Transcriptional mapping of early RNA from regions of the Shope fibroma and malignant rabbit fibroma virus genomes

153.53-69 (1986)

VIROLOGY

Transcriptional

Mapping of Early RNA from Regions of the Shope Fibroma and Malignant Rabbit Fibroma Virus Genomes

GARY F. CABIRAC,* JUSTIN J. MULLOY,? DAVID S. STRAYER,f STEWART SELL,? AND JULIAN L. LEIBOWITZT’ of Chemistry, University of Ca&fwnia, San Diego, La Jolla, Califmia 92093; tDepatiment of and Laboratory Medicine, University of Texas Health Science Center, Houston, Texas 77025; and of P&ho&l/, Yale University S&d of Medticin.e, New Haven, Connecticut 06510 fDepart,ment

‘Department

Pathology

Received September

20, 1985; accepted April

11, 1986

Malignant rabbit fibroma virus (MV) is a recombinant poxvirus derived from Shope fibroma virus (SFV) and rabbit myxoma virus (D. S. Strayer, E. Skaletsky, G. F. Cabirac, P. A. Sharp, L. B. Corbeil, S. Sell, and J. L. Leibowitz, 1983a, J. ImmunoL 130,399-404; W. Block, C. Upton, and G. McFadden, 1984, Virology 140,113-124). We report here the transcriptional mapping of early RNAs transcribed from the SFV sequences within MV and from the corresponding regions in SFV. Hybridization analysis and Sl nuclease mapping of RNA using viral DNA probes were used to define 5’ and 3’ ends of the various transcripts. The RNAs described here are transcribed in one direction in a densely arranged head to tail fashion similar to that described for some vacinia virus early transcriptional units. At late times of infection the early SFV RNAs are not detected whereas the early MV RNAs are present in minor amounts. The early SFV and MV transcripts range in size from 3170 to 425 nucleotides (nt) long. All of the longer transcripts are produced as a result of read through transcription. Three MV transcripts contain fused SFV and rabbit myxoma virus sequences due to transcription through the recombination junction region in the MV genome. Two other MV transcripts are transcribed from a unique initiation site near another recombination junction region resulting in RNAs that are composed of SFV sequences having unique 5’ ends. o 1986 Academic PWS, IN. INTRODUCTION

transcriptional modifications such as capping, methylation, and polyadenylation are contained within the infectious particle (for review see Moss, 1985). Since poxviruses replicate in the cytoplasm of the infected cell these enzymes are needed for such modifications. Detectable splicing of poxvirus transcripts does not occur during processing of early mRNA and most likely does not occur following transcription of late genes despite the poorly understood presence of 3’ heterogeneity in late mRNA (Cooper et ab, 1981). The only detailed transcriptional maps of poxvirus mRNA that exist are derived from a few viruses of the Orthopoxvirus genus (Wittek et al., 1980; Cooper et al., 1981; Golini and Kates, 1984; Mahr and Roberts, 1984a; Morgan and Roberts, 1984). Although studies of this genus has produced evidence of a unique transcriptional

Early transcription of the poxvirus genome can be defined as that which occurs before initiation of viral DNA synthesis. The RNA polymerase which carries out this transcription is a virus-encoded enzyme that is packaged within the virion. Synthesis of early mRNA by this polymerase begins prior to complete uncoating of the virus. DNA/RNA hybridization studies reveal that about 50% of the genome is transcribed prior to DNA replication (Kaverin et al, 1975; Boone and Moss, 19’78) and hybrid selection experiments indicate that these transcripts map throughout the genome (Belle Isle et al, 1981). In addition to the polymerase found within the virion, an array of enzymes responsible for post’ To whom dressed.

requests

for

reprints

should

be ad-

53

0042-6822/86 Copyright All rights

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0 1966 by Academic Press. Inc. of reproduction in any form reserved.

54

CABIRAC

strategy of the poxvirus genome, transcription by viruses of other poxvirus genera has not been investigated. Of particular interest is a group of poxviruses which cause tumors in their natural hosts. These include Yaba tumor virus and molluscum contagiosum, neither of which are classified into a specific genus, and viruses of the Leporipoxvirus genus. Recent work on various strains of Shope fibroma virus (SFV), a Leporipoxvirus, has shown that the genome of this virus possessesfeatures in common with those in the Orthopoxvirus genus. These include inverted terminal repeats and cross-linked termini (Wills et cd, 1983). We have isolated a poxvirus described elsewhere (Strayer et al, 1983a), which is a recombinant between two Leporipoxviruses, SFV and rabbit myxoma virus. It has been shown that the genome of this recombinant, designated malignant rabbit fibroma virus (MV), consists of rabbit myxoma virus sequences in which approximately 5 kb of DNA in each inverted terminal repeat has been replaced by SFV sequences (Block et aZ.,1984). MV produces a syndrome in infected rabbits clinically, pathologically, and immunopathologically distinct from those produced by SFV or rabbit myxoma virus (Strayer and Sell, 1983; Strayer et aZ., 1983b). As an initial step toward analyzing gene products encoded within this region of recombination and comparing transcriptional organization of a Leporipoxvirus with that of viruses belonging to the Orthopoxvirus genus, we have constructed a detailed transcriptional map of the early mRNAs synthesized from the region of recombination.

ET AL.

ate early viral RNA was isolated from cells infected in the presence of 100 pg/ml cycloheximide. Early RNA was prepared from cells infected in the presence of 40 pg/ml cytosine arabinoside and late RNA from cells infected in the absence of any inhibitors. RNA was extracted 12 hr postinfection for all conditions. RNA was denatured with glyoxal and electrophoresed through 1.5% agarose gels according to the method of McMaster and Carmichael (1977). The RNA was then transferred to 0.15 pm nitrocellulose essentially as described by Thomas (1980). All of the SFV viral DNA which was used for hybridization to RNA, probes A through H, shown in Fig. 2, was cloned into one of three plasmid vectors-pBR322, pMK16, or pUC19. MV DNA which spanned the recombinational junction region closest to the genome termini and the recombinational junction around the DdeI site in the left terminal region, i.e., the 1.8-kb BglII/PstI fragment, probe I, and the 0.73kb BglII/SmaI fragment, probe J, was cloned into pUC19. MV DNA which spanned the recombinational junction region around the KpnI site in the right terminal region, i.e., the 3.5-kb SmaI/SmaI fragment, probe K, was recovered from a preparative gel of digested viral DNA. All DNA fragments were labeled with 32Pby nick translation to a specific activity of 25 X lo8 cpm/pg (Rigby et al, 1977). Labeled DNA was hybridized to RNA at 42” for 20 to 24 hr in 50% formamide, 5X Denhardts solution (0.02% each of polyvinylpyrrolidone, Ficoll, and bovine serum albumin), 5X SSPE (1X = 0.15 M NaCI, 0.01 M PO1, pH 7.4, and 1 mM EDTA), 200 pg/ml denatured salmon sperm DNA, 100 pg/ml tRNA, and 0.1% SDS. Hybridized nitrocelMATERIAL AND METHODS lulose was washed twice with 2X SSPE, Cells and virus. RK-13 cells were grown 0.1% SDS at 25”, twice with 0.1X SSPE, in Dulbecco’s Modified Eagles Medium 0.1% SDS at 25”, and then once with 0.1X (DME, Flow Labs) supplemented with 10% SSPE, 0.1% SDS at 55”. 5’1nuclease mapping. The 3’ ends of DNA fetal bovine serum (FBS). The isolation of MV and the growth of all viruses has been fragments were labeled with 32Pby using described previously (Strayer et a& 1983a). alpha r2P]dCTP and T4 DNA polymerase Puri&xtion of RNA and northern anal- and the 5’ ends were labeled by using gamma [32P]ATP and T4 polynucleotide kiysis. Total cellular RNA was extracted from monolayers of virus-infected cells as nase. Labeled fragments were cut with the appropriate second restriction enzyme and described by Wittek et al. (1984). Immedi-

TRANSCRIPTIONAL

MAPPING

the desired fragment purified from a preparative low-melting-point agarose gel. Approximately 100 ng of labeled DNA probe, 5-10 pg of RNA isolated from infected cells, and 50 pg tRNA was dissolved in 10 ~1 of 80% formamide, 40 mM Pipes(Piperazine - NJ - bis[2 - ethanesulfonic acid]), pH 6.4,0.4 n/i NaCl, and 1 mM EDTA, and denatured at 70” for 15 min. DNA-RNA hybridization was carried out at 50” for 3 hr, then 0.2 ml of 0.28 M NaCl, 50 mM NaAcetate, pH 4.6, 4.5 mM ZnSOa, 20 pg/ml carrier salmon sperm DNA, and 500 U/ml Sl nuclease was added and incubated at 25” for 30 min. The reaction was stopped with 50 ~1 of 2.5 M ammonium acetate, 50 mM EDTA, and 200 pg/ml tRNA and then precipitated with isopropanol. Slresistant products and control samples were analyzed on 1.5 or 1.8% neutral and alkaline agarose gels. Neutral gels were run in a Tris-borate buffer system and alkaline gels were run as described by McDonnell (1977). Gels were fixed, dried, and exposed to Kodak XAR-5 film. Materials. All restriction endonucleases and DNA-modifying enzymes were obtained from Bethesda Research Laboratories or New England Biolabs. Cycloheximide and cytosine arabinoside were from Calbiochem. Nitrocellulose was from Schleicher & Schuell. Alpha [32P]dCTP and gamma [32P]ATP were from ICN Radiochemicals. The low-melting-point agarose was from Biorad. Pipes was from Research Organics Inc., and formamide was from Fluka. RESULTS

Northern anal&. The areas of the SFV and MV genomes relevant to the following transcriptional analysis are depicted in Fig. 1A. The figure shows restriction sites which define the SFV sequences in the MV genome and the corresponding sites in the SFV genome. Also shown are the relative locations of these regions in each virus genome, the extent of the inverted terminal repeats, and kilobase scales showing the distance between the genome termini and the regions of interest. Restriction mapping of the two viral genomes (Block et al.,

OF SFV AND

MV

55

1984) has revealed that the SFV/myxoma virus junction sequences closest to the termini of the MV genome are located within 150 bp of the ClaI site shown at both ends of the genomes. This junction is apparently the same in both the left and right terminus of MV. The ClaI site in the SFV genome is 5.8 kb from the end of the genome while the ClaI site in the MV genome is 6.1 kb from the end of the genome. The junction distal from the MV left terminus lies within 300 bp of the DdeI site. The total length of SFV sequences in the left terminus of MV then is approximately 4.0 to 4.5 kb long. The junction distal from the MV right terminus has not been precisely defined. The last identified restriction site which SFV and MV have in common is the KpnI site shown. As will be discussed below, we believe the SFV sequences in MV extend at least an additional 1.5 kb from this KpnI site. This region of uncertainty in the MV genome is shown in the figure by a hatched bar and question mark. To demonstrate the approximate number, sizes, and relative abundance of RNA species transcribed from the regions of interest in the SFV and MV genomes, total cellular RNA was isolated from virus-infected cells and analyzed by blot hybridization as described in Material and Methods. The probe used in this instance was derived from a cloned 6.‘7-kb SFV DNA fragment. The sequences represented by this DNA probe are shown in Fig. 1A by a bar below the right terminal region of the SFV map. Figure 1B shows the hybridization of this probe to RNA isolated from SFV- or MV-infected cells at immediate early, lane 1; early, lane 2; and late, lane 3, times of infection. This SFV probe hybridizes to what appears to be 9 to 10 different RNAs at early times of infection for both SFV and MV. The size range of these RNAs is between 3400 and 425 nt. As shown below, the intense band in the 1800- to 1600nt range of this autoradiogram actually represents four unique species. A comparison of SFV and MV immediate early RNAs, lane 1, demonstrates that a major 650-nt species present in SFV-infected cells is absent from MV-infected cells. The only difference observed between immediate

56

CABIRAC

ET AL.

A

r O\\-, PROBE

FIG. 1. Map of restriction sites which define the SFV sequences within the MV genome and northern analysis of RNA from SFV, MV, and rabbit myxoma virus-infected cells. (A) The restriction sites shown in the MV genome define the extent of SFV sequences (solid bars) within each terminal region of this genome. Open bars indicate rabbit myxoma virus sequences within this area of the MV genome. The left MV terminal region contains approximately 4 kb of SFV sequences extending from the ClaI site to the DdeI site. The right MV terminal region contains at least 5.5 kb of SFV sequences extending from the ClaI site to the KpnI site. The hatched region and question mark shown from the MV right terminal region indicate that an additional 1.5 kb of SFV sequences may exist in this area of the MV genome. Also shown for both SFV and MV are the lengths of the inverted terminal repeats (ITR) and kilobase scales showing the location of the restriction sites relative to the genome termini. The location of the pertinent sequences within each genome is shown in the top (SFV) or bottom (MV) scales. Both genomes are approximately 166 kb in length. (B) RNA

TRANSCRIPTIONAL

MAPPING

early RNA and late early RNA is quantitative. For both viruses these RNA species are more abundant at immediate early than at late early times of infection. At late times of SFV infection, lane 3, the species observed at early times are absent. However, this probe hybridizes to a broad range of heterogeneously sized RNAs in the high molecular weight range. Digestion of the late RNA samples with DNase-free RNase revealed that the high molecular weight species were not viral DNA intermediates (data not shown). Size heterogeneity has also been observed in vaccinia late mRNA, and it has been determined that this is due to heterogeneity of the 3’ ends of the viral mRNA (Cooper et aZ., 1981). Surprisingly, the early MV RNA species shown here are still present in minor amounts at late times of infection (lane 3). In addition, the high molecular weight, heterogeneously sized RNAs seen late in SFV infection are not detected late in MV infection. It is not clear whether the early MV transcripts are present late in infection due to increased stability of these RNAs in the MV-infected cell or to low levels of transcription of these species late in infection. Figure 1C shows the hybridization of the same SFV probe used in Fig. 1B to immediate early RNA isolated from MV and rabbit myxoma virus-infected cells. This hybridization was performed at the same stringency as that used in Fig. 1B in order to detect myxoma virus RNA species which cross hybridize with the SFV probe. As shown in the figure, under these hybridization conditions only two sizes of myxoma virus RNA species, 1700 and 1040 nt, hybridize to this SFV-specific probe. The fact that these two myxoma species were the same size as two of the MV species

OF SFV AND

MV

57

and that these in turn correspond to RNAs detected in SFV-infected cells illustrates the fact that the genomes of SFV and rabbit myxoma virus cross hybridize (Block et aL, 1984). These particular RNAs transcribed from the myxoma virus genome during infection probably encode polypeptides whose functions are the same or analogous to the corresponding polypeptides in SFV-infected cells. Despite this cross hybridization between SFV DNA and myxoma virus RNA, we did not change hybridization conditions during subsequent experiments but rather took this into consideration when interpreting results. Mapping of early RNAs. I. Hybridization with region-spec$c robes. The early RNAs transcribed from the recombination region in the MV genome and the corresponding region in the SFV genome were mapped to their respective genomes by using a combination of two methods. First, regionspecific probes derived from cloned DNA fragments were hybridized to RNA isolated from infected cells and second, RNA was subjected to Sl nuclease protection experiments. A rough map was derived from the hybridization results while the 5’ and 3’ ends of the transcripts were located using the Sl mapping data. The hybridization data will be presented in this section and the Sl data in a section below. Figure 2A shows: (1) the restriction sites which define the area of recombination (sites in boxes), (2) the restriction sites in the SFV and MV genomes used in the construction of the hybridization probes, and (3) the final transcriptional map of these regions of the two genomes. The transcriptional map is presented at this point in order to make interpretation of the hybridization and subsequent Sl data easier for the reader. The restriction sites which were

isolated from SFV and MV at immediate early (lane l), early (lane Z), and late (lane 3) times of infection was denatured with glyoxal and electrophoresed through 1.5% agarose, transferred to nitrocellulose, and hybridized to a cloned SFV DNA fragment which had been labeled with “P by nick translation as described in Materials and Methods. The SFV DNA represented by this probe is shown in panel A below the right terminal region of the SFV map. This same probe was hybridized to MV and rabbit myxoma virus early RNA (C). s2P-labeled i X HindIII/EcoRI and &X174 X TaqI DNA were used for molecular weight standards. A representative number of these standards are shown to the left of the figure. Numbers indicate length in nucleotides.

58

CABIRAC A

ET AL.

2950 2500 1600

2570 2160 425

1670

010

f

SW

SFV

left

MV.

2950 2570 ?I60 425

2560 -

1710

610

FIG. 2. (A) Restriction sites used to construct region-specific probes, transcriptional map of regions in MV and corresponding regions in SFV, and hybridization analysis of SFV and MV RNA using region-specific probes. Restriction sites in boxes shown below each line indicate the last site that SFV and MV have in common, i.e., the recombination junction. Solid thick and thin lines indicate SFV and rabbit myxoma virus sequences, respectively. The map of the right terminal region of MV has been inverted relative to its real orientation in the genome so that it can be lined up with the map of the left terminal region. The restriction sites shown above each line for SFV or MV indicate sites used to construct hybridization probes. These are lettered, A through K, above each respective restriction fragment. The open bar to the right of the KpnI site in probe K indicates the area of possible SFV sequences as described in Fig. 1. The mapped viral transcripts are shown above the restriction maps of SFV and each terminal region of MV. Solid thick and thin lines indicate unique and run-through transcripts, respectively, which are composed of SFV sequences. Open and broken lines indicate unique and run-through transcripts, respectively, which are composed of rabbit myxoma sequences. The numbers above each transcript refer to the size of each RNA in nucleotides. (B) Each DNA fragment represented by A through K was labeled by nick translation. Probes A-H were hybridized to immediate early RNA isolated from SFV (lane l)- and MV (lane 2)-infected cells. Probes I-K were hybridized to immediate early RNA extracted from myxoma virus-infected cells (lane 3) as well as to SFV (Lane 1) and MV (lane 2) immediate early RNA. The letter above each set of lanes corresponds to the fragments shown in (A). A through H were derived from SFV DNA while I through K were derived from MV DNA. The sizes of RNA detected with each probe as determined by comparison to the molecular weight standards described in Fig. 1, is given in Table 1.

used to construct the 12 DNA hybridization probes, A through K, are shown above the lines representing the genomes. Probes A through H were derived from SFV DNA while I through K were derived from MV

DNA. Shown below the lines in boxes are the restriction sites which were used in Fig. 1A to define the area of recombination in the MV genome and the corresponding regions of the SFV genome. Thick solid lines

TRANSCRIPTIONAL

MAPPING

B IA2

lE2

lB2

IF2

lC2

ID2

lG2

I”2

OF SFV AND

59

MV

RNA was isolated from virus-infected cells, electrophoresed through glyoxal gels, transferred to nitrocellulose, and then hybridized to the 32P-labeled DNA fragments described above. The results are shown in Fig. 2B. For probes A through K, lane 1 represents SFV RNA and lane 2 represents MV RNA, while lane 3 in I, J, and K shows rabbit myxoma virus RNA. A listing of RNAs detected with each specific probe is given in Table 1. For the sake of brevity, we will only discuss those DNA probes which produced dissimilar hybridization patterns with SFV and MV RNA. These are probes A, D, E, F, I, J, and K. The hybridization results obtained with probes B, C, G, and H are simply summarized in Table 1. The hybridized nitrocellulose filters were exposed for shorter periods without intensifying screens to produce sharp images of the signals in the 1600- to 1800-nt size range. This was needed to obtain accurate size determination of the various RNAs in this area. Longer exposures were necessary for visualization of some of the less abundant RNA species. Some faint signals in

TABLE VIRUS-SPECIFIC

RNAs

1

DETECTED

WITH DNA

PROBES

DNA

probe” FIG. 2-Continued.

indicate SFV sequences, thin lines indicate rabbit myxoma virus sequences in the MV genome, and the open thick line below probe K indicates the region of uncertainty discussed above. The arrows representing the viral transcripts are shown above each of the genome maps. Thick lines indicate those RNAs which terminate at the first available termination signal (as defined by the Sl mapping discussed below) while thin lines indicated “run through” transcripts. Solid lines represent SFV sequences and open or broken lines represent rabbit myxoma virus sequences.

RNAb

A B C D E F G H

3170, 2700, 1600, 650” 3170, 2500, 1670 3170, 2500, 1670 3300,” 2580,1670, 1040 3300,” 3000: 2580, 2470: 1710, 1600d 3300,” 3000,d 2580, 2470: 1710, 1600d 3300, 2950, 2570, 2160, 810, 425 2950, 2570, 2160, 1710, 810, 425

I J K

3170, 2700, 1600, llOOd 3300,” 3000,d 2580, 1710 3300, 2950, 25’70, 2160, 1710, 600d

DRefers to fragments shown in Fig. 2. *Sizes are given in nucleotides; end labeled h X HindIII/EcoRI and 9X174 X TaqI DNA was used as molecular weight standards. ’ RNA detected in SFV lane only. d RNA detected in MV lane only.

60

CABIRAC

the 4000 and 2000-nt size range are attributable to background hybridization to ribosomal RNA (for example, Fig. 2B, probes A, B, E, F, and I). These signals were not detected when SFV probes were hybridized to poly-A+ RNA isolated from mock or virus-infected cells (data not shown). The assignment of some RNA species to a specific probe was a constant problem since there are several RNAs transcribed from the region of recombination that are approximately the same size and, as discussed below, there are multiple species which are the result of the polymerase reading through transcriptional stop signals. These difficulties were resolved by Sl mapping. Probe A, which is derived from SFV DNA, spans the recombination junction region closest to the termini of the genome, i.e., the ClaI site shown in the box in Fig. 2A. This probe hybridizes to three sizes of RNA, 3170, 2700, and 1600 nt, from SFV (lane l)- and MV (lane 2)-infected cells, and to an additional 650-nt RNA in the SFV lane. Probe I, a 1.8-kb BglII/PstI MV fragment, roughly corresponds to the region covered by the SFV probe A. This probe hybridizes to the 3170, 2700, and 1600-nt RNAs detected with probe A. These are detected not only in SFV-and MV-infected cells but also in myxoma virus-infected cells (lane 3). This probe does not hybridize to the 650-nt SFV species. However, a minor llOO-nt species is detected in the MV and myxoma virus lanes. As will be shown below in the Sl nuclease mapping experiments, the three largest MV RNAs detected from this region span the area of recombination and are composed of SFV and myxoma virus sequences. The 650-nt SFV RNA and the 1100~nt MV and myxoma virus RNA map outside the region of recombination. This is probably a region in which the SFV and myxoma virus genomes have a low degree of homology since no cross hybridization is observed for these two RNA species. Probe E hybridizes to three different sized RNAs for each virus. Two of these, the 2580- and 1710-nt species, appear to be the same for both SFV and MV. However,

ET AL.

this probe also hybridizes to a 3300-nt species in the SFV lane which is absent in the MV lane, and a 3000-nt species in the MV lane which is not observed in the SFV lane. Probe F hybridizes to the same RNAs detected with probe E. Probe F contains the DdeI site shown in Fig. 2A and therefore should detect any MV transcripts which may contain SFV and myxoma virus sequences that are transcribed through the recombination junction in MV. We propose that there are no such SFV/myxoma fusion RNAs transcribed through this recombination junction in the left terminal region of MV. The data which support this is as follows: (1) Sl mapping results (discussed below) reveal that a transcription initiation site is located in the left terminal region of the MV genome within 30 bp of the DdeI site and that all transcription in this region is in a right to left direction. (2) A myxoma virus sequence-specific probe constructed from MV DNA did not detect any transcripts initiated within 1 kb to the right of the DdeI site in MV (data not shown). Therefore, the unique 3000-nt MV RNA cannot be derived by transcription from a unique MV initiation site within myxoma virus sequences to the right of the DdeI site and termination at a point to the left of the DdeI site in SFV sequences. This unique 3000-nt MV species is transcribed from an initiation site contained within SFV sequences in the right terminal region of the genome and terminated at a site which is different from the termination point of the unique 3300-nt SFV RNA. This conclusion is supported by the observation that upon longer exposure of the nitrocellulose this 3300~nt SFV species but not the 3000~nt MV species is detected with probe D (data not shown). Therefore the 3000-nt MV RNA is terminated at a point before the XhoI site at the junction between probes D and E while the 3300-nt SFV RNA terminates at a point past the XhoI site within the sequences of probe D. Why transcription of the 3000-nt MV RNA terminates at a different point than the 3300nt SFV RNA is not clear. The positions of these minor RNA species are not indicated on the transcriptional map (Fig. 2) for rea-

TRANSCRIPTIONAL

MAPPING

sons that are discussed below (see Sl nuclease mapping). Probe J, derived from MV sequences, hybridizes to the same SFV and MV RNAs that are detected with the SFV probe F but does not hybridize to any myxoma virus transcripts. Probe J, which spans the recombination junction region in the left MV terminus, and corresponds to the SFV probe F, extends from the BglII site to the SmaI site shown in Fig. 2A. The BglII site lies to the left of the SstI site (shown in the SFV map, Fig. 2A) and is common to both SFV and MV. The MV SmaI site lies to the right of the boxed DdeI site and is derived from myxoma virus sequences. This strengthens the idea that the unique 3000~nt MV transcript detected by probes E, F, and J is initiated at a point within SFV sequences, because if any initiation points existed to the right of the boxed DdeI site in the left region of MV, i.e., in myxoma sequences, then a myxoma virus transcript should be detected with this SFV/myxoma sequence-specific probe. Probe K, derived from MV sequences, hybridizes to the same SFV RNAs detected with probe H (see Table 1). These same RNAs plus a 600-nt species are seen in the MV and myxoma virus lanes, 2 and 3, respectively. Probe H extends only 0.57-kb from the SmaI site which is at the junction between probes G and H, while probe K extends 3.5 kb from this site. It is interesting that probe K hybridizes to only one RNA, the 600-nt species, from MV that is not detected with probe H. As will be shown in the Sl analysis of this area, this result most likely is due to initiation of these large SFV and MV transcripts from a region approximately 2 kb internal to the SmaI site. A preliminary map can be derived from this hybridization data. The 650-nt unique SFV transcript must be localized in the probe A region since probe B does not hybridize to it. This also applies to the 1600nt species detected in the SFV and MV lanes. The SFV and MV 3170-nt species is in the region covered by probes A, B, and C since it is not detected with probe D. The 2700-nt RNA which hybridizes to probe A

OF SFV AND

MV

61

must be transcribed from a point inside A since it is not detected with probe B. A 1670-nt RNA is transcribed from a region covered by probes B and C. The difference in size between this 1670-nt RNA and the 1600-nt RNA detected with probe A can be seen upon exposure of the nitrocellulose without an intensifying screen (not shown). A 2500-nt RNA is localized over the region covered by probes B, C, and D. The 1040nt RNA is detected only with probe D and therefore must be transcribed predominantly from this region. A 1710-nt RNA is localized in a region covered by probes E and F since no signal can be seen in this region with probe G and only a faint signal was seen with probe D. The 2580-nt RNA hybridizes to probes D, E, and F. The 810 and 425-nt transcripts hybridize mainly to probe G with minor hybridization to probe H and are therefore composed mostly of sequences from probe G. Another distinct RNA 1710 nt in length is transcribed from a region covered by SFV probe H and MV probe K since these two probes hybridize to this species while SFV probe G does not. The 2950-, 2570-, and 2160-nt RNAs detected with probes H and K must terminate at a point inside of probe G since they hybridize to this probe but not to probe F. II. Sl nuclease mapping. All the autoradiograms shown of Sl protection experiments were from alkaline DNA gels. However, representative samples from the entire region of recombination were run on neutral gels in order to detect splicing of transcripts. We found no evidence of mRNA splicing using this method. To date, this posttranscriptional modification has not been detected during poxvirus transcription. All of the SFV and MV RNAs detected in this work were transcribed toward the termini of the genomes, i.e., in a right to left direction in the left terminal region and a left to right direction in the right terminal region. As was briefly mentioned above, the Sl experiments indicate that most if not all transcriptional termination signals can be read through by the viral RNA polymerase. This can be seen in Figs. 3 through 5. The presence of large read-through transcripts have been de-

62

CABIRAC

ET AL.

1.9 WI WI n66.-.

G* Bgl II

Xhol E

I.0 Pst I

MV

FIG. 3. Sl nuclease mapping of immediate early RNA transcribed from the recombination junction region closest to the genome terminus. (A) Restriction sites are shown for SFV and MV which were used for construction of Sl hybridization probes. The restriction sites shown in bolder print are the same as those that defined the region-specific hybridization probes described in Fig. 2A. The boxed ClaI site shown for both SFV and MV is the last site shared by the two genomes as described in Fig. 1A. Sequences to the left of this site in MV are derived from rabbit myxoma virus. Each probe is shown by a thick line with a solid circle indicating the position of the =P-labeled end. Probes A-F were constructed using SFV DNA, G-I using MV DNA. Restriction sites outside the region shown which were used for construction of probes C, F, and I are indicated for each. Probes above the restriction maps, A-D for SFV and G for MV, were labeled at the 5’ end of the DNA fragment while those below, E and F for SFV and H and I for MV, were labeled at the 3’ end. The Sl protected fragments obtained with each probe are shown above the 5’ labeled probes or below the 3’ labeled ones. The thicker lines indicate the major (or in some cases the only) Sl protected fragment observed, thin lines indicate minor protected fragments. Sizes are given for each probe or protected fragment in kilobases. A line without a number next to it indicates that this particular protected fragment is the same size as the probe from which it was derived. A 5’ terminus of a protected fragment is shown with a short vertical line; a 3’ terminus with an arrow. Sl protected fragments unique to SFV or MV RNA are indicated for probes D, F, H, and I; all other Sl protected fragments were detected with SFV and MV RNA. (B) Autoradiograms of dried gels of Sl protected fragments. The letters above each set of lanes corresponds to the probes shown in (A). Sl protected fragments were derived after hybridization to SFV RNA (probes A-F and H, lane 1; probes G and I, lane 2), MV RNA (probes A-F and H, lane 2; probes G and I; lane l), or rabbit myxoma virus RNA (probes D and H, lane 3). Hybridization controls containing no viral RNA are shown in lane 3 (A-C, E-G, and I) or lane 4 (D and H). The far right lane in each case shows the DNA fragment used for hybridization to RNA (the fragment in F is not shown due to its position in the gel relative to the Sl protected fragments). Horizontal lines to the left of each autoradiogram indicate the position of the observed Sl protected fragments. The sizes of these fragments are indicated in panel A for each probe. Methods used for labeling DNA fragments and conditions of DNA-RNA hybridization and Sl nuclease digestion are described in Materials and Methods. Molecular weight standards are as described in Fig. 1.

TRANSCRIPTIONAL

MAPPING

E

1234

FIG. 3-Continued

tected in other studies of poxvirus transcription (Bajszar et aZ., 1983; Mahr and Roberts, 198413;Golini and Kates, 1984). Figure 3 shows Sl mapping of the region near the recombinational junction closest to the termini of the SFV and MV genomes, i.e., the ClaI site in Fig. 1A. The 5.8kb PstI/ XhoI fragment, designated probe F, is derived from SFV sequences and yields a prominent 0.6-kb Sl protected species when hybridized to both SFV (lane 1) and MV (lane 2) RNAs. In addition, hybridization of probe F with SFV RNA yields smaller amounts of a 2.1-kb Sl protected fragment while hybridization to MV RNA results in two, 1.5 and 1.4 kb, protected fragments. The opposite situation is observed with probe H, a 0.8-kb ClaI/BglII MV fragment. Two Sl protected fragments derived from SFV RNA and probe H (lane 1) have ends which map to approximately the same

OF SFV AND

MV

63

points as those from the Sl protected fragments derived from MV RNA and probe F (lane 2). Apparently these points map to a region of low homology between the SFV and myxoma virus genomes since RNA will only hybridize, and hence be protected from digestion with Sl (under the conditions used in these experiments as described in Material and Methods), if it has been transcribed from DNA which is exactly homologous to it. Probe D, the 1.9-kb BarnHI/ PstI fragment derived from SFV DNA produces Sl protected fragments when hybridized to SFV RNA (lane 1) but not when hybridized to MV or myxoma virus RNA (lanes 2 and 3, respectively). Again this is probably due to low homology between the sequences around the labeled end of the SFV probe and those in MV and myxoma virus RNA, since a probe constructed from MV DNA, probe G, produces a protected fragment when hybridized to MV RNA (lane 1) but not SFV RNA (lane 2). This MV probe is labeled at its BglII end which roughly corresponds to the location of the labeled BglII end of the SFV probe D. It should be pointed out that these two BglII sites are not necessarily the same in the SFV and MV genomes. The MV BglII site is located within myxoma virus sequences and hence this site could be conserved between the SFV and myxoma virus genomes. A 1.2-kb BglWBamHI fragment constructed from MV DNA, probe I, produces Sl protected fragments when hybridized to MV RNA (lane 1) but not SFV RNA (lane 2). This is most likely due to low homology between SFV and myxoma virus as mentioned above. SFV RNA (lane 1) and MV RNA (lane 2) produce identical Sl protected fragments when hybridized to probes A, B, C, and E. Figure 4 shows probes and Sl protected fragments in the internal region of recombination. The sequences in this area, delineated by the 2.7-kb XhoI/SstI fragment, should be identical in the SFV and MV genomes as shown by restriction mapping (Block et aZ., 1984). Therefore we did not expect to see any difference between Sl protected fragments derived from SFV RNA and MV RNA. This was observed for probes A, C, D, E, G, and H. However, when

CABIRAC

64

ET AL.

A

II XhOl egl

SSI I

Xho I

0.91~

0.494

A34

12

E

1234

12

F 123

12

0

34

12

C

G

12345

34

.G

1234

1234

D

Ii

5

5 dr

FIG. 4. Sl nuclease mapping in immediate early RNA transcribed from the middle region of recombination. (A) The region shown in the restriction map is common to both SFV and MV. Therefore all probes shown were constructed using only SFV DNA. As in Fig. 3, probes above the map are 5’ labeled and those below are 3’ labeled. The unique 1.4-kh MV protected fragment derived from probe

TRANSCRIPTIONAL

MAPPING

probe B, a 3.0-kb SFV XhoI/KpnI fragment labeled at the 5’ terminus of the XhoI site, was hybridized to SFV or MV RNA, a difference was observed. An extra Sl protected fragment can be seen in the MV lane (lane 2). This fragment is 1.4 kb long. If the map in Fig. 4 is superimposed on the map shown in Fig. 2A, it can be seen that the terminus of this MV protected fragment is approximately at the DdeI site, which is the last known site that SFV and MV have in common in the left terminal region of the MV genome. Figure 5 illustrates Sl mapping data from the internal junction regions of recombination in MV and the corresponding region in SFV. Probe B, which was constructed using SFV DNA, produces one Sl protected fragment when hybridized to SFV or MV RNA (panel B, lanes 1 and 2, respectively). No protected fragments are produced when this probe is hybridized to myxoma virus RNA (lane 3). This finding was unexpected since it was reported that the labeled BglI site of probe B is not present in MV (Block et aZ., 1984) and hence should be in the region of myxoma virus sequences. This suggests that the SFV sequences in the right terminal region of MV extend at least up to the BglI site. More restriction mapping and perhaps sequencing of this area of the SFV, MV, and myxoma virus genomes is needed to resolve this discrepancy. Probe F, which was constructed using MV DNA, produces a unique 0.3-kb MV protected fragment (lane 2) in addition to other common protected fragments when hybridized to SFV and MV RNA. No Sl protected fragments are observed when hybridized to myxoma virus RNA (lane 3). The terminus of the unique 0.3-kb MV fragment is located in the same

OF SFV AND

MV

65

position as the terminus of the unique 1.40kb MV protected fragment produced with probe B in Fig. 4. This terminus, which is located approximately at the recombination junction region in the left terminal region of the MV genome, corresponds to the unique MV transcriptional initiation point discussed above. SFV RNA (lane 1) and MV RNA (lane 2) produce identical Sl protected fragments when hybridized to probes A, C, D, and E. Transcriptional WUZ~. The resulting transcriptional maps of the SFV and MV RNAs discussed in this work are shown in Fig. 2A. Due to the number of transcripts, the uncertainty of some of the recombinational junction regions, and the presence of the read-through transcripts, this figure can only represent a best fit of the acquired data. We have classified the SFV and MV RNAs mapped here into two groupsunique and read-through transcripts. We define unique transcripts as those that are terminated at the first available termination signal while read-through transcripts are those that appear to be transcribed through a termination signal. It should be noted that there are initiation and termination points identified by Sl mapping that do not correspond to any of the transcripts shown in the figure. All of these points, however, were mapped from Sl protected fragments which were present in very minor amounts relative to the others. This was also observed during the transcriptional mapping of a region of the vaccinia genome (Golini and Kates, 1984). There are seven unique and eight readthrough SFV RNAs transcribed from the region studied, indicated in Fig. 2A by thick and thin lines, respectively. There are four unique and four read-through MV RNAs

B is indicated. (B) Autoradiogram of dried gels of Sl protected fragments. Letters correspond to probes shown in (A). Sl protected fragments were derived from SFV RNA (lane 1, A-H) or MV RNA (lane 2, A-E, G, and H). Probe F was not hybridized to MV RNA. Lane 5 in D, G, and H shows a longer exposure of the respective SFV lane. For A-H, G, and H, hybridization controls containing no viral RNA are shown in lane 3 while lane 4 shows the DNA fragment used for hybridization. Controls for probe F are shown in lanes 2 and 3. The lower molecular weight fragment in lane 4 of panel H is a contaminant of the probe. Early viral RNA does not hybridize to this contaminant. Horizontal lines to the left of each autoradiogram indicate the position of the observed Sl protected fragments. The sizes of these fragments are indicated in (A) for each probe.

CABIRAC

ET AL.

B A 1234

12!45

FIG. 5. Sl nuclease mapping of immediate early RNA transcribed from the internal recombination junction regions. (A) The boxed DdeI site shown is the last identified site in the left terminal region of the SFV and MV genomes that is shared. Sequences to the right of this site in MV are derived from rabbit myxoma virus. The boxed KpnI site is the last identified site in the right terminal

TRANSCRIPTIONAL

MAPPING

transcribed from the MV left terminal region described here and six unique and eight read-through transcripts from the right terminal region. Most of the unique transcripts are arranged in a densely packed head to tail fashion. However, the 650-nt SFV species has its initiation point located within the sequences of the 1600nt species. It should be noted that one RNA classified as a read-through transcript, the SFV and MV 3170~nt species, appears to have its own unique initiation point. The group of transcripts in the far right portion of the SFV map, the 2950-, 2570-, 2160-, and 1710-nt RNAs, are shown as being initiated at a point outside the SFV inverted terminal repeat region indicated in the figure (SFV ITR). These transcriptional initiation points were located by Sl mapping described above using SFV DNA from the right terminal region of the genome. The SFV right terminal sequences and not the left terminal sequences were used during construction of the Sl probes because the SFV sequences in the MV left terminal region fall well within the SFV inverted repeat while those in the MV right terminal region extend up to and perhaps past the SFV inverted terminal repeat sequences. As mentioned above, further work is needed to define the exact recombinational junction in the right terminal region of the MV genome. MV transcripts which are composed of SFV and myxoma virus sequences are shown in the map for both the left and right terminal regions of the MV genome (indicated in the figure by open or broken lines). The junction of SFV/myxoma se-

OF SFV AND

MV

67

quences of these MV RNAs is located in the region around the ClaI site. The two MV transcripts, 1600 and 2470 nt long, which are transcribed from the unique initiation point located at the DdeI site are also shown in the map of the left MV terminal region. These two RNAs correspond to the 1710- and 2580-nt SFV species shown in the SFV map. Initially during the hybridization analysis discussed above, the unique 1600- and 2470-nt MV RNAs were not readily detected. The 1600-nt species was obscured by the intense signal in this region of the autoradiogram (see Fig. 2B, probe E). Shorter exposure of the nitrocellulose revealed the additional RNA species in the MV lane and therefore it is indicated in Table 1. The signal from the 2470-nt RNA cannot be resolved from that of the 2580-nt RNA. However, using MV probes specific for sequences to the left and right of the DdeI site, we were able to confirm the existence of this MV RNA. The EOO- and 2470~nt MV RNAs should contain only SFV sequences and therefore represent truncated versions of the 1710- and 2580-nt SFV RNAs. However, DNA sequencing of the exact SFV/myxoma virus sequence junction and more detailed Sl mapping is needed to determine the 5’ sequence of these MV RNAs. The 1100~nt MV RNA which hybridized to probe I and the 600-nt MV RNA which hybridized to probe K (shown in Fig. 2B) are not shown since these appear to be transcribed from myxoma virus sequences. Also not shown in Fig. 2A is the unique 3300-nt SFV or the unique 3000-nt MV RNA detected with probes D, E, F, and G

region of the SFV and MV genomes that is shared. The dotted line indicates that the top SFV SmaI/ BglI fragment is contiguous with the BglII/SmaI fragment. Only the left MV terminal region map is shown since no Sl probes were constructed using DNA from the right MV terminal region. MV RNAs transcribed from the right terminal region were mapped using SFV right terminal DNA fragments. Probes are as described in Figs. 3 and 4. The unique 0.3-kb MV protected fragment derived from probe F is indicated. (B) Autoradiograms of dried gels of Sl protected fragments. Letters correspond to the probes shown in (A). Sl protected fragments were derived from SFV RNA (lane 1, A-F), MV RNA (lane 2, A-F), or rabbit myxoma virus RNA (lane 3, B and F). Hybridization controls containing no viral RNA are shown in lane 3, (A, C, D, and F) or lane 4 (B and E). The far right lanes show DNA fragments used for hybridization (fragments in lane 4 of A and C are not shown due to their position in the gel relative to the Sl protected fragments). Horizontal lines to the left of each autoradiogram indicate the position of the observed Sl protected fragments. The sizes of these fragments are indicated in (A) for each probe.

68

CABIRAC

shown in Fig. 2B. The exact location of the termini of these RNAs remains to be determined, but the fact that they are present in very low abundance has made it hard to do SO. The group of RNAs shown at the far right end of the MV right terminal region map, the 2950-, 2570-, 2160-, 1710-, 810-, and 425-nt species, are transcribed only from this region of the MV genome since these SFV sequences are absent in the MV left terminal region.

ET AL.

ripoxviruses. The significance of the difference between the amount of transcription from the terminal regions of vaccinia and SFV or MV is not clear at this time. Viable vaccinia and SFV mutants that have deletions in the terminal regions of their genomes have been isolated (Moss et aL, 1981; Strayer et al, 1984; Cabirac et al, 1985), which suggests that at least some polypeptides encoded from this region of these genomes are not required for replication in vitro. It is not yet possible to assign a function to any of the polypeptides DISCUSSION which may be encoded by the SFV or MV We report here the transcriptional map- mRNAs mapped here. ping of early RNA from two LeporipoxviAside from comparing transcription of ruses, SFV and MV. MV is a recently iso- the Leporipoxvirus genome to that of the lated recombinant poxvirus derived from Orthopoxviruses, the data presented in this SFV and rabbit myxoma virus. The RNAs work will be used to determine the role of detected are transcribed from the regions the SFV sequences during the replication of SFV sequences in the MV genome and of MV. MV’s unique biological characterthe corresponding regions of the SFV ge- istics may be attributable to the addition nome. SFV and MV transcription in this of SFV-specific gene products and/or to the region of the genome is entirely from one deletion from MV of rabbit myxoma virus strand of DNA. These transcripts map in gene products. In addition, we will try to determine if any polypeptides encoded a head to tail array. Thus, approximately 50% of the total available sequences in the from the terminal regions of the SFV and SFV inverted terminal repeat region de- MV genomes are responsible for the tuscribed in this work and the corresponding morigenic potential of these two viruses. region in MV are transcribed. The gaps be- At present it is not known if any gene products encoded from the terminal retween the 3’ and 5’ ends of these transcripts average about 100 bp in length although, gions of SFV, MV, or rabbit myxoma virus as described above, some unique tran- are important in tumor induction. It is inscripts have their 3’ or 5’ ends located in- teresting to note, however, that: (1) the ternally with respect to another unique vaccinia 19K polypeptide encoded within transcript. This is in contrast to the pat- the inverted terminal repeat region is tern of transcription seen in the approxi- highly homologous to epidermal growth mately 10 kb of sequences in the inverted factor and transforming growth factor I terminal repeats of vaccinia. The four (Reisner, 1985) and (2) that episomal plasmRNAs mapped to this region of the vac- mid DNA has been identified in the rabbit cinia genome account for transcription of cell nucleus which has homology to the in25% of the available sequences. These verted terminal repeat region of SFV RNAs are distributed such that there are (McFadden et d, 1984). If a protein similar large gaps between the genes encoding the to the vaccinia 19K polypeptide is encoded transcripts. They are also transcribed from from the inverted terminal repeat region both strands of viral DNA (Cooper et al., of the SFV or MV genome, it will be important to determine if such a protein plays 1981). However, the pattern of transcription described here for SFV and MV has a role in tumor induction by these two Lebeen described for other regions of the poripoxviruses. Also, the relationship bevaccinia genome (Cooper et al, 1981; Mahr tween the sequences in the terminal reand Roberts, 1984a; Morgan and Roberts, gions of the SFV genome and the endoge1984; Golini and Kates, 1984) and therefore nous host cell plasmid DNA may be does not appear to be unique to the Lepo- important in tumorigenesis.

TRANSCRIPTIONAL

MAPPING

ACKNOWLEDGMENTS We thank Dr. M. Neal Waxham and Dr. Brian Knoll for critical reading of this manuscript and Jackie Fagan and Ann Rose for preparation of the manuscript. This work was supported in part by a pre-doctoral fellowship from the National Cancer Cytology Center and by American Cancer Society Grant IM358. REFERENCES BAJSZAR,G., WITTEK, R., WEIN, J. P., and Moss, B. (1983). Vaccinia virus thymidine kinase and neighboring genes: mRNAs and polypeptides of wild type virus and putative nonsense mutants. J. ViroL 45, 62-72. BELLE ISLE, H., VENKATESAN,S., and Moss, B. (1981). Cell-free translation of early and late mRNAs selected by hybridization to cloned DNA fragments derived from the left 14 million to ‘72million daltons of the vaccinia virus genome. Virology 112,306-317. BLOCK,W., UPTON,C., and MCFADDEN,G. (1984). Tumorigenic poxviruses: Genomic organization of malignant rabbit virus, a recombinant between Shope flbroma virus and myxoma virus. Virology 140,113-124. BOONE,R. F., and MOSS,B. (1978).Sequence complexity and relative abundance of vaccinia mRNA is synthesized in vivo and in vitro. J. Viral 26,554-569. CARIRAC,G. F., STRAYER,D. S., SELL, S., and LEIBOWITZ,J. L. (1985). Characterization, molecular cloning, and physical mapping of the Shope fibroma genome. Virology 143,663-670. COOPER,J. A., WI?TEK, R., and Moss, B. (1981). Extension of the transcriptional and translational map of the left end of the vaccinia virus genome to 21 kilobase pairs. J. Viral 39,733-754. GOLINI, F., and KATES, J. R. (1984). Transcriptional and translational analysis of a strongly expressed early region of the vaccinia virus genome. J. vird 49,459-470. KAVERIN, N. V., VARICH, N. L., SURGAY,V. V., and CHERNOS,V. I. (1975). A quantitative estimation of poxvirus genome fraction transcribed as early and late mRNA. Virobgy 65,112-119. MAHR, A., and ROBERTS,B. E. (1984a). Arrangement of late RNAs transcribed from a 7.1 kilobase EcoRI vaccinia virus DNA fragment. J. Viral 49,510-520. MAHR, A., and ROBERTS,B. E. (1984b). Organization of six early transcripts synthesized from a vaccinia EcoRI DNA fragment. J. Viral. 49,497-509. MCDONNELL,M. W., SIMON,M. N., and STUDIER,F. W. (1977).Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J. Mel Biol 110,119-146. MCFADDEN, G., DELANGE, A. M., UPTON, C., BLOCK,

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W., and MACAULAY,C. (1984). “Shope fibroma virus: A model for tumorigenic poxviruses.” Fifth International Poxvirus/Iridovirus Meeting. (Abstract) MCMASTER, G. K., and CARMICHAEL,G. G. (1977). Analysis of single and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine organge. Proc. Nat1 Acad Sci. 74,43354838.

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THOMAS,P. S. (1980). Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Nat1 Acad Sci. 77,5201-5205. WILLS, A., DELANGE,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. WIT~EK, R., COOPER,J. A., BARBOSA,E., and Moss, B. (1980). Expression of the vaccinia virus genome: Analysis and mapping of mRNA encoded within the inverted terminal repetition. Cell 21,487-493. WITTEK, R., HANGGI, M., and HILLER, G. (1984). Mapping of a gene coding for a major late structural polypeptide on the vaccinia virus genome. J. viral. 49,371-378.