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Introduction to Retroviruses

Introduction to Retroviruses. Kathryn S. Jones, Ph.D. SAIC-Frederick/NCI-Frederick joneska@mail.nih.gov. I. Overview of retroviruses A. History B. Shared characteristics C. Classification II. Function of different regions of the retroviral genome A. Cis acting elements

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Introduction to Retroviruses

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  1. Introduction to Retroviruses Kathryn S. Jones, Ph.D. SAIC-Frederick/NCI-Frederickjoneska@mail.nih.gov I. Overview of retroviruses A. History B. Shared characteristics C. Classification II. Function of different regions of the retroviral genome A. Cis acting elements B. Gag proteins C. Pol proteins D. Env proteins III. Details of life cycle: A. Early stage B. Late stage

  2. General Introduction to Retroviruses Retroviruses - Ubiquitous; found in all vertebrates - Large, diverse family - Includes HIV, FIV and FeLV Definition and classification of retroviruses - Common features- structure, composition and replication - Distinctive life cycle: RNA-DNA-RNA - Nucleic acid is RNA in virus, and DNA in infected cell Transmission may be either: - Horizontal- by infectious virus (exogenous virus) or vertical- by proviruses integrated in germ cells (endogenous virus) - Can transmit either as free viral particle or (for some retroviruses) through cell-cell contact

  3. A Little Retrovirus History (part I) • - Francis Peyton Rous discovered the first retrovirus (cancer-causing chicken virus, RSV) in 1910. • Was derided at time. - Won Nobel prize for the work in 1966 (at age 87). • Prior to ~1970: • Retroviruses were “RNA tumor viruses” • Viruses able to cause cancer • Had RNA genome

  4. A Little Retrovirus History (part II) • Strange observations: • Infection could be stopped with DNA synthesis inhibitors • Transcription inhibitors blocked replication • Why so strange? • At time-“central dogma of molecular biology”:DNARNAProtein • So.. RNA couldn’t be template for DNA

  5. A Little Retrovirus History (part III) • 1960s: Howard Temin: suggested DNA “provirus” was • part of replication cycle:RNADNARNAProtein • - Originally derided • Won Nobel prize (with Baltimore) in 1970 after • they independently discovered RT activity in infected cells • 1980: Human T-cell leukemia virus discovered, • the first pathogenic human retrovirus. • 1982: Human immunodeficiency virus discovered. • 1990: First gene therapy trial involving the use of retroviral-based vectors in patient with a deficiency in adenosine deaminase (ADA). • 2006: Xenotropic murine leukemia-related virus discovered.

  6. Retrovirus Overview Enveloped virus with lipid bilayer and viral spike glycoproteins. Have outer matrix protein and inner core capsid containing viral genome. Genome: Two copies of single stranded positive-stranded RNA (8-10kb). All retroviruses contain gag, pol and env genes. Simple - only gag, pol, env Complex - additional genes involved in replication. Reverse transcriptase to generate DNA Viral genes are integrated into host genome. Progeny virus produced using host cell transcriptional and translational machinery.

  7. Env matrix capsid RNA Transmission EM Retroviruses Scanning EM 3D representation of HIV virion: http://www.mcld.co.uk/hiv/?q=3D%20HIV

  8. Retrovirus Classification Example Genus Genome Avian leukemia virus Alpharetrovirus Simple Betaretrovirus Mouse mammary tumor virus Simple Murine leukemia virus Feline leukemia virus Xenotropic murine leukemia-related virus Gammaretrovirus Simple Human T-cell leukemia virus Deltaretrovirus Complex Wall-eyed sarcoma virus Epsilonretrovirus Complex HIV, SIV, FIV Lentivirus Complex Human foamy virus Spumavirus Complex Yeast TY-3 Metavirus Drosophila melanogaster Gypsy Errantvirus

  9. Retrovirus genome is +RNA Retrovirus Genome (Diploid) • Ranges from 7-10 kb in size (1 copy) • Diploid: 2 copies/virion • Important in high recombination rate From Flint et al. Principles of Virology (2000), ASM Press

  10. PPT y ( Packaging Signal) PBS- primer binding site PPT- polypurine tract 5’m7GpppG AAAA 3’ R U5 gag pol env U3 R R - repeat sequence PBS U3 - promoter/enhancer U5 - reverse transcription/ integration. CA SU TM MA CA NC PRO RT IN MA-Matrix CA- Capsid NC- Nucleocapsid PRO- Protease RT- Reverse transcriptase IN- Integrase SU- surface envelope protein TM- transmembrane envelope protein.

  11. Genome of Simple vs. Complex Retroviruses

  12. Retroviral Structural genes Gene Proteins Function gag = group specific antigen (internal structural proteins) matrix (MA), binds envelope, organization capsid (CA), protects genome and enzymes nucleocapsid (NC) chaperones RNA, buds pol = polymerase enzymes reverse transcriptase + RNA to DNA RNAase H (RT) degrades template RNA protease (PR) maturation of precursors integrase (IN) provirus integration env = envelope proteins surface glycoprotein (SU) receptor binding transmembrane protein (TM) virus-cell fusion

  13. Gag proteins CA MA TM SU SU Gag protein: 1200-1800/virion; Gag-Pol protein: 100- 200/virion Matrix (MA)- involved in binding to envelope proteins- inner surface of membrane. Capsid (CA)-major protein of the shell; most abundant protein in the virion, forms core (fragile) Nucleocapsid (NC)- involved in RNA packaging and folding; also uncoating

  14. Pol proteins Protease (PR)- cleaves Gag and Pol polyproteins, required for virion maturation Reverse transcriptase (RT)-reverse transcribes the RNA genome, also has RNAseH activity. Has DNA polymerase activity that can use DNA or RNA as template. Integrase (IN)- inserts the dsDNA copy of the viral genome into the host cell chromosome.

  15. Protease • 10 kd, dimer • Cuts Gag polyprotein to MA,CA,NC • Aspartyl protease • Exquisite cleavage specificity • Major class of anti-HIV drugs are Protease Inhibitors

  16. Reverse Transcriptase RNA DNA

  17. Env proteins Surface glycoprotein (SU)- involved in receptor recognition Transmembrane glycoprotein (TM)- triggers the fusion of the viral and cellular membranes,

  18. LTR LTR gag pol env U3 R U5 U3 R U5 PBS (tRNA binding site) 2nd strand primer site DMS (dimer linkage site) packaging site transcription A cap gag pol env U3 R R U5 n Cis-acting Elements in Retrovirus Replication Cis acting sequences: important for Transcription of RNA genome and mRNAs for viral proteins (enhancer/promoter, cap site, polyadenylation sequences) Allowing full length (genomic) RNA to exit nucleus (RRE, CTE) 3. Reverse transcription (PBS, PPT, R U5) 4. Packaging genome (DMS, and packaging site [) Integrated proviral DNA genome RNA genome

  19. Retroviral Life Cycle Early events: from viral binding and entry until the time the DNA copy of the viral genome is integrated into the host cell’s chromosome Late events: From time when integrated provirus is expressed until virus has been released

  20. Retroviral Life Cycle: Binding and Fusion 4 • Virus binds to cell surface • Specific interactions occur between the Env proteins on the virus and specific host cell proteins (“receptors”) • Env proteins undergo conformational change, which results in the fusion of the viral and cellular membranes • Most use plasma membrane fusion by some use endocytosis and then fuse envelope with membrane of endosome

  21. Binding of Retroviruses to Target Cells HIV CD4 Immune system cell Co-receptor • Virus binds to specific receptors, via interaction with SU • Different retroviruses use different receptors • BUT small groups of viruses share receptors • Env proteins- undergo conformational change which allows TM to facilitate virus-cell fusion

  22. Ecotropic MLV out out in in C N N C CAT-1 XPR-1 (unknown function) (Cationic amino acid transporter) ALV-A ALV-B, -D, -E HIV CCR5 CXCR4 CD4 Tv-A CAR1 Examples of Retroviral Receptors Xenotropic/Polytropic MLV

  23. Integration of Provirus Provirus complexed with protein moves to nucleus – pre-integration complex • most retroviruses require cells going into mitosis for the breakdown of the nuclear membrane - productive infection only in dividing cells • HIV and related viruses can enter intact nuclei, so no need for cell division - can productively infect nondividing cells Integrase is still attached: cuts up the DNA of the cell and seals provirus in the gap • may lead to immediate expression of viral genes or little or no expression (latent infection) • when this cell divides so does the genomes and get daughter cells with viral genome • irreversible:advantage for vectors - can lead to insertional mutagenesis

  24. Latent vs. active infection In latent infection- retroviral genome is present but is not transcribing viral genome or mRNA for structural proteins.

  25. Retroviral Life Cycle: Transcription of Viral Genome If provirus is not latent, transcription of the provirus occus. This produces RNA for new retrovirus genomes and RNA that codes for the retrovirus capsid and envelope proteins.

  26. Two Pathways to Retroviral Assembly Capsid assembly occurs at the membrane during budding (most retroviruses) 2. Capsid presassembled in cytoplasm and then transported to plasma membrane: (Betaretroviruses: Type B/Type D; spumaretroviruses) ONE single A.A. change in MA (R55W) can convert M-PMV from type D to type C

  27. Retrovirus budding from a cell

  28. After Budding, Virus Goes from Immature to Mature Form Mature Form (after budding):-Core becomes more dense-Different retroviruses have different morphology in mature form

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