Tumorigenic poxviruses: Transcriptional mapping of the terminal inverted repeats of shope fibroma virus

VIROLOGY

158, 381-393

(1987)

Tumorigenic

Poxviruses: Transcriptional Mapping of the Terminal Inverted Repeats of Shope Fibroma Virus C. MACAULAY,

Department

of Biochemistry,

C. UPTON, AND G. MCFADDEN’

University

Received December

of Alberta, Edmonton, Alberta, Canada T6G 2H7

1, 1986; accepted

February

11I 1987

A composite transcriptional map for the entire 12.4-kb terminal inverted repeat (TIR) region of the Shope fibroma virus (SFV) genome has been determined. Northern blotting and Sl-nuclease mapping were used to determine the regions which are transcribed, their temporal relationships, as well as the transcriptional initiation sites. Sequences representing the entire TIR are transcribed into poly(A)+ mRNA at both early and late times in the infection. Fifteen transcriptional initiation sites were mapped, 12 within the TlRs and 3 within the unique sequences close to the junction between the right TIR and the unique internal sequences. Ten of the 12 transcriptional initiation sites within the TIR and 2 of the 3 sites outside the right TIR correspond to the 5’-ends of the major open reading frames (ORFs) Tl to T9 plus the SFV growth factor gene. The 3 other initiation sites map within ORFs but near potential start codons for shorter polypeptides. All the expressed ORFs are tandemly arranged and transcribed toward the hairpin terminus. At early times during SFV infection of cultured rabbit cells, transcription of each ORF gives rise to a transcript of distinct size, while at late times termination of transcription is imprecise and substantial read-through into downstream sequences occurs. These results are discussed in light of recent observations on the related recombinant leporipoxvirus, malignant rabbit fibroma virus, which suggest that one or more gene products from this region of the SFV genome are implicated 0 1997 Academic Press. Inc. in viral tumorigenicity.

birac et a/., 1985). Early studies reported that SFV can vary in its tumorigenicity and isolates can spontaneously lose their tumorigenic potential without loss of their infectivity (Febvre, 1962). This implies that the expression of the genetic information governing tumorigenicity is variable and is reminiscent of the observations with members of the orthopoxvirus genus that spontaneous DNA rearrangements in the viral genome, especially near the TIRs, occur with high frequency (McFadden and Dales, 1979; Dumbell and Archard, 1980; Moyer et a/., 1980; Esposito et al., 1981; Wittek and Moss, 1982; Archard et a/., 1984). SFV sequences important for tumorigenicity have been shown to have recombined into the genome of a related leporipoxvirus, myxoma virus (MYX), to create a novel tumorigenic poxvirus, designated malignant rabbit fibroma virus (MRV) (Strayer et al., 1983a, b; Block et al., 1985; Upton and McFadden, 1986b). Early during infection MRV has the capacity to induce the proliferation of fibroblasts in rabbit skin in a fashion similar to SFV, but at late times, unlike the benign syndrome associated with SFV, MRV causes a lethal and invasive disease reminiscent of myxomatosis (Strayer and Sell, 1983; Strayer et al., 1983a, b, c). Mapping and sequencing studies have indicated that the replacement of MYX sequences with a subset of SFV sequences which reside in an 8-kb stretch near the SFV termini is responsible for the tumorigenic phenotype of the recombinant MRV (Upton and McFadden, 1986b). The

INTRODUCTION Poxviruses are a family of eukaryotic DNA viruses noted for their large size and complex morphology. Members of the poxvirus family are characterized by having a large (160-200 kb) nonsegmented dsDNA genome with terminal inverted repeats (TIRs) and covalently crosslinked hairpin termini. The genome of vaccinia virus, the prototype of this family, has a coding capacity for more than 200 polypeptides, including all the enzymes required for the expression and replication of the viral genome, and more than 100 polypeptides are found within the virion itself (for reviews see Dales and Pogo, 1981; Holowczak, 1982; McFadden and Dales, 1982; Wittek, 1982; Moss, 1985). Poxviruses replicate exclusively within the cytoplasm of the infected host cell and this relative autonomy from the cell nucleus provides an attractive model system for studying the regulation of eukaryotic gene expression and DNA replication. Shope fibroma virus (SFV), a leporipoxvirus, is a prototype for members of the poxvirus family which induce substantial nonmalignant proliferation of target cells, usually of dermal origin, in their respective natural hosts (Shope, 1932; Febvre, 1962). The genome of SFV has been cloned into plasmid vectors and its physical map deduced (Wills et a/., 1983; DeLange et a/., 1984; Ca’ To whom requests for reprints should be addressed. 381

0042-6822187 Copyright All rights

$3.00

0 1997 by Academic Press, Inc. of reproduction in any form reserved.

382

MACAULAY,

UPTON,

major open reading frames (ORFs) within the SFV TIR, designated Tl through T9, have been deduced by DNA sequencing (Upton and McFadden, 1986a, b; Upton et al., submitted). In addition, it has recently been shown that sequences within the TlRs of SFV are homologous to an endogenous circular DNA species from uninfected host rabbit cells (Upton and McFadden, 1986a). For these reasons it is important to understand the expression of SFV genes encoded within the viral TIRs. This paper reports on the analysis of the regions of the SFV TIR that are transcribed during the course of an infection, as determined by Northern blotting and Sl -nuclease analysis, and correlates the transcriptional data with the locations of the major ORFs that have been determined by DNA sequencing. MATERIALS AND METHODS Cells and viruses SFV (strain Kasza) was obtained from the American Type Culture Collection. The monkey cell line BGMK was obtained from S. Dales (Department of Microbiology and Immunology, University of Western Ontario) and was grown in Dulbecco’s modified Eagle’s medium with 5% fetal calf serum. Growth and titration of the virus was described previously (Wills et al., 1983). RNA purification Semiconfluent BGMK cells were adsorbed with SFV for 3 hr at a multiplicity of infection of 1, fresh medium was added, and the infection was allowed to proceed. The infected cells were removed with 1X SSC (0.15 M sodium chloride and 0.015 Msodium citrate), pelleted, and then dissolved in 6 M guanidine thiocyanate, 5 mM sodium citrate, 0.5% sodium N-lauroylsarcosine and 0.1 M 2-mercaptoethanol. The mixture was homogenized in a Servall tissue homogenizer for 60 set and total cellular RNA was isolated by sedimentation through CsCl (Chirgwin eta/., 1979; Glisin eta/., 1974). The RNA pellet was dissolved in 0.1% sodium dodecyl sulfate (SDS),extracted with phenol:chloroform:isoamyl alcohol (49:49:2), and precipitated with ethanol. The pelleted RNA was finally resuspended in diethyl pyrocarbonate-treated water or 0.1% SDS. Late RNA, early RNA, and immediate-early RNA was isolated from cells infected in the absence of inhibitors, in the presence of arabinosyl cytosine (40 pg/ml), or in the presence of cycloheximide (100 pg/ml), respectively. Poly(A)+ RNA was prepared by chromatography on oligo(dT) cellulose (Aviv and Leader, 1972).

AND

MC FADDEN

Northern hybridizations Immediate-early, early, late, and uninfected cellular poly(A)+ RNA were denatured with dimethyl sulfoxide, reacted with glyoxal, and electrophoresed through a 1.O% agarose gel (McMaster and Carmichael, 1977). RNA was then blotted to a Pall Biodyne A nylon membrane in 20X SSC, dried, and baked in a 80” oven, according to the manufacturer’s recommendations. To serve as molecular weight markers, ribosomal RNA from uninfected BGMK cells was run on the outside lanes of the gel, sliced off, and stained with ethidium bromide. Cloning of the entire SFV TIR into M 13 vectors has been presented elsewhere (Upton and McFadden, 1986a, b; Upton et al., submitted) and strand-specific probes for both orientations were prepared as described by Hu and Messing (1982). The blots were prehybridized and hybridized in 50% formamide, 5x SE (20X SE is 3.6 M NaCl and 20 mM EDTA), 5x Denhardt’s solution, 25 mM sodium phosphate, pH 6.8, 250 pg/ml single-stranded calf thymus DNA, and 0.3% SDS, at 42”. Blots were washed in 0.1 X SSC and 0.1% SDS at 50” and then exposed to Kodak XAR-5 film with an intensifying screen at -70”. Sl-nuclease

mapping of transcriptional

start sites

The Berk and Sharp (1977) Sl mapping technique as modified by Weaver and Weissman (1979) was carried out as follows. Cloned SFV DNA (Wills et al., 1983) was digested with an appropriate restriction enzyme and the 5’-ends were dephosphorylated with calf intestinal phosphatase (Boehringer Mannheim) and then labeled with [T-~~P]ATP(DuPont NEN products), using T4 polynucleotide kinase (Pharmacia). The end-labeled DNA was digested with a second restriction enzyme and the desired end-labeled fragment was purified by electrophoresis in low-melting-point agarose (Langridge et al,, 1980). An equal amount of labeled DNA was precipitated with 50 pg of tRNA, immediate-early, or late total cellular RNA. The pellet was dissolved in 30 ~1of 80% formamide, 0.4 M sodium chloride, 0.04 M Pipes, pH 6.4, and 1 mM EDTA. DNA was denatured at 85” for 10 min and then hybridization was allowed to occur at 5 lo overnight (12-l 6 hr). The reaction was stopped by adding 300 ~1 of ice-cold Sl buffer (0.25 M NaCI, 30 mM KOAc, pH 4.5, 1 mM ZnSO,, and 5% glycerol) with 300-400 units of Sl -nuclease (Miles) and then incubated at 37’ for 1 hr. The solution was extracted with phenol:chloroform (1 :l) and the Sl -resistant material was precipitated with ethanol and then electrophoresed on a nondenaturing 1% agarose or 5% polyacrylamide gel. The gels were dried and exposed to Kodak XAR-5 film with an intensifying screen at -70”.

I

MAP OF SHOPE

FIBROMA

VIRUS TERMINAL

Northern blot analysis In order to determine the approximate number and size of transcripts from the SFV TIR, Northern blots of poly(A)+ RNA isolated from SFV-infected cells were used. The blots were hybridized with cloned strandspecific probes spanning the left TIR (Fig. l), and the profiles with each probe are shown in Fig. 2. From the data it is clear that, unlike the relatively simple profiles observed in the TlRs of vaccinia virus, the transcriptional pattern within the SFV TIR is quite complex (Wittek et a/., 1980; Cooper et al., 1981 a; Wittek et al., 1981; Venkatesan et a/., 1982). The entire TIR of SFV is transcribed, and numerous distinct transcripts can be seen with immediate-early or early RNA (lanes A, and B, respectively, in Fig. 2). Because of the complexity of those profiles, the detailed description of these blots and the assignment of the various transcripts to particular ORFs, will be addressed later. However, two important points can be noted. First, none of the probes hybridized to RNA from uninfected cells (not shown). Second, the probes used were strand specific and, with the exception of EA’(which maps at the TIR/unique sequence junction at the left terminus), only probes complementary to RNA transcribed toward the hairpin terminus hybridized to distinct transcripts. Probes complementary to RNA directed away from the hairpin terminus uniformly failed to hybridize to any distinct poly(A)+ species (not shown). Probe EA’appears

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383

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to hybridize to a single defined transcript directed away from the hairpin terminus, but whether this transcript initiates within or outside of the TIR is not yet known. Poxviral RNA synthesis has been divided into at least three temporal groups. Immediate-early transcription takes place prior to viral core uncoating and is possible because all the enzymes required for the synthesis of functional mRNA are packaged within the viral cores (for review see Moss, 1985). Immediate-early RNA can be isolated from poxvirus-infected cells by using inhibitors of protein synthesis such as cycloheximide. Early or delayed-early RNA is synthesized after the start of viral protein synthesis but prior to viral DNA replication and can be isolated by using inhibitors of DNA replication such as ara-C or hydroxyurea. Late RNA is synthesized after the start of viral DNA replication. By exposing the Northern blots for different lengths of time it appears that, in general, RNA isolated from cells infected in the presence of ara-C (lanes B, Fig. 2) is similar to RNA isolated from cells infected in the presence of cycloheximide (lanes A, Fig. 2) although the former class often appears in reduced amounts. Others have reported similar findings for immediate-early and early classes of RNA in both SFV and vaccinia (Cooper et al., 1981 b; Cabirac et al., 1986). Hybridization of the probes with poly(A)+ RNA isolated at late times in the infection (lanes C, Fig. 2) usually displays a heterogeneous smear of high-molecular-weight transcripts. This is probably caused by 3’ heterogeneity due to imprecise termination of late transcripts, which was first described

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FIG. 1. A physical map of the left (L) and right (R) terminal regions of the SFV genome, oriented with respect to their common BarnHI clones, and subclones of the BamHl fragments IT, E, and C. The approximate locations of the major ORFs, Tl through to the SFGF (see text), are shown as thick lines with the short vertical lines indicating the 5’-end of each ORF. The sequences donated by recombination into left (MRVL) or right (MRV-R) TlRs of the MRV genome are indicated by thin lines. The square brackets demonstrate the limits with which three of the four junctions between the SFV and MRV sequences have been mapped, while the fourth (vertical line) has been determined by sequencing to be within the &/II site, 13.5 kb from the right-hand end. B = BarnHI; Bg = &/II; BI = @/I; SII = Sstll; Sm = Smal; Pv = Pvull. Not all the sites for these restriction enzymes are indicated.

384

MACAULAY, UPTON, AND MC FADDEN I

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FIG. 2. Northern blotting analysis of poly(A)+ RNA from SFV-infected cells, hybridized with 3ZP-labeled strand-specific viral DNA probes. Immediate-early (lane A, 16 rg), early (lane B, 6 pg). and late (lane C, 2 pg) poly(A)+ RNA were denatured, reacted with glyoxal, electrophoresed through a 1% agarose gel, blotted, and hybridized with M 13 strand-specific DNA probes from the left TIR (see Fig. 1). Probe EAris complementary to RNA transcribed away from the hairpin terminus, while all the other probes are complementary to RNA transcribed toward the hairpin terminus. The origin and the rRNA markers from uninfected BGMK cells are indicated by arrows in the left margin.

in the case of vaccinia late mRNA (Cooper eta/., 1981 b; Mahr and Roberts, 198413; Weir and Moss, 1984; Weinrich and Hruby, 1986). Sl -nuclease

analysis

In order to more clearly define the regions of the SFV TIR that are transcribed during the course of an infection, the locations of the initiation sites for RNA transcribed toward the hairpin terminus were mapped by Sl -nuclease analysis. Figure 3i shows the probes used in map RNA initiation sites within the terminal 4.7 kb of the TIR and schematically illustrates the results of the Sl -protection experiments seen in Fig. 3ii. Probe A is 2.9 kb long, spans most of the terminal BarnHI clone pKBIT, and is asymmetrically 5’-end labeled at the Xhol site. A representative fluorogram is shown in Fig. 3ii (panel A). With immediate-early RNA(E), a protected fragment of approximately 0.53 kb is detected, but with late RNA (L) this protected fragment is more intense and additional protected fragments of 1.47 and 2.07 kb can be observed. From this data it is not possible to distinguish

whether the RNA which gives rise to the 1.47- and 2.07-kb protected fragments is transcribed only at late times in the infection or whether it is transcribed at both early and late times; but at early times the transcripts terminate prior to the 5’-end label at the Xhol site. To answer this and to more precisely locate the RNA start sites, probes B and C were constructed. Probe B is 1474 bp with the 5’-labeled end at the Xbal site (Fig. 3i). A 175-bp Sl-resistant fragment is detected with both immediate-early and late RNA but there is a high-molecular-weight smear where the larger protected fragment, indicated by the data from probe A, would be expected. This smear could be caused either by the excess probe needed to detect the 175bp fragment or by the relatively low hybridization temperature, which in this experiment was lowered from its usual 5 lo to 49” in order to detect the AT-rich 175bp fragment. If the hybridization with probe B is carried out at 51°, then the high-molecular-weight protected bands are more distinct (not shown). Probe C is 1 .O kb with the 5’-labeled end at the Sell site 2.6 kb from the viral terminus. A major 235-bp Slresistant fragment is detected with both immediate-

MAP OF SHOPE

FIBROMA

VIRUS TERMINAL

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analysis of the terminal 4.7 kb of the SFV TIR. (i) Schematically illustrates the 5’end-labeled DNA probes A through D FIG. 3. Sl-nuclease (thick lines) spanning the terminal BarnHI clones IT and 0. The labeled 5’-ends are indicated by filled circles. The thin lines represent the portion of the 5’end-labeled probes protected from Sl digestion by RNA (see (ii)), and the small vertical lines indicate the 5’-ends of the RNA. Barn = BarnHI; Bg = Bglll; Sa = SalI; SII = Sstll; Xb = Xbal; Xo = Xhol. (ii) Shows a fluorograph of Sl-nuclease-resistant 5’-32P-end-labeled DNA: RNA hybrids, separated on 1% agarose (probe A) or 5% acrylamide (probes B-D) gels. With probe C, the higher molecular weight protected fragments in lanes E and L map within ORFs 3C/D but are not shown in (i). Molecular weight markers (M) and 5’-end-labeled, HindIll-digested X DNA (panel A), or 5’end-labeled Hinfl-digested pBR322 DNA (panels B-D). The sizes in panel A are in kilobase pairs and those in panels BD are in base pairs. Lane P is the unhybridized probe run as a size marker; lanes E, L, and T are the probes hybridized with 50 pg of early RNA, late RNA, or control tRNA, respectively.

386

MACAULAY, UPTON, AND MC FADDEN

early and late RNA and in addition there are two highmolecular-weight protected fragments. These results indicate that the three Si -resistant fragments of 0.53, 1.47, and 2.07 kb detected by probe A, which correlate well with the beginnings of open reading frames Tl, T2, and T3A/B, respectively (see Fig. 6) are all expressed at both early and late times. We interpret these results to mean that at late times termination for the T2 and T3 transcriptional units is imprecise and in each case extends past the 5’-labeled site of probe A. The significance of the less intense high-molecular-weight protected fragments seen with probe C is not clear, but may represent minor initiations near the two small overlapping ORFs T3C and T3D (see the dashed arrow in Fig. 6). Probe D is 847 bp, with the 5’-labeled end at the Bglll site 3.9 kb from the viral terminus and spans most of the 1.2-kb BarnHI clone pKB0. As can be seen in panel D two protected fragments are detected using this probe. One of the Sl -resistant fragments is 3 15 bp and is protected with both immediate-early and late RNA. As shown in Fig. 6 this corresponds to an initiation site just upstream of ORF T4. A second fragment of 515 bp is detected only with immediate-early RNA and is not detected with late RNA. It is not clear whether the larger protected fragment indicates an early specific promoter for T4 or whether it is specific for a small (80aa) ORF located just upstream from T4. At this level of analysis this start site does not map precisely to the 5’-end of the small ORF, and it should be noted that there is at least one example of a gene in vaccinia virus which has, in tandem, both an early and late promoter (Cochran er al., 1985). Figure 4i illustrates the map locations of the probes which span the next 8.4 kb of the TIR and schematically represents the Sl results that are seen in Fig. 4ii. Probe A is 8.4 kb with the 5’-end labeled at the BamHl site and was chosen to survey transcription near the TIR boundary. Panel I shows a fluorogram of the Sl -resistant fragments derived from probe A. With immediateearly RNA a single Sl-resistant fragment of 1.2 kb is detected, while with late RNA at least five major protected fragments of 0.24, 1.2, 2.8, 3.7, and 5.3 kb are detected. In this hybridization an excess of probe was used and the gel was exposed long enough to detect the 1.2-kb fragment protected with immediate-early RNA. As a result, some of the minor bands seen in the hybridization with late RNA may not be actual transcriptional initiation sites, so only those initiation sites which have been confirmed with other shorter probes are indicated here and in Fig. 6. For example, in panel II a lesser amount of probe A was used and the Slresistant material was run out on a 5% acrylamide gel. Protected fragments of 240 and 1240 bp are seen,

whereas the intermediate minor fragment of about 700 bp observed in panel I is not. The Sl-resistant fragments of 1.2, 2.8, 3.7, and 5.3 kb detected with probe A in panel I correspond well with the presumptive initiating AUG codons of ORFs T5-T8, respectively (Fig. 6). The 240-bp Sl-resistant fragment generated with probe A is unusual in that it maps within a major ORF (T5). There are no other significant ORFs in this region except for T5, but two methionine codons in the same reading frame as T5 can be found within 30 nucleotides of this mapped initiation site. This gives rise to the possibility that the C-terminal portion of T5 is expressed as a separate polypeptide and so this initiation site is tenatively labeled as T5C. In vaccinia virus there are examples of overlapping transcripts where the initiation site for one gene is within the upstream transcript, indicating that poxvirus promoter regions can themselves be transcribed (Mahr and Roberts, 1984a; Golini and Kates, 1984; Earl et al., 1986). In order to confirm the other start sites seen with probe A, and to more precisely map their positions, probes B through E were constructed. As shown in Fig. 4i probe B is 660 bp long with the 5’-labeled end at the SalI site, 5.8 kb from the viral terminus. The 210-bp fragment protected with late RNA establishes a transcriptional start site at the beginning of ORF T5 (see Fig. 6). Probe C is 1.15 kb long, is 5’end labeled at the C/al site, and the position of the 1OObp Sl-resistant fragment observed with late RNA corresponds to the beginning of ORF T6. There are also two high-molecular-weight protected fragments detected with late RNA, both of which could potentially encode ORF T7. It is not yet clear why there are two resistant fragments in this region since with probe 0, which is 1.4 kb and 5’-end labeled at theXbal site, only a single major Sl-resistant fragment of 280 bp is observed at this site. With probe E, 427 bp and 5’-end labeled at the Ddel site, a 200-bp fragment is protected with late RNA and corresponds to the start of ORF T8 (see Fig. 6). With longer exposures all the Sl -resistant fragments that are observed in Fig. 4ii with probes BE can be detected with early RNA as well, albeit in substantially lesser amounts (not shown). This is also consistent with the data from the Northern blots (Fig. 2) where the distinct transcript sizes observed with immediate-early RNA can be tentatively assigned to each major open reading frame (see below). This indicates that, as in the case of the terminal region shown in Fig. 3, all the 5’-start sites mapped between 5 and 11 kb from the viral terminus are used at both early and late times, while at late times there is substantial readthrough into downstream sequences. Figure 5i illustrates the probes used to span the remainder of the TIR, the adjacent unique internal se-

MAP OF SHOPE

FIEROMA

VIRUS TERMINAL

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FIG. 4. Sl-nuclease analysis of the TIR of SFV between the BamHl site at 4.7 kb and the Smal site at 10.2 kb from the viral terminus. (i) Schematically illustrates the 5’-end-labeled DNA probes A through E (thick lines) with the 5’-ends indicated by filled circles. The thin lines illustrate the portion of the Sl -resistant fragments (see (ii)) and the small vertical lines indicate the Y-ends of the RNA. (ii) Shows representative fluorographs of Sl -nuclease-resistant 5’32P-end-labeled DNA:RNA hybrids, separated on 1% agarose (probe A, panel I) or 5% acrylamide (probes A-E, panel II) gels. Sizes of the protected fragments in panel I (probe A) are in kilobase pairs, while those in panel II (probes A-E) are in base pairs. In panel II (probes A-E) the signals for early RNA (lane E) are not observed at these exposures. Symbols for restriction enzymes and gel lanes are given in Fig. 3, plus the following: Cl = C/al; Dd = D&l; EC = EcoRI; Ps = fsrl; Sm = Smal.

388

MACAULAY,

UPTON,

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FIG. 5. Sl-nuclease analysis of the junction region between the TIR of SFV and the right-hand unique internal sequences. (i) Schematically illustrates the 5’end-labeled DNA probes A through D (thick lines) with the labeled 5’-ends indicated by filled circles. The thin lines illustrate the portion of the 5’-end-labeled probes protected by RNA (see (ii)), and the small vertical lines indicate the 5’-ends of the RNA. (ii) Shows representative fluorographs of Sl -nuclease-resistant 5’-“P-end-labeled DNA:RNA hybrids, separated on 1% agarose (panel A) or 5% actylamide (panels B-D) gels. Sizes of the protected fragments in panel A are in kilobase pairs; in panels B-D, base pairs. Symbols for restriction enzymes and gel lanes are given in Figs. 3 and 4, plus the following: BI = L?g/l.

quences from the right-hand end of the genome, and the schematic results of the Sl-nuclease protection experiments are shown in Fig. 5ii. The 8.8-kb probe A (Fig. 5ii, panel A), 5’-end labeled at the Bg/ll site 9.5 kb from the viral terminus, is from the left end of the genome. A major Sl -resistant fragment of 0.49 kb is observed at late times, and corresponds to the start of ORF T8 (see also Fig. 4ii, panel E). A second minor protected fragment of 1.36 kb is also found with late

RNA. Identical results are obtained if the probe, 5’-end labeled at the same Bgfll site, is from either the left or the right end of the viral genome (not shown). Probe B (2.04 kb) was constructed to confirm the position of the 1.36 kb Sl-resistant fragment seen with probe A, and is 5’-end labeled at the C/al site 10.2 kb from the hairpin terminus. Using probe B, a major Sl-resistant fragment of 525 bp is seen with both early and late RNA, and a fragment of 400 bp is protected only with

MAP OF SHOPE FIBROMA

VIRUS TERMINAL

early RNA (Fig. 5ii, panel B). Although the start sites determined by probes A and B are slightly different, this is most likely due to mobility differences in the two gel systems. These start sites map in an area of the SFV TIR containing four small ORFs (Tn A-D, see below). Probes C and D were constructed to test for transcripts which map to the open reading frames T9-R at the TIRIunique sequence junction at the right terminus (Upton and McFadden, 1986b) and the SFV growth factor gene (SFGF) (Chang et al., 1987) in the unique right-end sequences. Probe C is 1224 bp, 5’-end labeled at the Ddel site near the end of the TIR and traverses the junction between the TIR and the right-hand unique internal sequences. A minor Sl -resistant fragment of 520 bp is detected with late RNA and thus places the start site about 85 bp upstream from the first AUG of T9-R. This 520-bp fragment was rather difficult to detect and appeared to be underrepresented when compared to the other start sites mapped in the TIR. With an equivalent probe end labeled at the same Ddel site, but instead spanning the junction between the TIR and the left-hand unique internal sequences, no protected fragments were observed (not shown). This indicates that T9-R, but not T9-L, is transcribed, although the resulting transcript is present at steadystate levels lower than those for the other ORFs in the TIR. Probe D is 568 bp and 5’-end labeled at the C/al site 12.8 kb from the right SFV hairpin terminus. A Sl-resistant fragment of 5 10 bp is detected with both early and late RNA, while a fragment of 240 bp is detected only with late RNA. These start sites are upstream to the SFGF ORF, which is homologous to epidermal growth factor (EGF), transforming growth factor-a (TGF(Y), and vaccinia virus growth factor (VGF) (Chang et al., 1987). SFGF is the first ORF which lies entirely in the unique sequences outside the right TIR (Fig. 6).

Composite SFV TIR transcriptional

map

The genomic organization of the TlRs of SFV, including the major ORFs and the transcriptional initiation sites, is illustrated in Fig. 6. There is a good correlation between the beginning of each major ORF deduced by DNA sequencing of the TIR and a transcriptional initiation site determined by Sl analysis. In fact, at this level of analysis, the transcriptional initiation sites for ORFs Tl, T2, T3A/B, T4, T5C, T5, T6, T7, and T8 are all within 30 nucleotides of the presumptive initiating codon. Excluding the SFV growth factor gene, all the mapped transcriptional start sites are within 100 nucleotides of a candidate AUG of an ORF. It has been shown forvaccinia virus that the transcriptional initiation sites of early genes (Venkatesan et a/., 1981, 1982;

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Weir and Moss, 1983; Earl et al., 1986) and of late genes (Weir and Moss, 1984; Bertholet et al., 1985; Rose1 and Moss, 1985; Hit-t et al., 1986; Weinrich and Hruby, 1986) are within 100 nucleotides of the presumptive initiating AUG and in many cases are within 15 nucleotides. The 5’-untranslated sequences in poxvirus transcripts seem to be rather short and the promoters themselves are not as extensive in length as those for most eukaryotic genes. For both early and late genes the promoter sequences contained within approximately 30 nucleotides upstream of the RNA start site can temporally regulate the initiation of transcription at the correct site both in vitro and within the genome of a recombinant virus, although not always with maximum efficiency (Cochran et al., 1985; Bertholet et a/., 1986; Hanggi et al., 1986). The SFGF is somewhat unusual in that the major start site detected at both early and late times is 350 bp upstream from the first AUG, while the closest start site, detected only at late times, is 75 bp upstream from the AUG. It is possible that the level of detection is not sufficiently sensitive to detect the closest start site at early times, but this issue remains to be resolved. In vaccinia the RNA start site for VGF (an early gene) is 54 bp upstream from the first AUG (Venkatesan et a/., 1982). The initiation site corresponding to T9-R is 85 nucleotides upstream from the first AUG of the ORF in the unique right-end sequences and oriented toward the TIR. It is shown as a dotted line in Fig. 6 because it was present only as a minor species and it is much less abundant than ORFs T6 and T8, which are both closely related to T9-R in terms of sequence (Upton and McFadden, 198613).We have presented arguments elsewhere that during the evolution of the viral genome the left SFV TIR was copied directly from the right TIR with the latter serving the role as the master template (Upton and McFadden, 1986b). As a result, the N-terminal portion of T9-R, including its promoter, was decapitated by the transposition event, leaving the left copy (T9-L) transcriptionally silent (Fig. 6). There is considerable homology at the amino acid level between the putative gene products of T6, T8, and T9-R, suggestive of an ancestral gene triplication event (Upton and McFadden, 1986b). If T9-R, T6, and T8 are in fact functionally equivalent, then the T9-R transcript may not be required in substantial amounts since ORFs T8 and T6 are both transcribed at high levels. It is interesting to note that in the Boerlage strain of SFV there is a deletion in part of the T6 ORF (Upton and McFadden, 1986b). Thus at least the T6 gene product is not necessary for viability, although it may effect the pathology of the lesions in rabbits (Strayer et a/., 1984). All the major ORFs described here are transcribed constitutively, that is, at both early and late times in the infection. It was much easier to detect transcrip-

390

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UPTON,

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FIG. 6. The composite transcriptional map of the SFV TIR and flanking regions. The restriction enzyme symbols are given in Figs. 3 and 4. The major ORFs Tl through to the SFGF were determined by DNA sequencing of the entire TIR plus the flanking unique sequences (see text). T9-R spans the junction between the TIR and the right-hand unique internal sequences, while T9-L is a subset of this ORF found within the left TIR (Upton and McFadden, 1986b). The transcriptional initiation sites, determined by Sl -nuclease mapping, are indicated by bent arrows.

tional initiation sites with late RNA than with early RNA because of the progressive increase in the amount of these transcripts as the infection proceeded. Since Slnuclease analysis measures the steady-state levels of a particular transcript in the infected cell, it is not clear whether the increase in the amount of Sl-resistant mRNA:DNA hybrids at late times compared to early times is due to increases in the rate of transcription, in RNA stability, in the amount of viral DNA template transcribed, or all of these. The fact that all the major ORFs are expressed at early times is illustrated by the data in lanes A and B on the Northern blots (Fig. 2). Starting at the junction of the TIR and moving toward the hairpin terminus in Fig. 2, probe EArshows a single transcript directed away from the hairpin terminus. Like other constitutively expressed transcripts, at late times it is associated with a high-molecular-weight smear, probably due to imprecise termination. Probe EA’ detects two major transcripts in this region oriented toward the hairpin terminus. Half of this probe is derived from the viral TIR and half is from the unique internal sequences at the left end, and so the transcripts de-

tected by blotting cannot be unambiguously assigned to either T9 or SFGF. Probe EFdetects two major small transcripts corresponding to one or more of the Tn ORFs, most likely TnB and TnC, since TnA lies entirely within EEand 80 bp of TnD lies within EA(Figs. 1 and 6) but neither species was detected by the corresponding probes. Probe EEdetects a single major transcript which would correspond to the ORF T8 transcript. Probe EC detects two major transcripts, the smallest of which corresponds to ORF T7, while the larger is most likely made up of two transcripts, almost identical in size, encoding ORFs T6 and T8. Probe ED detects transcripts for ORFs T5 and T6 (484aa and 508aa, respectively) which appear to have similar sizes. Probe 0 identifies three transcripts encoding sequences for the ORFs T4, T5, and T5C. The most intense transcript detected by probe lTBcorresponds to a transcript initiating within the TBA-D ORFs, while the minor species corresponds to ORF T4. Probe IT6also detects two very faint species of size similar to the two major bands seen with probe ITA.This is unexpected in view of the fact that the T2 transcript should be pres-

MAP OF SHOPE

FIBROMA

VIRUS TERMINAL

ent in both blots since half of the T2 ORF lies in ITAand half in IT’. An alternative explanation is that transcripts corresponding to Tl and T2 (258aa and 325aa, respectively) may be found in the smaller heterogeneous species seen with probe ITA.These tentative assignments remained to be confirmed by in vitro translation of hybrid- and size-selected mRNA. With longer exposures, a number of higher molecular weight species (between 18 and 28 S) are seen with early RNA on the Northern blots. This phenomenon can be particularly noted with probes ITA,ED, EC, EE, and EFand similar results have been seen on Northern blots of vaccinia early RNA (Cooper ef al., 1981 b). There are at least two possible explanations for these transcripts. One is that the high-molecular-weight transcripts are due to the partial read-through into downstream sequences at early times. Although it is known that at late times imprecise termination results in highmolecular-weight smears on Northern blots (Wittek, 1982) at early times termination is thought not to be random; however, it may still be only partially efficient. Thus a small fraction of the transcripts may not terminate after the first encoded ORF but instead readthrough into the downstream sequences to the next available termination site. This would result in a higher molecular weight but nevertheless distinct transcript size, as opposed to the heterogeneous smear due to imprecise termination seen at late times. A similar phenomenon has been seen with vaccinia transcripts from two separate early gene clusters (Golini and Kates, 1984; Bajszar et a/., 1983). One of these clusters includes the TK gene of vaccinia, but it is not clear if the longer TK transcript is functional (Mahr and Roberts, 1984a). Others have also reported read-through transcripts with SFV early RNA (Cabirac et a/., 1986). A consensus sequence (TlIlTNT) found at the 3’-end of vaccinia virus early genes regulates transcriptional termination in vitro (Rohrmann et a/., 1986; Yuen and Moss, 1986). This same sequence is found exclusively at the 3’-end of all the major SFV TIR ORFs excluding SFGF (Upton et a/., submitted). Although the function of this sequence in SFV has not yet been confirmed as an early terminator, its specific location at the 3’end of all the SFV TIR ORFs further strengthens the conclusion that they are all bona fide early genes. A second possibility is that these high-molecularweight transcripts represent splicing precursors or processing intermediates. No evidence for the splicing of intervening sequences has yet been found for poxvirus mRNA. Capped mRNA has been found to correspond to early initiation sites determined by Sl analysis in the left 21 kb of the vaccinia genome (Wittek et a/., 1980, 1981; Cooper et a/., 1981 b; Venkatesan et a/., 1981; Venkatesan and Moss, 1981), but it should be noted that this kind of study has only been done with

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early genes and not yet with late genes or with clustered tandem arrays of genes. One vaccinia late gene (11 K) has, however, been found to possess its own promoter within 30 bp of the transcriptional initiation site, and this promoter can function in chimeric constructs in recombinant vaccinia virus (Bertholet et al,, 1986; Hanggi et a/., 1986). Thus the possibility that Sl -nuclease analysis might under some circumstances detect sites for mRNA processing rather than transcriptional initiation cannot be rigorously excluded, and more work is required to address this question. DISCUSSION The DNA sequences within and around the TlRs of poxviruses are associated with a number of intriguing phenomena. They have been shown to be highly recombinogenic in cowpox (Archard et al., 1984) monkeypox (Dumbell and Archard, 1980; Esposito et al., 1981) rabbitpox (Moyer eta/., 1980) and vaccinia virus (McFadden and Dales, 1979; Wittek and Moss, 1982). Limited TIR variability has been observed in SFV by the presence of a deletion within the TIR (ORF T6) in strain Boerlage (Upton and McFadden, 1986b) and in the formation of a recombinant leporipoxvirus, MRV, in viva by the replacement of TIR sequences from MYX with TIR sequences from SFV (Block eta/., 1985; Upton and McFadden, 1986b). This recombinant MRV virus induces a novel disease profile in rabbits and produces invasive fibromas that are histopathologically distinct from either the benign fibromas induced by SFV or the invasive myxomatous lesions induced by MYX (Strayer and Sell, 1983; Strayer et a/., 1983a, b, c). Since the major genetic background of MRV appears indistinguishable from MYX, this would suggest that the sequences in or around the TlRs of SFV have some role in determining the tumorigenic properties of both MRV and SFV. In order to understand these phenomena it is essential to characterize the viral genes in this region, and it is to this end that the TIR sequences of SFV that are transcribed during the course of an infection have been determined and the sites for the initiation of transcription mapped. This work complements and extends the recent analysis of leporipoxvirus transcription in a subset of the TIR regions which are common to both SFV and MRV (Cabirac et al., 1986). Northern blot and Sl analysis indicates that the majority of SFV TIR sequences are transcribed at both early and late times during an infection. Twelve sites for the initiation of transcription have been mapped within the TIR: 10 of these sites are located at the 5’end of the major ORFs (Tl, T2, T3A/B, TBC/D, T4, T5, T6, T7, T8, and TnB/C), and the remaining two sites map within ORFs.

392

MACAUIAY,

UPTON,

Three sites for the initiation of transcription were mapped outside the right TIR. The first site lies just 5’ to T9-R (dotted line in Fig. 6), which traverses the junction between the right TIR and the unique internal sequences. The other two start sites map 5’ to SFGF, a gene with sequence homology to EGF, TGF-a, and VGF &hang et al., 1987). The stat-t site 75 bp from the first AUG of the SFGF gene is seen only at late times in these experiments while a second initiation site 350 bp upstream is seen at both early and late times. The relationship of these sites with functional SFGF mRNA is being investigated. These studies indicate that the TIR region of the SFV genome is densely packed with a tandem array of ORFs, all of which are transcribed from the same strand reading toward the hairpin terminus. The only ORFs which physically overlap are T3C with T3D and TnC with TnD. This efficient utilization of space also continues into the unique right sequences which includes the SFGF gene and a second ORF for which transcription data has yet to be obtained. This arrangement of tandemly arrayed ORFs may be true outside the left TIR as well because Northern blot analysis with probe EAr(Fig. 2) indicates that there is at least one transcript in this region directed away from the hairpin terminus. Thus the genetic organization of the TIR of SFV is very different from the organization of the members of the ot-thopoxvirus genus. For example, the vaccinia TIR only contains four genes, two transcribed toward the hairpin terminus and two transcribed away from the hairpin terminus (Wittek eta/., 1980, 1981). On the other hand, sequences outside the left TIR of vaccinia are more efficiently utilized for coding (Cooper et al., 198 1b), and several other regions of the vaccinia genome have been found to contain tandem arrays of ORFs expressed at early and/or late times (Golini and Kates, 1984; Mahr and Roberts, 1984a, b; Weir and Moss, 1984; Niles et a/., 1986; Weinrich and Hruby, 1986). All the ORFs in the TIR of SFV are transcribed constitutively but it is not yet known whether the translation products of these ORFs are expressed at early or late times, or both. In order to investigate both the translational regulation of gene expression and the functions of the gene products from these ORFs, both in vitro translation of mRNA transcribed from the TIR and reagents for the identification of the individual gene products are required. To date, SFGF is the only ORF in this region for which there is clear homology to a known family of gene products (Chang et al., 1987). Since MRV and SFV both encode SFGF plus a subset of SFV TIR genes (Block et a/., 1985; Upton and McFadden, 1986b; Chang et a/., 1987), analysis of the expression of these genes should prove instructive in the elucidation of poxvirus tumorigenesis in general.

AND

MC FADDEN

ACKNOWLEDGMENTS This work was supported in part by the Alberta Heritage Foundation for Medical Research (AHFMR) and by an operating grant from the National Cancer Institute of Canada. G.M. is an AHFMR Scholar, C.M. holds an AHFMR studentship, and C.U. is a postdoctoral fellow. We are grateful to R.A. Maranchuk and A. Wills for excellent technical assistance and to W. Chang for helpful discussions.

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STRAYER,D. S., CABIRAC, G., SELL, S., and LEIBOWIT~.J. L. (1983a). Observations on the culture and histopathologic characteristics of a new virus-induced rabbit tumor. 1. Nat/. Cancer Inst. 71,91-l 04. STRAYER,D. S., and SELL, S. (1983). lmmunohistology of malignant rabbit fibroma virus-a comparative study with rabbit myxoma virus. f. Nat/. Cancer inst. 71, 105-l 16. STRAYER,D. S., SKALETSKY,E., and LEIBOWIIZ, J. L. (198313). Immunologic dysfunction during viral oncogenesis. I. Nonspecific immunosuppression caused by malignant rabbit fibroma virus. 1. /mmunol. 131, 2595-2600. STRAYER,D. S., SKALETSKY,E., CABIRAC, G. F., SHARP, P. A., CORBEIL, L. B., SELL, S., and LEIBOWIZ, J. L. (1983c). Malignant rabbit fibroma virus causes secondary immunosuppression in rabbits. J. Immunol. 130,399-404. STRAYER,D. S., SKALETSKY,E., and SELL, S. (1984). Strain differences in Shope fibroma virus. An immunopathologic study. Amer. J. Pathol. 116, 342-358. UPTON, C., and MCFADDEN, G. (1986a). DNA sequence homology between the terminal inverted repeats of Shope fibroma virus and an endogenous cellular plasmid species. Mol. Cell. Biol. 6, 265276. UPTON, C., and MCFADDEN, G. (1986b). Analysis of viral DNA sequences implicated in the tumorigenicity of Shope fibroma virus and malignant rabbit virus. Virology 152, 308-321. VENKATESAN,S., BAROUDY, B. M., and Moss, B. (1981). Distinctive nucleotide sequences adjacent to multiple initiation and termination sites of an early vaccinia virus gene. Cell 25, 805-813. VENKATESAN,S., GERSHOWITZ,A., and Moss, B. (1982). Complete nucleotide sequence of two adjacent early vaccinia virus genes located within the inverted terminal repetition. J. Viral. 44, 637-646. VENKATESAN,S., and Moss, B. (1981). In vitro transcription of the inverted terminal repeats of the vaccinia virus genome: Correspondence of initiation and cap sites. J. Viral. 37, 738-747. WEAVER,R. F., and WEISSMAN,C. (1979). Mapping of RNA by a modification of the Berk-Sharp procedure: The 5’ terminal of 15s /3globin mRNA have identical map coordinates. Nucleic Acids Res. 7, 1175-l 193. WEINRICH, S. L., and HRUBY, D. E. (1986). A tandemly-oriented late gene cluster within the vaccinia virus genome. Nucleic Acids Res. 14, 3003-3016. WEIR, J. P., and MOSS, B. (1983). Nucleotide sequence of the vaccinia virus thymidine kinase gene and the nature of spontaneous frameshift mutations. J. Viral. 46, 530-537. WEIR, J. P., and Moss, B. (1984). Regulation of expression and nucleotide sequence of a late vaccinia virus gene. 1. Viral. 51, 662669. WILLS, A., DE~NGE, A. M., GREGSON,C., MACAULAY, C., and MCFADDEN, G. (1983). Physical characterization and molecular cloning of the Shope fibroma virus DNA genome. Virology 130, 403-414. WITTEK, R. (1982). Organization and expression of the poxvirus genome. Experientia 38, 285-3 10. WITTEK,R., COOPER,J. A., BARBOSA,E., and Moss, B. (1980). Expression of the vaccinia virus genome: Analysis and mapping of the mRNAs encoded within the inverted terminal repeats. Ce// 21, 487-493. WITTEK, R., COOPER,J. A., and Moss, B. (1981). Transcriptional and translational mapping of a 6.6-kilobase pair DNA fragment containing the junction of the terminal repetition and unique sequences at the left end of the vaccinia virus genome. 1. l&o/. 39, 722-732. WITTEK,R., and MOSS, B. (1982). Colinearity of RNAs with the vaccinia virus genome: Anomalies with two complementary early and late RNAs results from a small deletion or rearrangement within the inverted terminal repetition. 1. Viral. 42, 447-455. YUEN, L., and Moss, B. (1986). Multiple 3’ ends of mRNA encoding vaccinia growth factor occur within a series of repeated sequences downstream of T clusters. J. Viral. 60, 320-323.