May 17,1993

SMALL DNA VIRUSES

Harold Varmus

Relevant chapters in Fields et al. VIROLOGY, 1990.

Challberg and Kelly, Animal virus DNA replication. Ann. Rev. Biochem. 58:671,1989.
Fanning. Simian virus 40 large T antigen: The puzzle, the pieces, and the emerging
picture. J. Virol. 66:1289, 1992.

Hurwitz et al. The in vitro replication of DNA containing the SV40 origin. J.Biol.Chem.
26518043, 1990.

Matthews and Shenk. Adenovirus virus-associated RNA and translational control.
J.Viro1. 655657, 1991.

Prives. The replication functions of SV40 T antigen are regulated by phosphorylation.
Cell 61:735, 1990.

A Selection of Important 0 riginal Papers

(Also see papers assigned for Discussion Groups on Papilloma virus replication.)

Challberg and Kelly. Adenovirus DNA replication in vitro. PNAS 76:655,1979.
Dana and Nathans. Bidirectional replication of simian virus 40 DNA. PNAS 69:3097,
1972.

Lambert et al. A transcriptional repressor encoded by BPV- 1 shares a common carboxyl-

terminal domain with the E2 transactivator. Cell 50: 69,1987.

Li and Kelly. Simian virus 40 DNA replication in vitro. PNAS 81:6973,1984.
Samulski et al. Targeted integration of AAV into human chromosome 19.  EMBO J.

10:3941, 1991.

Shortle et al. Mutational analysis of the SV40 replicon: Pseudorevertants of mutants with
a defective replication origin. PNAS 76:6128,1986.

Stewart et al. Image reconstruction reveals the complex molecular organization of
adenovirus. Cell 67: 145, 199 1.

Tamanoi and Stillman. Initiation of adenovirus DNA replication in vitro quires a specific
DNA sequence. PNAS 80:6446, 1983.

Tsurimoto et al. Sequential initiation of lagging and leading strand synthesis by two
different polymerase complexes at the SV40 DNA replication origin. Nature 346534,
1990.
Weinberg et al. Reconstitution of simian virus 40 DNA replication with purified proteins.
PNAS 8753692, 1990.

COMPARATIVE VIROLOGY: A PREVIEW

Parvos Polyomas PaDillomas Adenos

PARVOVTRUSES

E.C

c

PATHOGENIC AGENTS: B19 VIRUS (ANEMIA OF NEONATES)

MANY VETERINARY DISEASES

GROWTH DEPENDENT UPON HELpERVIRU(ADEN0VIRUS
-===

OR HERPESVIRUS FOR ADENOASSOCIATED VIRUSES) OR

S_PHASE1(AUTONOMOUS PARVOS, SUCH AS MVM, H1,ADV)

GENOME IS SINGLE STRANDED DNA OF CA. 5 KB

MINUS OR PLUS STRANDS (MV)
ms ONLY (MVM)

-

PALINDROMIC ENDS (100-300 NT)

CODING REGIONS AND GENE EXPRESSION

TWO LARGE NON-OVERLAPPING FRAMES

EXPRESSED VIA 1 TO 3 PROMOTERS AND ALTERN. SPLICING

-

c

-

AAV

REP PROTEIN AS TRANSACTIVATOR??

     LINKED TO DNA (IN VIRION)

     ROLE IN REPLICATION?? MVM

& a

B 19

CAP

I
2
3
4 -
5
6 -

A

I

A

2-

Transcriptional maps of three patvoviruses

. ..

REPLICATION STRATEGY

o REQUIRES S PHASE, BUT DOES NOT INDUCE IT

o USES HOST DNA POLYMERASES

THEpRIMEm LOMER PROBLEM;

PALINDROMC ENDS PROVIDE DNA PRIMER

                       P C c, f
a V EF
        VW

- )G

8T-G BC

E Fbar

CLEAVAGE OF HAIRPINS, EXTENSION REGENERATES ENDS

V EF

SECOND STRAND BY DISPLACEMENT SYNTHESIS

fA

ROLLING HAIRPIN IN SOME CASES

FLIP-FLOP OF PALINDROMES

I

halrpn
lormallon

Y

1 *bva'*

L

1 blCL

.. .

Model for AAV DNA replication.

AAV: LATENT INFECTION, INTEGRATION, VECTORS

LATENT STATE FREQUENT AFTER HELPER-FREE INFECTION

GENOME INTE

OFIEN ON CHR. 19 Q 13.4-TER WITHIN 100 BP

REP GENE REQUIRED FOR ADEN0 RESCUE, NOT FOR LATENCY

P

(NOT AN INTEGRASE)

RESCUE MJMICKED BY RECOVERY OF GENOME FROM

RECOMBINANT PLASMIDS IN TRANSFECED CELLS

AM> IN CELL EXTRACTS

AAV VECTORS: ONLY ENDS REQUIRED
1

HIGH EFFICIENCY

ADVANTAGE OF CHR. 19 TARGETING?

LIMITED CARRYING CAPACITY

HELPER CELLS AVAILABLE

I-

I POLYOkIAVIRUSES )

EXAMPLES AND PATHOLOGY

Virus Host soecles

Mouse -
Mouse

Monkey -

  Hamster papovavirus (Hap\/) Hamster
ASimlan vacuolating virus 40 (SV40)

  Lymphotroplc papovavlrus (LPV) Monkey
  Simian agent 12 (SA12) Baboon
ABKV Human

--p JCV Human =-+
  Rabbit kidney vacuolatlng virus (RKV)   Raobit
Budgerlgar fledgling disease virus Budgerigar

.(

- \ STRUCTURE OF PARTI~L~PSEUDO-EQUIVALENCE)

L

, GENOME LAYOUJ

c

- -

-- -

__c_

._- --

rOIkRVIEW OF LIFE CYCLE

-

I

2

FIG. 3. Replication cycle of polyoma and SV40. Steps in
the replication cycle are indicated by numbers as follows:
1. adsorption of virions to the cell surface: 2. entry by
endocytosis: 3, transport to the cell nucleus: 4, uncoat-
ing; 5, transcription of early region mRNAs: 6. translation
of early proteins (T antigens); 7. viral DNA replication; 8,
transcription of late region mRNAs; 9, translation of late
proteins (virion proteins): 10. assembly of progeny virus
particles.

9 DNA IS CIRCULAR AND SUPERCOILED

QS DEFINITION OF ORIGIN

-

PULSE-CHASE + RESTRICTION MAPPING
@-#-

@

EARLY AND LATE WAS FROM DIFFERENT STRANDS o

cj ENHANCERS

Id. INTEGRATION OF VIRAL DNA (ALBEIT IRREGULAR)

9 HELPER CELLS (COS CELLS: CV-1 WITH ORI MUTANT DNA)

et- .:--

IN VITRO DNA SYNTHESIS: T AG AND ORI DEPENDENCE

I_MULTIPLE ROLES OF T ANTIGE

- __-~-

-

Finger

(302.320)

NLS

(1 26-1 32)

Host Range

(682-708) ,

I.
I Polcr. D53

'I I

I lGIlba3G

PHYSIOLOGY: STIMULATE HOST DNA SYNTHESIS
-

g TRANSFORM CELLS)

4

INHIBIT SYNTHESIS OF EARLY RNA

- 1 I

INITIATE SYNTHESIS OF VIRAL DNA

- 7

ENHANCE SYNTHESIS OF LATE RNA

v

HELPER FUNCTION FOR DEFECTIVE ADEN0

BIOCHEMISTRY: BINDS TO ORIGIN (AS HEXAMER)

-

wsz -

SV40 DNA

E mRNA

01 I

REPL-INACTNE

TARGET FOR PHOSPHORYLATION (MULTIPLE

U -

KINASES) AND DEPHOSPH. (PEA .....)

b-&t+lJ

BINDS TO RB PROTEIN AND P53

Y

BINDS HOST-SPECIFIC DNA POL-ALPHA

-

NUCLEAR LOCALIZATION SIGNAL

-- a

ATP-DEPENDENT HELICASE

-

- ._

-

-

REPLICATION OF SV40 DNA IN VIT

e

--_ - -. - __ -_. __ -

LI AND KELLY: REPLICATION IN CRUDE EXTRACT

Idly OM-DEPENDENT

T AG-DEPENDENT

BI-DIRECTIONAL AND FULL LENGTH

KELLY, STILLMAN, HURWrrZ ET AL:

REOUIR ED FOR INITIATION: ORI, T A-G~INDING, ELICASE)
           7 --:

/ SS-DNA BP (RF-A, RP-A)

--a

iqlM

dCfloJ

DNA POL AL.PHA/PRIMASE -_ -. -~ -,

. vt@

(STIM. BY PP2A, ACTS ON T AG)

. ,? -s,vA

----

ORIGIN -

9

ORIGIN

RZDNA POL -_ DELTA/PCNA

-- --- 3

-

-

- -.

DNA-DEP. ATPASE (RF-C)

-

y-- -

TOPOISOMERASE I

-

--

c __-

_-

'. PAPILLOMA VIRUSES 7

MANY STRAINS IN MAN (OVER 60) AND ANIMALS

-

(FAVORED: BPV-1, HPV-16 AND -18, HPV-6 AND -11)

PATHOLOGY: BENIGN TUMORS (PAPILLOMAS) AND

-

- --

-

MALIGNANT TUMORS (CARCINOMAS)

OFSKINANDMEMBRANES

.+)r\c- --

HUMAN CERVICAL CARCI~OMA

t

GROWTH PROPERQS; VIRUS PRODUCED ONLY IN DIFF.

KERATINOCYTES WHERE L1 AND L2 EXPRESSED

AND REPLICATION OF VIRAL DNA IS "VEGETATIVE"

GENOME REPLICATES AS EPISOME IN SOME CELL LINES,

-

=Z%--&W

IN BASAL EPITHELIUM (MAINTAINED AS 50-400 COPIES).

/

TRANSCRIPTIONAL CONTROL BY PAPILLOMAVIRUSES

GENOME ORGANIZATION: EARLY AND LATE

UNIDIRECTIONAL TRANSCRIPTION

MULTIPLE PROMOTERS

LONG CONTROL REGION:

L

The bovine papillomavirus type 1 genome.

1

I

I10 1 (48Kd) ~~`~~~=r

REGULATED (+ AND -) BY E2 PROTEINS

-

2

comsrvsd N-miM1 corned C.tarmmd

domain domain

TRANSACTIVATION

DNA BINDING
DIMERIZATION

MULTIPLE COPIES OF E2 BINDING SITE (ACCN6GGT)

EARLY REGION SUFFICIENT FOR TRANSFORMATION

(MECHANISMS TO BE DISCUSSED IN FINAL WEEK)

\.DNA REPLICATION \

REQUIRES ON AND El AND E2 PRODUCTS

       HOST DNA POLYMERASE

U -

(SEE DISCUSSION GROUPS)

INFLUENCE OF OTHER GENES ON COPY NUMBER?

ON VEGETATIVE REPLICATION IN KERATINOCYTES?

REPLICATION COORDINATED WITH S PHASE

INTEGRATION RARE EVENT

."

' ADENOVIRUSES 1

1 -

L

MANY SMS - IN HUMANS AND OTHER MAMMALS, BIRDS

(BEST STUDIED: HUMAN ADEN0 2,5, 12,18)

-- h

AEh -2.4

-

PATHOLOGY: PHARYNGITIS, CONJUNclTvITIS

P

TUMORS (RODENTS INFECTED WITH HUMAN AD)

PARTICLE: COMPLEX NUCLEOCAPSID

.-

L-- -

Hexon

-

L-

-

-7e n t o n b as e>
,'Mociated protein
-+FErl

- Core protein

- Hexon associated protein

- Core protein

, Hexon associated protein

  groups of nine hexons

- - Protein specific for

ADENOVIRUS GENOME

L-_ 4

DS LINEAR DNA, CA. 40 KE3, WITH TERM. PROTEIN

pronase

C'3.i

C SA

1

Proteins

IX

13K  21K
E6K   16K
32K   55K

RNAs

I.4K
14.5K

core PTNase non-rirlon

n n

52- omton hexon 33 K

. . ..

IOK flow

IOOK pZII 16K X

13.6K

I2i 3

..
..
..

..
..
..
..

..
..
..
..

..
..
..
..
..
..

r

LI

--

IR
VA

3

-+
- --
--

[h 5
0 IO 20 30 40 50 60 70 , 80

        5
       I 3
9c 5;

r -strand 3'
'-strand 5'1 __._I .... I .... I ._._ I .... I .... I __.. I .... I .... I .... I .... I .... I .... I .... I _... 1 ....~....~....1... ,

RNAs

- -3

i

Prorclns

r

\ OVERVIEW OF ADENOVIRUS REPLICATION CYCLE I

ENTRY VIA ENDOSOMES, RECEPTOR NOT KNOWN

IMMEDIATE EARLY (ElA)
EARLY (ElB, E2, E3, E4, MLP)

DNA REPLICATION
LATE (STRUCTURAL GENES VIA MLP)

-

-

-

E1A REGULATES OTHER EARLY GENES VIA E2F

1

I88

eo 121 140

40

pkqphqGGkq~

DNA syntkesls J of mttosls virus early genes locolirotion

E2 ESSENTIAL FOR D-N: TERM PROT, DNA PgL.

'

-- -_ - - - - ssfle-L+4P!

(ElA AND ElB ARE ONCOGENIC...SEE LATER LECTS.)

LATE M-RNAs (MONOCISTRONIC) MADE BY DIFF. SPLICING

- -A

AND POLYADENYLATION FROM ONE PROMOTER-->

XYZ (e.g. HEXON)

.-"p"--AAA

PRINCIPLES OF ADENOVIRUS DNA REPLICATION

4

i

3 VIRAL DNA POLYMERASE (PRODUCT OF E2 GENE)

-

t

@ ORIGINATENDS

-e=.

@ ENDS L=-- ARE INVERTED REPEATS

- -4

--P c&

e

P_RIMER IS VIRUS-CODED PROTEIN,

j/

Rorm

9 -krs as Prtm
         Srram

/ fm

1

LINKED TO SENDS BY SER-dCMP,

ENCODED BY E2

&

PROCESSED BY VIRAL PROTEASE 5.d

3

ck5' .. +

3 5 -E

DISPLACEMENT (TYPE I> AND REPLACEMENT (II) SYNTHESIS

-

4

I

ADENOVIRUS DNA REPLICATION IN VITRO7

O

FIRST SUCCESSFUL IN VITRO SYSTEM:

ADEN0 DNA WITH TP

INFECTED CELL EXI", dNTPS, ATP

USE BUDR TO SHOW SYNTHESIS

OBSERVE INTERMEDIATES BY EM

@ INITIATIONINVlTRO

p DEFINITION OF ON FROM CUT PLASMKD

@ PURIFICATION OF FACTORS FROM INF'ECTED CELL EXTRACT:

- P140: VlRALDNAPOL

- P80: PRE-TERMINAL PROTEIN (PRIMER)

U

- NF1 AND NJ? III FROM UNINFECTED NUCLEAR EXTRACT

- -

FOR INITIATION

-

- W2 (VIRAL SS DNA BINDING PROTEIN) AND NF II (TOPO?)

L-. -

FOR ELONGATION

-

OTHER ILLUMINATING OR PROVOCATIVE FEATURES

vzi->55KD AND 19 KD PROTEINS

55KD PROMOTES EGRESS OF VTRAL RNA FROM NUCLEUS

--

AND BLOCKS EGRESS OF HOST RNA

(ALSO BWS P53 .... PER LATER LECTURES)

19KD LIMITS DNA DEGRADATION AND CYTOTOXICITY

0

iFwb~~~~~ HOST RESPONSES

(E.G. BIND EGF RECEPTOR7 INHIBIT LYSIS BY TNF,

BINDS MHC CLASS I IN ER)

-

rwt'ROMOTE ACTION OF E2F ON E2 REGION

VP2 V/m

ROMOTE LATE TRANSLATION BY I

POLE Picoh

PREVENTING ACTION OF IFN-DTDUCED DAI KINASE

rc I qo NT

.'

0

INTERFERON

DAI

\ CDP

eIF-2-CDP

eE-2 a(P) *CDP
+GEF

eIF-2-CTP TTWPED GEF

.

INITIATION INITIATION BLOU<ED

- -- -

ADENOVIRUS VECTORS

- __ __ .

- --_

--

e EFFICIENT ENTRY, REPLICATION, AND EXPRESSION (VIA MLP)

-

p e I us

McC

~__

B DELIVERY DOES NOT REQUIRE REPLICAmG CELLS,

WIDE HOST RANGE (RECEPTOR) FOR ADENOVIRUS

6 DEFECTIVE OR INACTIVATED ADENOVIRUS ALSO USED FOR

GENE DELIVERY VIA ADENORECEPTOR AND ENDOSOMES

(DNA-POLYLYSINE-ADENOVIRUS COMPLEX)

+

-

---_

--

-

r-L/-=--- -

REPRISE: WHICH OF THESE VIRUSES IS MOST USEFUL

FOR UNDERSTANDING CELLULAR DNA SYNTHESIS?

>

,'

5
P ---\ _- --\ - -- L-- ~ -

- --.

<- -_

-.-- -\, ----Le -- -A- _- --.

--

PARVOS: USE HOST POLYMERASE, BUT DNA SELF-PRIMES AND

ENDS NOT RELEVANT TO CELL TELOMERES

ADENOS: USE VIRAL POLYMERASE, PROTEIN PRIMER,
=+Es?=
     ATYPICAL ORIGIN, CONTINUOUS SYNTHESIS

POLYOMAS: T AG/ORI RELATIONSHIP MAY HAVE NO EQUAL,

AND REPLICATION IS UNCONTROLLED .....

BUT REPLICATION FORK (LEADING AND LAGGING

STRANDS), USE OF HOST ENZYMES AND FACTORS,

INTERSECTION WITH CELL CYCLE COMPONENTS

IMPLY STRONG SIMILAJUTES WITH HOST REPLIC.

PAPILLOMAS: CONSIDER THIS QUESTION IN DISCUSSION ....

-

L--

__ -- --