-1- I Many of the functional attrihtes of retroviruses IXIW amar to depend upon a bmain of several hundred base mirs present at both e&s.of viral DNA, %is dsmain, referred to hereafter as the long terminal repeat or L,T,R., is created during synthesis of retroviral DNA Sy the remarkable fupn of sequences derived o from both the SI and 3' ends of viral RIA. Since the discovery of LTfis abut 3 years ago, much has been learned ahout their synthesis, structure, and multiple functicns particularly with recambinant rxvA techniques. LTRs mntain regula- tory inforrnti.cn crucial to the orrterlv progress of the virus life cycle, They can affect transcriptimal activity of viral or heterologous BJA, aparently by multiple mechanism, and these effects may he instrumental in oncoqenesis. 1 6 My plrpct;e here is to provide a brief review of these and other aspects of WRS, saying mre about what LTRs ckr than abt hod they are synthesized, will begin with a schematic view of the life cycle which gnphasizes the oziqin and role of the LTB in the replicative mechanism, (First slide). The single stranded T3NA suhunit of all replication-oompetent retroviruses contains three ding domains: however, these genes am7 their products will. not concern us further here. I direct yrwr attention instead first to a short sequence, called / R, represented as a solid box and present at both ends of viral RNA, and then to shaded and open bxes which represent those sequences - called US and U3 - mip to the 5' and 3' ends of viral RNA but repeated in DNA, by virtue of J: their imlusicm in LTRS. the smp of tcdav's dismssion but the product is a linear duplex with LTRs aompas& of US, R, and U3, the synthesfs of an LTR is that the LTR rkrmain is detenninefi by prhing sites, Thus, the first strand of UNA is primed by A host tREiA position at what will lxcom the outer boundary of US: ard the smnd strand is initiated at a site Viral WA is synthesized in a series of steF bemd rhe important feature to remember in thinking about REPLICATION OF RETROVIRUSES 1 PROPERTIES OF LTRs -FLANKED BY PRIMING SITES FOR VIRAL DNA. -TERMINATE WITH INVERTED REPEATS. -ENCODE INTEGRATION SITES 2 BP FROM EACH END. -INCLUDE SITES AND SIGNALS FOR INITIATION AND POLYADENYLATION OF RNA. btyrrtian sit. STRUCTURE OF AN LTR *,sit* -2- Linear DNA can circularize to generate molecules with one or with two LTRs as stwrWn here, or interesting aberrations of these structures. 1L, prduccl an integrated provirus, one of these unintegrated forms - which one, is not kmmn - is joimd covalently to a host chrcmoscsne. Three facts apar to be fairly firm abut retraviral integration: (1) there is little or no preference for inteqration sites in the host genome: (2) integration sites in viral DNA are invariant, situated close to the ends of LTRs; and (3) a short sequence of host TXTA - 4, 5, or 6 bp, demnding apparently upcn the virus strain - is &p?-icated at the insertion site, forminq a direct repeat which flanks the provir-us. Ihe general structure of the pmrirus is appropriate for its function as a tenplate for synthesis of mnpermeated viral RNA, which my in turn be spliced to form subgenomic mRNks. likely to figure prominently in the initiation, termination, or polyadenvlation of transcr ipts . It is also aparent from the cliaqram that LTRs are Nwleotide sequencing of cloned LTRS from several retroviruses has con- firmed some predicted features and revealed me unexpected ones. These are listed on the next slide (Fig. 2) and will be shown schematically on the one to follow (Fig. 3). primer? and on the other by a potvpurine tract that probably encodes the primer €or the second IXR strand. often imperfect and ranqe from 5 to 20 or so bp's in length. are invariably 2 kp's from each end, and the interior of the LTRs contains recoqnizable sites and sequences for initiation and m3var7envlatim of IFJA con- formirn tc tkK? km structure of viral Ms. LTRs are bounded on one side by the bonding site for host tRNA LTRs end with short, inverted repeats: these are fnteqration sites -3- next slide (Fig. 3) sbws a more anatmica1 version of these findinqs. Mations across the tcp refers to features of relevant to the 5' LTR (ire., the upstream or left hand LTR): the inteqration site; a Hogness Gold!xrg box abut 30 kp from the probable initiation site for transcription; and the tRNA bindinq site. stream) LTR: the potpurine primer site before the boundarv; a canonical siqnal for ply A arlditicn abut 20 kp before the 3' end of R, aM.3 the integration site 2 kp from tk end. tk 5' si* of the cap site - as it is in my viruses - some viruses encode this signal within R, creating a need to suppress this signal in the 5' LTR; how this is done is not km.) Insignia alonq the hattan refer to features relevant to the 3' (down- (Although I have sham the polyadenvlation signal here on At this point, I muld like to mnsider sane experimental evic'ence for the functimal attributes of LTRS listed on the next slide (Fig. 4). I will dismss some recent work supprting the following contentions: (1) LTRs can promte transcription, as d&uced from experiments in which cloned LTRs have been joined to cloned heterologous qenes - in vitro, I- then reintroduced to cultured cells; (2) LTF& have mrly defined properties which can enhance the efficiency of KNA transformaticn after microinfection of such recanbinants; (3) E!?Rs directly mediate certain regulatory events such as glucocortiooid con- trol of mouse marmtary tumor virus RNA synthesis: (4) LTRs can affect the transcriptional activity of adjacent cellular genes, regardless of the arrangement of viral and host IF&, and such effects my eon- tribute to or#x>qenesis, for example, in avian bursal lymphomas; (5) the behavior of LTRs may be modified by their chrm-1 oontext; and In particular, (6) LTRs can act as sites for hml.qous recanbination, Eaciiitatinq the exci- sim of proviral RJA from the chrmm. PROPERTIES OF LTRs -CAN ACT AS PROM~TERS FOR LINKED GENES. -ENHANCE EFFICIENCY OF DNA TRANSFORMATION AFTER MI~ROINJECT~ON~ -MEDIATE TRANSCRIPTIONAL REGULATION, e.g. BY GLUCOCORTICOID HORMONES. -AUGMENT TRANSCRIPTIONAL ACTIVITY OF ADJACENT CELLULAR GENES, e,g. IN BURSAL LYMPHOMAS. -ACTIVITY INFLUENCED BY CONTEXT OF FLANKING DNA. -MEDIATE PROVIRAL EXCISION BY HOMOLOGOUS RECOMBINATION. AN LTR CAN PROMOTE TK EXPRESSION TK TRANSFORMATION AFTER MICROINJECTION 21.. pRSV.ATK A PR'" B u.1 . 1 <0.1'; I pATK \ \ Pfl( 0.8% TK EXPRES~ION PRO~~~TED EY ASV' LTR HSV Th PRbBE 12 * i ASV LTR Us PROBE 12 F 4Sy LTR u3 PROBE .12 -4- To test the promoter activity of the LTR of Rous sarc&na virus, Paul Ludw and Mario Capaxhi microinjected the two plasmicfs illustrated on this slide (Fig. 5) into tk- mise L cells. Both plasmids mntain a herpes tk qene, devoid of its own promoter, and a cloned fracynent of retroviral DNA containing the LTR. In plasmid A, the LTR and the - tk gene are in the same transcriptional orienta- tim: in plasmid B, the tk fragment has been inverted, As tabulated at the hot- tom of the slide, a large proportion of cells receiving plasmid A were biochemi- tally transformed to a tk - phenotype, hut few or none of those receivinq plasmid B - or a plasmid with only the defective tk gene - were transformed. inglv, the frequency of transformation was significantly higher with plasmid A than with a plasmid (e containing an intact - tk gene kt no LTR. retun to this point in a moment. - + Interest- - I shall To ask whether cells transformed by plasmid A were expressing the tk gene via the LTR, polyadenylated !WA from these cells was analyzed by gel electro- phoresis and molecular hybridization, from cells transformed by p1.asmid A is present in lane 1, gene and US - but not for U3 - detect a stable RMS, species of the anticipated length from the transformed cells, a result consistent with initiation of syn- thesis within the LTR, near or at the end of U3. trol cells transformed by a nonclefective tk gene, without LTRs. (Next slide; Fig. 6.) In each panel, RNA Probes for the tk I Lane 2 contains PNA from can- -- To investiate the surprisingly high efficiency of transformation by plasmids containing LTRs, Paul am? Mario next injected cells with new plasmids (next slide; Fig. 7) containing the same fraqment of Rrxls sarCClmd virus DNA but a fully went herpes - tk gene in either orientation. tim to a - tk' phenotype was nm obtained using either plasmid; there was a greater than 20-fold increase Over the frequency observed using a plasmid con- taining only a competent tk qene and no LTR. Efficient transforma- This emriment has also been - AN LTR INDIRECTLY ENHANCES TK TRA~SFORMAliON TK TRANSFORMATION AFTER MICRO INJECTION PTK 0.8.. pRSV.TK A 23% pRSV-TK 6 19% TK EXPRESSION FROM AN MMTV LTR RESPONDS TO GLUCOCORTICOIDS -+ POSITIONS OF ALV PROVIRUSES WHKH ENHANCE EXPRESSION OF C-MYC -5- performed with plasmids bearinq an RW CNA fragment containing only L'rR sequences, with virtually identical resu1.t.s. We do not know the mechanism of this enhancement. It could operate U~R replicatim of free plasmids, upon integration, or upon transcriptiat. If the effect is upcn inteqration, it does not involve simply the donation of a gooil integratim site in the LTR, since these plasmids do not preferentially integrate at the site usd during natural viral infection, effect is upm transcriptim, it is more mysterious than the simple provision of a superior promter, since the effect is observed with the _. tk gene in both orimtatims. will refer - mt entirely for h~ous effect - tm the provision of a gocrd pro- moter as the force of an I;TR and to more elusive enhancirq effects as the charm of an WR. Similarly, if the Tb distinguish these two types of transcriptimal mechanisms I Bcpression of the muse mammary tmr virus (WfIV) genome has been km for many years to be regulated at the transcripticnal level by qlucocortimii! hormones. interactims with the LTR itself (next slide; Fiq. 8), Jdvl Majors has con- strwted plasmids with mst of an LTR, cloned from a steroidally-respcnsive MMTV provirus, linked to a herpes - tk gene without its promoter. Analysis of PNA from L cells into which this DNA was introduced [as a calcium phosphate precipitate] shclws a dramatic imrease in the amount of -_c tk EWA after addition of home. Based on its size, this RN?4 is likely (but not yet proven) fm be initiated within the MWW LTR. labaratorits mare clearly lccalize the site of initiation within the LTR. agree that some conpxnent of the LTR is likely to mediate the hormone response. To ask whether the force of MMTV LTR is hormonally modulated by Similar experiments performed with ~~ L"Rs in other All We have been discussing the effects of LTRs upn expression of heterologous genes in unnatural settinqs, after constructim of recanbinants -_- in vitro. But -6- the mast dramatic evidence for such effects has emerqed from recent studies of in3uctim of R cell. Lyqhomqs after infection of birds by avian takosis virus (or ALV). Viruses of this type, unlike agents such as REV, produce tmrs slmly, ib mt transform cultured cells, and lack a viral anrxsgene. Xktbugh virus-irducett tumors contain ACV m, the cx%A is often deranqed so that oodinq domiins are absent or unexpressed, However, viral DNA in each tumor is gene erally found in a c- reqim of the host genome - a region identified by Hay- ward, Astrin anif their colleagues as t3-mvc, the cellular homologue and predeces- sr to the oncdqent3 of the transforming rt?trCrVirus, K29,* Mxeover, the level of e-% IWA is markedly elevated in such tumors, what is responsible for the enhanced expression, nforee" or "&arm" or sawthing else? 9.) Tn all of the tmrs reported by Rayward et al - and in over half of those stuctied in our lab by Greg Pap - an AI;V Lm is position& on the 5' side (or - (Next slide; Fiq, _- upstream) c-~ - sbown here as an interrupted cellular gene with two introns - in the same transcriptimal orientation. fn this arranqement, as sham at the b3p of the szide, the LTR can be predicted to act as an efficient promoter for e-=. Antf, in fact, Hayward's group - and later, ours - found U5 (but not U3) sequences linked to c-z sequences, in stable transcripts that are probab?y the spliced pralucts of the putative primary transcripts diagrd here. Hakffver , in other tumors examined by Greg, "charm" seems to play a me significant role than "force". a truncated provirus, mntaining little mre than an LTR, is positioned on the In one tumorv as diagramed in the middle panel, 3' side of e-=, in the same transcripticnal orientati.cn, transcripts in this tumor contain U3, but not U5 ~equenms; we do not km whether the normal em - pronnter is empIoyecl to overproduce c-~ case, the 5' side of c-rn - htl in the trp~czsite transcriptjmal orientation, "he abundant c-mw= - in this In several other tumors (hvttan panel) Greq has found an ALV provirus on Again, -7- expressim of emyc is enhanced, but c-mvc - transcripts contain neither U3 nor US. c-myc into flanking cellular DXA. In at least one of those cases, an LTR promotes transcriptim away from --- How general are such phenanenon? Llo they have equivalents in human canc- ers? Or in tumors induced by other viruses lackinq oncogenes? Roe1 Nusse has recently used the strategy outlined in the next slide (Fig. 10) to assess the relevance of the AtV finclings to carcimqenesis by ~JWIV. First he used viral probes to analyze Ecx, - RI restriction digests of tumor ClNA, to identify the rare tLrmor (T1with only a single provirus in addition to the several endogenous pro- viruses in normal mouse DNA (N). was then cloned, a probe containing only single copy cellular sequences was prepared, and many tumors were analyzed for novel fragments - such as these - irdicative of imertims or rearrangements of this such as that in T2 - indica- tive of insertions or rearrangements of this unidentified regim of the host genome. to define a transcriptionally active dmain in this reqicn or to identify unusual transcripts containing LTR sequences. "he right hand host-viral junction fragment Thus far, at least 20% of mmry tumors are affected, but we have yet I wuld like to make two final brief points abut LTRs, derived from yet another experimental mtext exemplifying insertion mutagenesis by retroviruses. (Next slide; Fig. 11.) In these experiments, a rat cell line (called R31) - transformed by a single RSV provirus - was mrphologically reverted at low fre- quency after superinfectim by murine leukemia virus, a non transforming retro- virus, when the latter's provirus was inserted between the prcmoter and the d- ing domain or between the splice sites (11) for the Rsv transforming gene, src. - In the two studied examples, both the RSV and MLV proviruses are in the same transcriptional orientatim and the 5' RW LTR remains transcriptionally active. Yet the first (5') ML,V LTR do~s act to terminate or polyzdenylate the RW- SEARCHING FOR INTEGRATION SITES INVOLVED IN CARCINOGENESIS BY MMTV Cloned RI Frymen9 (19hb) N Ti 12 L FLANKING PR~BE INSERTION MUTATION AND EXCISION SUMMARY 1. LTRs CONTAIN SEQUENCES WHICH DETERMINE IMPORTANT STEPS IN THE VIRUS LIFE CYCLE. 2. LTRs MAY INFLUENCE TRANSCRIPTION BY MULTIPLE MECHANISMS ("FORCE" VS "CHARM") 3. LTRs MAY PROMOTE ONCOGENESIS BY ACTIVATING HOST GENES. 4. LTRs MAY SERVE AS SITES FOR HOMOL0,GOUS RECOMBINATION. -8- initiated transcripts, the 3' MLV LTR does not promote emression of - src, and no stable transcripts of - src are found. retransformtion occurs sptaneouslv at law frequency, with reappearance of normal - src mRNA and protein. MLV LTR remains, suggestirq that homologous reconbination between LTRS; has oocured. "intron", wirbut apparent influence over transcription or processinq. results irdicate two things (1.) that the functions of L?'Rs may be influenced by tbir positim (or context) in host chromosomes, and (2) that LTRs may olay a phenotypically-important role as sites for hcmohqous crossinq over. In the second of these two mutants, Most of the MLV provirus is now gone, but a single The residual MLV LT? behaves like a neutral sequence in the src - These In sumnary then (last slide; Fig. 12) I wish to leave you with four points about LTRS: (1) they contain sequences with important roles in lye cvcle (2) they influence transcription by multiple mechanisms (3) they may promote neoplasia by activating host genes (4) they may elininate proviruses by providing recanbinational sites.