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1 I. Overview of retroviruses A. History B. Shared characteristics C. Classification II. Function of different regions of the retroviral genome A. Cis.

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Presentation on theme: "1 I. Overview of retroviruses A. History B. Shared characteristics C. Classification II. Function of different regions of the retroviral genome A. Cis."— Presentation transcript:

1 1 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 Introduction to Retroviruses Kathryn S. Jones, Ph.D. SAIC-Frederick/NCI-Frederick

2 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 3 A Little Retrovirus History (part I) - Won Nobel prize for the work in 1966 (at age 87). - Francis Peyton Rous discovered the first retrovirus (cancer-causing chicken virus, RSV) in Was derided at time. Prior to ~1970: Retroviruses were RNA tumor viruses –Viruses able to cause cancer –Had RNA genome

4 4 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 couldnt be template for DNA A Little Retrovirus History (part II)

5 5

6 6 – 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.

7 7 Retrovirus Overview Enveloped virus with lipid bilayer and viral spike glycoproteins. 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. Have outer matrix protein and inner core capsid containing viral genome. Viral genes are integrated into host genome. Progeny virus produced using host cell transcriptional and translational machinery. Reverse transcriptase to generate DNA

8 8 Retroviruses Scanning EM Transmission EM Env matrix capsid RNA 3D representation of HIV virion:

9 9 Retrovirus Classification Genus Example Genome Alpharetrovirus Avian leukemia virus Mouse mammary tumor virus Murine leukemia virus Feline leukemia virus Xenotropic murine leukemia-related virus Wall-eyed sarcoma virus HIV, SIV, FIV Human foamy virus Betaretrovirus Gammaretrovirus Deltaretrovirus Epsilonretrovirus Lentivirus Spumavirus Simple Complex Simple Complex Human T-cell leukemia virus Metavirus Yeast TY-3 Errantvirus Drosophila melanogaster Gypsy

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

11 11 AAAA 3 CA RU5U3R 5m 7 GpppG gagpolenv ( Packaging Signal) PBS PPT MACANC PRORTIN SUTM MA-Matrix CA- Capsid NC- Nucleocapsid PRO- Protease RT- Reverse transcriptase IN- Integrase SU- surface envelope protein TM- transmembrane envelope protein. PBS- primer binding site PPT- polypurine tract R - repeat sequence U3 - promoter/enhancer U5 - reverse transcription/ integration.

12 12 Genome of Simple vs. Complex Retroviruses

13 13 Retroviral Structural genes GeneProteinsFunction 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 RNAaseH (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

14 14 Gag proteins 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 Gag protein: /virion; Gag-Pol protein: /virion CA MA SU TM SU

15 15 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. Pol proteins

16 16 10 kd, dimer Cuts Gag polyprotein to MA,CA,NC Aspartyl protease Exquisite cleavage specificity Major class of anti-HIV drugs are Protease Inhibitors Protease

17 17 Reverse Transcriptase RNADNA

18 18 Env proteins Surface glycoprotein (SU)- involved in receptor recognition Transmembrane glycoprotein (TM)- triggers the fusion of the viral and cellular membranes,

19 19 gagpol env RU3U5RU3U5 PBS (tRNA binding site) DMS (dimer linkage site) packaging site 2nd strand primer site LTR gagpol envR U5 U3 cap A n R transcription Cis-acting Elements in Retrovirus Replication Cis acting sequences: important for 1.Transcription of RNA genome and mRNAs for viral proteins (enhancer/promoter, cap site, polyadenylation sequences) 2.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

20 20 Retroviral Life Cycle Late events: From time when integrated provirus is expressed until virus has been released Early events: from viral binding and entry until the time the DNA copy of the viral genome is integrated into the host cells chromosome

21 21 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

22 22 HIV Immune system cell CD4 Co-receptor Binding of Retroviruses to Target Cells 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

23 23 Examples of Retroviral Receptors out in N C CAT-1 (Cationic amino acid transporter) Ecotropic MLV out in N C XPR-1 (unknown function) Xenotropic/Polytropic MLV ALV-AALV-B, -D, -E CAR1Tv-A CD4 CCR5 CXCR4 HIV

24 24 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

25 25 Latent vs. active infection In latent infection- retroviral genome is present but is not transcribing viral genome or mRNA for structural proteins.

26 26 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. Retroviral Life Cycle: Transcription of Viral Genome

27 27 1.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 Two Pathways to Retroviral Assembly

28 28 Retrovirus budding from a cell

29 29 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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