Retroviruses

1. Retroviruses Replication cycle Envelope glycoproteins Receptors Entry Integration Expression Endogenous retroviruses HIV reverse transcription and viral evolution Phylogenetic trees

2. The Family Retroviridae

3. Simple Retroviruses MLV gag pol env e.g. murine leukemia virus (MLV)

4. Complex Retroviruses gag-pol-env-tat-rev-nef-vif-vpr-vpu HIV-1 gag-pol-env-tat-rev-nef-vif-vpr-vpx HIV-2/SIV gag-pol-env-tax-rex-HBZ HTLV Complex retroviruses have additional genes that are described as regulatory or accessory.

5. From “Retroviruses” CSHL Coffin, Hughes,Varmus A. HIV B. Beta retrovirus, MMTV C. Alpha , ALV

6. tRNA membrane TM ENV SU capsid CA reverse transcriptase RT matrix MA POL protease PR nucleocapsid NC integrase IN RNA genome Structure of retrovirus particles GAG

7. R U5 U3 R RNA genome • Two copies of viral RNA are in each virus particle. • 5'-Cap-R-U5---PBS--DLS------GAG-POL-ENV------U3-R-AAAAAA-3' • Cap and poly A • R repeats • U5, U3 unique regions at each end • PBSprimer binding site • (Psi) packaging signal DLS dimerization site

8. Replication Cycle Virion and cell membranes fuse and virus infects cell 2 Virions bind receptors on cell surface 1 RTC: reverse transcriptase complex 3 Reverse transcription to form viral DNA 4 PIC: pre-integration complex integrase Integration into chromosome 5 Proviral DNA mRNAs 7 6 Virion RNA proteins Virus particles bud from cell membranes 8 Virus particles mature and become infectious 9

9. DD Receptors for retroviruses Pit-1 FeLV-B GALV CAT-1 MLVe GLUT-1 HTLV-I HTLV-II CD4 CCR5/CXCR4 HIV/SIV CD134 CXCR4 TNF-R family FIV TVB TNF-R family ALV-B ALV-D ALV-E TVA LDLR-like ALV-A

10. Retroviral trafficking A viral substructure is released and begins trafficking to sites of integration. The RTCs and PICs exploit microtubules to transport towards the chromosomes for integration.

11. Retroviruses and dividing cells Simple retroviruses e.g. MLV, cannot infect non-dividing differentiated cells. HIV and other lentiviruses can infect non-dividing, differentiated cells e.g. macrophages, with the RTC/PIC trafficking through the nuclear membrane

12. Host proteins involved in early events after HIV entry Host ProteinProposed function FEZ1, dynein & microtubules Transport of RTCs/PICs Cyclophilin A uncoating Pin1 uncoating PDZD8 uncoating Importin-7 Nuclear import Transportin-3 Nuclear import LEDGF/p75 (transcription coactivator) Tethering of integrase to chromatin

13. Integration The retroviral cDNA in the PIC becomes integrated into host DNA. No specific site for integration. Integration catalyzed by INTEGRASE.

14. INTEGRATION 1) Two ends held together in the PIC AATG TTAC CATT GTAA 2) 3’ processing OH CA GTAA AATG AC HO

15. TGAC ACTG 3) Host DNA attacked ACTG TGAC

16. ACTG CA GTAA AATG AC TGAC 4) Gap repair ACTG TG AC CA GT TGAC ACTG TGAC Note:flanking sequences now repeated HIV has 5-base duplication MLV, FeLV have 4-base duplication ALV has 5-base duplication

17. Retrovirus integration sites

18. Synthesis of retroviral mRNA Full length HIV mRNA in virions mRNA synthesis RNA pol II HIV-1 LTR region/promoter

19. Simple retrovirus make two mRNAs GAG-POL

20. Complex Retroviruses Complex retroviruses have additional genes that are described as regulatory or accessory.

21. Tax ---tax transactivates transcription. ---Acts on HTLV LTR, stimulating transcription. ---Binds GC rich regions on the LTR adjacent to CRE sites. Then interacts with CREB transcription factors bound to these adjacent CRE sites. ---However, tax has major effects on other transcription factors e.g. NFB. ---Upregulates NFB by stimulating dissociation with IB

22. CDK9 P-TEFb complex Cyclin T TAT RNA pol II mRNA P DNA without tat, mRNA transcription is curtailed P P Tat TAR( tat responsive element) Secondary RNA structure at the start of mRNAs

23. Rev/Rex ---rev and rexare functionally equivalent. ---rev and rex regulate splicing of viral mRNAs. ---confer nuclear export of mRNAs before they are fully spliced.

24. RRE GAG/POL AAAA HIV-1 mRNAs and the rev response element ENV Early mRNAs TAT AAAA REV AAAA NEF AAAA NEF Late mRNAs RRE GAG/POL AAAA GAG/POL VIF AAAA VIF VPR AAAA VPR VPU AAAA VPU/ENV ENV

25. REV Rev increases the production of unspliced or singly spliced mRNAs that code for late mRNAs. Stem loop II Rev binding site Rev binds to a specific site on the RRE and then oligomerizes around the first bound molecule. rex has the same function for HTLV.

26. HIV Simian beta-retroviruses Constitutive transport Element: CTE Rev Crm1 TAP nucleoporins nucleoporins 5S ribosomal RNA export path mRNA export path

27. Translation of Gag and Gag/Pol precursor proteins Gag is translated as a long precursor protein. 5% of Gags are made as a Gag/Pol precursor. Translational readthrough e.g. MLV Frame shift e.g. HIV Proteins produced: Gag Gag/Pol GAG POL ENV

28. Virion maturation Viral protease cleaves the Gag and Gag/Pol precursor proteins into mature MA, CA, NC Gag proteins and RT, IN, PR enzymes. Virion maturation is essential for the virion to be infectious.

29. Endogenous Retroviruses (ERVs) ---Proviruses that become integrated into the chromosomes of germ cells can be passed down vertically via the sperm or egg DNA. ---The human genome is littered with 1000s of proviral sequences and make up about 8% of the genome. ---These endogenous retroviruses (ERVs) represent archived or 'fossil' evidence of past retroviral infections.

30. ERV Classification and Taxonomy Class I gamma-related Class II beta-related Class III spuma-related Note: Endogenous lentiviruses shown in rabbits and lemurs but not humans.

31. ERV Classification and Taxonomy ---No sensible taxonomy system. ---Named after host tRNA involved in reverse transcription e.g. HERV-W (class I), HERV-K (class II), HERV-L (class III) etc. ---Sometimes ERVs are arbitrarily named by amino acid motifs e.g. HERV-FRD, in their sequences (class I).

32. ERV Families Once integrated into the germline, endogeneous retroviruses tend to proliferate. So a single germ line infection can give rise to family of closely related elements

33. Colonization of primates by ERVs from; Bannert and Kurth, Annu. Rev. Genom. Human Genet. 2006. 20-39

34. HERV K106 ---recent acquisition about 150,000 years ago. probably after the emergence of modern humans. ---defective.

35. Nearly all endogenous retroviruses are dead ---Generally endogenous retroviruses are inactive and open reading frames have become interrupted by acquisition of many stop codons as well as deletions. ---Methylation also plays a role. ---Many are in the form of a single LTR. ---Mice, cats sheep, Koalas and pigs carry endogenous proviruses that can give rise to fully infectious retroviruses.

36. Some ERV genes are open despite ancient origins ---HERV W env (syncytin) ---HERV-FRD env (syncytin 2) ---expressed in the placenta ---believed to be involved in the formation of the syncytiotrophoblast

37. The syncytiotrophoblast is at the junction of maternal and fetal tissues From Gude et al. 2004. Thrombosis Res. 114:397-407

38. ERVs have been associated with: autoimmune disease Cancers Virus particles budding from a human teratocarcinoma cell line

39. Diseases associated with endogenous retroviruses FeLV and leukemia in cats ---exogenous FeLV-A recombines with endogenous FeLV sequences to give rise to the leukemogenic FeLV-B. ---FeLV-B recombinants use a different receptor and have a broader tropism ---leukemias caused by insertional mutagenesis