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 ,' 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-- __ -- --