MolecularImmunology, Vol.29,No. 5,pp. 573-582, 1992 Printedin Great Britain.
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0161-5890/92 $5.00 +O.OO
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AND STRUCTURE OF SERUM gp70 AS AN ACUTE PHASE PROTEIN IN NZB MICE
KAZUHIROSHIGEMOTO,* SACHIHOKUBO,* YOHJIROITOH,*? GENSHUTATE,* SETSUKOHANDA* and NAOKIMARUYAMA*~ Department of Molecular Pathology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173 and tDepartment of Biology, Faculty of Science, Okayama University, Okayama 700, Japan (First received 7 August 1991; accepted in revisedform 28 October 1991)
Abstract-A cDNA corresponding to a serum gp70 synthesized as an acute phase protein in mouse hepatocytes was cloned and analyzed. This cloned cDNA had the characteristics of an cndogenous xenotropic murine leukemia virus. Synthesized oligo-DNA specific for this cDNA reacted strongly with liver RNA derived from NZB mice injected with LPS as a trigger of an acute phase inflammatory response. There was also low level of gp70 in the kidney in response to LPS injection. The LTR structure of the cDNA showed that this clone is the immediate precursor of an infectious xenotropic virus in the proposed evolutionary scheme of murine leukemia virus.
INTRODUCTION (NZB x NZW)FI hybrid mice spontaneously develop severe autoimmune disease manifested most notably as immune complex glomerulonephritis. A variety of immune complexes are deposited in their diseased glomeruli, including complexes containing a 70,000 M, glycoprotein (gp70) similar to the envelope protein of retroviruses (Yoshiki et al., 1974). Virtually all strains of mice have in their circulation gp70 molecules free from any association with viral particles. However, only those mice genetically predisposed to autoimmune disease from natural antibody against the gp70 with resultant immune complexes and glomerulonephritis (Izui et al., 1981; Izui et al., 1987; Maruyama et al., 1983b; Nakai et al., 1983). Serum gp70 is the only definitive endogenous antigen which is known to evoke immune complex glomerulonephritis. Therefore, analysis of the structure of serum gp70 may contribute to an understanding of the critical epitope(s) responsible for overproduction of natural antibody in autoimmune disease. Although this serum gp70 may be a product of an endogenous proviral env gene in virtually all strains of mice, the regulation of its expression is controlled in a manner characteristic for several inbred strains of mice. Serum gp70 is induced by such stimuli of acute phase proteins (APP) as lipopolysaccharide (LPS), poly I-poly C and turpentine oil, however, no significant amount of gag gene products or reverse transcriptase activity has been detected in sera of mice injected with LPS (Hara et al., 1981; Hara et al., 1982). The precise reason for the absence of gag and pal gene products has not been reported. As with other APP, gp70, is mainly produced by hepatocytes. In addition, several host genetic systems govern the induction of serum gp70 as an APP. We have already demonstrated that the production of serum To whom correspondence should be addressed.
gp70 is controlled by an H-2-linked gene or gene cluster, which we have designated as the Sgp-1 system (Maruyama and Lindstrom, 1983; Shigemoto et al., 1989), and Sgp-2 linked with the Hbb locus on chromosome 7 (Maruyama et al., 1983~). The results of tryptic peptide analysis suggested that this serum gp70 is related to a protein of xenotropic virus isolated only from NZB and (NZB x NZW)Fl mice and identical to that detected in sera from the mice without LPS-injection (Elder et al., 1977). In contrast, serum gp70 is distinct from the thymocyte differentiation antigen, GIX, although they share immunological and biochemical properties. Prior segregation studies demonstrated that the expression of GIX antigen is determined by two unlinked genes, designated Gv-1 and Gv-2 (Stockert et al., 1971). Our previous study demonstrated that Gv-1 might also be responsible for regulating the amounts of serum gp70 (Maruyama et al., 1983~). These findings suggested similarities between the genetic regulation of both env gene products. Levy and coworkers reported the coordinate regulation of multiple proviral genomes by the Go-1 locus (Levy et al., 1982; Repaske et al., 1983; Levy et al., 1985~; Policastro et al., 1989). Whether those endogenous retrovirus products induced by Gv-1 are identical to serum gp70 remains questionable. To clarify these questions, we studied the expression and induction of RNA transcripts encoding gp70 in liver tissues of NZB mice injected with LPS. From their livers, we isolated cDNA clones of transcripts for gp70 and analyzed their complete nucleotide sequence. Based on comparison with the env sequence of infectious NZB xenotropic virus, we prepared synthetic probes, which can specifically detect LPS-induced transcripts, and studied its expression in five different tissues from LPS injected NZB mice. The evolutional relationships of endogenous viral structures are also discussed. 573
K. SHIGEMOTO
574 MATERIALS
AND METHODS
Mice and LPS stimulution NZB male mice were obtained from Charles-River Japan Inc. All mice tested were 556 weeks old. Lipopolysaccharide (LPS) purified from Escherichia coli 0111: B4 was purchased from Sigma. Twenty-five micrograms of LPS was dissolved in sterile saline and injected intraperitoneally into test mice in a final volume of 0.2 ml. Several organs including liver, kidney, brain, thymus, and spleen were excised from the mice 0, 6, 9 and 12 hr after LPS injection, and frozen quickly in liquid nitrogen. Preparation
of polyadenylated
RNA
[poly(A)+
+ RNA]
Total RNA was prepared from frozen mouse tissues by the guanidine hydrochloride extraction procedure (Chirgwin et al., 1982). Poly(A)+ + RNA was obtained by passing total RNA through an oligo (dT)-cellulose chromatography column. Probes and labeling The envelope-specific clone, pXenv was originally provided by Dr M. Martin (NIAD, Bethesda). This clone has a 455 bp fragment derived from the 5’ terminus of the NFS-Th-I xenotropic provirus (Buckler et al., 1982) and is reactive with products of the xenotropicand MCF-enzl genes. The ecotropic specific probe, pEcB4, was originally developed by Dr H. W. Chan (Chan et al., 1980). Rat cDNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was provided by Dr F. M. Piechaczyk. The Molony murine leukemia viral genome (pMLV-1) was used to prepare gag and pol regions specific probes (Levy et al., 1982). Two subcloned DNA segments for gag and pol region were designated as pMo@R and pMoP”‘, respectively. pMo”“R and pMoP”’ span PstI (750 nt) to PcuII (1434 nt) and Hind111 (4894 nt) to Kpn I (5576 nt), respectively. Double stranded DNA probes of high specific radioactivity were prepared by using a random priming kit (Boehringer Mannheim) with [M-“P]dCTP ( > 6000 Cijmmol, Amersham Corp.). An oligonucleotide probe (pGP24,,,,. details in Results) was synthesized by the phosphoramide method of an Applied Biosystems 380-A DNA synthesizer. Endlabeling was done with [y -3’P]ATP ( > 5000 Ci/mmol, Amersham Corp.) using T4 polynucleotide kinase for Northern hybridization analysis. Northern
hybridization
analysis
Total RNA (20 pg) was denatured in 50% formamide, 6% formaldehyde, 20 mM MOPS (morpholinepropanesulfonic acid) (pH 7.0) at 55°C for 15 min and electrophoresed through 1% agarose gels containing 6% formaldehyde, 20 mM MOPS (pH 7.0), 5 mM sodium acetate, 1 mM EDTA. RNA transfer to a nitrocellulose filter was carried out in 1 M ammonium acetate (pH 7.4). Blotted filters were prehybridized overnight at 42°C in 50% (v/v) formamide, 5 x Denhardt solution (1 x Denhardt solution = 0.02% each BSA, Ficoll, and 5 x SSPE [l x SSPE=O.l8M polyvinylpyrolidone),
et ul.
NaCl, 10 mM NaH,PO, (pH 7.4), 1 mM EDTA (pH 7.4)], 0.1% SDS, and sonicated denatured herring sperm DNA (100 pgg/ml). Hybridizations were done overnight at 42°C in the same hybridization mixture containing the labeled probe at a final concn of 3 x lo6 cpm/ml. The filters were washed twice at 55°C in 2 x SSC, 0.1% SDS for 30 min and twice at 55°C in 0.1 x SSC, 0.1% SDS for 30 min. Prehybridization and hybridization with labeled synthesized oligonucleotide probe was carried out at 45°C in the same hybridization mixture as described above. Autoradiography was performed at -70°C with Kodak XAR-5 film with Cronex Lightning-Plus intensifying screens. Construction of a liver cDNA library and isolation characterization of cDNA clones
and
The cDNA library was constructed according to the procedure previously described (Huynh et al., 1985). Briefly, poly(A)+ RNAs from livers of NZB mice were fractionated by sucrose gradient centrifugation to concentrate the gp70 mRNA. A fraction containing 3.2 kb poly(A)+ RNA reactive with pXenv was identified and used to produce cDNA which was ligated with ,?gtlO. After addition of the ligated DNA to bacteriophage lamda DNA packaging mix (Gigapack; Stratagene), phage were plated on E. coli C6OOhJI, and plaques were transferred to nitrocellulose filter for probe screening. Isolated cDNA clones were subcloned into pUC18 and used for further characterization. Sequence analysis of cDNA clones subcloned into the M 13 cloning vector was done by the dideoxy chain termination method with [x5Sldeoxyadenosine 5’-(a-thio)-triphosphate. RESULTS
Northern hybridization analysis of endogenous env-related transcripts in livers and other tissues of LPS stimulated NZB mice. To determine the tropism of gp70 induced by LPS in several tissues, we performed Northern hybridization analysis (Fig. 1). Total RNAs were isolated from the livers, thymuses, spleens, kidneys, and brains of NZB mice given LPS or saline injections. RNAs were electrophoresed and hybridized with pXenv. The most marked env-related transcription was observed in the livers of LPS injected animals and was represented by two bands (3.4 and 7.2 kb). When comparing the relative amounts of transcripts by densitometer, the increase of liver env transcript was more than 70-fold by 9 hr after LPS injection in comparison with untreated mice. In kidneys, 3.4 kb RNA transcription increased more than 3-fold 9 hr after LPS-injection. Only a trace amount of enL’-related RNA transcript was seen in thymus before LPS injection. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was used to confirm that comparable amounts of RNA were present in all lanes. As shown in the bottom of Fig. 1, there was no marked variation of the amounts of GAPDH transcript. The probe, pXenv, reacts with both xenotropic and MCF env sequences but not with the ecotropic specific sequence.
Serum gp70 as an APP
LPS (-)
575
LPS (+)
pxenv
,7.2 kb 28s -
3.4 kb
t8S -
GAPDH 18S---
Fig. 1. Induction of retroviral env-related transcripts by LPS in various organs. Five to six week-old maIe NZB mice were injected intraperitoneaI~y with 25 ,ug of LPS or saline, LPS (+) or LPS (-); respectively. Tissue samples were taken 9 hr after LPS injection. Total RNAs were prepared from liver, thymus, spleen, kidney and brain tissues of both groups. Twenty micrograms of each total RNA were analyzed by Northern hybridization using pXenv. The same filter was stripped and reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe to demonstrate that equal amounts of RNA had been loaded.
These findings suggest that enu-related transcription does not derive from an endogenous ecotropic proviral genome. To confirm the absence of ecotropic specific transcripts in test tissues, we used an ecotropic MuLVspecific probe (pEc-B4) in duplicate experiments. No ecotropic-specific transcription was evident in any of these tissues (data not shown). Kinetics of endogenous MuL V-related RNA transcription in liver after LPS i~je&tion We then examined the kinetics of induction of envrelated RNA transcripts by LPS in NZB livers (Fig. 2). Total RNAs were extracted from the livers 0, 3, 6 and 9 hr after LPS injection, and Northern hybridization anaiysis followed using pXenv as a probe. Values for both 7.2 and 3.4 kb ena-related RNAs increased within 3 hr of LPS injection. The expression of 3.4 kb transcription was maximum at 9 hr, decreased slightly by 12 hr and returned to the baseline 24 hr after LPS injection (data not shown). Since the induction of gag- and pal-gene products by LPS has not been detected in the liver by using several assay systems, we examined the transcription of both genes. Northern hybridization analyses of total liver RNAs were performed using the gag and pal specific probes from Molony murine leukemia virus (MoMuLV) (pMo@ and pMP*‘, respectively). The results indicated that gag- and pal-related 7.2 kb RNA transcripts, which correspond to full-length genomic RNA, were induced and reached a maximum 9 hr after LPS injection (Fig. 3). The expression of 3.4 kb transcripts was also very weakly induced in the same manner as
7.2 kb RNA transcripts (invisible in Fig. 3). This finding indicates that the gag- and pal-regions can be activated at the transcriptional level. Isolation and nucleotide sequence analysis ofcDNA clones ~or~e*~po~dingto env-related RNA transcripts induced by LPS injection To analyze the nucleotide sequence structure and determine whether induced env-related RNA is translatable into gp70, cDNA clones representative of LPS-induced erzu-related RNA were selected and characterized. Oh 3h
6h
9h
pXenv
‘e -
7.2kb 3.4kb
GAPDH 18S-c Fig. 2. Time course of env-related transcript expression in the liver of LPS injected NZB mice. The livers were excised at 0, 3, 6, and 9 hr after LPS injection. Twenty-micrograms of total RNA were used for Northern hybridization analysis with pXenv. The same filter was stripped and reprobed with GAPDH probes.
K. SHIGEMOTO c’t 01.
576
28s 18s
-
7.2kb
-
3.4kb
-
Oh
3h
6h
9h
Oh
3h
6h
9h
Fig. 3. Induction of gag- andpol-transcripts by LPS injection in livers of NZB mice The livers were excised at 0, 3, 6, and 9 hr after LPS injection. Twenty micrograms of total RNA were hybridized with pMog“g and pMoP”‘.
A total of 6 x lo5 recombinant igt 10 phages constructed from size fractionated poly(A)+ RNA of NZB liver were screened with pXenv. Approximately 300 positive signals were detected in the first screening. Finally, we established two independent cDNA clones corresponding to the enu region. Both clones, which were designated pGP24 and pGP68, had approximately 3 kb inserts and were characterized by sequencing analysis. The complete nucleotide sequence of pGP24 (Fig. 4) was determined in reference to the genetic structure of MO-MuLV. Clone pGP24 has a 2935-bp insert and starts 13 bp downstream from the cap site in the R region of the MO-MuLV genome. It has a single open reading frame encoding 644 amino acids occurring after an ATG initiation codon, and splicing follows the pattern observed for MO-MuLV em gene mRNA. In the 3’ region of the cDNA, pGP24 contains U3, R sequences and 3’ terminal oligo(A) tracts, indicating that it represents the 3’ end of retroviral mRNA. Since the pXenv probe does not differentiate the unique sequence of xenotropic virus from that of MCF virus, we compared three segments of amino acid sequence to discrimate pGP24 from MCF virus according to the definition previously reported (Khan, 1984). The 3’ end of the pol gene fragment in pGP24 had a deletion of four amino acids in comparison with MCF247 (Fig. 5A) (Holland et al., 1983). This deletion is identical with that of infectious xenotropic viruses (NZB-IU-6 and NFS-Th-1) (O’Neill et al., 1985) and AKR ecotropic virus (Lenz et al., 1982). However, the nucleotide sequence on either side of this deletion in pGP24 is more like that of the infectious xenotropic viruses than of AKR ecotropic MuLV DNA. Comparison of the 5’ env region of the cloned pGP24 and other known sequences is shown in Fig. 5B and C. Insertion of 4 amino acids (Fig. 4A and Fig. 5B) and deletion of one amino acid (Fig. 4B and Fig. SC) in pGP24 were identical with that of infectious xenotropic viruses but not with MCF247. These findings indicate that the gp70 induced as an acute phase protein in hepatocytes is derived from endogenous xenotropic virus. The struc-
ture of pGP24 was aligned with the MO-MuLV as shown in Fig. 6. The other clone, pGP68, has a 3.3 kb insert and starts at a point that is equivalent to 15 bp downstream from the cap site of the MO-MuLV genome. The nucleotide sequence of pGP68 is identical to that of pGP24 excluding a 362 bp inserted fragment between the splicing donor and acceptors site of pGP24. The nucleotide sequence of this inserted region was compared with MO-MuLV genome sequence and could be aligned with the gug and pol region of MO-MuLV (Fig. 6). This may indicate that pGP68 is alternatively spliced from the endogenous xenotropic viral genome and represents LPS-induced 3.4 kb mRNA detected by Northern hybridization analysis using gag specific probes. pGP68 cDNA does not contain the fragment corresponding to that of pMoP”’ used in Northern hybridization analysis (Fig. 3). Thus, there may be different types of altenative splicing. We further compared the structures of pGP24 and the infectious endogenous xenotropic virus NZB-IU-6 previously reported (O’Neill et al., 1985). In the em coding region, 19 nucleotide substitutions were recognized but no insertions or deletions of nucleotides. The amino acid sequence of pGP24 was identical to the comparable portion of NZB-IU-6 gene in 640 out of 644 amino acids (99% homology). The assignment of the U3, R and U5 regions as well as functional domains for TATA and polyadenylation signal boxes were identical those of NZB-IU-6 except for the CCAAT sequence. In addition, both the NZB-IU-6 and our cDNA LTR sequences had the tandem direct repeat CAGCCC found upstream from the TATA box in xenotropic but not ecotropic or MCF viral LTR sequences (fin Fig. 4). However, neither NZB-IU-6, pGP24 nor pGP68 has the 190 bp transposon-like insert (Khan and Martin, 1983) upstream from the CCAAT-like box (e in Fig. 4) in the LTRs (e in Fig. 4). The most notable difference a deletion of 14 nucleotides is in the U3 LTR region of NZB-IU-6 and MCF247 in comparison with pGP24 and others (Fig. 7).
Serum gp70 as an APP
These nucleotide sequences indicate that clones pGP24 and pGP68 are derived from a xenotropic virus. Northern probes
hybridization
analysis with synthetic
DNA
To confirm the dominant expression of enu-related RNA, which we have cloned as a cDNA, we performed Northern hybridization analysis using synthesized oligonucleotide probes. According to the difference in U3 LTR between pGP24 and NZB-IU-6, as shown in Fig. 7, we also designed a 29-bp pGP24,,, probe to distinguish them from the characterized infectious NZB xenotropic virus sequence (a in Fig. 7). Northern hybridization analyses were applied to the total RNAs isolated
577
from liver, thymus, spleen, kidney and brain tissues of LPS stimulated and unstimulated NZB mice (Fig. 8). A remarkable increase of env-related 3.4 kb and 7.2 kb RNA transcripts was detected in the livers and a 3.4 kb transcript was found faintly in the kidneys of LPSinjected mice (invisible in Fig. 8) by using pGP24,,,. These findings and the cDNA sequence analysis indicate that our cDNAs represent LPS-induced enu-related transcripts and encode serum gp70 products. DISCUSSION There are many copies of MuLV-related sequences in the mouse genome. Some of these proviral genomes
578
cxuxxxc-~GAcII;AGIcG crlws-8
2340 2400
Fig. 4(B) Fig. 4. Complete DNA sequence of pGP24. The location (nucleotide numbers) of significant MuLV sequences are as follows: U5 region in LTR, -412 to -343; splicing site, -278; U3 region in LTR, 1968 to 2317; R region in LTR, 2318 to 2442; CAT box, 2287 to 2291; TATA box, 2344 to 2350; polyadenylic acid signal, 2420 to 2425. The locations of env initiation and termination were deduced by analogy with MO-MuLV proviral DNA sequences. Underlined italics indicate potential glycosylation sites. Arrows indicate the predicted sites of cleavage. Numbers without parentheses indicate nucleotide positions. Amino acid positions are shown inside parentheses. (a) Xenotropic virus specific sequence (Refer to Fig. 5B), (b) Deletion (1) specific for xenotropic virus (refer to Fig. 5C), (c) Synthesized oligonucleotide probe corresponding sequence (refer to Fig. 7 and 8), (d) Sequence deleted in L62 (refer to Discussion), (e) Deletion (A) of 190 bp insert (transposon-like element) characteristic for endogenous proviruses (refer to Discussion) (Khan and Martin, 1983) (f) 6 bp duplication (refer to Discussion) (Khan and Martin, 1983).
are responsible for the expression of env gene products, i.e. serum gp70, GIX antigen, and Fv-4 (Ikeda and Sugimura, 1989). Among these gene products, serum gp70 has been regarded as a major nephritogenic endogenous antigen in immune complex glomerulonephritis of New Zealand mice (Yoshiki er al., 1974; Maruyama et al., 19836). In this study we examined the characteristics of gp70 synthesized in the liver and
its enhancement by LPS in the acute phase of inflammation. We have examined the induction of env-related RNA by LPS and found that the major site of serum gp70 synthesis was in hepatocytes with a minor degree of induction in the kidney (Fig. 1). This induction corresponds to the gp70 expressed in tubular epithelium (Lerner et al., 1976). This result suggests that the
Serum gp70 as an APP A. pGP24 -14 (nt)PAGTA----WKV10 (nt) NZB-IU-6 PAGTA----WKV MCF247 PAGTASGPTWKV AKR EC0 PIKPS----WRV
B. pGP24 NZB-IU-6 MCF247 AKR EC0
1983; Bosze et al., 1986; Laigret et al., 1988), we have not yet detected the genetic element responsible for gp70 induction in hepatocytes. Their is still a possibility of novel consensus sequence for IL-6 responsiveness in LTR of pGP24 or pGP68 cloned by us. A recent study on the gene product of truncated MuLV proviral genome suggested another possibility. Ikeda and Sugimura (1989) examined the biological function of a S-flanking cellular sequence adjacent to Fv-4’ allele. Fv-4 is a mouse gene which controls susceptibility to infection by ecotropic murine leukemia virus (MuLV). They cloned part of an endogenous MuLV associated with the resistance allele of the Fv-4 gene (Fv-4’). The whole structure of Fv-4’ allele shows the truncated structure lacking a 5’ LTR of MuLV. This fragment of approximately 13 kb including the whole Fv-4’ genome (1 Fv4- 17) was transfected to NIH3T3 cells, resulting in greater expression of Fv-4’ env gene product when compared with the transfection of 1.5 kb 3’ flanking with the Fv-4’. This result suggested that the S-flanking region of IIFv4- 17 contains transcriptional regulatory elements enhancing the expression of Fv-4’ transcript, splice donor sites, and/or untranslated exon(s). By the same token, the transcript expressed in the NZB liver could have similar regulatory elements in the cellular sequence adjacent to the proviral genome encoding serum gp70. This sort of element may enhance the production of gp70 in hepatocyte. The induction of serum gp70 in the liver and the expression of the thymocyte differentiation antigen, GIX, in the thymus share same common features. The expression of GIX antigen with gp70 properties is regulated by two unlinked loci, Gv-1 and Gv-2 (Stockert et al., 1971). These loci also seem to affect the induction of serum gp70 in the liver. A 129GIX+ murine strain showed higher basal levels of serum gp70 (20 pg/ml in males) than a congenic 129GIX- strain (1.8 yg/ml), yet both developed increases of serum gp70 in response to LPS. Since only Go-1 should differ between these substrains, the amounts of serum gp70 might be regulated by this locus. Moreover, Sgp-2, the locus responsible for gp70 induction as an APP, is located on chromosome 7, as is Gv-2. These findings not only suggest some common mechanisms for the expression of env gene products
(NFS-Th-11
81 VGDYWDDPEPDIGDGCRTPG 100 VGDYWDDPEPDIGDGCRTPG (NFS-Th-11 IGDDWDE----TGLGCRTPG (El1 GPSYWGPPCCS-GSSDSTPG t IGLEYRAPFSPPP]
C. pGP24 NZB-IU-6 MCF247
161 KDQGPCYDSS-VSSGVQQATP180 KDQGPCYDSS-VSSGVQQATP fNFS-Th-II QNQGPCYDSSAVSSDIKGATP (El, L5111
Fig. 5. Comparison of the deduced amino acid sequences of pGP24, MCF247, NZB-IU-6, and AKR ecotropic (AKR ECO) viruses in three segments of the env region. Designations of cloned DNA with identical sequences are shown in parentheses. El and L511 are reported by Levy (Levy et al., 19856). (A) Deletion of 4 amino acids in 3’ pol region overlapped with 5’ env region of pGP24. Although pGP24 has no open reading frame in the pal region, these four sequences were aligned to compare the 3’ pal region. This segment maps between 1 nt and 2 nt (1st codon of env region) in Fig. 4. (B) Insertion of xenotropic specific sequence in pGP24. This segment corresponds to the segment “a” in Fig. 4. Number indicates amino acid position shown in Fig. 4. (C) Single amino acid deletion specific for xenotropic virus in pGP24. Number indicates amino acid position shown in Fig. 4. existence of gp70 in renal tubuli tion of serum components.
is not due to reabsorp-
Hara and colleagues (1982) reported that serum gp70 behaves like an APP in several respects and its expression is controlled by a mechanism similar to that of other APPs. There are several reports that some APPs are induced by various cytokines including IL-6. Generally APPs induced by this cytokine have IL-6 responsive element in their genomes (Heinrich et al., 1990). We recently examined the effect of IL-6 on the production of gp70 in hepatocytes. The primary hepatocyte culture with recombinant IL-6 showed the enhancement of the gp70 synthesis (Itoh et al., 1992). Our data suggests that LTR as probable promoter of pGP24 or pGP68 may have IL-6 responsive element. Although several reports have described the tissue specific replication of MuLV restricted by the LTR structure (DesGroseillers et al.,
gag
LTR pls
Pd
-Ul-
LTR
OPfO
p30 P’2
579
p15E
PI0
z
7
pGP24
I------__
pGP68
-t,
Y
,8-x,
------_--_________------
”
____--------
,.-------__________,-___------
Fig. 6. Alignment of two cloned endogenous xenotropic viral sequence, pGP24 and pGP68, with whole MO-MuLV genome. Broken lines represent spliced regions. A linear map of murine retrovirus genome and gag and env probes (hatched bars) used in Northern hybridization analysis in Fig. 2 are also shown.
K.
580 2061
pGP24
SHIGEMOTO
2120 TACTAGGACA
a
TACAAGGAAG
TTCAGTLTAA
NZB-R-6
. . . ..AA...
..TG......
..A.[
MCF247
. . . . .. . . . . ..T.......
..A.[
E2 L62 B-77
cltul
I..
..T.......
. . ..G.....
I . . . . . . . ..C.. A......... . . . . . . . . . . . . . . . . ..T. . . . . . . . . . . . ...G..... .. . .. . . ..G C...G..... . . ..a..... . . . . . . .. . . . . . . . . . . . . . ...G..T.. .,........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . ..C.......... e b
. . . . . .. . . . . . ..G.....
b'
Fig. 7. Comparison of LTR sequences. The deletion of 14 bp is indicated as brackets. The segment, b and b’ indicates a 6 bp duplication (refer to Discussion) immediately upstream from TATAA box of B-77 (endogenous provirus sequence) (Khan and Martin, 1983). E2 and L62 are described by Levy (Levy et al., 1985b, 1987).
but also raise the question of whether serum gp70 and GIX antigen are identical. Although the serum gp70 in 129GIX was not well-characterized, no structural difference was found among various mouse strains by tryptic peptide analysis (Elder rt al., 1977). Levy and colleagues (Levy et al., 1985a) determined the structure of endogenous retrovirus derived from various tissues of the 129GIX strain. They characterized cDNA clones preRNA of several tissues of pared from poly(A)+ 129GIX+ mice. Those cDNAs encoded a part of gp70 which is determined by Ga - 1-responsive endogenous retrovirus loci (Levy et al., 1985b; Levy et al., 1987). We have now aligned the amino acid sequences of two cDNA clones, El and L511 derived from epididymis and liver, respectively, with pGP24 (Fig. 5B and C). The amino acid sequence of pGP24 is identical with that of the xenotropic proviral genome. In contrast, El and L511 were characteristic of MCF virus. Of the structural differences in the regulatory region recognized between pGP24 and the Gu- l-responsive endogenous retroviruses, the most notable was a 39-bp deletion in the 129GIX+ mouse liver derived clone, L62, compared with pGP24 (d in Fig. 4). Levy and colleagues (1985a) suggested the action of a regulatory element, encoded by Go-l, functioning in tram to control the normal expression
129GIX+
of endogenous
retroviral
and GIX-- mouse strains.
sequences
in congenic
These findings
indi-
LPS (-)
cate that the Gv-1 system affects the induction of both the MCF viral genome and the xenotropic proviral genome encoding serum gp70. There is no report, including our preliminary study, on the existence of gag- or pol-gene products in the liver. However, we showed that LPS induced gag- and pol-related RNA in the liver (Fig. 3). Based on the nucleic acid analysis of cloned cDNA (pGP68), the truncated gagand pol-related transcripts lay adjacent to the enc-gene. These gag- and pol-related transcripts did not have an open reading frame. These findings suggest that the induction of gag- and pol-related transcripts by LPS results from enr-related transcription, as for pGP68. This would account for the absence of gag- and pol-gene products in the liver. However a conflicting result was shown in our study. The pGP68 contains the partial codes of the pol gene, but not pMoJ”“. The induction of pol gene in the hepatocytes after LPS injection was detected by pMoP”’ probe (Fig. 3). There are following two explanations. (1) The expression of pMd”” corresponding RNA due to different types of alternative splicing. (2) The activation of multiple units encoding pol gene corresponding to pMd”’ probe. Temin (1980) has proposed that retroviruses evolved from cellular moveable genetic elements. Since serum gp70 is synthesized in hepatocytes and secreted into the circulation, we expected that serum gp70 might be an
LPS (+)
-7.2
kb
-3.4
kb
Fig. 8. Induction by LPS of xenotropic and pGP24 eno-related RNAs in the NZB liver. The source of total RNAs was the same as noted in Fig. 1. Northern hybridization analysis was performed using pGP24,,,., synthetic probes mentioned in Materials and Methods. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was used to confirm that comparable amounts of RNA were present in all lanes.
Serum gp70 as an APP
evolutionally ancestral protein of retroviruses. However, its structure is not remarkably distinct from other retroviruses reported. Khan and Martin (1983) analyzed the sequence and compared LTRs associated with infectious xenotropic, ecotropic, and MCF proviruses. The three segments of LTR compared were: (1) 14 bp duplicated sequence (b and b’ in Fig. 7); (2) insertion or deletion of the highly conserved transposon-like sequence (190 bp) and the adjoining region (e in Fig. 4) (Khan and Martin, 1983); (3) 6 bp duplication (f in Fig 4). Based on their analyses, they proposed a possible evolutionary scheme relating endogenous and infectious MuLV LTRs (Khan and Martin, 1983). The LTR of pGP24 has a 14 bp duplicated sequence as a hallmark of the endogenous group (B-77, E2 and L62 in Fig. 7). Because pGP24 LTR is missing a highly conserved 190 bp insertion, this clone was regarded as “mature”. The 6 bp duplication is also a definitive marker of xenotropic virus in concert with several hallmarks of the enu region as shown in Fig. 5. These hallmarks may mean that pGP24 is a hypothetical immediate precursor of infectious xenotropic virus or precursor of the ecotropic virus via separate lineage in the evolutionary scheme (Khan and Martin, 1983). Acknowledgements-We thank Professor Toshikazu Shirai, Juntendo University, and Professor Masaru Taniguchi, Chiba University for their discussion and support during our investigation. The excellent editorial assistance of MS Phyllis Minick and technical assistance of MS Yoshie Urano are gratefully acknowledged. This study was performed through Special Coordination Fund of the Science and Technology Agency of the Japanese Government, and Sandoz Pha~aceutical Japan. REFERENCES Bosze Z., Thiesen H.-J. and Charnay P. (1986) A transcriptional enhancer with specificity for erythroid cells is located in the long terminal repeat of the Friend murine leukemia virus. EMBO J. 5, 1615-1623. Buckler C. E., Hoggan M. D., Chan H. W., Sears J. F., Khan A. S., Moore J. L., Hartley J. W., Rowe W. P. and Martin M. A. (1982) Cloning and characterization of an envelopespecific probe from xenotropic murine leukemia proviral DNA. J. Viral. 41, 228-236. Ghan H. W., Bryan T., Moore J. L., Staal S. P., Rowe W. P. and Martin M. A. (1980) Identification of ecotropic proviral sequences in inbred mouse strains with a cloned subgenomic DNA fragment. Proc. natn. Acad. Sci. U.S.A. 77,5779-5783. Chirgwin J. M., Przybyla A. E., MacDonald R. J. and Rutter W. J. (1982) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. B~oehem~tr~ 18, 5294-5299. DesGroseillers L., Rassart E. and Jolicoeur P. (1983) Thymotropism of murine leukemia virus is conferred by its long terminal repeat. Proc. natn. Acad. Sci. U.S.A. 80,4203-4207. Elder J. H., Jensen F. C., Bryant M. N. and Lemer R. A. (1977) Pol~orphism of major envelope glycoprotein (gp70) of murine C-type viruses: multi-gene family. Nature (London) 267, 23-28. Hara I., Izui S., McConahey P. J., Elder J. H., Jensen F. C. and Dixon F. J. (1981) Induction of high serum levels of retroviral env gene products (gp70) in mice by bacterial
581
lipopolysaccharide. Proc. natn. Acad. Sci. U.S.A. 78, 4397440 1. Hara I., Izui S. and Dixon F. J. (1982) Mu&e serum glycoprotein gp70 behaves as an acute phase reactant. J. exp. Med. 155, 345-357. Heinrich P. C., Caste11J. V. and Andus T. (1990) Interleukind and the acute phase response. Biochem. J. 265, 621-620. Holland C., Wozney J. and Hopkins N. (1983) Nucleotide sequence of the gp70 gene of murine retrovirus MCF247. J. Viral. 47, 413420. Huynh T. V., Young R. A. and Davis R. W. (1985) Constructing and screening cDNA libraries in lgtl0 and Igtll, p. 49-78. In DNA cloning Vol. I: A Practical Approach (Edited by D. M. Glover). IRL Press Limited, Oxford, England. Ikeda H. and Sugimura H. (1989) Fv-4 resistance gene: a truncated endogenous murine leukemia virus with ecotropic interference properties. 1. Virol. 63, 5405-5412. Itoh Y., Maruyama N., Kitamura M., Shirasawa T., Shigemoto K. and Koike T. (1992) Induction of endogenous retroviral gene product (SU) as an acute phase protein by IL-6 in murine hepatocytes. In press. Izui S., McConahey P. J., Clark J. P., Hang L. M., Hara I. and Dixon F. J. (1981) Retroviral gp70 immune complexes in NZB x NZW F2 mice with murine lupus nephritis. J. exp. Med. 154, 517-528. Izui S., McConahey P. J. and Dixon F. J. (1987) Increased spontaneous polyclonal activation of B lymphocytes in mice with spon~neous autoimmune disease. J. Immun. 121, 2213-2219. Khan A. S. and Martin M. A. (1983) Endogenous murine leukemia proviral long terminal repeats contain a unique 190-base-pair insert. Proc. natn. Acad. Sci. U.S.A. 80, 2699-2703. Khan A. S. (1984) Nucleotide sequence analysis establishes the role of endogenous murine leukemia virus DNA segments in formation of recombinant mink cell focus-forming murine leukemia viruses. J. Virof. 50, 864-871. Laigret F., Repaske R., Boulukos K., Robson A. B. and Khan A. S. (1988) Potential progenitor sequences of mink cell focus-foxing (MCF) murine leukemia viruses: ecotropic-, xenotropic-, and MCF-related viral RNAs are detected concurrently in thymus tissues of AKR mice. J. Viral. 62, 376-386. Lerner R. A., Wilson C. B., Del Villano, B. C., McConahey P. J. and Dixon F. J. (1976) Endogenous oncornaviral gene expression in adult and fetal mice: Quantitative, histologic, and physiologic studies of the major viral glycoprotein, gp70. J. exp. Med. 143, 151-166. Lenz J., Crowther R., Straceski A. and Haseltine W. A. (1982) Nucleotide sequence of the Akv env gene. J. Virol. 42, 519-529. Levy D. E., Lemer R. A. and Wilson M. C. (1982) A genetic locus regulates the expression of tissue-specific mRNAs from multiple transcription units. Proc. natn. Acad. Sci. U.S.A. 79, 5823-5827. Levy D. E., Lerner R. A. and Wilson M. C. (1985a) The Go-1 locus coordinately regulates the expression of multiple endogenous murine retroviruses. Ceti 41, 289-299. Levy D. E., Lerner R. A. and Wilson M. C. (1985B) Normal expression of ~l~o~hic endogenous retroviral RNA containing segments identical to mink cell focus-forming virus. J. Virol. 56, 691-700. Levy D. E., McKinnon R. D., Brolaski M. N., Gautsch J. W. and Wilson M. C. (1987) The 3’ long terminal repeat of a
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transcribed yet defective endogenous retroviral sequence is a competent promoter of transcription. J. Virol. 56, 691-700. Maruyama N. and Lindstrom C. 0. (1983) H-2-linked regulation of serum gp70 production in mice. Immunogenetics 17, 507-521.
Maruyama N., Lindstrom C. O., Sato H. and Dixon F. J. (1983a) Serum gp70 production regulated by a gene on murine chromosome 7. Immunogenetics 18, 365-372. Maruyama N., Furukawa F., Nakai Y., Sasaki Y., Ohta K., Ozaki S., Hirose S. and Shirai, T. (1983b) Genetic studies of autoimmunity in New Zealand mice. IV. Contribution of NZB and NZW genes to the spontaneous occurrence of retroviral gp70 immune complexes in (NZB x NZW)F, hybrid and the correlation to renal disease. J. Immun. 130, 740-746.
Nakai Y., Maruyama N., Ohta K., Yoshida H., Hirose S. and Shirai T. (1983) Genetic studies of autoimmunity in New Zealand mice. Association of circulating retroviral gp70 immune complex with proteinuria. Zmmun. Lett. 2, 53-58. O’Neill R. R., Buckler C. E., Theodore T. S., Martin M. A. and Repaske R. (1985) Envelope and long terminal repeat
sequences of a cloned infectious NZB xenotropic murine leukemia virus. J. Viral. 53, 100-106. Repaske R., O’Neil R. R., Khan A. S. and Martin M. A. (1983) Nucleotide sequence of the env-specific segment of NFS-Th1 xenotropic murine leukemia virus. J. Viral. 46, 204-211. Policastro P. F., Fredholm M. and Wilson M. C. (1989) Truncated gag products encoded by Gv-l-responsive endogenous retrovirus loci. J. Virof. 63, 413&4147. Shigemoto K., Maruyama N., Itoh Y., Kubo S. and Koike T. (1989) Murine leukemia virus gene product as an acute phase protein: Complete suppression of serum gp70 synthesis in hepatocytes of B1O.S mice. C/in. exp. Immun. 78, 484487. Stockert E., Old L. J. and Boyse E. A. (1971) The GIX system: A cell surface allo-antigen associated with murine leukemia virus. J. exp. Med. 133, 13341355. Temin H. M. (1980) Origin of retroviruses from cellular moveable genetic elements. Cell. 21, 5999600. Yoshiki T., Mellors R. C., Strand M. and August J. T. (1974) The viral envelope glycoprotein of murine leukemia virus and the pathogenesis of immune complex glomerulonephritis of New Zealand mice. J. exp. Med. 140, 1011-1027.
EXPRESSION
0161-5890/92 $5.00 +O.OO
0 1992PergamonPressLtd
AND STRUCTURE OF SERUM gp70 AS AN ACUTE PHASE PROTEIN IN NZB MICE
KAZUHIROSHIGEMOTO,* SACHIHOKUBO,* YOHJIROITOH,*? GENSHUTATE,* SETSUKOHANDA* and NAOKIMARUYAMA*~ Department of Molecular Pathology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173 and tDepartment of Biology, Faculty of Science, Okayama University, Okayama 700, Japan (First received 7 August 1991; accepted in revisedform 28 October 1991)
Abstract-A cDNA corresponding to a serum gp70 synthesized as an acute phase protein in mouse hepatocytes was cloned and analyzed. This cloned cDNA had the characteristics of an cndogenous xenotropic murine leukemia virus. Synthesized oligo-DNA specific for this cDNA reacted strongly with liver RNA derived from NZB mice injected with LPS as a trigger of an acute phase inflammatory response. There was also low level of gp70 in the kidney in response to LPS injection. The LTR structure of the cDNA showed that this clone is the immediate precursor of an infectious xenotropic virus in the proposed evolutionary scheme of murine leukemia virus.
INTRODUCTION (NZB x NZW)FI hybrid mice spontaneously develop severe autoimmune disease manifested most notably as immune complex glomerulonephritis. A variety of immune complexes are deposited in their diseased glomeruli, including complexes containing a 70,000 M, glycoprotein (gp70) similar to the envelope protein of retroviruses (Yoshiki et al., 1974). Virtually all strains of mice have in their circulation gp70 molecules free from any association with viral particles. However, only those mice genetically predisposed to autoimmune disease from natural antibody against the gp70 with resultant immune complexes and glomerulonephritis (Izui et al., 1981; Izui et al., 1987; Maruyama et al., 1983b; Nakai et al., 1983). Serum gp70 is the only definitive endogenous antigen which is known to evoke immune complex glomerulonephritis. Therefore, analysis of the structure of serum gp70 may contribute to an understanding of the critical epitope(s) responsible for overproduction of natural antibody in autoimmune disease. Although this serum gp70 may be a product of an endogenous proviral env gene in virtually all strains of mice, the regulation of its expression is controlled in a manner characteristic for several inbred strains of mice. Serum gp70 is induced by such stimuli of acute phase proteins (APP) as lipopolysaccharide (LPS), poly I-poly C and turpentine oil, however, no significant amount of gag gene products or reverse transcriptase activity has been detected in sera of mice injected with LPS (Hara et al., 1981; Hara et al., 1982). The precise reason for the absence of gag and pal gene products has not been reported. As with other APP, gp70, is mainly produced by hepatocytes. In addition, several host genetic systems govern the induction of serum gp70 as an APP. We have already demonstrated that the production of serum To whom correspondence should be addressed.
gp70 is controlled by an H-2-linked gene or gene cluster, which we have designated as the Sgp-1 system (Maruyama and Lindstrom, 1983; Shigemoto et al., 1989), and Sgp-2 linked with the Hbb locus on chromosome 7 (Maruyama et al., 1983~). The results of tryptic peptide analysis suggested that this serum gp70 is related to a protein of xenotropic virus isolated only from NZB and (NZB x NZW)Fl mice and identical to that detected in sera from the mice without LPS-injection (Elder et al., 1977). In contrast, serum gp70 is distinct from the thymocyte differentiation antigen, GIX, although they share immunological and biochemical properties. Prior segregation studies demonstrated that the expression of GIX antigen is determined by two unlinked genes, designated Gv-1 and Gv-2 (Stockert et al., 1971). Our previous study demonstrated that Gv-1 might also be responsible for regulating the amounts of serum gp70 (Maruyama et al., 1983~). These findings suggested similarities between the genetic regulation of both env gene products. Levy and coworkers reported the coordinate regulation of multiple proviral genomes by the Go-1 locus (Levy et al., 1982; Repaske et al., 1983; Levy et al., 1985~; Policastro et al., 1989). Whether those endogenous retrovirus products induced by Gv-1 are identical to serum gp70 remains questionable. To clarify these questions, we studied the expression and induction of RNA transcripts encoding gp70 in liver tissues of NZB mice injected with LPS. From their livers, we isolated cDNA clones of transcripts for gp70 and analyzed their complete nucleotide sequence. Based on comparison with the env sequence of infectious NZB xenotropic virus, we prepared synthetic probes, which can specifically detect LPS-induced transcripts, and studied its expression in five different tissues from LPS injected NZB mice. The evolutional relationships of endogenous viral structures are also discussed. 573
K. SHIGEMOTO
574 MATERIALS
AND METHODS
Mice and LPS stimulution NZB male mice were obtained from Charles-River Japan Inc. All mice tested were 556 weeks old. Lipopolysaccharide (LPS) purified from Escherichia coli 0111: B4 was purchased from Sigma. Twenty-five micrograms of LPS was dissolved in sterile saline and injected intraperitoneally into test mice in a final volume of 0.2 ml. Several organs including liver, kidney, brain, thymus, and spleen were excised from the mice 0, 6, 9 and 12 hr after LPS injection, and frozen quickly in liquid nitrogen. Preparation
of polyadenylated
RNA
[poly(A)+
+ RNA]
Total RNA was prepared from frozen mouse tissues by the guanidine hydrochloride extraction procedure (Chirgwin et al., 1982). Poly(A)+ + RNA was obtained by passing total RNA through an oligo (dT)-cellulose chromatography column. Probes and labeling The envelope-specific clone, pXenv was originally provided by Dr M. Martin (NIAD, Bethesda). This clone has a 455 bp fragment derived from the 5’ terminus of the NFS-Th-I xenotropic provirus (Buckler et al., 1982) and is reactive with products of the xenotropicand MCF-enzl genes. The ecotropic specific probe, pEcB4, was originally developed by Dr H. W. Chan (Chan et al., 1980). Rat cDNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was provided by Dr F. M. Piechaczyk. The Molony murine leukemia viral genome (pMLV-1) was used to prepare gag and pol regions specific probes (Levy et al., 1982). Two subcloned DNA segments for gag and pol region were designated as pMo@R and pMoP”‘, respectively. pMo”“R and pMoP”’ span PstI (750 nt) to PcuII (1434 nt) and Hind111 (4894 nt) to Kpn I (5576 nt), respectively. Double stranded DNA probes of high specific radioactivity were prepared by using a random priming kit (Boehringer Mannheim) with [M-“P]dCTP ( > 6000 Cijmmol, Amersham Corp.). An oligonucleotide probe (pGP24,,,,. details in Results) was synthesized by the phosphoramide method of an Applied Biosystems 380-A DNA synthesizer. Endlabeling was done with [y -3’P]ATP ( > 5000 Ci/mmol, Amersham Corp.) using T4 polynucleotide kinase for Northern hybridization analysis. Northern
hybridization
analysis
Total RNA (20 pg) was denatured in 50% formamide, 6% formaldehyde, 20 mM MOPS (morpholinepropanesulfonic acid) (pH 7.0) at 55°C for 15 min and electrophoresed through 1% agarose gels containing 6% formaldehyde, 20 mM MOPS (pH 7.0), 5 mM sodium acetate, 1 mM EDTA. RNA transfer to a nitrocellulose filter was carried out in 1 M ammonium acetate (pH 7.4). Blotted filters were prehybridized overnight at 42°C in 50% (v/v) formamide, 5 x Denhardt solution (1 x Denhardt solution = 0.02% each BSA, Ficoll, and 5 x SSPE [l x SSPE=O.l8M polyvinylpyrolidone),
et ul.
NaCl, 10 mM NaH,PO, (pH 7.4), 1 mM EDTA (pH 7.4)], 0.1% SDS, and sonicated denatured herring sperm DNA (100 pgg/ml). Hybridizations were done overnight at 42°C in the same hybridization mixture containing the labeled probe at a final concn of 3 x lo6 cpm/ml. The filters were washed twice at 55°C in 2 x SSC, 0.1% SDS for 30 min and twice at 55°C in 0.1 x SSC, 0.1% SDS for 30 min. Prehybridization and hybridization with labeled synthesized oligonucleotide probe was carried out at 45°C in the same hybridization mixture as described above. Autoradiography was performed at -70°C with Kodak XAR-5 film with Cronex Lightning-Plus intensifying screens. Construction of a liver cDNA library and isolation characterization of cDNA clones
and
The cDNA library was constructed according to the procedure previously described (Huynh et al., 1985). Briefly, poly(A)+ RNAs from livers of NZB mice were fractionated by sucrose gradient centrifugation to concentrate the gp70 mRNA. A fraction containing 3.2 kb poly(A)+ RNA reactive with pXenv was identified and used to produce cDNA which was ligated with ,?gtlO. After addition of the ligated DNA to bacteriophage lamda DNA packaging mix (Gigapack; Stratagene), phage were plated on E. coli C6OOhJI, and plaques were transferred to nitrocellulose filter for probe screening. Isolated cDNA clones were subcloned into pUC18 and used for further characterization. Sequence analysis of cDNA clones subcloned into the M 13 cloning vector was done by the dideoxy chain termination method with [x5Sldeoxyadenosine 5’-(a-thio)-triphosphate. RESULTS
Northern hybridization analysis of endogenous env-related transcripts in livers and other tissues of LPS stimulated NZB mice. To determine the tropism of gp70 induced by LPS in several tissues, we performed Northern hybridization analysis (Fig. 1). Total RNAs were isolated from the livers, thymuses, spleens, kidneys, and brains of NZB mice given LPS or saline injections. RNAs were electrophoresed and hybridized with pXenv. The most marked env-related transcription was observed in the livers of LPS injected animals and was represented by two bands (3.4 and 7.2 kb). When comparing the relative amounts of transcripts by densitometer, the increase of liver env transcript was more than 70-fold by 9 hr after LPS injection in comparison with untreated mice. In kidneys, 3.4 kb RNA transcription increased more than 3-fold 9 hr after LPS-injection. Only a trace amount of enL’-related RNA transcript was seen in thymus before LPS injection. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was used to confirm that comparable amounts of RNA were present in all lanes. As shown in the bottom of Fig. 1, there was no marked variation of the amounts of GAPDH transcript. The probe, pXenv, reacts with both xenotropic and MCF env sequences but not with the ecotropic specific sequence.
Serum gp70 as an APP
LPS (-)
575
LPS (+)
pxenv
,7.2 kb 28s -
3.4 kb
t8S -
GAPDH 18S---
Fig. 1. Induction of retroviral env-related transcripts by LPS in various organs. Five to six week-old maIe NZB mice were injected intraperitoneaI~y with 25 ,ug of LPS or saline, LPS (+) or LPS (-); respectively. Tissue samples were taken 9 hr after LPS injection. Total RNAs were prepared from liver, thymus, spleen, kidney and brain tissues of both groups. Twenty micrograms of each total RNA were analyzed by Northern hybridization using pXenv. The same filter was stripped and reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe to demonstrate that equal amounts of RNA had been loaded.
These findings suggest that enu-related transcription does not derive from an endogenous ecotropic proviral genome. To confirm the absence of ecotropic specific transcripts in test tissues, we used an ecotropic MuLVspecific probe (pEc-B4) in duplicate experiments. No ecotropic-specific transcription was evident in any of these tissues (data not shown). Kinetics of endogenous MuL V-related RNA transcription in liver after LPS i~je&tion We then examined the kinetics of induction of envrelated RNA transcripts by LPS in NZB livers (Fig. 2). Total RNAs were extracted from the livers 0, 3, 6 and 9 hr after LPS injection, and Northern hybridization anaiysis followed using pXenv as a probe. Values for both 7.2 and 3.4 kb ena-related RNAs increased within 3 hr of LPS injection. The expression of 3.4 kb transcription was maximum at 9 hr, decreased slightly by 12 hr and returned to the baseline 24 hr after LPS injection (data not shown). Since the induction of gag- and pal-gene products by LPS has not been detected in the liver by using several assay systems, we examined the transcription of both genes. Northern hybridization analyses of total liver RNAs were performed using the gag and pal specific probes from Molony murine leukemia virus (MoMuLV) (pMo@ and pMP*‘, respectively). The results indicated that gag- and pal-related 7.2 kb RNA transcripts, which correspond to full-length genomic RNA, were induced and reached a maximum 9 hr after LPS injection (Fig. 3). The expression of 3.4 kb transcripts was also very weakly induced in the same manner as
7.2 kb RNA transcripts (invisible in Fig. 3). This finding indicates that the gag- and pal-regions can be activated at the transcriptional level. Isolation and nucleotide sequence analysis ofcDNA clones ~or~e*~po~dingto env-related RNA transcripts induced by LPS injection To analyze the nucleotide sequence structure and determine whether induced env-related RNA is translatable into gp70, cDNA clones representative of LPS-induced erzu-related RNA were selected and characterized. Oh 3h
6h
9h
pXenv
‘e -
7.2kb 3.4kb
GAPDH 18S-c Fig. 2. Time course of env-related transcript expression in the liver of LPS injected NZB mice. The livers were excised at 0, 3, 6, and 9 hr after LPS injection. Twenty-micrograms of total RNA were used for Northern hybridization analysis with pXenv. The same filter was stripped and reprobed with GAPDH probes.
K. SHIGEMOTO c’t 01.
576
28s 18s
-
7.2kb
-
3.4kb
-
Oh
3h
6h
9h
Oh
3h
6h
9h
Fig. 3. Induction of gag- andpol-transcripts by LPS injection in livers of NZB mice The livers were excised at 0, 3, 6, and 9 hr after LPS injection. Twenty micrograms of total RNA were hybridized with pMog“g and pMoP”‘.
A total of 6 x lo5 recombinant igt 10 phages constructed from size fractionated poly(A)+ RNA of NZB liver were screened with pXenv. Approximately 300 positive signals were detected in the first screening. Finally, we established two independent cDNA clones corresponding to the enu region. Both clones, which were designated pGP24 and pGP68, had approximately 3 kb inserts and were characterized by sequencing analysis. The complete nucleotide sequence of pGP24 (Fig. 4) was determined in reference to the genetic structure of MO-MuLV. Clone pGP24 has a 2935-bp insert and starts 13 bp downstream from the cap site in the R region of the MO-MuLV genome. It has a single open reading frame encoding 644 amino acids occurring after an ATG initiation codon, and splicing follows the pattern observed for MO-MuLV em gene mRNA. In the 3’ region of the cDNA, pGP24 contains U3, R sequences and 3’ terminal oligo(A) tracts, indicating that it represents the 3’ end of retroviral mRNA. Since the pXenv probe does not differentiate the unique sequence of xenotropic virus from that of MCF virus, we compared three segments of amino acid sequence to discrimate pGP24 from MCF virus according to the definition previously reported (Khan, 1984). The 3’ end of the pol gene fragment in pGP24 had a deletion of four amino acids in comparison with MCF247 (Fig. 5A) (Holland et al., 1983). This deletion is identical with that of infectious xenotropic viruses (NZB-IU-6 and NFS-Th-1) (O’Neill et al., 1985) and AKR ecotropic virus (Lenz et al., 1982). However, the nucleotide sequence on either side of this deletion in pGP24 is more like that of the infectious xenotropic viruses than of AKR ecotropic MuLV DNA. Comparison of the 5’ env region of the cloned pGP24 and other known sequences is shown in Fig. 5B and C. Insertion of 4 amino acids (Fig. 4A and Fig. 5B) and deletion of one amino acid (Fig. 4B and Fig. SC) in pGP24 were identical with that of infectious xenotropic viruses but not with MCF247. These findings indicate that the gp70 induced as an acute phase protein in hepatocytes is derived from endogenous xenotropic virus. The struc-
ture of pGP24 was aligned with the MO-MuLV as shown in Fig. 6. The other clone, pGP68, has a 3.3 kb insert and starts at a point that is equivalent to 15 bp downstream from the cap site of the MO-MuLV genome. The nucleotide sequence of pGP68 is identical to that of pGP24 excluding a 362 bp inserted fragment between the splicing donor and acceptors site of pGP24. The nucleotide sequence of this inserted region was compared with MO-MuLV genome sequence and could be aligned with the gug and pol region of MO-MuLV (Fig. 6). This may indicate that pGP68 is alternatively spliced from the endogenous xenotropic viral genome and represents LPS-induced 3.4 kb mRNA detected by Northern hybridization analysis using gag specific probes. pGP68 cDNA does not contain the fragment corresponding to that of pMoP”’ used in Northern hybridization analysis (Fig. 3). Thus, there may be different types of altenative splicing. We further compared the structures of pGP24 and the infectious endogenous xenotropic virus NZB-IU-6 previously reported (O’Neill et al., 1985). In the em coding region, 19 nucleotide substitutions were recognized but no insertions or deletions of nucleotides. The amino acid sequence of pGP24 was identical to the comparable portion of NZB-IU-6 gene in 640 out of 644 amino acids (99% homology). The assignment of the U3, R and U5 regions as well as functional domains for TATA and polyadenylation signal boxes were identical those of NZB-IU-6 except for the CCAAT sequence. In addition, both the NZB-IU-6 and our cDNA LTR sequences had the tandem direct repeat CAGCCC found upstream from the TATA box in xenotropic but not ecotropic or MCF viral LTR sequences (fin Fig. 4). However, neither NZB-IU-6, pGP24 nor pGP68 has the 190 bp transposon-like insert (Khan and Martin, 1983) upstream from the CCAAT-like box (e in Fig. 4) in the LTRs (e in Fig. 4). The most notable difference a deletion of 14 nucleotides is in the U3 LTR region of NZB-IU-6 and MCF247 in comparison with pGP24 and others (Fig. 7).
Serum gp70 as an APP
These nucleotide sequences indicate that clones pGP24 and pGP68 are derived from a xenotropic virus. Northern probes
hybridization
analysis with synthetic
DNA
To confirm the dominant expression of enu-related RNA, which we have cloned as a cDNA, we performed Northern hybridization analysis using synthesized oligonucleotide probes. According to the difference in U3 LTR between pGP24 and NZB-IU-6, as shown in Fig. 7, we also designed a 29-bp pGP24,,, probe to distinguish them from the characterized infectious NZB xenotropic virus sequence (a in Fig. 7). Northern hybridization analyses were applied to the total RNAs isolated
577
from liver, thymus, spleen, kidney and brain tissues of LPS stimulated and unstimulated NZB mice (Fig. 8). A remarkable increase of env-related 3.4 kb and 7.2 kb RNA transcripts was detected in the livers and a 3.4 kb transcript was found faintly in the kidneys of LPSinjected mice (invisible in Fig. 8) by using pGP24,,,. These findings and the cDNA sequence analysis indicate that our cDNAs represent LPS-induced enu-related transcripts and encode serum gp70 products. DISCUSSION There are many copies of MuLV-related sequences in the mouse genome. Some of these proviral genomes
578
cxuxxxc-~GAcII;AGIcG crlws-8
2340 2400
Fig. 4(B) Fig. 4. Complete DNA sequence of pGP24. The location (nucleotide numbers) of significant MuLV sequences are as follows: U5 region in LTR, -412 to -343; splicing site, -278; U3 region in LTR, 1968 to 2317; R region in LTR, 2318 to 2442; CAT box, 2287 to 2291; TATA box, 2344 to 2350; polyadenylic acid signal, 2420 to 2425. The locations of env initiation and termination were deduced by analogy with MO-MuLV proviral DNA sequences. Underlined italics indicate potential glycosylation sites. Arrows indicate the predicted sites of cleavage. Numbers without parentheses indicate nucleotide positions. Amino acid positions are shown inside parentheses. (a) Xenotropic virus specific sequence (Refer to Fig. 5B), (b) Deletion (1) specific for xenotropic virus (refer to Fig. 5C), (c) Synthesized oligonucleotide probe corresponding sequence (refer to Fig. 7 and 8), (d) Sequence deleted in L62 (refer to Discussion), (e) Deletion (A) of 190 bp insert (transposon-like element) characteristic for endogenous proviruses (refer to Discussion) (Khan and Martin, 1983) (f) 6 bp duplication (refer to Discussion) (Khan and Martin, 1983).
are responsible for the expression of env gene products, i.e. serum gp70, GIX antigen, and Fv-4 (Ikeda and Sugimura, 1989). Among these gene products, serum gp70 has been regarded as a major nephritogenic endogenous antigen in immune complex glomerulonephritis of New Zealand mice (Yoshiki er al., 1974; Maruyama et al., 19836). In this study we examined the characteristics of gp70 synthesized in the liver and
its enhancement by LPS in the acute phase of inflammation. We have examined the induction of env-related RNA by LPS and found that the major site of serum gp70 synthesis was in hepatocytes with a minor degree of induction in the kidney (Fig. 1). This induction corresponds to the gp70 expressed in tubular epithelium (Lerner et al., 1976). This result suggests that the
Serum gp70 as an APP A. pGP24 -14 (nt)PAGTA----WKV10 (nt) NZB-IU-6 PAGTA----WKV MCF247 PAGTASGPTWKV AKR EC0 PIKPS----WRV
B. pGP24 NZB-IU-6 MCF247 AKR EC0
1983; Bosze et al., 1986; Laigret et al., 1988), we have not yet detected the genetic element responsible for gp70 induction in hepatocytes. Their is still a possibility of novel consensus sequence for IL-6 responsiveness in LTR of pGP24 or pGP68 cloned by us. A recent study on the gene product of truncated MuLV proviral genome suggested another possibility. Ikeda and Sugimura (1989) examined the biological function of a S-flanking cellular sequence adjacent to Fv-4’ allele. Fv-4 is a mouse gene which controls susceptibility to infection by ecotropic murine leukemia virus (MuLV). They cloned part of an endogenous MuLV associated with the resistance allele of the Fv-4 gene (Fv-4’). The whole structure of Fv-4’ allele shows the truncated structure lacking a 5’ LTR of MuLV. This fragment of approximately 13 kb including the whole Fv-4’ genome (1 Fv4- 17) was transfected to NIH3T3 cells, resulting in greater expression of Fv-4’ env gene product when compared with the transfection of 1.5 kb 3’ flanking with the Fv-4’. This result suggested that the S-flanking region of IIFv4- 17 contains transcriptional regulatory elements enhancing the expression of Fv-4’ transcript, splice donor sites, and/or untranslated exon(s). By the same token, the transcript expressed in the NZB liver could have similar regulatory elements in the cellular sequence adjacent to the proviral genome encoding serum gp70. This sort of element may enhance the production of gp70 in hepatocyte. The induction of serum gp70 in the liver and the expression of the thymocyte differentiation antigen, GIX, in the thymus share same common features. The expression of GIX antigen with gp70 properties is regulated by two unlinked loci, Gv-1 and Gv-2 (Stockert et al., 1971). These loci also seem to affect the induction of serum gp70 in the liver. A 129GIX+ murine strain showed higher basal levels of serum gp70 (20 pg/ml in males) than a congenic 129GIX- strain (1.8 yg/ml), yet both developed increases of serum gp70 in response to LPS. Since only Go-1 should differ between these substrains, the amounts of serum gp70 might be regulated by this locus. Moreover, Sgp-2, the locus responsible for gp70 induction as an APP, is located on chromosome 7, as is Gv-2. These findings not only suggest some common mechanisms for the expression of env gene products
(NFS-Th-11
81 VGDYWDDPEPDIGDGCRTPG 100 VGDYWDDPEPDIGDGCRTPG (NFS-Th-11 IGDDWDE----TGLGCRTPG (El1 GPSYWGPPCCS-GSSDSTPG t IGLEYRAPFSPPP]
C. pGP24 NZB-IU-6 MCF247
161 KDQGPCYDSS-VSSGVQQATP180 KDQGPCYDSS-VSSGVQQATP fNFS-Th-II QNQGPCYDSSAVSSDIKGATP (El, L5111
Fig. 5. Comparison of the deduced amino acid sequences of pGP24, MCF247, NZB-IU-6, and AKR ecotropic (AKR ECO) viruses in three segments of the env region. Designations of cloned DNA with identical sequences are shown in parentheses. El and L511 are reported by Levy (Levy et al., 19856). (A) Deletion of 4 amino acids in 3’ pol region overlapped with 5’ env region of pGP24. Although pGP24 has no open reading frame in the pal region, these four sequences were aligned to compare the 3’ pal region. This segment maps between 1 nt and 2 nt (1st codon of env region) in Fig. 4. (B) Insertion of xenotropic specific sequence in pGP24. This segment corresponds to the segment “a” in Fig. 4. Number indicates amino acid position shown in Fig. 4. (C) Single amino acid deletion specific for xenotropic virus in pGP24. Number indicates amino acid position shown in Fig. 4. existence of gp70 in renal tubuli tion of serum components.
is not due to reabsorp-
Hara and colleagues (1982) reported that serum gp70 behaves like an APP in several respects and its expression is controlled by a mechanism similar to that of other APPs. There are several reports that some APPs are induced by various cytokines including IL-6. Generally APPs induced by this cytokine have IL-6 responsive element in their genomes (Heinrich et al., 1990). We recently examined the effect of IL-6 on the production of gp70 in hepatocytes. The primary hepatocyte culture with recombinant IL-6 showed the enhancement of the gp70 synthesis (Itoh et al., 1992). Our data suggests that LTR as probable promoter of pGP24 or pGP68 may have IL-6 responsive element. Although several reports have described the tissue specific replication of MuLV restricted by the LTR structure (DesGroseillers et al.,
gag
LTR pls
Pd
-Ul-
LTR
OPfO
p30 P’2
579
p15E
PI0
z
7
pGP24
I------__
pGP68
-t,
Y
,8-x,
------_--_________------
”
____--------
,.-------__________,-___------
Fig. 6. Alignment of two cloned endogenous xenotropic viral sequence, pGP24 and pGP68, with whole MO-MuLV genome. Broken lines represent spliced regions. A linear map of murine retrovirus genome and gag and env probes (hatched bars) used in Northern hybridization analysis in Fig. 2 are also shown.
K.
580 2061
pGP24
SHIGEMOTO
2120 TACTAGGACA
a
TACAAGGAAG
TTCAGTLTAA
NZB-R-6
. . . ..AA...
..TG......
..A.[
MCF247
. . . . .. . . . . ..T.......
..A.[
E2 L62 B-77
cltul
I..
..T.......
. . ..G.....
I . . . . . . . ..C.. A......... . . . . . . . . . . . . . . . . ..T. . . . . . . . . . . . ...G..... .. . .. . . ..G C...G..... . . ..a..... . . . . . . .. . . . . . . . . . . . . . ...G..T.. .,........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . ..C.......... e b
. . . . . .. . . . . . ..G.....
b'
Fig. 7. Comparison of LTR sequences. The deletion of 14 bp is indicated as brackets. The segment, b and b’ indicates a 6 bp duplication (refer to Discussion) immediately upstream from TATAA box of B-77 (endogenous provirus sequence) (Khan and Martin, 1983). E2 and L62 are described by Levy (Levy et al., 1985b, 1987).
but also raise the question of whether serum gp70 and GIX antigen are identical. Although the serum gp70 in 129GIX was not well-characterized, no structural difference was found among various mouse strains by tryptic peptide analysis (Elder rt al., 1977). Levy and colleagues (Levy et al., 1985a) determined the structure of endogenous retrovirus derived from various tissues of the 129GIX strain. They characterized cDNA clones preRNA of several tissues of pared from poly(A)+ 129GIX+ mice. Those cDNAs encoded a part of gp70 which is determined by Ga - 1-responsive endogenous retrovirus loci (Levy et al., 1985b; Levy et al., 1987). We have now aligned the amino acid sequences of two cDNA clones, El and L511 derived from epididymis and liver, respectively, with pGP24 (Fig. 5B and C). The amino acid sequence of pGP24 is identical with that of the xenotropic proviral genome. In contrast, El and L511 were characteristic of MCF virus. Of the structural differences in the regulatory region recognized between pGP24 and the Gu- l-responsive endogenous retroviruses, the most notable was a 39-bp deletion in the 129GIX+ mouse liver derived clone, L62, compared with pGP24 (d in Fig. 4). Levy and colleagues (1985a) suggested the action of a regulatory element, encoded by Go-l, functioning in tram to control the normal expression
129GIX+
of endogenous
retroviral
and GIX-- mouse strains.
sequences
in congenic
These findings
indi-
LPS (-)
cate that the Gv-1 system affects the induction of both the MCF viral genome and the xenotropic proviral genome encoding serum gp70. There is no report, including our preliminary study, on the existence of gag- or pol-gene products in the liver. However, we showed that LPS induced gag- and pol-related RNA in the liver (Fig. 3). Based on the nucleic acid analysis of cloned cDNA (pGP68), the truncated gagand pol-related transcripts lay adjacent to the enc-gene. These gag- and pol-related transcripts did not have an open reading frame. These findings suggest that the induction of gag- and pol-related transcripts by LPS results from enr-related transcription, as for pGP68. This would account for the absence of gag- and pol-gene products in the liver. However a conflicting result was shown in our study. The pGP68 contains the partial codes of the pol gene, but not pMoJ”“. The induction of pol gene in the hepatocytes after LPS injection was detected by pMoP”’ probe (Fig. 3). There are following two explanations. (1) The expression of pMd”” corresponding RNA due to different types of alternative splicing. (2) The activation of multiple units encoding pol gene corresponding to pMd”’ probe. Temin (1980) has proposed that retroviruses evolved from cellular moveable genetic elements. Since serum gp70 is synthesized in hepatocytes and secreted into the circulation, we expected that serum gp70 might be an
LPS (+)
-7.2
kb
-3.4
kb
Fig. 8. Induction by LPS of xenotropic and pGP24 eno-related RNAs in the NZB liver. The source of total RNAs was the same as noted in Fig. 1. Northern hybridization analysis was performed using pGP24,,,., synthetic probes mentioned in Materials and Methods. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was used to confirm that comparable amounts of RNA were present in all lanes.
Serum gp70 as an APP
evolutionally ancestral protein of retroviruses. However, its structure is not remarkably distinct from other retroviruses reported. Khan and Martin (1983) analyzed the sequence and compared LTRs associated with infectious xenotropic, ecotropic, and MCF proviruses. The three segments of LTR compared were: (1) 14 bp duplicated sequence (b and b’ in Fig. 7); (2) insertion or deletion of the highly conserved transposon-like sequence (190 bp) and the adjoining region (e in Fig. 4) (Khan and Martin, 1983); (3) 6 bp duplication (f in Fig 4). Based on their analyses, they proposed a possible evolutionary scheme relating endogenous and infectious MuLV LTRs (Khan and Martin, 1983). The LTR of pGP24 has a 14 bp duplicated sequence as a hallmark of the endogenous group (B-77, E2 and L62 in Fig. 7). Because pGP24 LTR is missing a highly conserved 190 bp insertion, this clone was regarded as “mature”. The 6 bp duplication is also a definitive marker of xenotropic virus in concert with several hallmarks of the enu region as shown in Fig. 5. These hallmarks may mean that pGP24 is a hypothetical immediate precursor of infectious xenotropic virus or precursor of the ecotropic virus via separate lineage in the evolutionary scheme (Khan and Martin, 1983). Acknowledgements-We thank Professor Toshikazu Shirai, Juntendo University, and Professor Masaru Taniguchi, Chiba University for their discussion and support during our investigation. The excellent editorial assistance of MS Phyllis Minick and technical assistance of MS Yoshie Urano are gratefully acknowledged. This study was performed through Special Coordination Fund of the Science and Technology Agency of the Japanese Government, and Sandoz Pha~aceutical Japan. REFERENCES Bosze Z., Thiesen H.-J. and Charnay P. (1986) A transcriptional enhancer with specificity for erythroid cells is located in the long terminal repeat of the Friend murine leukemia virus. EMBO J. 5, 1615-1623. Buckler C. E., Hoggan M. D., Chan H. W., Sears J. F., Khan A. S., Moore J. L., Hartley J. W., Rowe W. P. and Martin M. A. (1982) Cloning and characterization of an envelopespecific probe from xenotropic murine leukemia proviral DNA. J. Viral. 41, 228-236. Ghan H. W., Bryan T., Moore J. L., Staal S. P., Rowe W. P. and Martin M. A. (1980) Identification of ecotropic proviral sequences in inbred mouse strains with a cloned subgenomic DNA fragment. Proc. natn. Acad. Sci. U.S.A. 77,5779-5783. Chirgwin J. M., Przybyla A. E., MacDonald R. J. and Rutter W. J. (1982) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. B~oehem~tr~ 18, 5294-5299. DesGroseillers L., Rassart E. and Jolicoeur P. (1983) Thymotropism of murine leukemia virus is conferred by its long terminal repeat. Proc. natn. Acad. Sci. U.S.A. 80,4203-4207. Elder J. H., Jensen F. C., Bryant M. N. and Lemer R. A. (1977) Pol~orphism of major envelope glycoprotein (gp70) of murine C-type viruses: multi-gene family. Nature (London) 267, 23-28. Hara I., Izui S., McConahey P. J., Elder J. H., Jensen F. C. and Dixon F. J. (1981) Induction of high serum levels of retroviral env gene products (gp70) in mice by bacterial
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Maruyama N., Lindstrom C. O., Sato H. and Dixon F. J. (1983a) Serum gp70 production regulated by a gene on murine chromosome 7. Immunogenetics 18, 365-372. Maruyama N., Furukawa F., Nakai Y., Sasaki Y., Ohta K., Ozaki S., Hirose S. and Shirai, T. (1983b) Genetic studies of autoimmunity in New Zealand mice. IV. Contribution of NZB and NZW genes to the spontaneous occurrence of retroviral gp70 immune complexes in (NZB x NZW)F, hybrid and the correlation to renal disease. J. Immun. 130, 740-746.
Nakai Y., Maruyama N., Ohta K., Yoshida H., Hirose S. and Shirai T. (1983) Genetic studies of autoimmunity in New Zealand mice. Association of circulating retroviral gp70 immune complex with proteinuria. Zmmun. Lett. 2, 53-58. O’Neill R. R., Buckler C. E., Theodore T. S., Martin M. A. and Repaske R. (1985) Envelope and long terminal repeat
sequences of a cloned infectious NZB xenotropic murine leukemia virus. J. Viral. 53, 100-106. Repaske R., O’Neil R. R., Khan A. S. and Martin M. A. (1983) Nucleotide sequence of the env-specific segment of NFS-Th1 xenotropic murine leukemia virus. J. Viral. 46, 204-211. Policastro P. F., Fredholm M. and Wilson M. C. (1989) Truncated gag products encoded by Gv-l-responsive endogenous retrovirus loci. J. Virof. 63, 413&4147. Shigemoto K., Maruyama N., Itoh Y., Kubo S. and Koike T. (1989) Murine leukemia virus gene product as an acute phase protein: Complete suppression of serum gp70 synthesis in hepatocytes of B1O.S mice. C/in. exp. Immun. 78, 484487. Stockert E., Old L. J. and Boyse E. A. (1971) The GIX system: A cell surface allo-antigen associated with murine leukemia virus. J. exp. Med. 133, 13341355. Temin H. M. (1980) Origin of retroviruses from cellular moveable genetic elements. Cell. 21, 5999600. Yoshiki T., Mellors R. C., Strand M. and August J. T. (1974) The viral envelope glycoprotein of murine leukemia virus and the pathogenesis of immune complex glomerulonephritis of New Zealand mice. J. exp. Med. 140, 1011-1027.
