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Introduction to Retroviruses

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


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 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.

8 Retroviruses Transmission EM matrix Env RNA capsid
Scanning EM Schematic cross section of retroviral particle. Retroviruses: Core- surrounded by an envelope. “Enveloped virus”. Core- thought to be icosohedral- not true. Inside core: Dimer RNA genome Enzymes and other proteins required for replication 3. OUTSIDE the core-Envelope Derived from the lipid bilayer of the cell 2 proteins encoded by the virus- Env- SU/TM 3D representation of HIV virion:

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

10 Retrovirus Genome (Diploid)
Retrovirus genome is +RNA 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

11 PBS- primer binding site PPT- polypurine tract
( Packaging Signal) PPT PBS- primer binding site PPT- polypurine tract 5’m7GpppG R U5 gag pol env U3 R AAAA 3’ 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.

12 Genome of Simple vs. Complex Retroviruses

13 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

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

15 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. 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-

16 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

17 Reverse Transcriptase

18 Env proteins SU: larger -primary antigen against which neutralizong antibodies are made -important for interacting with specific host receptors TM: -anchors SU/TM to membrane has fusion peptide After SU binds: conformational change; fusion peptide goes into membrane of target cell Surface glycoprotein (SU)- involved in receptor recognition Transmembrane glycoprotein (TM)- triggers the fusion of the viral and cellular membranes,

19 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 gag pol env R U3 U5 PBS (tRNA binding site) DMS (dimer linkage site) packaging site 2nd strand primer site LTR RNA genome gag pol env R U5 U3 cap A n transcription

20 Retroviral 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 chromosome Late events: From time when integrated provirus is expressed until virus has been released

21 Retroviral Life Cycle: Binding and Fusion
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 4

22 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

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

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 24

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

26 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.

27 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) 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

28 Retrovirus budding from a cell

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 Retroviruses Change Morphology When Bud from Cell: Immature Form (when it buds):-Core is spherical Mature Form (after budding): -Core becomes more dense -Different retroviruses have different morphology in mature form

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