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Lateral DNATransfer M E C H A N I S M S A N D C O N S E Q U E N C E S

Frederic Bushman The Salk Institute, La Jolla, California

COLD SPRING HARBOR LABORATORY PRESS

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Lateral DNA Transfer: Mechanisms and Consequences © 2002 by Cold Spring Harbor Laboratory Press

FIGURE 6.1. A retroviral particle. The lipid bilayer is shown as a gray ring. Outside theparticle are trimers of envelope protein, indicating the Su and Tm components. Justunderneath the lipid bilayer lies the matrix protein (white spheres). The capsid protein(green) forms the viral capsid structure. In the case of HIV, the capsid is bullet-shaped(capsid morphology differs among retroviruses). Inside the capsid are two copies ofthe viral RNA (dark green ribbons) bound to the nucleocapsid protein (small whitespheres). Also contained in the capsid are the viral enzymes (reverse transcriptase, darkgray; protease, white; and integrase, light green).

EnvSu

Tm

MA

RT

PR

RNA

IN

CA NC

Membrane




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A C

DB


FIGURE 6.2. Electron micrographs of some retroviral particles.(A) Murine leukemia virus budding. (B) Mature murineleukemia virus. (C) Lentivirus budding. (D) Mature lentivirus.(Reprinted, with permission, from Coffin et al. 1997.[15])




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FIGURE 6.3. Model for the organization of the HIV capsid. (A) Thebullet-shaped capsid is built up from hexagonal “tiles” comprisingcapsid protein hexamers. Introduction of a few pentagonal defectsin the lattice allows the formation of the closed capsid structure.(B) Electron micrograph of an HIV particle illustrating the capsidmorphology. (Reprinted, with permission, from Ganser et al. 1999[©American Association for the Advancement of Science].[23])

A B




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Src

env

pol

progag

tatnefpolpro

gag env

revvpu

vifvpr

taxpro

pol

gag env

rex

HTLV

HIV

RSV

A

B

C

D

ψ

ψ

ψ

ψ

env

polprogag

MLV


FIGURE 6.4. Some retroviral genomes. Flanking DNA is shown in gray, viralcoding regions in green. The R region in the LTR is shown black. The onco-gene of RSV, src, is also shown black. Ψ indicates the packaging site in theRNA. (Adapted from Coffin et al. 1997.[15])




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FIGURE 6.5. Retroviral phylogeny. The gene names in gray indicatewhere new genes were acquired in different retroviral lineages. Virusnames are as follows: MLV, murine leukemia virus; FeLV, felineleukemia virus; HERV-C, human endogenous retrovirus C; WDSV,walleye dermal sarcoma virus; HFV, human foamy virus; HIV-1,human immunodeficiency virus type-1; HIV-2, human immunodefi-ciency virus type-2; EIAV, equine infectious anemia virus; VMV,Visna-Maedi virus; MPMV, Mason-Pfizer monkey virus; MMTV,mouse mammary tumor virus; HERV-K, human endogenous retro-virus K; IAP, intracisternal A particle; ASLV, avian sarcoma-leukosisvirus; BLV, bovine leukemia virus; HTLV-1, human T-cell leukemiavirus type 1; HTLV-2, human T-cell leukemia virus type 2. (Modified,with permission, from Coffin et al. 1997.[15])




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Receptorbinding

Preintegrationcomplex

Entry

Reversetranscription

Integration

Nucleus

Transcription Assembly

TranslationMaturation

Budding


FIGURE 6.6. The retroviral life cycle. A retroviral particle (top) initially binds to a cell-surface receptor,leading to fusion and entry of the viral core into the cell cytoplasm. Fusion can take place either at themembrane or after endocytosis of the bound viral particle, depending on the type of retrovirus. Followingentry, the viral RNA is reverse-transcribed, yielding a double-stranded DNA copy of the viral genome.The resulting preintegration complex then migrates to the nucleus and integrates the viral cDNA into ahost chromosome. The resulting provirus can then be inherited during cell division like a normal cellulargene. The provirus also serves as a transcriptional template for synthesis of viral RNA. Viral RNAs serveas mRNAs for protein synthesis, and the full-length viral RNA also can become packaged and serves asthe genome for the next generation of virions. Assembly takes place at the membrane (in the case of typeC retroviruses and lentiviruses). Budding yields the immature viral particle in which the viral proteins arepresent as polyprotein precursors. Proteolysis directed by the virus-encoded protease yields the maturevirion.




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R

R

U5

U5U3

gag

gag

env

env

Reverse transcriptionintegration

U3

U3 U5R

pol

pol

RU5 gag

envU3

R

pol

RNA

RNA

DNA

TranscriptionProvirus


FIGURE 6.7. Information flow during retroviral replication, illustrating the dif-ferences in the LTRs between the RNA and DNA forms.




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FIGURE 6.8. Pathway of reverse transcription. (A) The viral RNA is packaged with a tRNA moleculebound to the primer-binding site (PBS). After entering an appropriate target cell, reverse transcription ini-tiates (B) with the extension of the tRNA 3´ end to the 5´ end of R. The viral genome is identical insequence to the mRNA; in the jargon of virology, this is the plus strand. The complementary cDNA is theminus strand. The growing cDNA chain is then transferred (C) to the 3´ end of the RNA genome andextended the length of the RNA (D). The RNA is degraded concomitant with cDNA synthesis. An RNAfragment remains bound at the polypurine tract (PPT), which serves as the primer for plus-strand cDNAsynthesis. Plus-strand synthesis extends to the tRNA primer (E), then jumps to the other end of the cDNAand pairs using complementary sequences in the PBS (F). Extension of both 3´ ends in the cDNA to theends of the genome yields the complete linear viral cDNA (G). (Adapted from Varmus and Brown1989.[49])

ppt

pptRNA

primerU3 R U5 PBS

ppt U3 R U5U3 R U5 PBS

U3 R U5 PBS

PBS

ppt U3 R U5 PBSPBS

ppt U3 R U5 PBSPBS

ppt U3 R

tRNAprimer

RNA

DNA

ppt U3 RR U5 PBS

R U5 PBS

tRNAprimer

RNA

A

B

C

D

E

F

G

RNA

DNA

DNA

DNA

DNA

RNA




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Viral DNA

Preintegrationcomplex

Terminalcleavage

Host celltarget DNA

Strandtransfer

Gap repair

A

B

D

C


FIGURE 6.9. Pathway of cDNA integration. (A) Following reverse transcription, theviral cDNA (green) remains bound with viral and cellular proteins (dark gray) thatdirect integration. (B) Prior to integration, nucleotides are removed from each 3´DNA end (typically two), probably to remove heterogeneous additional basessometimes added by reverse transcriptase. (C) The recessed 3´ ends are then joinedto protruding 5´ ends in the cellular target DNA. (D) The DNA gaps and two-baseoverhang at the host viral DNA junction are then repaired, probably by host cell gaprepair enzymes, to yield the integrated provirus.




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FIGURE 6.10. Schematic diagram of the RSV integrase enzyme.

Zn-bindingdomain

H

1 286

H C C D

RSV integrase

D E

DNA-bindingdomain

Catalyticdomain




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A B


FIGURE 6.11. (A) X-ray structure of a fragment of HIV integrase showing the catalytic andDNA-binding domains. Rendered from coordinates generated by Chen et al. (2000) (14).(B) X-ray structure of a fragment of RSV integrase showing the catalytic and DNA-bind-ing domains. Image rendered from coordinates generated by Yang et al. (2000) (53).Monomers in the dimer are shown in green and gray. The three catalytic residues areshown in black.




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FIGURE 6.12. Transcription factor-binding sites in two retroviral U3 regions. Retroviral transcriptioninitiates at the border between U3 and R in the upstream LTR and terminates at the border between Rand U5 in the downstream LTR. The U3 region is rich in binding sites for cellular transcription factorsthat promote efficient transcription of the proviral genome. Some of the known binding sites in the U3regions of Mo-MLV and HIV are shown. (Portion redrawn, with permission, from Coffin et al.1997.[15])

U3

UCRBP NF-1 Ets

MCREFGR

Direct repeats

Promoter(+)

Enhancer(+)

EtsELP bHLH

bHLH C/EBPCBFU3

Mo-MLV LTR

GenomicDNA

Provirus

R U5

FactorA

U5R U3 U5R

TATA

100 bp

NFAT-1

COUPTF

GR

NRE (+/–) Enhancer(+)

(–) (–)

Ets TCF-1 AP2

USF-1 Sp-1LBP-1NF-ΚB TAR

RNA

U3

HIV-1 LTR

R U5

TATA

PBS(–)

GenomicDNA

Viral RNA




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FIGURE 6.13. Assembly and budding. The viral Gag proteins associate with membranes by interactionswith the amino-terminal MA domain. Env proteins are separately routed through the endoplasmicreticulum and Golgi apparatus to the cell surface. RNA is bound by the nascent particle, primarily bythe NC domain of Gag. Assembly of Gag monomers deforms the membrane, allowing the virus to budout from the membrane.

GagGagPol

GenomicRNA

GenomicRNA




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MA

CA

NC

PR

RT

IN

MA

CA

NC

MA

CA

NC

MACA

NC

MAMA MA

CA

PR RT

INNC NC

NC NC

RT

NC

NC

CA CACA

CACA

MA MA MA


FIGURE 6.14. Maturation. The initially budded particle contains the Gag andGag-Pol polyprotein precursors (left). Following budding, action of the viralprotease converts the polyprotein precursors to the individual proteins active insubsequent steps of the viral life cycle (right). Maturation is accompanied by for-mation of the mature viral core.




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SH3Active

c-Src

c-Srcinactiveform

v-Src

SH2

γ 527

SH1

SH3Inactive SH2

γ 527

SH1

Y416

P

Y416

Y416

Y527

Y416

P

P

SH3 SH2 SH1

SH3

SH2KinaseSH1

P


FIGURE 6.15. Regulation of the c-Src protein and disregulation in v-Src. The activeform of c-Src (top) is phosphorylated at Y416 but not at Y527. Dephosphorylation ofY416 and phosphorylation of Y527 inactivates the enzyme (middle). v-Src lacks the Y527inhibitory site and so is constitutively active. Phosphorylation of Y527 results in bind-ing to the SH2 domain, which inhibits the kinase activity and so effects negative regu-lation (bottom). (Adapted from Coffin et al. 1997.[15])




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RSV

AMV

Pr76Pr180Pr68

Pr76Pr180

p60 Src

p45Gag-Myb-Env

gag pol env

∆envv-mybpolgag

src

p75Gag-ErbAgp65 ErbB

AEV-ES4∆envv-erb Bv-erb A∆gag

Ab-MLV∆envv-abl∆gag

p160Gag-Abl

p55 Fos

FBJ-MSV∆envv-fos


FIGURE 6.16. Some acute transforming viruses and the proteins they encode. OnlyRous sarcoma virus (RSV) is capable of independent replication; the others requirehelper viruses to supply proteins for replication. White rectangles indicate codingregions for viral proteins, black rectangles, oncogenes. Abbreviations: AMV, avianmyeloblastosis virus; AEV, avian erythroblastosis virus; Ab-MLV, Abelson murineleukemia virus; FBJ-MSV, Finkel-Biskis-Jenkins mouse sarcoma virus. (Modified andredrawn, with permission, from Coffin et al. 1997.[15])




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A

B

C

U3 U5R U3 U5Rgag pol env

U3

or

U5R U3 U5R

U3 U5R

U3 R

AAA

gag pol env

c-onc

c-onc

D

EU3 U5R U3 U5R

AU3 U5R ∆gag

U5R ∆gag

gag pol env

env

c-onc

c-onc

c-onc

c-onc

U5R

AAA

or

and

∆gagU5R

U3 U5R

polAAA

∆gagU5R

U3 Renv

c-oncAAA

∆gag

onc∆gag

U5R

polAAA

gagU5R


FIGURE 6.17. Possible mechanisms by which retroviruses pick up oncogenes. DNA isshown as thick light green lines, RNA as thin dark green lines. (A) Oncogene acquisi-tion begins when a retrovirus integrates upstream of a proto-oncogene. A transcript isthen formed containing both a retroviral LTR sequence and oncogene sequences (B).Such transcripts can form either by readthrough of the viral RNA or by a deletion inthe DNA. These transcripts are packaged with proteins supplied by helper viruseselsewhere in the genome. (C) The readthrough transcript, or a deleted transcripttogether with an intact genome, is then packaged. Recombination during reverse tran-scription allows the formation of a cDNA containing the oncogene flanked by LTRs (Dand E). In the presence of an intact helper virus, the resulting genome can be packagedand transferred to new cells. (Redrawn, with permission, from Coffin et al. 1997.[15])




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pol env

env

ψ gag ψ gag

ψ gene x

Retrovirus

Retroviralvector

ψ gene x

pol env

polgag


FIGURE 6.18. Retroviral vectors. Normal retrovirus infection (top) involves pack-aging the wild-type genome, which directs synthesis of the viral proteins. Forretroviral vectors (bottom), separate DNAs encode the viral proteins and thegenome that becomes packaged. Consequently, the genome transferred to the target cell does not encode the viral proteins and so cannot spread farther. The neteffect is to install only the engineered “gene x” into target cells.




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FIGURE 6.19. Mechanisms of proto-oncogene activation by provirus insertion. DNAis shown as thick light green lines, RNA as thin dark green lines. (A) Structure of aproto-oncogene locus, showing a transcriptional control region, three exons, and themRNA synthesized. (B) Promoter insertion. Insertion of a provirus in the same ori-entation as the proto-oncogene can lead to production of a readthrough transcript,potentially altering the rate of transcription or the stability of the message. (C)Enhancer insertion. This mechanism typically involves a retrovirus in opposite ori-entation to the proto-oncogene, which increases the rate of transcription initiation.(D) Readthrough transcript formation. Transcripts initiated in the left LTR are fusedby RNA splicing to the proto-oncogene exons. This can alter the rate of transcription,the stability of the message, or the nature of the encoded protein. (Adapted fromCoffin et al. 1997.[15])

envpolgag

Promoterinsertion Exon 1R R

R R

R R

Exon 2 Exon 3

env pol gag

Enhancerinsertion Exon 1 Exon 2 Exon 3

Proto-oncogeneRegulatory

region Exon 1 Exon 2 Exon 3

Read-throughtranscriptformation Exon 1 Exon 2 Exon 3

B

A

C

D




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FIGURE 6.20. Insertion of a HERV element upstream of an amylase gene in humansresults in parotid (salivary gland) expression. This allows partial digestion of somestarches in the mouth, resulting in a sweet taste. (Adapted from Ting et al. 1992.[48])

LTR LTR

Amylase genepromoter

Amylaseexons

HERV

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