Presentation on theme: "Introduction to Retroviruses"— Presentation transcript:
1Introduction to Retroviruses Kathryn S. Jones, Ph.D. SAIC-Frederick/NCI-FrederickI. Overview of retrovirusesA. HistoryB. Shared characteristicsC. ClassificationII. Function of different regions of the retroviral genomeA. Cis acting elementsB. Gag proteinsC. Pol proteinsD. Env proteinsIII. Details of life cycle:A. Early stageB. Late stage
2General Introduction to Retroviruses - Ubiquitous; found in all vertebrates- Large, diverse family- Includes HIV, FIV and FeLVDefinition 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 cellTransmission 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
3A 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).Found (in 1910) that hens could be transmitted to fowl of the same inbred stock by injecting a submicroscopic agent extractable from tumor cells ; this discovery gave rise to the idea that a virus can cause cancer causation. Although his research was derided at the time, subsequent experiments vindicated his thesis, and he received belated recognition in 1966 when he was awarded (with Charles B. Huggins) the Nobel Prize.Prior to ~1970:Retroviruses were “RNA tumor viruses”Viruses able to cause cancerHad RNA genome
4A Little Retrovirus History (part II) Strange observations:Infection could be stopped with DNA synthesis inhibitorsTranscription inhibitors blocked replicationWhy so strange?At time-“central dogma of molecular biology”:DNARNAProteinSo.. RNA couldn’t be template for DNA
6A Little Retrovirus History (part III) 1960s: Howard Temin: suggested DNA “provirus” waspart of replication cycle:RNADNARNAProtein- Originally deridedWon Nobel prize (with Baltimore) in 1970 afterthey independently discovered RT activity in infected cells1980: 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.
7Retrovirus OverviewEnveloped 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, envComplex - additional genes involvedin replication.Reverse transcriptase to generate DNAViral genes are integrated into host genome.Progeny virus produced using host cell transcriptional and translationalmachinery.
8Retroviruses Transmission EM matrix Env RNA capsid Scanning EMSchematic cross section of retroviral particle.Retroviruses:Core- surrounded by an envelope. “Enveloped virus”. Core- thought to be icosohedral- not true.Inside core:Dimer RNA genomeEnzymes and other proteins required for replication3. OUTSIDE the core-EnvelopeDerived from the lipid bilayer of the cell2 proteins encoded by the virus- Env- SU/TM3D representation of HIV virion:
10Retrovirus Genome (Diploid) Retrovirus genome is +RNARanges from 7-10 kb in size (1 copy)Diploid: 2 copies/virionImportant in high recombination rateFrom Flint et al. Principles of Virology (2000), ASM Press
13Retroviral Structural genes Gene Proteins Functiongag = group specific antigen (internal structural proteins)matrix (MA), binds envelope, organizationcapsid (CA), protects genome and enzymesnucleocapsid (NC) chaperones RNA, budspol = polymerase enzymesreverse transcriptase + RNA to DNARNAase H (RT) degrades template RNAprotease (PR) maturation of precursorsintegrase (IN) provirus integrationenv = envelope proteinssurface glycoprotein (SU) receptor bindingtransmembrane protein (TM) virus-cell fusion
14Gag proteins CA MA TM SU SU Gag protein: /virion; Gag-Pol protein: /virionMA- membrane associated- has Has myristic acid (characteristic of proteins on internal surface of membrane. IMPORTANT for 1. assembly of core, 2. Binding Env.CA- hydrophobic, form the major internal structural feature of virions- core shell. Core it forms- is somewhat fragile, so can dissolve.NC- small basic protein- interacts with genomic RNA- has zinc finger (“zinc knuckle”) that interact with RNAMatrix (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
15Pol proteinsProtease (PR)- cleaves Gag and Pol polyproteins, required for virion maturationReverse 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- all function as enzymes.PR- viral protein that cuts the polyprotein precursors into individual proteins. Recognize short stretches of aa- are similar to cellular aspartic proteases. Acts late in assembly (during/after budding). Causes the changes in the core.RT and IN- 2 enzymes that are critical for the early stage of infection. RT- contains 2 activities-
16Protease 10 kd, dimer Cuts Gag polyprotein to MA,CA,NC Aspartyl proteaseExquisite cleavage specificityMajor class of anti-HIV drugs are Protease Inhibitors
18Env proteinsSU: larger-primary antigen against which neutralizong antibodies are made-important for interacting with specific host receptorsTM:-anchors SU/TM to membranehas fusion peptideAfter SU binds: conformational change; fusion peptide goes into membrane of target cellSurface glycoprotein (SU)- involved in receptor recognitionTransmembrane glycoprotein (TM)- triggers the fusion of the viral and cellular membranes,
19Cis-acting Elements in Retrovirus Replication Cis acting sequences: important forTranscription 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 genomegagpolenvRU3U5PBS (tRNA binding site)DMS (dimer linkage site)packaging site2nd strand primer siteLTRRNA genomegagpolenvRU5U3capAntranscription
20Retroviral Life Cycle Early events: Late events: from viral binding and entry until the time the DNA copy of the viral genome is integrated into the host cell’s chromosomeLate events:From time when integrated provirus is expressed until virus has been released
21Retroviral Life Cycle: Binding and Fusion Virus binds to cell surfaceSpecific 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 membranesMost use plasma membrane fusion by some use endocytosis and then fuse envelope with membrane of endosome4
22Binding of Retroviruses to Target Cells HIVCD4Immunesystem cellCo-receptorVirus binds to specific receptors, via interaction with SUDifferent retroviruses use different receptorsBUT small groups of viruses share receptorsEnv proteins- undergo conformational change which allows TM to facilitate virus-cell fusion
24Integration of Provirus Provirus complexed with protein moves to nucleus – pre-integration complexmost retroviruses require cells going into mitosis for the breakdown of the nuclear membrane- productive infection only in dividing cellsHIV and related viruses can enter intact nuclei, so no need for cell division- can productively infect nondividing cellsIntegrase is still attached: cuts up the DNA of the cell and seals provirus in the gapmay 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 genomeirreversible:advantage for vectors- can lead to insertional mutagenesis24
25Latent vs. active infection In latent infection- retroviral genome is present but is not transcribing viral genome or mRNA for structural proteins.
26Retroviral 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.
27Two 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)Envelope glycoproteins and the pol-encoded enzymes are required for the production of infectious progeny virions, expression of Gag proteins alone is generally sufficient for the assembly and release of non-infectious, virus-like particles (VLPs).Retrovirus assembly itself usually takes place at either of two subcellular locations (Gof): for type C retroviruses, which include the alpharetroviruses, gammaretroviruses, and lentiviruses, the assembly of electron-dense structures occurs at the plasma membrane. For type B and D retroviruses, assembly takes place in the cytosol and the assembled intracytoplasmic particles (known as A-type particles) traffic to the plasma membrane where they bud from the cell.ONE single A.A. change in MA (R55W) can convert M-PMV from type D to type C
29After Budding, Virus Goes from Immature to Mature Form Mature Form (after budding): -Core becomes more dense -Different retroviruses have different morphology in mature formRetroviruses Change Morphology When Bud from Cell:Immature Form (when it buds):-Core is sphericalMature Form (after budding): -Core becomes more dense -Different retroviruses have different morphology in mature form