JOURNAL OF VIROLOGY, Apr. 1990, p. 1839-1843 0022-538X/90/041839-05$02.00/0
Vol. 64, No. 4
Pattern of Transcription of the Genome of Equine Infectious Anemia Virus SILVI NOIMAN,1 ABRAHAM YANIV,1 LEVANA SHERMAN,1 STEVEN R. TRONICK,2* AND ARNONA GAZIT' Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel,1 and Laboratory of Cellular and Molecular Biology, National Cancer Institute, 9000 Rockville Pike (37-1E24), Bethesda,
Maryland 208922 Received 15 August 1989/Accepted 13 December 1989
The pattern of expression of the equine infectious anemia virus (EIAV) genome in a persistently infected canine cell line was determined. Five EIAV-specific transcripts (8.2, 5.0, 4.0, 2, and 1.8 kilobases [kb]) were detected by using subgenomic restriction enzyme fragments of EIAV DNA and EIAV-specific oligonucleotides as probes. The 8.2-kb mRNA could be shown to represent viral genomic RNA, whereas the smaller transcripts were generated by splicing events. Evidence was obtained that indicated that each subgenomic RNA species shared a common 5'-splice donor. The 5.0-kb mRNA was found to be expressed at relatively low levels, was difficult to detect consistently, and appeared to be generated by a single splicing event which linked the 5' exon to the 3' region of pol. The 4.0-kb transcript was concluded to be the env mRNA on the basis of its hybridization pattern with the various probes and its abundance. The 2-kb species was found to be multiply spliced and was encoded by sequences derived from orf2 but was not detected by probes representing 3'-envl3'-orf sequences. The 1.8-kb species was shown to consist of sequences representing orl, part of oaJ2, and the 3'-orflenv and may represent the message for the EIAV trans-activator gene. Equine infectious anemia (EIA) is a chronic recrudescent disease of horses characterized by symptoms of fever, anemia, glomerulonephritis, and uremia. Extensive replication of the etiologic agent, equine infectious anemia virus (EIAV), occurs in target macrophages (7, 8). Infection of horses with EIAV can result in the rapid appearance of symptoms, and the afflicted animals may either die shortly thereafter or survive but suffer periodic clinical episodes (7, 8). The final outcome of recurrent disease may be fatal; alternatively, the animals may survive but become chronic carriers of EIAV. The rapid onset and cyclical nature of EIA thus make it different from other lentivirus infections which are characterized by a chronic degenerative and slowly progressive disease course (22). Lentiviral infections are also characterized by virus persistence and spread in the face of a significant host immune response (8, 22). Although the basis for persistence is unknown, one possible mechanism may involve restriction of viral gene expression which could enable virus-infected cells to evade elimination by the immune system. The genomes of lentiviruses have been shown to encode regulatory proteins that are thought to play a role in these complex virus-host interactions (10, 22, 35). The products of the tat, rev, and nef genes of human immunodeficiency virus (HIV) have been shown to regulate viral gene expression (1-4, 10, 15-17, 19, 25, 30, 39, 41). Other HIV genes encode proteins such as vif (24, 27, 29, 40, 43), which affects the yields of infectious virus; vpx (21, 23, 49), which influences the ability of the virus to replicate in peripheral blood lymphocytes (20); and vpu, which may be required for efficient virus replication and virion maturation (44). The function of another gene, vpr (47), is not known. The EIAV genome appears to be less complex than those of other lentiviruses in that only three open reading frames other than gag, pol, and env have been identified. One of these short open reading frames, located between pol and env, encodes *
Corresponding author.
trans activator (14, 26, 38). In the present study, as one approach towards determining the products of the EIAV genome that regulate its expression, we have determined the pattern of EIAV-specific RNAs in a chronically infected cell line. Five species of EIAV-specific mRNAs were detected and included genomic RNA (8.2-kilobases [kb]), the env mRNA (4.0 kb) and three multiply spliced subgenomic transcripts 5.0, 2.0, and 1.8 kb in size. For these studies, a canine thymus cell line, Cf2Th (ATCC CRL 1430), was grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum. Total cellular RNA was isolated by the guanidine isothiocyanate method, pelleted through cesium chloride (9), and further purified by extraction with phenol-chloroform. Poly(A)+ RNA was then selected by oligo(dT)-cellulose affinity chromatography (5), and 5-,ug samples were electrophoresed in 1% agaroseformaldehyde horizontal gels at 150 V (28). The 28S and 18S rRNAs from a poly(A)- RNA sample as well as commercially available standards (Bethesda Research Laboratories, Bethesda, Md.) were used as size standards. Following electrophoresis, gels were treated with 50 mM NaOH for 30 min, then with 0.1 M Tris hydrochloride (pH 7.0) for 30 min, and finally with 1Ox SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 30 min. The RNAs were transferred to GeneScreen Plus Membranes (Du Pont, Wilmington, Del.) in 1Ox SSC for 18 h. After being washed with 2x SSC at room temperature, the membranes were hybridized with nick-translated DNA probes or riboprobes generated from EIAV subclones or with end-labeled oligonucleotide probes. Membranes were prehybridized for 4 h at 40°C in a solution containing 50% formamide, 5x SSC, 4x Denhardt solution (lx Denhardt solution is 0.02% polyvinylpyrrolidine, 0.02% Ficoll, 0.02% bovine serum albumin), 0.5% sodium dodecyl sulfate (SDS), 0.1% sodium PP1, 200 p.g of single-stranded salmon sperm DNA per ml, and 150 ,ug of tRNA per ml. Hybridization was carried out for 24 h with 5 x 106 cpm of 32P-labeled nick-translated DNA or riboprobes per ml at 42°C. After hybridization, membranes were a
1839
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J. VIROL.
NOTES
I
I
I
I
et3O
4 000
2000 I
1 Iswinulilillsl [1s1
I
Kb
G
I
I
--
I
env LTR
Dal puf
I
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a]^
I LTR
1.
-
iw
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A
12
PROBES de,
s
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-.
, l o4-.
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TRANSCRIPTS
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8
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40 I
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FIG. 1. Analysis of EIAV-specific transcripts. A schematic representation of the EIAV genome (26, 37, 42) is displayed at the top of the figure. The hybridization probes are depicted underneath as double-headed arrows (A through D, restriction fragments or riboprobes as described in the text and Table 1) or by vertical bars (synthetic oligonucleotides). Structures of the transcripts as deduced from this experiment are shown (solid lines), and their sizes (in kilobases) are indicated to the right. Representative Northern (RNA) blots are shown at the bottom. The numbers above the lanes refer to the probe used. The ensure the validity of lack of detection of a transcript by the various probes, the blots were stripped of probe and then rehybridized with probe C, which recognized all 5 RNAs. Differences in migration of the same RNA species in some of the lanes are because some RNA preparations were run in different gels.
washed twice for 5 min at room temperature in 2x SSC, twice for 30 min at 60°C in 2x SSC-1% SDS, and twice for 30 min at room temperature in 0.1 x SSC. For hybridization with the oligonucleotide probes, membranes were prehybridized for 4 h at 42°C in a solution containing 6x SSC, lx Denhardt solution, 0.5% SDS, 250 ,ug of single-stranded salmon sperm DNA per ml, 200 ,ug of tRNA per ml, and 0.05% sodium PPi, followed by hybridization at 42°C with the same solution containing 5 x 106 cpm of 32P-end-labeled oligonucleotide probe. After hybridization, filters were washed twice for 20 min at room temperature with 2x SSC-0.5% SDS and then twice at 50°C in 1 x SSC-1% SDS-0.5% sodium PP1 and autoradiographed at -70°C with intensifying screens. A probe representing the entire EIAV genome (probe A) (Fig. 1, Table 1) was prepared from EIAV clone p1369 (26, 48) by digestion with MIuI, which cuts once within each long terminal repeat (LTR). Following gel electrophoresis and recovery by electroelution (31) or DEAE-cellulose chromatography (46), the isolated fragment was nick translated with [a-32P]dCTP. To increase the sensitivity of the hybridization assays, we made use of single-stranded, uniformly labeled antisense DNA or RNA probes. Appropriate regions of EIAV 1369 DNA were subcloned into pGEM (probes B and C) or M13 (probe D) vectors (Fig. 1, Table 1) in an orientation that would allow labeling of the antisense strand. To
TABLE 1. Summary of hybridization results Probe
Subcloned probes A B C D
Sequence
8.2 kb
mRNA present 2.0 5.0 4.0 kb kb kb
1.8 kb
157-8065 3600-4460 4890-5338 5775-6511
+ + + +
+
+
+
+
+ + +
+ +
+ +
+
430-459 460-489 3255-3284 3510-3539 3540-3569 5095-5124 5153-5182 5176-5205 5212-5240 5283-5312 5312-5341 5435-5464 7201-7233 7421-7450 7750-7779
+ + +
+
+
+
+
+ + + + + + + + + +
+ + + + + + + + +
Oligonucleotide probes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
+ + + + + + + + + + + +
+ + +
+
+ + + +
NOTES
VOL. 64, 1990 SA 15080 15095(6) G TGTACCATTAACCA P
V
L
T
R
T
L
X
L
I
X P N end pol
TGTTGQ
T *
V
L
L
Q
orf1 -> SA
15153(7) GGAAGCAAGACCCAACTA_
SA
SDJ15176(8)
I
CTC S L
G
I
_T N X Q R L X
A
I
R
A
P
N
I C
Y
Q
L
C
T
L
R
15211(9)
TGATTACCTCQGATGCTTQTT_r Y
D
L
D
A
S
L
R
X
K
15283(10)
SD
TAT CCAACAAGGALGACAACCTCAATATTTGTTATALGGTTTG^AThATG Q Q G R Q P Q Y L L * MG L F orf2 ->
45312(11)
AAI_sGG_CCA K G
G
V
T
W 8 AASS M V S I A
S F
CAGGGG
MG Y
G
G
G
S
I
Q P
G G
Z
G
env ->
AATCTCAACCCCTATTACCCAACAGTCAGAAAAATCTAAGTGTGAGGAGAACAC S Q P L L P N S Q K N L S V R R T Q I 8 T P I T Q Q S IX S X C Z I N T
45435(12) AATGTTTCAACCTTATTGTTATAATAATGAAGTAAGCA C
Q
F
M
N
J
L
P
I
Y
I
V
C
Y
I
N
A M T V R T A W Q N R N D S K N S M A I S
GAAaAAGraAGAGACCAAGAACTGA.AAGaAGAATCTTAAAGaAGAAA R
X
I T
Q
X
I
A
R
D
X
Q
X
I
*
M N
L
XI I
S
X
I Z
K
FIG. 2. Nucleotide sequence, predicted open reading frames, and putative splicing sites in the region between pol and env. The starting positions of oligonucleotide probes (underlined) are shown along with the probe number which follows in parenthesis. The respective 3' and 5' ends of the predicted pol and env open reading frames are shown as well as those of orfl and orf2. Putative splice donor (SD) and splice acceptor sites (SA) are also shown.
label specific regions, pGEM plasmids were linearized by digesting at an appropriate restriction site within the EIAV insert (Table 1). Riboprobes were synthesized from pGEM subclones by using SP6 polymerase and [a-32P]UTP (3,000 Ci/mmol). Single-stranded DNA probes were prepared from M13 subclones by using DNA polymerase (Klenow fragment) and [a-32P]dCTP (3,000 Ci/mmol). Oligonucleotides were synthesized by using a Biosearch Model 8700 synthesizer and were end labeled with [_y-32P]ATP (3,000 Ci/mmol) and T4 polynucleotide kinase (6). The location of each probe within the EIAV genome is indicated in Table 1 and Fig. 1. Nucleotide sequence analysis of EIAV clones revealed the presence of open reading frames predicted to encode gag, pol, and env gene products (26, 37, 42): two short open reading frames between pol and env, designated here as orf1 and ocr2 (nucleotides 5123 to 5271 and nucleotides 5283 to 5480, respectively) (26, 37), and a third, designated 3'-orf, that overlaps the 3' end of env (nucleotides 7233 to 7638) (26, 37, 42). (orfl, or2, and 3'-orf have been referred to as Si, S2, and S3, respectively [37].) Nucleotide numbering from positions 1 to 5345 is based on the sequence published by our laboratory (26). In this sequence, there are four G residues beginning at position 5345, whereas in the sequence published by Rushlow et al. (37), there are five. This resulted in differences between the two sequences in the length of or12 and the start of the env open reading frame. The nucleotide sequence of an infectious EIAV clone (unpublished data) in this region is identical to that of Rushlow et al. (37). Thus, nucleotide numbering and translation of the sequence, after position 5345, is based on the addition of a fifth G (Fig. 2).
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Polyadenylated RNAs isolated from canine thymus cells persistently infected with EIAV and from uninfected cells were first analyzed by using a probe representing the entire EIAV genome (Fig. 1, probe A). Five RNA species were detected only in infected cells, and their sizes were 8.2, 5.0, 4.0, 2.0, and 1.8 kb. The 8.2-kb RNA species hybridized with all probes used (Table 1, Fig. 1), strongly suggesting that it represented the viral genomic RNA. Retroviral subgenomic transcripts all initiate within the 5' LTR at the first nucleotide of R. A splice donor for the first exon is generally located just downstream of the 3' end of the 5' LTR. On the basis of nucleotide sequence data (26, 42), a consensus splice donor is located at nucleotide 459. To test whether this site might serve as the splice donor for the various species of EIAV mRNAs, two oligonucleotides were synthesized, one which covered the putative consensus splice donor (nucleotides 430 to 459) and one (probe 2, nucleotides 460 to 489) which covered the 5' edge of the putative first intron. Probe 1 hybridized with all mRNA species, whereas probe 2 hybridized only to the 8.2-kb genomic mRNA (Fig. 1). These data indicate that nucleotides 1 to 459 likely form the 5' end of each EIAV-specific mRNA. The 5.0-kb transcript, which was difficult to detect consistently, was less abundant than the other species, but in some experiments it gave a clear signal with the genomic probe (A) and some oligonucleotide probes, probe 1 in particular. Thus, the 5.0-kb mRNA hybridized with probes representing part of the pol gene (B) and the middle part of the genome (probes C, 6, 7, 8, 9, and 10) and with probes representing the env region (probes 11, 12, D, 13, 14, and 15 [Table 1, Fig. 1]). Because of the problems cited above, conclusions regarding the generation of this species must be made with caution, but the results would indicate that a single splicing event generates the 5.0-kb transcript in which the leader joins a long exon starting at the 3' part of the pol gene and extends continuously to the 3' LTR. The 4.0-kb mRNA was not detected by probes representing sequences upstream of position 5153 (with the exception of probes 1 and A) but readily hybridized to all probes for downstream sequences (Table 1, Fig. 1). Since the 4.0-kb mRNA hybridized to probe 7 (nucleotides 5153 to 5182), but not to probe 6 (nucleotides 5095 to 5124), this suggested that the splice acceptor site for this mRNA may be located between nucleotides 5124 and 5182, where there is a single splice acceptor site at position 5134, upstream of the putative env initiation codon (position 5312) (Fig. 2). The 2.0-kb mRNA hybridized with probes representing or12 (C, 10, and 11) and very faintly with the env probe D but not with probes derived from sequences upstream (probes 3, 4, 5, B, 6, 7, 8, and 9) or downstream (probes 12, 13, 14, 15) (Fig. 1 and 2). These data suggest that this mRNA is generated by multiple splicing events. The first splicing event links the 5' leader to or12 sequences (probes 10 and 11), and since probe 12 did not hybridize, an intron may be present between position 5435 (probe 12) and probe D sequences (nucleotides 5775 to 6511) (Fig. 2). The next splice would link probe D sequences to the LTR (data not shown). The 1.8-kb mRNA hybridized with 3' pol (probe C), with orfl and 3' sequences of orf2 (probes 7, 8, 9, and 12), and with 3' env probes (probes 14 and 15) (Table 1, Fig. 1). There was no detectable hybridization with probes representing the pol gene (probes 3, 4, 5, B, and 6), with the probe representing the 5' region of or12 (probes 10 and 11), or with probes representing the middle part of the env gene (probes D and
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NOTES
13). Thus, this mRNA may be triply spliced and could direct the synthesis of the product encoded by orfl (Fig. 1 and 2). We have demonstrated here that the pattern of transcription of the EIAV genome is similar to those of other lentiviruses. For example, HIV type 1 (HIV-1) directs the synthesis of 9.3-kb genomic and 4.3-kb env mRNAs as well as 1.8-kb multiply spliced mRNAs that encode regulatory proteins (4, 34, 36). Additional mRNAs of 5.5, 5.0, (36) and 2.0 (4) kb have also been reported. In visna virus-infected cells, the genomic (9.4-kb) RNA is accompanied by singly spliced 5.0- and 4.3-kb (doublet) transcripts in addition to doubly spliced mRNAs of 1.8 and 1.5 kb (12). One of the 4.3-kb species is likely the env message (12). Similar observations (species of 9.4, 4.8, 4.3, 3.7 [env], 1.6, and 1.2 kb) were made by others (45). The 1.8 (1.6)-kb species was shown to encode a trans-activating protein and a putative rev product as well (11, 18, 32). Each of the EIAV transcripts was found to share the same 5' exon, whose splice donor site probably corresponds to a consensus sequence upstream of the start of the gag open reading frame at position 459 (26, 42). The hybridization data indicated that the two largest mRNAs, approximately 5.0 and 4.0 kb in size, were spliced once, whereas the smaller transcripts (2.0 and 1.8 kb) were multiply spliced. The 4.0-kb mRNA likely represents the env transcript, on the basis of its pattern of hybridization and relatively high level of expression. A 5.0-kb mRNA was synthesized at low levels and was found to be formed by splicing of the common 5' exon to 3' pol sequences. The hybridization data suggested that it was continuously transcribed from this point to the polyadenylation site. The identity of the gene that encodes this mRNA is unknown. As noted above, 5-kb mRNAs have been detected in HIV-1 (36) and visna virus (12, 45) infected cells, and it was suggested that they encode the vif product. Interestingly, the EIAV genomes characterized to date lack such an open reading frame. The 5-kb transcript found here in EIAV-infected cells might represent the equivalent of vif with its exon(s) embedded within the 3' terminus of pol and in env rather than between pol and env as in other lentiviruses. Efforts are under way to isolate the cDNA for this message in order to characterize it in detail. The 2-kb mRNA hybridized with or12- but not orflspecific probes, suggesting that it contributes sequences to the orf2 product. The predicted amino acid sequence of orf2 shows no significant similarity to that of any known protein. The position of orf2 suggests that it could be analogous to rev. A regulatory role for the 2-kb mRNA is suggested by our findings that it is preferentially expressed at high levels early after infection (S. Noiman, A. Gazit, 0. Tori, L. Sherman, T. Miki, S. R. Tronick, and A. Yaniv, in press). Work is under way to examine the effects of orf2 mutations on gene expression in cells transfected with an infectious EIAV clone. We previously speculated (26) that orfl might encode the tat gene of EIAV (13, 38) on the basis of a short stretch of amino acid sequence similarity to a highly conserved region of the HIV-1 tat gene. Our recent studies using in vitro mutagenesis, cDNA cloning, and antisense oligonucleotide inhibition of tat activity support this conclusion (Gazit et al., submitted). Since all of the orfl-specific probes hybridized with the 1.8-kb but not with the 2.0-kb species, we propose that the 1.8-kb mRNA encodes tat. The 3' orf lacks an initiation codon but is preceded by a consensus splice acceptor sequence (33). Thus, it could provide a 3' tat coding exon or 3' untranslated sequences, depending upon the upstream splice donor(s). Alternatively, it may also contrib-
ute sequences to another protein. Thus, the 1.8-kb band might consist of more than one species. We are very grateful to S. Aaronson for support.
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35. Peterlin, B. M., and P. A. Luciw. 1988. Molecular biology of HIV. AIDS 2:S29-S40. 36. Rabson, A. B., D. F. Daugherty, S. Venkatesan, K. E. Boulukos, S. I. Benn, T. M. Folks, P. Feorino, and M. A. Martin. 1985. Transcription of novel open reading frames of AIDS retrovirus during infection of lymphocytes. Science 229:1388-1390. 37. Rushlow, K., K. Alse, G. Stiegler, S. L. Payne, R. C. Montelaro, and C. J. Issel. 1986. Lentivirus genomic organization:the complete nucleotide sequence of the em'l gene region of equine infectious anemia virus. Virology 155:309-321. 38. Sherman, L., A. Gazit, A. Yaniv, T. Kawakami, J. E. Dahlberg, and S. R. Tronick. 1988. Localization of sequences responsible for tri-ans-activation of the equine infectious anemia virus long terminal repeat. J. Virol. 62:120-126. 39. Sodroski, J., W. C. Goh, C. Rosen, A. Dayton, E. Terwilliger, and W. Haseltine. 1986. A second post-transcriptional transactivator gene required for HTLV-III replication. Nature (London) 321:412-417. 40. Sodroski, J., W. C. Goh, C. Rosen, A. Tartar, D. Portetelle, A. Burny, and W. Haseltine. 1986. Replicative and cytopathic potential of HTLV-III/LAV with sor gene deletions. Science 231:1549-1553. 41. Sodroski, J., R. Patarca, C. Rosen, F. Wong-Staal, and W. Haseltine. 1985. Location of the tri-ans-activating region on the genome of human T-cell lymphotropic virus type 111. Science 229:74-77. 42. Stephens, R. M., J. W. Casey, and N. R. Rice. 1986. Equine infectious anemia virus gag and pol genes: relatedness to visna and AIDS virus. Science 231:589-594. 43. Strebel, K., D. Daugherty, K. Clouse, D. Cohen, T. Folks, and M. A. Martin. 1987. The HIV 'A' (sor) gene product is essential for virus infectivity. Nature (London) 328:728-730. 44. Strebel, K., T. Klimkait, and M. A. Martin. 1988. A novel gene of HIV-1. vpii, and its 16-kilodalton product. Sciencc 241:
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Vol. 64, No. 4
Pattern of Transcription of the Genome of Equine Infectious Anemia Virus SILVI NOIMAN,1 ABRAHAM YANIV,1 LEVANA SHERMAN,1 STEVEN R. TRONICK,2* AND ARNONA GAZIT' Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel,1 and Laboratory of Cellular and Molecular Biology, National Cancer Institute, 9000 Rockville Pike (37-1E24), Bethesda,
Maryland 208922 Received 15 August 1989/Accepted 13 December 1989
The pattern of expression of the equine infectious anemia virus (EIAV) genome in a persistently infected canine cell line was determined. Five EIAV-specific transcripts (8.2, 5.0, 4.0, 2, and 1.8 kilobases [kb]) were detected by using subgenomic restriction enzyme fragments of EIAV DNA and EIAV-specific oligonucleotides as probes. The 8.2-kb mRNA could be shown to represent viral genomic RNA, whereas the smaller transcripts were generated by splicing events. Evidence was obtained that indicated that each subgenomic RNA species shared a common 5'-splice donor. The 5.0-kb mRNA was found to be expressed at relatively low levels, was difficult to detect consistently, and appeared to be generated by a single splicing event which linked the 5' exon to the 3' region of pol. The 4.0-kb transcript was concluded to be the env mRNA on the basis of its hybridization pattern with the various probes and its abundance. The 2-kb species was found to be multiply spliced and was encoded by sequences derived from orf2 but was not detected by probes representing 3'-envl3'-orf sequences. The 1.8-kb species was shown to consist of sequences representing orl, part of oaJ2, and the 3'-orflenv and may represent the message for the EIAV trans-activator gene. Equine infectious anemia (EIA) is a chronic recrudescent disease of horses characterized by symptoms of fever, anemia, glomerulonephritis, and uremia. Extensive replication of the etiologic agent, equine infectious anemia virus (EIAV), occurs in target macrophages (7, 8). Infection of horses with EIAV can result in the rapid appearance of symptoms, and the afflicted animals may either die shortly thereafter or survive but suffer periodic clinical episodes (7, 8). The final outcome of recurrent disease may be fatal; alternatively, the animals may survive but become chronic carriers of EIAV. The rapid onset and cyclical nature of EIA thus make it different from other lentivirus infections which are characterized by a chronic degenerative and slowly progressive disease course (22). Lentiviral infections are also characterized by virus persistence and spread in the face of a significant host immune response (8, 22). Although the basis for persistence is unknown, one possible mechanism may involve restriction of viral gene expression which could enable virus-infected cells to evade elimination by the immune system. The genomes of lentiviruses have been shown to encode regulatory proteins that are thought to play a role in these complex virus-host interactions (10, 22, 35). The products of the tat, rev, and nef genes of human immunodeficiency virus (HIV) have been shown to regulate viral gene expression (1-4, 10, 15-17, 19, 25, 30, 39, 41). Other HIV genes encode proteins such as vif (24, 27, 29, 40, 43), which affects the yields of infectious virus; vpx (21, 23, 49), which influences the ability of the virus to replicate in peripheral blood lymphocytes (20); and vpu, which may be required for efficient virus replication and virion maturation (44). The function of another gene, vpr (47), is not known. The EIAV genome appears to be less complex than those of other lentiviruses in that only three open reading frames other than gag, pol, and env have been identified. One of these short open reading frames, located between pol and env, encodes *
Corresponding author.
trans activator (14, 26, 38). In the present study, as one approach towards determining the products of the EIAV genome that regulate its expression, we have determined the pattern of EIAV-specific RNAs in a chronically infected cell line. Five species of EIAV-specific mRNAs were detected and included genomic RNA (8.2-kilobases [kb]), the env mRNA (4.0 kb) and three multiply spliced subgenomic transcripts 5.0, 2.0, and 1.8 kb in size. For these studies, a canine thymus cell line, Cf2Th (ATCC CRL 1430), was grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum. Total cellular RNA was isolated by the guanidine isothiocyanate method, pelleted through cesium chloride (9), and further purified by extraction with phenol-chloroform. Poly(A)+ RNA was then selected by oligo(dT)-cellulose affinity chromatography (5), and 5-,ug samples were electrophoresed in 1% agaroseformaldehyde horizontal gels at 150 V (28). The 28S and 18S rRNAs from a poly(A)- RNA sample as well as commercially available standards (Bethesda Research Laboratories, Bethesda, Md.) were used as size standards. Following electrophoresis, gels were treated with 50 mM NaOH for 30 min, then with 0.1 M Tris hydrochloride (pH 7.0) for 30 min, and finally with 1Ox SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 30 min. The RNAs were transferred to GeneScreen Plus Membranes (Du Pont, Wilmington, Del.) in 1Ox SSC for 18 h. After being washed with 2x SSC at room temperature, the membranes were hybridized with nick-translated DNA probes or riboprobes generated from EIAV subclones or with end-labeled oligonucleotide probes. Membranes were prehybridized for 4 h at 40°C in a solution containing 50% formamide, 5x SSC, 4x Denhardt solution (lx Denhardt solution is 0.02% polyvinylpyrrolidine, 0.02% Ficoll, 0.02% bovine serum albumin), 0.5% sodium dodecyl sulfate (SDS), 0.1% sodium PP1, 200 p.g of single-stranded salmon sperm DNA per ml, and 150 ,ug of tRNA per ml. Hybridization was carried out for 24 h with 5 x 106 cpm of 32P-labeled nick-translated DNA or riboprobes per ml at 42°C. After hybridization, membranes were a
1839
1840
J. VIROL.
NOTES
I
I
I
I
et3O
4 000
2000 I
1 Iswinulilillsl [1s1
I
Kb
G
I
I
--
I
env LTR
Dal puf
I
KII
WY L_i,,
a]^
I LTR
1.
-
iw
f>aO
A
12
PROBES de,
s
II 3
-.
, l o4-.
Ma
C
D
11 I
ID
4 5
I __
-
TRANSCRIPTS
_
*.
5
WM
-
V-
'I w
6
7t
0 8,
_" A
i .;
Om
8
9.
40 I
T ...
..
a
d
4 0
-
2.0
-
i
04 "
X 0
i
4s
a
FIG. 1. Analysis of EIAV-specific transcripts. A schematic representation of the EIAV genome (26, 37, 42) is displayed at the top of the figure. The hybridization probes are depicted underneath as double-headed arrows (A through D, restriction fragments or riboprobes as described in the text and Table 1) or by vertical bars (synthetic oligonucleotides). Structures of the transcripts as deduced from this experiment are shown (solid lines), and their sizes (in kilobases) are indicated to the right. Representative Northern (RNA) blots are shown at the bottom. The numbers above the lanes refer to the probe used. The ensure the validity of lack of detection of a transcript by the various probes, the blots were stripped of probe and then rehybridized with probe C, which recognized all 5 RNAs. Differences in migration of the same RNA species in some of the lanes are because some RNA preparations were run in different gels.
washed twice for 5 min at room temperature in 2x SSC, twice for 30 min at 60°C in 2x SSC-1% SDS, and twice for 30 min at room temperature in 0.1 x SSC. For hybridization with the oligonucleotide probes, membranes were prehybridized for 4 h at 42°C in a solution containing 6x SSC, lx Denhardt solution, 0.5% SDS, 250 ,ug of single-stranded salmon sperm DNA per ml, 200 ,ug of tRNA per ml, and 0.05% sodium PPi, followed by hybridization at 42°C with the same solution containing 5 x 106 cpm of 32P-end-labeled oligonucleotide probe. After hybridization, filters were washed twice for 20 min at room temperature with 2x SSC-0.5% SDS and then twice at 50°C in 1 x SSC-1% SDS-0.5% sodium PP1 and autoradiographed at -70°C with intensifying screens. A probe representing the entire EIAV genome (probe A) (Fig. 1, Table 1) was prepared from EIAV clone p1369 (26, 48) by digestion with MIuI, which cuts once within each long terminal repeat (LTR). Following gel electrophoresis and recovery by electroelution (31) or DEAE-cellulose chromatography (46), the isolated fragment was nick translated with [a-32P]dCTP. To increase the sensitivity of the hybridization assays, we made use of single-stranded, uniformly labeled antisense DNA or RNA probes. Appropriate regions of EIAV 1369 DNA were subcloned into pGEM (probes B and C) or M13 (probe D) vectors (Fig. 1, Table 1) in an orientation that would allow labeling of the antisense strand. To
TABLE 1. Summary of hybridization results Probe
Subcloned probes A B C D
Sequence
8.2 kb
mRNA present 2.0 5.0 4.0 kb kb kb
1.8 kb
157-8065 3600-4460 4890-5338 5775-6511
+ + + +
+
+
+
+
+ + +
+ +
+ +
+
430-459 460-489 3255-3284 3510-3539 3540-3569 5095-5124 5153-5182 5176-5205 5212-5240 5283-5312 5312-5341 5435-5464 7201-7233 7421-7450 7750-7779
+ + +
+
+
+
+
+ + + + + + + + + +
+ + + + + + + + +
Oligonucleotide probes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
+ + + + + + + + + + + +
+ + +
+
+ + + +
NOTES
VOL. 64, 1990 SA 15080 15095(6) G TGTACCATTAACCA P
V
L
T
R
T
L
X
L
I
X P N end pol
TGTTGQ
T *
V
L
L
Q
orf1 -> SA
15153(7) GGAAGCAAGACCCAACTA_
SA
SDJ15176(8)
I
CTC S L
G
I
_T N X Q R L X
A
I
R
A
P
N
I C
Y
Q
L
C
T
L
R
15211(9)
TGATTACCTCQGATGCTTQTT_r Y
D
L
D
A
S
L
R
X
K
15283(10)
SD
TAT CCAACAAGGALGACAACCTCAATATTTGTTATALGGTTTG^AThATG Q Q G R Q P Q Y L L * MG L F orf2 ->
45312(11)
AAI_sGG_CCA K G
G
V
T
W 8 AASS M V S I A
S F
CAGGGG
MG Y
G
G
G
S
I
Q P
G G
Z
G
env ->
AATCTCAACCCCTATTACCCAACAGTCAGAAAAATCTAAGTGTGAGGAGAACAC S Q P L L P N S Q K N L S V R R T Q I 8 T P I T Q Q S IX S X C Z I N T
45435(12) AATGTTTCAACCTTATTGTTATAATAATGAAGTAAGCA C
Q
F
M
N
J
L
P
I
Y
I
V
C
Y
I
N
A M T V R T A W Q N R N D S K N S M A I S
GAAaAAGraAGAGACCAAGAACTGA.AAGaAGAATCTTAAAGaAGAAA R
X
I T
Q
X
I
A
R
D
X
Q
X
I
*
M N
L
XI I
S
X
I Z
K
FIG. 2. Nucleotide sequence, predicted open reading frames, and putative splicing sites in the region between pol and env. The starting positions of oligonucleotide probes (underlined) are shown along with the probe number which follows in parenthesis. The respective 3' and 5' ends of the predicted pol and env open reading frames are shown as well as those of orfl and orf2. Putative splice donor (SD) and splice acceptor sites (SA) are also shown.
label specific regions, pGEM plasmids were linearized by digesting at an appropriate restriction site within the EIAV insert (Table 1). Riboprobes were synthesized from pGEM subclones by using SP6 polymerase and [a-32P]UTP (3,000 Ci/mmol). Single-stranded DNA probes were prepared from M13 subclones by using DNA polymerase (Klenow fragment) and [a-32P]dCTP (3,000 Ci/mmol). Oligonucleotides were synthesized by using a Biosearch Model 8700 synthesizer and were end labeled with [_y-32P]ATP (3,000 Ci/mmol) and T4 polynucleotide kinase (6). The location of each probe within the EIAV genome is indicated in Table 1 and Fig. 1. Nucleotide sequence analysis of EIAV clones revealed the presence of open reading frames predicted to encode gag, pol, and env gene products (26, 37, 42): two short open reading frames between pol and env, designated here as orf1 and ocr2 (nucleotides 5123 to 5271 and nucleotides 5283 to 5480, respectively) (26, 37), and a third, designated 3'-orf, that overlaps the 3' end of env (nucleotides 7233 to 7638) (26, 37, 42). (orfl, or2, and 3'-orf have been referred to as Si, S2, and S3, respectively [37].) Nucleotide numbering from positions 1 to 5345 is based on the sequence published by our laboratory (26). In this sequence, there are four G residues beginning at position 5345, whereas in the sequence published by Rushlow et al. (37), there are five. This resulted in differences between the two sequences in the length of or12 and the start of the env open reading frame. The nucleotide sequence of an infectious EIAV clone (unpublished data) in this region is identical to that of Rushlow et al. (37). Thus, nucleotide numbering and translation of the sequence, after position 5345, is based on the addition of a fifth G (Fig. 2).
1841
Polyadenylated RNAs isolated from canine thymus cells persistently infected with EIAV and from uninfected cells were first analyzed by using a probe representing the entire EIAV genome (Fig. 1, probe A). Five RNA species were detected only in infected cells, and their sizes were 8.2, 5.0, 4.0, 2.0, and 1.8 kb. The 8.2-kb RNA species hybridized with all probes used (Table 1, Fig. 1), strongly suggesting that it represented the viral genomic RNA. Retroviral subgenomic transcripts all initiate within the 5' LTR at the first nucleotide of R. A splice donor for the first exon is generally located just downstream of the 3' end of the 5' LTR. On the basis of nucleotide sequence data (26, 42), a consensus splice donor is located at nucleotide 459. To test whether this site might serve as the splice donor for the various species of EIAV mRNAs, two oligonucleotides were synthesized, one which covered the putative consensus splice donor (nucleotides 430 to 459) and one (probe 2, nucleotides 460 to 489) which covered the 5' edge of the putative first intron. Probe 1 hybridized with all mRNA species, whereas probe 2 hybridized only to the 8.2-kb genomic mRNA (Fig. 1). These data indicate that nucleotides 1 to 459 likely form the 5' end of each EIAV-specific mRNA. The 5.0-kb transcript, which was difficult to detect consistently, was less abundant than the other species, but in some experiments it gave a clear signal with the genomic probe (A) and some oligonucleotide probes, probe 1 in particular. Thus, the 5.0-kb mRNA hybridized with probes representing part of the pol gene (B) and the middle part of the genome (probes C, 6, 7, 8, 9, and 10) and with probes representing the env region (probes 11, 12, D, 13, 14, and 15 [Table 1, Fig. 1]). Because of the problems cited above, conclusions regarding the generation of this species must be made with caution, but the results would indicate that a single splicing event generates the 5.0-kb transcript in which the leader joins a long exon starting at the 3' part of the pol gene and extends continuously to the 3' LTR. The 4.0-kb mRNA was not detected by probes representing sequences upstream of position 5153 (with the exception of probes 1 and A) but readily hybridized to all probes for downstream sequences (Table 1, Fig. 1). Since the 4.0-kb mRNA hybridized to probe 7 (nucleotides 5153 to 5182), but not to probe 6 (nucleotides 5095 to 5124), this suggested that the splice acceptor site for this mRNA may be located between nucleotides 5124 and 5182, where there is a single splice acceptor site at position 5134, upstream of the putative env initiation codon (position 5312) (Fig. 2). The 2.0-kb mRNA hybridized with probes representing or12 (C, 10, and 11) and very faintly with the env probe D but not with probes derived from sequences upstream (probes 3, 4, 5, B, 6, 7, 8, and 9) or downstream (probes 12, 13, 14, 15) (Fig. 1 and 2). These data suggest that this mRNA is generated by multiple splicing events. The first splicing event links the 5' leader to or12 sequences (probes 10 and 11), and since probe 12 did not hybridize, an intron may be present between position 5435 (probe 12) and probe D sequences (nucleotides 5775 to 6511) (Fig. 2). The next splice would link probe D sequences to the LTR (data not shown). The 1.8-kb mRNA hybridized with 3' pol (probe C), with orfl and 3' sequences of orf2 (probes 7, 8, 9, and 12), and with 3' env probes (probes 14 and 15) (Table 1, Fig. 1). There was no detectable hybridization with probes representing the pol gene (probes 3, 4, 5, B, and 6), with the probe representing the 5' region of or12 (probes 10 and 11), or with probes representing the middle part of the env gene (probes D and
1842
J. VIROL.
NOTES
13). Thus, this mRNA may be triply spliced and could direct the synthesis of the product encoded by orfl (Fig. 1 and 2). We have demonstrated here that the pattern of transcription of the EIAV genome is similar to those of other lentiviruses. For example, HIV type 1 (HIV-1) directs the synthesis of 9.3-kb genomic and 4.3-kb env mRNAs as well as 1.8-kb multiply spliced mRNAs that encode regulatory proteins (4, 34, 36). Additional mRNAs of 5.5, 5.0, (36) and 2.0 (4) kb have also been reported. In visna virus-infected cells, the genomic (9.4-kb) RNA is accompanied by singly spliced 5.0- and 4.3-kb (doublet) transcripts in addition to doubly spliced mRNAs of 1.8 and 1.5 kb (12). One of the 4.3-kb species is likely the env message (12). Similar observations (species of 9.4, 4.8, 4.3, 3.7 [env], 1.6, and 1.2 kb) were made by others (45). The 1.8 (1.6)-kb species was shown to encode a trans-activating protein and a putative rev product as well (11, 18, 32). Each of the EIAV transcripts was found to share the same 5' exon, whose splice donor site probably corresponds to a consensus sequence upstream of the start of the gag open reading frame at position 459 (26, 42). The hybridization data indicated that the two largest mRNAs, approximately 5.0 and 4.0 kb in size, were spliced once, whereas the smaller transcripts (2.0 and 1.8 kb) were multiply spliced. The 4.0-kb mRNA likely represents the env transcript, on the basis of its pattern of hybridization and relatively high level of expression. A 5.0-kb mRNA was synthesized at low levels and was found to be formed by splicing of the common 5' exon to 3' pol sequences. The hybridization data suggested that it was continuously transcribed from this point to the polyadenylation site. The identity of the gene that encodes this mRNA is unknown. As noted above, 5-kb mRNAs have been detected in HIV-1 (36) and visna virus (12, 45) infected cells, and it was suggested that they encode the vif product. Interestingly, the EIAV genomes characterized to date lack such an open reading frame. The 5-kb transcript found here in EIAV-infected cells might represent the equivalent of vif with its exon(s) embedded within the 3' terminus of pol and in env rather than between pol and env as in other lentiviruses. Efforts are under way to isolate the cDNA for this message in order to characterize it in detail. The 2-kb mRNA hybridized with or12- but not orflspecific probes, suggesting that it contributes sequences to the orf2 product. The predicted amino acid sequence of orf2 shows no significant similarity to that of any known protein. The position of orf2 suggests that it could be analogous to rev. A regulatory role for the 2-kb mRNA is suggested by our findings that it is preferentially expressed at high levels early after infection (S. Noiman, A. Gazit, 0. Tori, L. Sherman, T. Miki, S. R. Tronick, and A. Yaniv, in press). Work is under way to examine the effects of orf2 mutations on gene expression in cells transfected with an infectious EIAV clone. We previously speculated (26) that orfl might encode the tat gene of EIAV (13, 38) on the basis of a short stretch of amino acid sequence similarity to a highly conserved region of the HIV-1 tat gene. Our recent studies using in vitro mutagenesis, cDNA cloning, and antisense oligonucleotide inhibition of tat activity support this conclusion (Gazit et al., submitted). Since all of the orfl-specific probes hybridized with the 1.8-kb but not with the 2.0-kb species, we propose that the 1.8-kb mRNA encodes tat. The 3' orf lacks an initiation codon but is preceded by a consensus splice acceptor sequence (33). Thus, it could provide a 3' tat coding exon or 3' untranslated sequences, depending upon the upstream splice donor(s). Alternatively, it may also contrib-
ute sequences to another protein. Thus, the 1.8-kb band might consist of more than one species. We are very grateful to S. Aaronson for support.
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