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I. Overview of retrovirusesA. HistoryB. Shared characteristicsC. Classification

II. Function of different regions of the retroviral genome A. Cis acting elementsB. Gag proteinsC. Pol proteins D. Env proteins

III. Details of life cycle:A. Early stageB. Late stage

Introduction to RetrovirusesKathryn S. Jones, Ph.D.

SAIC-Frederick/NCI-Frederickjoneska@mail.nih.gov

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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

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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 1910.-Was derided at time.

Prior to ~1970:Retroviruses were “RNA tumor viruses”

–Viruses able to cause cancer–Had RNA genome

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• Strange observations:–Infection could be stopped with DNA synthesis inhibitors–Transcription inhibitors blocked replication

• Why so strange?–At time-“central dogma of molecular biology”:DNARNAProtein –So.. RNA couldn’t be template for DNA

A Little Retrovirus History (part II)

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A Little Retrovirus History (part III)

1960s: Howard Temin: suggested DNA “provirus” waspart of replication cycle:RNADNARNAProtein- 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.

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Retrovirus OverviewRetrovirus 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, envComplex - 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 translationalmachinery.

Reverse transcriptase to generate DNA

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RetrovirusesRetroviruses

Scanning EM

Transmission EM

Envmatrix

capsid RNA

3D representation of HIV virion: http://www.mcld.co.uk/hiv/?q=3D%20HIV

3D representation of HIV virion: http://www.mcld.co.uk/hiv/?q=3D%20HIV

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Retrovirus ClassificationGenus Example Genome

Alpharetrovirus Avian leukemia virus

Mouse mammary tumor virus

Murine leukemia virusFeline leukemia virusXenotropic murine leukemia-related virus

Wall-eyed sarcoma virus

HIV, SIV, FIV

Human foamy virus

Betaretrovirus

Gammaretrovirus

Deltaretrovirus

Epsilonretrovirus

Lentivirus

Spumavirus

Simple

Complex

Simple

Simple

Complex

Complex

Complex

Human T-cell leukemia virus

Metavirus Yeast TY-3

Errantvirus Drosophila melanogaster Gypsy

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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

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AAAA 3’

CA

R U5 U3 R5’m7GpppG gag pol env

( Packaging Signal)

PBS

PPT

MA CA NC

PRO RT IN

SU TM

MA-MatrixCA- CapsidNC- NucleocapsidPRO- ProteaseRT- Reverse transcriptaseIN- Integrase

SU- surface envelope proteinTM- transmembrane envelope protein.

PBS- primer binding sitePPT- polypurine tract

R - repeat sequence

U3 - promoter/enhancer

U5 - reverse transcription/ integration.

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Genome of Simple vs. Complex Retroviruses

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Retroviral Structural genes

GeneProteins Functiongag = gag = ggroup specific roup specific aantintiggen (internal structural proteins)en (internal structural proteins)

matrix (MA), binds envelope, organizationcapsid (CA), protects genome and enzymesnucleocapsid (NC) chaperones RNA, buds

pol = pol = polpolymerase enzymesymerase enzymesreverse transcriptase + RNA to DNARNAase H (RT) degrades template RNAprotease (PR) maturation of precursorsintegrase (IN) provirus integration

env = env = envenvelope proteinselope proteinssurface glycoprotein (SU) receptor binding transmembrane protein (TM) virus-cell fusion

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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: 1200-1800/virion; Gag-Pol protein: 100- 200/virion

CA

MA

SU

TM SU

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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

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• 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

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Reverse Transcriptase

RNA DNA

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Env proteins

Surface glycoprotein (SU)- involved in receptor recognition

Transmembrane glycoprotein (TM)- triggers the fusion of the viral and cellular membranes,

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gag pol env RU3 U5R U3U5

PBS (tRNA binding site)DMS (dimer linkage site)

packaging site

2nd strand primer site

LTR LTR

gag pol env RU5 U3cap A nR

transcription

Cis-acting Elements in Retrovirus ReplicationCis acting sequences: important for1. 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

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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 cell’s chromosome

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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

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HIV

Immunesystem 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

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Examples of Retroviral Receptors

out

inN CCAT-1

(Cationic amino acid transporter)

Ecotropic MLV

out

inN CXPR-1

(unknown function)

Xenotropic/Polytropic MLV

ALV-A ALV-B, -D, -E

CAR1Tv-A CD4 CCR5CXCR4

HIV

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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

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Latent vs. active infection

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

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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

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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

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Retrovirus budding from a cell

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After Budding, Virus Goes from Immature to Mature Form

Mature Form (after budding):Mature Form (after budding):-Core becomes more dense-Core becomes more dense-Different retroviruses have different morphology in -Different retroviruses have different morphology in mature formmature form