruses. 1-virus integrates near a gene (c-gene) 2-transcription of the viral RNA copy #1 (deletion) 3- transcription of the viral RNA copy #2 (no-deletion) 4-packaging of both copies of the viral RNA

27. 27 Figure 22.10 Why is the addition of v-onc into the viral genome a problem? U3U5 U3U5

28. 28 Figure 30.14 Human retroviruses?

29. 29 Figure 30.13 Replication-defective virus

30. 30 Figure 22.12 Yeast Ty elements resemble retroviruses What features are similar between retroviruses and Ty elements? Translation of TyA and TyB involves stop codon suppression and frame shift. TyA and TyB are similar to gag and pol. Transposition of Ty is through an RNA intermediate. Direct repeat delta (  ) elements at both ends of Ty like LTR. No Env genes ….so non-viral particles are generated.

31. 31 22.7 Yeast Ty Elements Resemble Retroviruses Ty transposons have a similar organization to endogenous retroviruses. Ty transposons are retroposons, with a reverse transcriptase activity. –They transpose via an RNA intermediate (splicing reaction) Figure 22.13

32. 32 22.8 Many Transposable Elements Reside in Drosophila melanogaster Copia is a retroposon that is abundant in D. melanogaster. Figure 22.15 What features are similar between retroviruses and Copia elements? Copia is a single gene, similar to gag and pol. Transposition of Copia is through an RNA (messenger) Direct repeat elements (LTR-like) at both ends. No Env genes so non-viral particles are generated.

33. 33 Some retroposons directly resemble retroviruses in their use of LTRs. Others do not have LTRs. Other elements can be found that were generated by an RNA- mediated transposition event; But they do not themselves code for enzymes that can catalyze transposition. Figure 22.17 No Env. genes? Viral vs Non-viral particles Mammalian genomes Yeast D. Melanogastes Human

34. 34 22.9 Retroposons Fall into Three Classes Retroposons of the viral superfamily are transposons that mobilize via an RNA that does not form an infectious particles. Figure 22.16

35. 35 Transposons and retroposons constitute almost half of the human genome. SINES: short interspersed elements LINES: long interspersed elements

36. 36 22.11 Processed Pseudogenes Originated as Substrates for Transposition A processed pseudogene (not active) is derived from an mRNA sequence by reverse transcription. (i.e., LINES) Figure 22.19: Reverse transcription + insertion = pseudogene.

37. 37 Figure 22.21: LINES proteins are cis-acting. LINES a-6,500 bp long b-terminal repeat in A/T-rich region c-Two ORF (1/2) -RNA binding protein -RT -endonuclease Functional transposon

38. 38 22.10 The Alu Family Has Many Widely Dispersed Members (SINES) A major part of repetitive DNA in mammalian genomes consists of repeats of a single family: –organized like retro-transposons –No Rev. Trans. –Alu I sensitivity <300 bp repeats –derived from RNA polymerase III transcripts Sequence related to 7SL RNA, a comp of SRP/SRP- Receptor. (A)n= 5' AG/CT 3';