REVIEW ARTICLE

Retroviruses and AutoimmunityIwao Sekigawa, Hitoshi Ogasawara*, Hiroshi Kaneko*, Takashi Hishikawa* and Hiroshi Hashimoto*

Abstract

The investigation of humanretroviruses has showndra-matic progress since the discovery of human immunodefi-ciency virus (HIV). These studies have also contributed tothe exploration of the role of retroviruses, including endog-enous retroviruses, in the induction of autoimmune diseasessuch as systemic lupus erythematosus (SLE). This reviewdescribes the potential role of retroviruses in autoimmu-nity, based on recent findings including our own results.(Internal Medicine 40: 80-86, 2001)

Key words: human immunodeficiency virus, human endog-enous retrovirus, systemic lupus erythematosus

Introduction

Retroviruses, including endogenous retroviruses, have re-peatedly been suggested as etiological factors for autoimmunerheumatic diseases such as SLE (1-4). Extensive studies per-formed during the last decade on the molecular biology of HIV-1 and human T cell leukemia virus (HTLV)-l have providedboth the intellectual framework and the molecular methods totest whether human retroviruses may be involved in the patho-genesis of a wide spectrum of autoimmune phenomena. In ad-dition, marked advances in our knowledge of human endog-enous retroviruses (HERV)have contributed to the investiga-tion of their role in the pathogenesis of humanautoimmunediseases. Onthe basis of recent results obtained at our labora-tory, we here review the virological and immunological prop-erties of human retroviruses that may be involved in the induc-tion of autoimmunity, especially the SLE- and HIV-relatedautoimmunephenomena.

Retroviruses and Autoimmunity

Retroviruses are plus strand RNAviruses with a modeofreplication in which the RNAgenome is transcribed into DNAby reverse transcriptase (RT) and is integrated into the hostgenome as a provirus. Twoidentical elements (the long termi-

nal repeats: LTRs) are reconstituted at the 5' and 3' ends of theprovirus, which contain the promoter, enhancer, and repressorsequences for tissue-specific expression. All genera ofretroviruses encode three basic genes, which are expressed andprocessed as polyproteins: gag encoding the core proteins, polencoding the RT, and env encoding the envelope proteins. Whileexogenous retroviruses such as HIVand HTLVcan reproduceviral RNAand viral particles from their proviral DNA, endog-enous retroviruses have generally lost the competence to repli-cate viral proteins because of several interrupters, includingtermination codons, deletions, frameshift mutations, or me-thylation sites. Endogenousretroviruses are knownto exist notonly in animals, but also in the genomic DNAof humans; ap-proximately 20 HERVfamilies have been reported to date, allof which are inherited in a Mendelian fashion (3, 5-8). Theprecise origin of HERVremains unclear, but certain HERV-related sequences can be traced back to the time of the diver-gence of Old and NewWorld monkeys (25-30 million yearsago) (7, 9). HERVare estimated to compose up to 0.1-0.6% ofhumanDNAand thus contribute substantially to the architec-ture of the human genome (10). Recent studies have revealedthat certain HERVcan undergo transcriptional and translationalactivation that results in HERVprotein expression and HERVparticle formation (3, 6, 1 1).The possibility that retroviruses may play an etiologic rolein various autoimmune diseases (such as rheumatoid arthritis,Sjogren's syndrome, mixed connective tissue diseases, inflam-matory neurologic diseases, and SLE) has long been discussed(3). A role for retroviruses as an etiologic factor in SLE is sup-ported by the following evidence: 1) the importance of endog-enous retroviruses in mouse models of SLE (12-14); 2) thedetection of antibodies, antigens, and sequences for animal andhumanretroviral components in the organs and serum of pa-tients with autoimmune diseases such as SLE (15-23); 3) elec-tron microscopic detection of unknownretroviral particles inthe organs of SLE patients (24); 4) the finding that viral com-ponents derived from endogenousor exogenousretrovirusescan induce the immune abnormalities observed in SLE (25-27); and 5) the similarity of autoimmune manifestations andimmune dysregulation between patients with SLEand thoseinfected with known human retroviruses such as HIV-1 (4, 28).

From the Department of Medicine, Juntendo University Izu-Nagaoka Hospital, Shizuoka and *the Department of Internal Medicine and Rheumatology,Juntendo University School of Medicine, Tokyo

Reprint requests should be addressed to Dr. Iwao Sekigawa, the Department of Medicine, Juntendo University Izu-Nagaoka Hospital, 1 129 Nagaoka, Izu-Nagaoka-cho, Tagata-gun, Shizuoka 4 1 0-2295

80 Internal Medicine Vol. 40, No. 2 (February 2001)

Retroviruses and Autoimmunity

HIV and AutoimmunityThe mechanisms of autoimmunity in HIV- 1 infection havebeen aggressively debated. Several autoantibodies, includingantinuclear antibody, anti-cardiolipin antibody, anti-plateletantibody, and anti-lymphocyte antibody can be detected in theserum of HIV-infected patients. T cell and B cell dysfunctionassociated with HIV-1 infection is knownto be similar to thatobserved in SLE, including T cell activation (implied by theexpression of HLAclass II molecules), T cell anergy (indi-cated by a decrease of the mixed lymphocyte reaction (MLR)and by a decline of mitogen/antigen-mediated interleukin (IL)-2 production and cell proliferation), and polyclonal B cell acti-vation (PBA) involving cytokines such as IL-6 and tumor ne-crosis factor (TNF)-oc (4). CD4+T cell anergy (a weak responseor unresponsiveness to antigen specific or nonspecific stimuli)is known to be closely related to its activation in SLE patients(29). HIV-1 infection is initiated by binding between HIV-1envelope glycoprotein (gpl20) and CD4 molecules (Dl do-main) on T cells, although HIValso employs receptors forchemokines as co-receptors. HIV-1 gpl20 is known to play animportant role in the immunedysregulation associated withHIV- 1 infection, including the SLE-like phenomena describedabove, either directly or via the stimulation of cytokine pro-duction (30-41). The features of gp l20-mediated immune dys-function are summarized in Table 1, based upon reported re-sults including our own. As shownin this table, someaspectsof gpl20-mediated T and B cell dysfunction are enhanced bycytokines, especially TNF-oc, derived from gp l 20-stimulatedmacrophages. Various events affecting in CD4+T cells, such asloss of CD4molecules (due to down-modulation or apoptosis),T cell receptor (TCR) inactivation, and blocking of conjuga-tion between CD4and the major histocompatibility complex(MHC),as well as CD4+T cell anergy related to the activation

(Table 1), mayinfluence the antigen recognition process andmay be a significant factor in the decline of self-tolerance andthe resultant induction of autoimmunity by HIV infection inaddition to PBA.Interestingly, the changes of in vivo and/or invitro cytokine and chemokine productions with the progres-sion of HIV-1 infection are similar to those occurring in SLEwith an increase of disease activity, including an increase inthe production of T helper type 2 (Th2) cytokines and RANTES(regulated-upon activation, normal T expressed and secreted),as well as a decrease in the production ofT helper type 1 (Thl)cytokines and monocyte chemoattractant protein- 1 (MCP- 1)(42-45). Such changes of cytokine and chemokine productionare also regulated through stimulation by HIV envelope glyco-protein (46, 47). Furthermore, antibodies to CD4 moleculesare known to exist in 10-20% of HIV-1-infected patients andin 20-30% of SLE patients (48, 49). In SLE patients, theseantibodies can induce T cell anergy (49). Fluctuations in theproduction of cytokines, chemokines, and such autoantibodiesmaybe linked to the occurrence of SLE-like autoimmune phe-nomena in HIVinfection.In contrast, epidemiological studies have revealed that HIVinfection and SLE are rarely seen in the same patient. In fact,only 20 patients with a diagnosis of both SLE md HIV infec-tion,have been reported to date (50). Wepreviously reportedthat serum levels of IL-16 in SLE patients were significantlyhigher than in normal volunteers and that there wasa strongcorrelation between the IL- 16 level and the disease activity ofSLE (51). IL-16 is a CD8+T cell-derived cytokine that usesCD4 as its receptor (52) and can inhibit HIV-1 infection invitro (53). This cytokine also induces activation and anergy ofCD4+ T cells observed in SLE (52, 54, 55). Although severalpossibilities have been proposed, we think that IL-16 may havea protective effect against HIV infection in SLE patients andmaycontribute to the low incidence of the coexistence of bothdiseases (50).

Table 1. Immune Abnormalities Mediated by the Binding of HIV-1 gp!20 to CD4g p 1 2 0 -sti mu la te d ce ll I mm u n e a bn o r m al i t i e s ( r e f er e n c e s) T N F - c c d e p e n d e n c y

T ce ll 1. A ct iv a ti on o fp 56 l ck ( 35 , 37 , 4 1) (- )

2 . D o w n - m o d u l a t i o n o f C D 4 ( 3 5 , 4 1 ) (+ )(in te rn aliz atio n )

3 . CD 4 + T c el l a ct iv a ti on (4 1) (+ )( H LA c la s s I I e x pr e ss i o n)

4. CD 4 + T ce l l a n er g y ( 3 0, 4 1)

( s up p re s si o n o f I L- 2 p r od uc t i on ) (+ )( i nh i b i ti o n of M L R) (- )

5. CD 4 + T c el l a p op t os i s ( 34 , 3 9 ) (+ )6. T CR i na c t iv a t io n (3 7 ) (- )7 . B lo c k i n g o f b i n d i n g b e t w e e n C D 4 a n d M H C ( 3 1 ) (- )

M a c ro p h ag e 1 . C yt ok i ne p r od uc t io n ( IL -6 , I L- 1 0, T N F- oc , e tc . ),wi th re la ted T c ell ( 38 , 3 9, 41 ) (+ )an d B ce l l a bn o rm a li t ie s ( P BA ) ( 3 2, 33 , 3 6 ) (+ )

2 . D e c r e a s e o f C D 4 e x p r e ss i o n o n m a c ro p h a g e s ( 4 0 ) (+ )

Internal Medicine Vol. 40, No. 2 (February 2001 ) 81

Sekigawa et al

HERV and SLE

HERVclone 4-1 is a memberof the HERV-Efamily that iswidely distributed in the genomic DNAof Japanese individu-als. As it is one of the most completely understood HERVwithrespect to structure and other characteristics (5), we have beeninvestigating this clone as a possible causative agent of SLE.Clone 4-1 shows sequence homology with Moloney murineleukemia virus (MuLV) and is approximately 8.8 kb in size,with open reading frames in the gag, pol, and env regions (Fig.1). This HERVsequence was originally detected in human brainfragments and in a human DNAlibrary using African greenmonkey DNAas a probe that hybridized to MuLVand endog-enous baboon virus (BaEV) (5). Recent studies using humansequence data banks have shownthat there are approximately85 copies of clone 4-1 at 7 to ll integration sites in humanchromosomes (8, 56, 57). Clone 4-1 transcripts and gene prod-ucts have been reported in syncytiotrophoblasts and in the vas-cular endothelium of some human placentas (1 1).Although the transcription and translation of HERVclone

4- 1 is not observed in peripheral blood lymphocytes (PBL) from

normal individuals, we have detected messenger RNAfor theclone 4-1 gag region in PBL from SLE patients (Fig. 2A). De-tection was positive by both the standard polymerase chain re-action (PCR) and the real-time quantitative PCR (RQ-PCR;TaqManmethod), which can analyze the quantity of mRNAand is more sensitive than RT-PCR(58). Furthermore, the gagregion antigen (p3Oa*) was detected in PBL from SLE patients,but not in PBLfrom normal controls (Figs. 2B and 2C). Sup-porting this finding, we have previously reported the detectionof serum antibodies to recombinant clone 4-1 gag products(p3Oa*) in approximately 50% of SLE patients, but not in nor-mal controls (23). Similar data to those for the clone 4-1 gagregion have also been obtained for the env region, althoughthese results are not described here. These findings suggest thatthe transcription and translation of clone 4-1 may occur in SLEpatients, but not in normal individuals. In fact, our quantitativeanalysis using RQ-PCRrevealed a decrease of clone 4- 1 mRNAin SLE patients after steroid therapy (data not shown). Thus,steroid therapy may inhibit the transcription of HERVand thisinhibition (probably due to suppression of the promoter region)may be important for the efficacy of steroids in controlling SLE,in addition to an immunosuppressive effect on lymphocytes.

__I I I I I, ,. LTR gag pol env LTR

clone4-1 I I I 1_ I I

1 1,042 2,650 6,233 8,309 8,806

ORF1 CZD p3°gaS (i-630"2'650) P!5E (7,628-8,316)ORF2P,

ORF3' i 1

0RF4 ORF5 C=». .1630 ?Qis ORF6 à"------* .

i^u Z,yid QRF7 CZ3

original sequence TGA TAA - TAG -\ TGA |SLE1 TGG GAA GAG~ jTGAj

SLE 2 CGG GAA GAG | TGA |SLE 3 CGG GAA GAG -J TGA J

i ic hromosome ll CGG GAA GAG J TGA !I 1 I 1 I 1 i j

^1,891 2,011 2,374 2,647

"^-^ Hpl5 ^~'*~

chromosomell ( BUI H |B ^Cft B11^)

Figure 1. General structure of clone 4-1 and various genomic DNAsequences. The sequences between nt 1,630and nt 2,913 were analyzed. The changes to the original stop codon sequences in the SLEpatients are shown bysolid boxes and the conserved sequence is shown by a dotted box. The prototype sequence of clone 4-1 is based ondata from Repaske et al (5). The consensus sequence of clone 4-1 in chromosomell is modeled from the GenBankdatabase and the location of clone 4-1 in chromosome ll is based on data from Taruscio et al (56, 57). Numbersindicate the nucleotide numbersand ORFmeans open reading frame.

82 Internal Medicine Vol. 40, No. 2 (February 2001)

Retroviruses and Autoimmunity

M l 2 3 4 5 6 7 8

bp1 QCQ - -** aJMIl^ML --à"å --̂ ^j^g^g^.

1,078 HH^^^^H^ ^^^å å W^g^ !

603 ^^^^^^^^H|310 HH^^^^^^^^^^^^^^^^lHfi ; A

Figure 2. Detection of clone 4-1 mRNAand p3&"s antigen in PBL. An approximately 600 bp fragment of the clone 4-1 gag regionwas amplified by RT-PCR using two gag primers (primer 1; nt 1,651-1,668, and primer 2; nt 2,238-2,250), (SLE patientswithout steroid treatment; lanes 1-5, normal individuals; lanes 6-8, Lane M; molecular weight marker) (A). PBL were stainedwith goat anti-MuLV pW*"* antibody, which reacted with recombinant clone 4-1 P308"8 as reported previously (23), and withfluorescein isothiocyanate (FITC)-labeled rabbit F(ab')2 anti-goat inimunoglobulin (Cappel Laboratories, West Chester, PA) asthe secondary antibody. A positive reaction was observed in SLE PBL(B), but not in normal PBL (C).

Interestingly, other immunosuppressants (cyclosporin A andFK506) have been shown to inhibit the promoter activity ofHIV provirus integrated into the human genome, thus suppress-

ing its transcription (59).Wehave found that 5-aza-deoxycytidine, a demethylating

agent, causes a quantitative increase of clone 4-1 gag mRNA

Internal Medicine Vol. 40, No. 2 (February 2001) 83

Sekigawa et al

(detected by RQ-PCR) in normal control lymphocytes (datanot shown). Thus, methylation of DNAmay be important forinhibiting clone 4- 1 gene transcription in normal individualsand preventing the production of its proteins, and this may alsobe true for other HERV(60). With reference to this point, somedrugs such as procainamide and hydralazine, which have ademethylating effect, are knownto occasionally trigger an SLE-like illness, especially in genetically predisposed individuals(6 1). These drugs maypromote the transcription of endogenousantigen-related genomic DNA, including HERVDNA, throughdemethylation and thus elicit autoimmunephenomena.Inter-estingly, however, the stimulation of mitogens such as li-popolysaccharide (LPS), concanavalin A (Con A), and phyto-hemagglutinin (PHA) does not induce a quantitative increaseof clone 4- 1 mRNAfrom normal PBLin vitro (data not shown).This indicates that clone 4-1 expression is not the consequenceof cell activations, though SLE-lymphocytes are known to beactivated in vivo (62).The prototype clone 4-1 gag region reported by Repaske etal has four stop codons between nt 1,630 and nt 2,650 (5). Ouranalysis using direct sequencing revealed that clone 4-1 DNAfrom several SLEpatients showedchanges at the same nucle-otides when compared with the original sequence (Fig. 1) (58).In all three SLE patients studied, adenine (A) at nt 1,893 waschanged to guanine (G), thymine (T) at nt 2,011 was changedto G, and T at nt 2,374 was also changed to G. As a result ofthese variations, three of the four stop codons in the gag regionof clone 4-1 were inactivated (Fig. 1). Our computer search ofcurrent entries in sequence libraries (GenBank database) re-vealed >90% sequence homology between genomic DNAfromthe SLEclone 4-1 gag region and the consensus sequence ofclone 4-1 located on chromosome ll of normal individuals,including inactivation of these stop codons, unlike clone 4-1located on the other chromosomes(Fig. 1). Wecannot com-pletely rule out the possibility that the virus detected by ourPCRprimers was not actually clone 4- 1 , because there are manymembers of the HERV-Efamily that are genetically similar tothis clone (6, 8). However, more precise information on theintegration sites of clone 4-1 in human chromosomes maybe-comeavailable as a result of the recent completionof the hu-man genomeproject. Despite this limitation, our findings raisethe possibility that the clone 4-1 transcribed in SLE patientsmay be derived from chromosome ll, and that inactivation ofstop codons maycontribute to the translation and productionof gag proteins. Chromosomell is known to contain a breakpoint, which is initiated by several activators including X-rays(56, 57). This is also interesting in view of the potential role ofenvironmental factors in the induction of SLE, because endog-enous retroviruses may be activated by various stimuli such asX-rays, ultraviolet light, chemical agents, bacteria and othermicroorganisms, hormones, and cytokines (3, 63, 64).Certain components derived from endogenous retroviruses,such as pl5E (which is a specific sequence of ^nv-encodedtransmembrane (TM) proteins and is conserved among virusesfrom several species, including murine, feline, and humanretroviruses), can induce the immuneabnormalities observed

in SLE lymphocytes, including the inhibition of IL-2 produc-tion and suppression of the lymphocyte proliferative response(25-27), although the mechanisminvolved is still unclear. TheTMenvelope proteins of clone 4-1 share amino acid sequencehomology with pl5E of the retroviruses from several species(25, 26). Our recent studies have indicated that synthetic clone4-1-derived pl5E peptides can induce CD4+ T cell activationand anergy, production of several cytokines (including IL- 1 6),and cytokine-related PBAin vitro (data not shown). Such CD4+T cell abnormalities may contribute to the loss of self-toler-ance and the induction of SLE-related autoimmune phenom-ena (including autoantibody production) in cooperation withPBA(62) and in addition to the role of molecular mimicry be-tween certain retroviral components and autoantibodies, whichis also reported to be important for autoantibody development(17, 18, 22).

Several reports have shownan important role for activatedCD8+ T cells in the induction of SLE, in addition to CD4+ Tcells. The number of CD4+T cells is generally decreased inSLE patients when compared with that of normal individualsand this phenomenon is inversely correlated with the increaseof activated CD8+ T cells (implied by HLA-DRexpression)and with the rise in the serum level ofIL-16 (55, 65). Also, theremoval of activated CD8+T cells and the loss of factors pro-duced by these cells normalizes CD4+T cell abnormalities suchas decreased in vitro IL-2 production in SLE (66, 67). Thus,activated CD8+T cells and/or the cytokines derived from thesecells seem to influence both the decrease of CD4+T cell num-bers and the impairment of CD4+T cell function in SLE (55).In general, endogenous antigens such as HERVare presentedwith HLAclass I molecules and are recognized by CD8+Tcells (68). Therefore, CD8+ T cells may be activated in SLEthrough the recognition of translated HERVproducts expressedon the cell surface and these activated CD8+T cells may thenproduce various cytokines, including IL- 16. This mechanismmay also be important in HERV-related autoimmunity, in ad-dition to the immune abnormalities promoted by the incuba-tion of cultured lymphocytes with clone 4-1 pl5E peptide, asdescribed above.Virus-infected cells are eliminated by CD8+cytotoxic T lym-phocytes (CTL) and/or natural killer (NK) cells. Recent evi-dence has indicated that the binding of killer cell inhibitoryreceptor (KIR) on CD8+cells with E antigen of HLAclass Imolecules on virus-infected cells can inhibit the cytolytic ac-tivity of CD8+cells against infected cells (69). Cytomegalovi-rus (CMV)infection is reported to occasionally promote theexacerbation of SLE (70), and a glycoprotein homologous withHLAclass I antigens is encoded by CMVand is known to be aligand of KIR (71). This mechanism may be related to the in-hibition of the killing of CMVby CTL and/or NK cells. Ourcomputer search using the GenBankdatabase revealed an ex-tremely high level of sequence homology between the clone 4-1 gag region and the E antigen (-91% homology) (72), whichmay contribute to the escape of clone 4-1 from killing by CTLand/or NKcells and to the production of clone 4-1 viral pro-teins in vivo.

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Conclusion

Retroviruses seemto be one of the most plausible causativeagents for various autoimmune diseases like SLE based onexperimental data from humans and animals, although therehave also been negative reports regarding their role in suchdiseases (73). HTLV-1 can induce several autoimmune mani-festations as does HIV-1 (74), though these findings are notdescribed here. In addition to autoimmune disorders, the po-tential role of HERVas a causative factor of other humandis-eases such as cancer and diabetes has been postulated, althoughthis is still controversial (3, 6). More accurate information onHERVwill soon be revealed by the ongoing human genomeproject. Investigation of the role of clone 4-1 in other autoim-mune diseases besides SLE, such as rheumatoid arthritis (75),as well as studies on the pathogenic influence of other HERVin SLE, should be performed to clarify the etiology of thesediseases and to develop new therapeutic strategies.

Acknowledgements: We wish to thank Dr. T. Naito, Dr. S. Lee, Dr. H.Akimoto (Juntendo University), Dr. N. Maruyama (Tokyo Metropolitan Insti-tute of Gerontology), Prof. N. Yamamoto(Tokyo Medical and Dental Univer-sity), and Dr. Y. Kaneko (Ajinomoto Co., Inc.) for helpful discussion of themanuscript.

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