Journal of A utoimmunity (1992) 5 (Supplement A), 61-66

A r e E n d o g e n o u s R e t r o v i r u s e s I n v o l v e d in H u m a n A u t o i m m u n e Disease?

N o r m a n Talal, E l i e z e r F l e s c h e r a n d H o w a r d D a n g Clinical Immunology Section, Audie L. Murphy Memorial Veterans Hospital and the Department of Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, T X 78284-7874, U S A

A role for v i r u s e s i n the e t i o p a t h o g e n e s i s o f h u m a n a u t o i m m u n e diseases has l o n g b e e n s u s p e c t e d b u t has n o t y e t b e e n p r o v e n . I n S j i i g r e n ' s s y n d r o m e (SS), t h e r e is c o n t i n u i n g e x p e r i m e n t a l s u p p o r t f o r the p o s s i b l e i n v o l v e m e n t of E p s t e i n - B a r r v i r u s . S i n c e the a d v e n t o f A I D S , t h e r e is also g r e a t i n t e r e s t i n r e t r o v i r u s e s a n d a u t o i m m u n e disease. We p r e v i o u s l y r e p o r t e d t h a t 30% o f SS p a t i e n t s a n d 36% of s y s t e m i c l u p u s e r y t h e m a t o s u s (SLE) p a t i e n t s h a v e s e r u m a n t i b o d i e s to the p24 gag p r o t e i n o f HIV-1. We n o w r e p o r t t h a t two m e c h a n i s m s classic for r e t r o v i r u s e s ( m o l e c u l a r m i m i c r y a n d i m m u n o s u p p r e s s i o n ) m a y b e o p e r a t i v e i n SS a n d SLE. T h e p24 gag p r o t e i n s h a r e s a p r o l i n e - r i c h e p i t o p e w i t h the S m n u c l e o p r o t e i n to w h i c h m a n y SLE p a t i e n t s h a v e a n t i b o d i e s . T h e i m p a i r e d l y m p h o c y t e a c t i v a t i o n seen i n p e r i p h e r a l b l o o d T cells i n SS p a t i e n t s is also seen i n a h u m a n T cell l l n e i n f e c t e d w i t h a n A - t y p e r e t r o v i r a l p a r t i c l e l i n k e d to SS. M a n y s t u d i e s suggest t h a t e n d o g e n o u s r e t r o v i r a l s e q u e n c e s a r e i m p o r t a n t i n i m m u n o r e g u l a t i o n . We n o w s u g g e s t t h a t e n d o g e n o u s r e t r o v i r a l s e q u e n c e s m a y also b e i m p o r t a n t i n the etiology a n d p a t h o g e n e s i s of SS a n d SLE.

Introduction A l t h o u g h n u m e r o u s factors ( i n c l u d i n g genetic, infectious, e n d o c r i n e a n d p s y c h o n e u r o i m m u n o l o g i c a l ) c o n t r i b u t e to the p a t h o g e n e s i s of a u t o i m m u n e disease, a c o n c e p t u a l b r e a k t h r o u g h to help e x p l a i n the etiology of these diseases is b a d l y n e e d e d . A f t e r all, a u t o i m m u n i t y can be physiological i n certain c i r c u m s t a n c e s . These studies were supported by the General Medical Research Service of the Veterans Administration, and USPHS grant no. 1R01 DE09311-01, and the RGK Foundation. Correspondence to: Norman Talal, MD, Professor of Medicine and Microbiology, Head, Division of Clinical Immunology, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7874, USA. 61 0896-8411/92/0A0061 + 06 $03.00/0

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Autoimmunity and autoimmune disease are not all the same. T h e genetic predisposition to autoimmune disease has been well studied. We know far less about the role of infectious agents aside from the important phenomena of shared epitopes and molecular mimicry [1]. Ever since the appearance of A I D S and the discovery of the A I D S virus, retroviruses have captured attention as possible causative agents in autoimmune rheumatic diseases [2-4], multiple sclerosis and the chronic fatigue syndrome [5]. T h e group of endogenous retroviruses have recently emerged as possible contributors to the etiology of human autoimmune diseases [6]. T h e thesis of this communication is that systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and Sj6gren's syndrome (SS) are disorders in which endogenous retroviral sequences are expressed and act pathologically. A considerable portion of the mammalian genome is made up of these retroviral sequences whose function is unknown. A recent series of articles has clearly linked expression of endogenous retroviruses in mice to Mls (minor lymphocyte stimulating) genes [7-10]. T h e Mls determinants act as superantigens and simultaneously associate with M H C class II molecules and all T cell receptors encoded by one or more V~ gene segments. Positive or negative selection o f t cells in the thymus by these determinants controls T cell repertoire selection, Tolerance is established through self-deletion of T cells by these regulatory elements. Our interest in viruses and autoimmunity began over two decades ago with the observation that some SS patients, after many years of benign disease, go on to develop lymphoid malignancies [11]. T h e first virus implicated in SS was the Epstein-Barr virus (EBV) [12]. A role for EBV still remains an attractive possibility. EBV could be a cofactor, along with retroviruses in disease pathogenesis. T h e evidence implicating retroviruses in SS is based on both laboratory as well as clinical findings. Similar immunological abnormalities are seen in both primary S S and A I D S patients [13]. For example, increased amounts o f C D 5 + B cells occur in SS [14] as well as in A I D S patients [ 15]. L y m p h o c y t e abnormalities of transmembrane signalling revealed by exposure to P M A and ionomycin occur spontaneously in SS [ 16] and can be induced in normal peripheral blood lymphocytes by exposure in vitro to HIV-1 [17]. T h e r e are now many examples of arthritic and dermatological symptoms occurring in subjects infected with H I V - 1 including an S S-like disease [ 18, 19]. Results

We undertook a study, by Western blot analysis, to search for antibodies to HIV-1 antigens in the sera of SS and S L E patients [20, 21]. Finding antibodies to p24 gag proteins in 30% of patients, we then co-cultured salivary gland biopsy material from SS patients with a human T cell line (H9) and found, by electron microscopy, an intracisternal A-type retroviral particle [22, 23]. We have now extended our studies to demonstrate (1) molecular mimicry in the form of a shared proline-rich epitope on p24 gag of HIV-1 and the nucleoprotein autoantigen Sm, and (2) defects in transmembrane signalling including protein kinase C activation in T cells containing the intracisternal A-type retroviral particle. Antibodies to gag proteins in S S

Antibodies to gag proteins of HIV-1 were found in 14 of 47 patients with primary SS [20] compared to one of 120 normal subjects. T w o patients also reacted to p17

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gag. Specificity was confirmed by reactivity with recombinant p24. T h e r e was no reactivity with envelope proteins of HIV-1 or with any protein of H T L V - 1 . Clinically, these 14 patients showed a broad spectrum of disease but, like HIV-infected subjects presenting with an SS-like illness [18, 19], they lacked anti-Ro(SS-A) and anti-La(SS-B). By contrast, anti-Ro(SS-A) and anti-La(SS-B) are seen in 50-75% of primary S S patients.

Antibodies to gag proteins in S L E We conducted a search for other diseases in which patients have antibodies to p24 gag. T w e n t y - t w o of 61 S L E patients (36%) produced antibodies to p24 gag [21]. These antibodies were also found in 19 of 115 (16%) patients with systemic sclerosis (particularly when diffuse cutaneous disease was present). By contrast, such antibodies were not seen in adult rheumatoid arthritis, polymyositis, chronic fatigue syndrome or leprosy. In SLE, 20 of the 22 patients making antibodies to p24 gag (91%) also expressed an Ig idiotype (Id 4B4) previously identified on a human/human monoclonal anti-Sm antibody [24]. Id 4B4 localized to the C D R 2 region of the Ig H chain of the monoclonal antibody and was conserved between species. T h e gene for this Id was cloned from 4B4 and found to be entirely germline [25]. Molecular mimicry Eight of the 21 S L E patients reacting to p24 gag also had antibodies to Sm, suggesting cross-reactivity between the retroviral gag protein and the nucleoprotein. Evidence to support this hypothesis came from competitive inhibition experiments demonstrating that Sm inhibited the binding of p24 gag. Anti-Id 4B4 antibodies were also able to inhibit p24 binding. T h e monoclonal 4B4 was able to bind both Sm and p24 gag [21]. T h e possibility of a shared epitope between the Sm B/B' subunits and p24 gag protein was investigated. Using recombinant fusion proteins prepared by D r Sally Hoch, we showed localization of 4B4 binding to the proline-rich C-terminal region of BIB' [26]. Both Sm binding and p24 binding by the monoclonal antibody 4B4 were inhibited by polyproline and related molecules. S L E sera were similarly inhibited by polyproline [26].

Transmembrane signalling defects in S S lymphocytes and in retrovirus-infected H9 cells We attempted to isolate, by co-culture with human T cells (H9), a retrovirus from salivary gland homogenates prepared from SS patients [22, 23]. A human intracisternal A-type retroviral particle was seen by electron microscopy in the human T cell line co-cultured with these homogenates. Reverse transcriptase activity was also demonstrated in these infected cell lines. T cells containing the A-type retroviral particle had reduced activation of protein kinase C and mobilization of calcium in response to P M A and ionomycin [27]. Culture supernatants of neither infected nor non-infected cells support B cell growth. Both cell types sustained cytocidal damage at the same levels ( > 500 nM) of herbimycin (a specific protein-tyrosine kinase inhibitor). T cells from the peripheral

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blood of SS patients are also deficient in their ability to produce I L - 2 and to proliferate in response to P M A and ionomycin [28]. Discussion

We have presented evidence [20, 21] implicating retroviruses in human systemic autoimmune disease (SS, SLE) and are currently extending these observations by pursuing two lines of investigation: (1) the molecular mimicry, based on a prolinerich epitope, shared by HIV-1 gag proteins and Sm, and (2) defects in transmembrane signalling in a human T cell line carrying an intracisternal retroviral A-type particle linked to SS. T h e proline-rich epitope shared between p24 gag of HIV-1 and Sm is reminiscent of numerous examples of molecular mimicry involving viruses and autoantigens. Particularly pertinent is the study by Query and Keene [29] in which M C T D serum was used to clone a human e D N A encoding a 70 kDa snRNP. T h e deduced amino acid sequence of this autoantigen was homologous to a p30 gag antigen of Moloney M u L V , baboon endogenous virus and other retroviruses. T h e authors suggested that the anti-U t R N P autoantibodies in the patient's serum were produced in response to a cross-reactive antigenic determinant specified by a consensus sequence present in both the 70 kDa autoantigen and the endogenous retroviral gag protein. A defect in transmembrane signalling is probably common to many chronic inflammatory and autoimmune diseases including SS [28]. H o w it relates to the intracisternal A-type retroviral particle is an important question which we are currently studying. Immunosuppression linked to retroviral infection has been related to the envelope protein p l S E and can be mimicked by a synthetic peptide CKS-17 linked to BSA [30, 31]. T h e immune effects of p l 5 E and C K S - 1 7 - B S A include inhibition of I L - 2 secretion, N K cell function and interferon-y production. C K S - 17-BSA also inhibits protein kinase C activity, as seen also for the viral infected H9 cells. T h e r e are many mechanisms by which retroviruses can induce autoimmunity, including polyclonal B cell activation, cytokine dysregulation and molecular mimicry with formation of autoantibodies to lymphocyte cell surface proteins including M H C molecules. Almost all endogenous retroviruses are non-infectious. Perhaps 5 % of the mammalian genome arises through reverse transcription of retroviral sequences. T h e first human endogenous retrovirus sequence was cloned 10 years ago [32]. T h e linkage of endogenous retroviral sequences to the Mls locus suggests the importance of r'etroviruses in immunoregulation and their potential importance in autoimmune disease [7-10]. For example, in the MuLV-related murine acquired immunodeficiency syndrome, which is characterized by polyclonal T and B cell proliferation, immunodeficiency and late B cell lymphoma, M u L V p30 gag proteins expressed on B cells act as superantigens causing selective expansion of T cells bearing V~5 [33]. T h e r e is a unique 8.4 K b R N A transcript derived from a mink cell focus-forming (MCF) M u L V expressed in all autoimmune but not in normal mice as early as 1 day of age [34]. A new human endogenous retroviral D N A related to H T L V - 1 and H T L V - 2 has been isolated from T cells obtained from a patient with mixed cryoglobulinemia [35]. Our studies, still incomplete, suggest that two mechanisms classic

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for r e t r o v i r u s e s ( m o l e c u l a r m i m i c r y a n d i m m u n o s u p p r e s s i o n ) m a y be o p e r a t i v e in s y s t e m i c a u t o i m m u n e r h e u m a t i c diseases s u c h as SS a n d S L E . B o t h t h e s e m e c h a n i s m s a p p e a r to i n v o l v e t h e e x p r e s s i o n o f viral p r o t e i n s . H o w ever, it is p o s s i b l e t h a t s o m e i n d i v i d u a l s c a r r y e n d o g e n o u s r e t r o v i r a l s e q u e n c e s in t h e i r g e n o m e in close p r o x i m i t y to critical m a m m a l i a n genes. T h e r e g u l a t i o n o f t h e e x p r e s s i o n o f t h e s e genes m i g h t b e a l t e r e d , y i e l d i n g a b n o r m a l cellular f u n c t i o n w i t h o u t viral e x p r e s s i o n . T o test this p o s s i b i l i t y it will b e n e c e s s a r y to i d e n t i f y t h e p o s s i b l e d y s r e g u l a t e d genes in cells t a k e n f r o m p a t i e n t s a n d s e q u e n c e t h o s e genes a l o n g w i t h t h e i r 3' a n d 5' flanking r e g i o n s .

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