WHY STUDY RETROVIRUSES?

* PATHOGENIC CO~SEQUE~CE~: A HISTORY OF DISCOVERY ...

EQUINE: ANEMIA 1904 (EIAV)

CHICKEN LEUKOSIS 1907 (ALV)

CHICKEN SARCOMA 1911 (RSV)

MOUSE M~~RY CARCINOMA 1930's (MMTV)

MOUSE LEUKEMIA 1951 (MLV)

ADULT T CELL LEUIEMIA/LYMPHOMA 1980 (HTLV)

ACQUIRED ~~~NODEFI~~~CY S~ROME 1983 (HN)

(OTHER PATHOLOGY INCLUDES ARTHRITIS, NEUROLOGICAL, DISEASES,

OSTEOPE~OSIS, MANY CANCERS, ETC.)

*UNUSUAL LIFE CYCLE

TEMINS PROVIRUS HYPOTHESIS:

THE CLUES: ~~ITORS OF DNA SYNTHESIS AND OF DNA

-DIRECTED RNA SYNTHESIS BLOCK INFECTION OF AN RNA VIRUS

THE EVIDENCE:  REVERSE TRANSCRIPTASE IN VIRIONS

           TS MUTANTS OF POL GENEi

DNA IN INFEXTED CELLS (HYBRIDIZATION.

TRANSFECTION, CLONING)

Trans-
lation

binding

Pre-genome

complex

transcription

RNA
processng

WHY STUDY RETROVIRUSES?

* WIDE DISTRIBUTION IN NATURE ... THE SEVEN CLASSES

ALV'"

NXV

WP.

ALV-R.lr1.d

BLV-HTLV

HSRV

MLV
HERVC

tal. rev. etc

3-i

HIV-1
HIV-2
Vlsna
ElAV

SMRV

new em

MMN
HERV-K

IAP

RSV

BLV
HTLV.1
HTLV-2

-

CI

(OTHER MEANS OF CLASS~CA~ON: - PATHOLOGY, PARTICLE TYPE, HOST)
~S~SSIO~ BLOOD, MILK (MMTV, HTLV), ~ONGE~~ INFECTION,

-

VIA GERM LINE ~E~~GENOUS PROV~USES)

* EVOLUTIONARY IMPLICATIONS OF ENDOGEWOUS PROVIRUSES,

9

OTHER RETRO-ELEMENTS (NEXT LECTURE)

* NOVEL STRATEGIES FOR EXPRESSION (TAT, REV, FR~ESHI~ING)

* ONCOGENIC ~IECHA~IS~S:

-

TRANSDUCTION

* UTILITY AS GENETIC VECTORS

I *

c

RETROVIRAL GENOMES ....

HO~O~I~~, DIMER LINKAGE, ASSOCIATED tRNA

WU-ewPO~ tw,
hcccNLy QRM@?&

"3' J% .'

IMPLICATIONS OF "PSEUDODIPLOIDY": HETEROZYGOSIS, RECOMBINATION

-

2 Rlt)R, S;.bdqr -4P  1 ?RQVtRUS

e----- _- ~ - -

CIS ACTIVE: SITES FOR PRIMING RT AND FOR INTEGRATION (ATT SITES);

      PROMOTERS, ENHANCERS, AND RNA PROCESSING SIGNALS
        (SPLICING AND POLY~E~TI~~;

-

TRANSLATION, DIMERIZATION, PACKAGING SIGNALS.

3'SS PPTlATT U3 R

'\I I

R US ATTPBS PSI 5'SS DLS

.\f J 3'

5 A& 1 / A/-@,

1,

t t t t t'

GAG POL ENV

TRANS ACTIVE:

GAG----> MATRIX, CAPSID, NUCLEOCAPSID

POL----> PROTEASE, RT, INXEGRASE

ENV-----> SURFACE, TRANSMEMBRANE GP

( IDTOSYNCRATIC ELEMENTS:

- -.I

TRANSCRIPTIONAL REGULATORS: TAX, TAT, BEL GENES AND TARGETS

REGULATORS OF RNA METABOLISM: REX AND REV AM) TARGETS

--- -

- -

OTHER LENTIVIRUS GENES: MEF, VIF, VPU, VPR, VPX

--LI-Ip

SUPERANTIGEN (SAG) GENE OF MMTV; dUTP-ASES OF LENTIVIRUSES

4= P

TMNSDUCED GENES (MOSTLY ONCOGENES)

r

P17

MA

  rev A .....
4 4 tat .. .

. ..................................... * ..'..'..
               . \:. .

.......:
. .....

j: VITl'

ooooOoO pll P5l p32

PT

PR

ooooooQ

I1 I IN gp120 gP41

-_

su TM

viral 00-

genomic -
RNA

DETERMINANTS OF VIRUS ENTRY AND HOST RANGE

  ENTRY MEDIATED BY VIRAL SURFACE ENV PROTEIN AND VARIETY OF

CELL SURFACE RECEPTORS WITH WIDE RANGE OF NORMAL FUNCTIONS ...

2

II 0

INFECTION BLOCKED BY: ABSENCE OF RECEPTOR OR CO-FACTOR

c"-

HOMOLOGOUS INTERFERENCE, DOWN REG.

ANTIBODY AGAINST SU-ENV OR RECEPTOR

SOLUBLE RECEPTOR

ytfhs: HIV GALV/FLV-B MLV-ECO RSV-A

?TRANSPORTER BASIC AA
            PERMEASE

Qs&wd; cD4

(REQUIRES

COFACTOR)

?

LDL-RELATED a

SURFACE INTERACTIONS ARE MAJOR DETERMINANT OF VIRAL HOST RANGE

e

3

AND TISSUE TROPISM

RESTRICTION CAN BE OVERCOME BY FORCED FUSION,

\\

PROVIRAL TRANSFECTION, AND PSEUDOTYPE FORMATION

--

- - - _- ___

EARLY EVENTS: THE NUCLEOPROTEIN COMPLEX

A PRODUCT OF ENTRY AND UNCOATING

Fmmme UNCERTAIN

VEmCLE: FOR REVERSE TRANSCRIPTION IN CYTOPLASM, TO MAKE

FULL-LENGTH LINEAR DUPLEX WITH LONG TER~~~ RllPEATS

~IGRAT~~~ TO THE NUCLEUS:
----

* EQUIREMENT FOR NUCLEAR MEMBRANE BREAKDOWN IN MITOSIS (MLV)

VS. DIRECT ENTRY cHnr>

* PRES- DEAD ENDS:  CIRCLES WITH ONE OR TWO LTRS

INTEGRATION MACHINE FOR INSERTING DNA INTO CHROMOSOh4ES

4--V*L-+

HOST RESTRICTION VIA NPC:  SOME FV-I ALLELES ramxrcnr

---- 'y.sB:

GROWTH OF SOME STRAINS OF MLV (VIA CAPSID DETERMINANT),

OPERATING AFIER EirflsRY AMD BEFORE ~G~~ON ....

w

PRINCIPLES OF RET~OVIR~L REVERSE TRANS~RIP~IO~

* PROPERTIES OF ENZY ME:.... RNASE H AND DNA POLYMERASE

HOLOENZYME (INTRINSIC ~~~~:P~R, DISRUPTED VIRIONS)
ISOLATED ENZYME (EROM VIRIONS, MADE IN BACTERIA)

-

* HETEROD~ERS (HIV, RSV) VS. ~ONO~ERS (ML'V)

RT RNASE H IN

RSV

MLV

* TE~P~ATES AND PRIMERS

NATURAL PRIMERS: tRNA FOR FIRST (MINUS)

POLYPURINE OLIGO-RNA FOR PLUS STRAMD

4
PPr

EXOGENOUS TEMPLATE-PRIMERS: mRNA-OLIGO dT

GAPPED DNA
POLY rA: dT OR POLY rC: dG

* JUMPS, METASTABILITY, ERRORS

* INHIBITORS: NUCLEOSIDE ANALOGS, OTHERS

8flV

env

Duplex Linear Viral DNA

-

:SA.:.

......... .

...... i...

U3 R U5

[ ztaggered cleavage

mt cell uNAl) Y IN

3'
5'

Cellular 5'
DNA 3'

mw=Rtf%

I Joining (Le., strand transfer) by IN 3 ow

-

f *-a,

TG 7 ...... 3'

......

5'

Q+:+

5'

3' 4R!2 gag - env

of mismatched

      dinucleotides
enzymes -fill-in of gaps

I'

Short dlrect repeats
in cellular DNA

Integrated Provirus

-

PRINCIPLES OF RETROVIRAL INTEGRATION

4

THE BEST UNDERSTOOD RECOMBINATION EVENT IN EUKARYOTIC CELLS

MACHINERY: NUCLEOPROTEIN COMPLEX,

PRECISION AT VIRAL ENDS, MANY SITES IN TARGET
NO REQUIREMENT FOR ENERGY SOURCE
ESSENTIAL, FOR REPLICATION AND PERSISTENCE

7

c-

ASSAYS:

-

INFECTED CELLS .... MAPPING, CLONING, PCR

IN VITRO ... NPC VS. PURIFIED INTEGRASE AS SOURCE OF ACTIVITY

-

_-

--

iE

-

ROLE OF NUCLEOSOMES

int'n

a

DNA

HELIX

NUCLEOSOME
  CORE

+

*-CA,,

+

30

CI 1 L

+

o

30

i I-

l6 I

CONTROL OF PROVIRAL TRANSCRIPTION

1) HOST TRANSCRIPTION FACTORS INTERACT WITH TARGETS IN U3
o L -

* DETERMINES LEVEL OF EXPRESSION
* INFLUENCES TISSUE TROPISM AND PATHOGENIC TARGET

--

MLV: ERYTHRO VS. T CELL LEUKEMIA

MMTV: MAMMARY VS. T CELL TUMORS

2) VIRAL FACTORS AS TRANSCRIPTIONAL CO-ACTIVATORS

-

b

* TAX OF HTLVBLV

* BEL OF FOAMY VIRUSES

=15;.

3) VIRAL FACTORS AS REGULATORS VIA VIRAL RNA TARGET

C-T-

* TATITAR OF HN 9 Qj/
* ABSOLUTE REQUIREMENT FOR VIRUS PRODUCTION

-

* ROLE IN TEMPORAL CONTROL OF LATE EVENTS (AND LATENCY?)

-

I I 1 I

I I I I

          1
100 200 30(

0

-500 -400 -300 -200 -100

I I I 1 I

1

20 40 60 80 loo

0

EXON I EXON II

Un

RNA blndlng
Nuclear localization

Acidic domain Cysteine domaln Basic domain

fyNMsrS

ncreased Initiation

Efficiency

RNA Pol I1
Transcription
Complex

&eased Elongation

Efficiency

Cellular Factors

I\

Cellular Factors

Nascent
mRNA

I

\

[GI w] TAR

RNA Pol I1
Transcription
Complex

              GG
Loop - U G
             CA
              CG
              GC
              AU

w 'A U

BULGE cuG

GC

CG

AU
UA
UA

STEM ___) E

UA
CG
UG
CG

c::;

GC

HIV-1 TAR

GG
UG
CA

CG
CG
GO
A0

'A U
GC
AU
'G C
GC
UA

UG c

G
UG
CG

GC

U C
CA
CG

CG GC +140

AU

+1 +14 G c

m7G pppG...AGAG AG GACUCUCACC UGCUUCEUUA

UA

AU
GC
CG
AU
GC
GC

uc
AU

GO
A UG
GG

CG
CG
U

HIV-2 / SlVmac TAR

PROCESSING OF RETROVIRAL RNA

CAPPING 5' ENDS

POLYADENYLATION:

RESPONDING TO SIGNALS FROM 3' (NOT 5') LTR

SPLICING AND TRANSPORT TO CYTOPLASM:

@  MAINTAINING AN APPROPRIATE RATIO OF RNA's

-

GENOME (GAG mRNA) vs ENV mRNA vs  OTHER mRNA
-- rcrs -

(N.B.: PRE-mRNA IS GENOME AND&,SO mRNA)

a FOR SIMPLE GENOMES: CIS SIGNALS SUFFICE

0 FOR COMPLEX GENOMES:

APPEARANCE OF INCOMPLETELY SPLICED mRNA IN

CYTOPLASM GOVERNED BY REX OR REV VIA RXRE OR RRE

(Y~Qwkc WA
NORWUWS

NlV

RV +

u IV
RN-

I I        I 1        I 1        I
0 20       40 60       80 100      120

EXON I EXON II

I II

Multlmerizatlon

I

I 1

RNA binding
Nuclear locallzatlon
  n

HIV-1 Rev N
(W -

Bask domaln Leucine domain

V

COOH

2) MINUS.ONE FRAMESHIFTING---ONCE (RSV, HIY) OR TWICE (MMTV, HTLV)

L

SIGNALS: SHIFTY SITE, RNA STRUCTURE 3' OF SITE, SPECIAL tRNA

--1

MECHANISM: SIMULTANEOUS SLIPPAGE AFTER DECODNG YYZ
7-
FRE UENCY: 3% TO 20%

OTHER VENUES: CORONOVIRUSES, RETROTRANSPOSONS, YEAST DS

&

-

RNA, BACTERIAL TRANSPOSONS, PLANT VIRUSES, E.COL1 DNA X.....

NO EUKARYOTIC HOST GENES AS YET

RULES GOVERNING RETROVIRAL ASSEMBLY

CAPSID-CAPSID INTERACTIONS DRIVE ASSEMBLY OF CORE

--

GAG PRECURSOR SUFFICES
I
INTERACTIONS WITH CA PORTION OF GAG-POL INCLUDE POL

-

MATRIX DETERMINES SITE OF ASSEMBLY

-

ACETYLATIONA4YRISTYLATION REQUIRED TO GET TO MEMBRANE

MATRIX VARIANTS FOR ASSEMBLY AT PLASMA MEMBRANE (MOST),

CYTOSOL (TYPE D), OR ER (MUTANTS)

NUCLEOCAPSID REGION INTERACTS WITH PSI SITE:ON VIRAL RNA,

-

AND FACILITATES DIMERIZATION ?

RT PROMOTES INCLUSION OF APPROPRIATE TRANSFER RNA (PRIMER)

w

ENV PROTEINS MODIFIED, OLIGOMERIZED IN ER AND GOLGI

-

BUDDING AT SITE OF EiW PROTEIN: MOST HOST TM PROTEINS EXCLUDED

I

HOW DOES CORE RECOGNIZE ENV-RICH PLASMA MEMBRANE?

MATURATION: DIMERIZATION OF PROTEASE REQUIRED FOR ACTIVITY,
- -

CLEAVES GAG AND GAG-POL AT SPECIFIC SITES

COINCIDENT WITH CORE CONDENSATION, INFECTIVITY, RT ACTIVITY

Flaps

/\

Dimer
Interface

P4 F3 F2 P1

Gly - Thr - Ser - Cys -Tyr
Pro - Pro - Tvr - Val - Gly
Pro: Val - Val - Ala - Met
Ile - Ala - A:a - Ala - Met
Gln - Pro - Leu - Ile' -Met
Fro - Pro - Ata - Val - Ser
Arg - A:a - Tnr - Val  -Leu
Thr - Pne- Gin - fila -Tyr
Ser - Pm - Leu - Pne - Aia

Val - Ser - Gln - Asn-Tyr
Lys - A& - fiq - Val - Leu
Thr -Ala - Tnr - Ile -Met
Arg- Pro - Gly - Asn- Pne
Glu- Arg - Gin - Ab -Asn
Val - Ser - Pne - Asn- Pne
Cys- Tnr - Leu - Asn- Pne
lie -Arg - Lvs - Ile -Leu

P1' P2' FJ"'

His - Cys - Giy
Ser - Giy - Leu
Pr:, - Val - Val
Ser - Ser - Ala
Ala - Val - Val
Leu - Ata - Me!
Tnr - Val - +%a
Pro - Leu - A:g
Giy - Ile - Sei

I

Pro - Ile  - Val
Ala - Giu  - Ala

Leu,- Gln - Se:
Pne - Leu - Giy
Pro - Gtn - lie
Pro - Ile - Ser
Pne - Leu -  AS^

Me1 - Gln - Aq

lmmatu re
Virus Particle

Mature
Virion

        IN
-p66/51 RT

        NC

`PI7 MA

~~gp120 su
000 -

gp41 TM

* Cap

Poly-A Tail

Viral Genomic
RNA

A
+ tRNA Primer
3

ode1 for virion assembly. II

8
4

RETROVIRAL GENETICS

ewnee- - GcP,$CT :

MPLEMENTATION HE!#% w-u r

3

-

HELPER GENOMES FOR GAG, POL, ENV COMPLEMENTATION FG f?. @ %,em@

GNV PSEUDOTYPES (BETWEEN ANY TWO

e RETROVIRUSES AND VSV) AFFECT HOST RANGE

WH13

via,+

Tb4PbJ

RATES SIMILAR TO OTHER RNA VIRUSES

WH13 WH14

HIGH FREQUENCY (TOO HIGH FOR GENETIC MAPPING!)

HETEROZYGOTES ARE OBLIGATE INTERMEDIATES

infect D17 cells

{infect D17 cells

Recombinant proviruses Recombinant proviruses

were not observed

INSERTION MUTATIONS (ACTIVATING OR INACTIVATING)

ENDOGENOUS PROVIRUSES

LEVELS OF COMPETANCE

* ENCODE INFECTIOUS VIRUS (MMTV, XENOTROPIC MLV, RD 114 ETC)
* ENCODE VlRAL PROTEINS ONLY (GAG OR ENV)

* NO PRODUCTS

NUMBERS AND ORIGINS

* RECENT ENTRY (POST-SPECIATIO N)... VARIATION AMONG

INDIVIDUALS, 0-10 COPIES

(ECOTROPIC MLV, MMTV, ALV)

* ANCIENT SURVIVORS .... UNIFORM IN SPECIES, MAY BE 100 TO 1000

  COPIES (XJ5NOTROPIC MLV, IAP'S, ENDOGENOUS HUMAN PVS)

.L----

OBSERVED GERM LINE INSERTIONS

- 3
r-

* SOME VIRUS-PRODUCING MOUSE LINES

* INFECTION OF PRE-IMPLANTATION EMBRYOS

* ??INFECTION OF MALE GERM CELLS

MUTAGENIC POTENTIAL , Expma,PtF5'

* IN GERM --pi----- LINE: -- HPRT, DILUTE (REVERTS BY LTR-LTR EXCISION)

COLLAGEN ALPHA 1A (MOV 13), OTHER DEVELOPMENTALS

* MOVEMENT IN SOMATIC CELLS:

IAP INTO PROTO-ONCOGENES

-