Viral persistence: Parameters, mechanisms and future predictions

Virology 344 (2006) 111 – 118 www.elsevier.com/locate/yviro

Viral persistence: Parameters, mechanisms and future predictions Michael B.A. Oldstone * Molecular and Integrative Neurosciences Department and Department of Infectology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA Received 10 August 2005; accepted 10 September 2005

Abstract For a virus to persist, it must actively curtail the host’s antiviral immune response. Here, we review the conceptual basis by which this can occur and discuss the subsequent fate of differentiated cells infected over long periods of time. We also consider how the compromised antiviral immune response can be revigorated or replaced with a potent response that purges the virus and thereby terminates persistent infection. D 2005 Elsevier Inc. All rights reserved. Keywords: Persistent infection; T cell function; Dendritic cell function; Immune clearance; Differentiated function

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunologic tolerance and T cell exhaustion/tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . What mechanism(s) allows viruses to disrupt immune responses thereby allowing the virus to persist? . . . Persisting virus can alter the differentiation function of a specialized cell in the absence of killing that cell Termination of persistent viral infection to cure related diseases . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction One of the remarkable advances in modern virology is the realization that persistent viral infections exist and are common. Hence, understanding the principles by which persistence is initiated and maintained, as well as the pathologic consequences of continued virus replication in a host over its life in terms of causing disease provides research areas of high significance as well as opportunities for challenging investigation. The three foundations upon which the understanding of persistent infection rests are, first, that the host’s immune response fails to form or fails to purge virus from the infected host. Thus, viral persistence is synonymous with evasion of the hosts immunologic surveillance system. Recent advances have * Fax: +1 858 784 9981. E-mail address: [email protected]. 0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2005.09.028

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shed light on the cellular and molecular players involved. Second, viruses can acquire unique component(s) or strategies of replication. That is, viruses can regulate expression of both their own genes and host genes to achieve residence in a nonlytic state within the cells they infect. Third, the type of diseases that persisting viruses cause are often novel and unexpected. The focus in this case is on the ability of a virus to infect differentiated or specialized host cells virtually over the life time of the host. The continuous replication of a viral, i.e., foreign gene, in a differentiated cell can selectively disorder the functions of that cell without destroying it. Several examples are viruses that interfere with the ability of neurons to make neurotransmitters, that block endocrine cells from making hormones or that prevent immune cells from producing inflammatory cytokines and/or killing molecules like perforin, etc. The result is a disturbance in the host’s biologic equilibrium. Thus, one important direct effect of persistent

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virus replication is to disorder the normal homeostasis of the host and thereby cause disease without destroying (killing) the infected cell. For example, a virally caused neurotransmitter defect of neurons altering cognitive learning and yielding behavioral disorders. Similarly, persistent infection can promote disorders in synthesis or degradation of hormones leading to diabetes, growth hormone or thyroid dysfunction with disorders of growth and metabolism. Associated disorders in synthesis or release of cytokines, antibodies and other molecules made by immune cells can lead to either immunosuppression on the one hand or hyperimmune/autoimmune responses on the other. Thus, solving the puzzle of viral persistence fulfils not only an intellectual pursuit but also the pragmatic purpose of uncovering the possible infectious etiology for a number of human diseases of unknown etiology. Additionally, combating diseases known to stem from persistent infections like hepatitis B and C, human retroviral and measles, etc., is work that remains incomplete. This review describes selected viral strategies, several of which were uncovered primarily in the author’s laboratory. First is an overview to define how viruses deregulate the host’s immune system to allow persistence to occur, second, selected examples depict how, once infection is initiated, continually replicating virus can alter the function of a differentiated cell, disturb homeostasis and cause disease and, third, some strategies available to terminate persistent infections and thereby treat these disorders are discussed. Immunologic tolerance and T cell exhaustion/tolerance Over 50 years ago, about the time Virology began as a journal, to explain observations concerning natural infections of mice with lymphocytic choriomeningitis virus (LCMV) and results of Ray Owen’s study of chimeric cattle, Macfarlane Burnett and Frank Fenner postulated clonal elimination (deletion) of immunocompetent cells as the basis for immunologic tolerance to persisting viruses and self antigens (Burnet and Fenner, 1949). Thus, immunologic tolerance was defined as a state of specific refractoriness in responding to a virus (antigen) following prior exposure to that antigen. The concept of immunologic tolerance to viruses was then used to explain persistent infection in adults after exposure to virus in utero or at birth. The resultant clinical picture first observed for LCMV and later for retro and other viruses was viral invasion before or at birth with retention of infectious virus throughout the animals’ lives, usually in the absence of freely circulating antibody. Later came the discovery of CD8 cytotoxic T cells and CD4 helper T cells combined with observations of patients having genetic or acquired defects of their B cell (humoral immunity) or T cells (cellular immunity) compartment. These findings were complimented by studies in mice where various B and T cell subsets or their effector molecules were genetically or chemically deleted. The overall interpretation of these clinical and experimental observations was that T cells played a commanding role in controlling most viral infections, whereas antibodies and complement were most often essential for combating bacterial infections (reviewed in Whitton and

Oldstone, 2001). Further support for this concept came from detailed studies in cell and molecular biology, indicating that the processing of MHC class I molecules, which are predominantly responsible for CTL recognition, was associated with organisms like viruses that required intracellular replication. In contrast, MHC class II molecules responsible primarily for CD4/B cell responses were associated with organisms that replicated extracellularly. However, it is unlikely that clonal elimination plays a role in removing antiviral B cells or T cells with infection in utero or at birth. First, in persistent virus infection that begins in utero, at birth or during adulthood, the lack of freely circulating antibody originally observed in natural rodent models of LCMV and retrovirus infections was primarily a technical defect; that is, instead of traveling free in the blood, antibodies were bound (complexed) to virus and viral antigens forming virus – antibody immune complexes (Oldstone and Dixon, 1968; Oldstone et al., 1972). Since persistent infections are characterized by an excess of virus/viral antigens, it is not surprising that free antibodies would have been difficult to detect. Interestingly, such circulating virus –antiviral immune complexes are infectious, contribute to maintaining persistence and often redirect the infection to cells that otherwise would not be susceptible because they lack receptors for the virus but do express Fc, C1 or C3 receptor molecules (reviewed Whitton and Oldstone, 2001). Fc- and complement-expressing cells are able to bind circulating infectious complexes and thereby support viral entry. Furthermore, detection of virus –antiviral immune complexes can be used as a sign of persistent infection in most if not all such infection of humans and animals (Whitton and Oldstone, 2001). Correspondingly, virus-specific T cells do not undergo irreversible clonal elimination as first shown with LCMV (Jamieson and Ahmed, 1988). Once persistent virus is removed, for example by adoptive immunotherapy with T memory cells (Oldstone et al., 1986), the recipient mice can regenerate LCMVspecific antiviral CTL (Jamieson and Ahmed, 1988). The inability to detect antiviral T cell responses (Moskophidis et al., 1993; Penna et al., 1991) despite more recent evidence from tetramer analysis that T cells are present during some persistent infections (Gallimore et al., 1998; Ou et al., 2001; Wherry et al., 2003; Zajac et al., 1998) coupled with the fact that adoptive transfer of memory immune T cells cleared virus from persistently infected mice (Oldstone et al., 1986) led to the observation that T cells are present in persistently infected hosts but not responsive or able to clear the infection and the concept of T cell exhaustion or functional impairment. The germane question is whether these unresponsive T cells are destined never to function again or, in contrast, can eventually be retrieved or manipulated to become antiviral T cells. David Brooks, in a series of recent studies (Brooks et al., submitted for publication), documented that, during the initial stages of infection, i.e., the first 5 days, adult mice that would later become persistently infected generated robust CD8- and CD4-specific anti-LCMV T cells. However, 4 days later (day nine), neither the CD8 nor CD4 T cells any longer acted as effectors as judged by their inability to lyse virus-infected targets and secrete TNF or IL-2 when in

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contact with virus or virus-infected cells. Correspondingly, there is a decrease in interferon-g secretion. Transfer of such nonresponder T cells into infection-free syngeneic mice restored their specific antiviral T cell functions (Brooks et al., submitted for publication). In contrast, Brooks et al. (submitted for publication) found that transfer of responder-specific antiviral T cells into mice persistently infected with virus turned off their virus-specific responses. Subsequent removal of these unresponsive cells from the viral environment, either by culturing them or by use of antiviral drug to lower viral titers in the host by as little as one log, restored their antiviral function (Brooks et al., submitted for publication). Thus, exhaustion/tolerance of T cells is not a hard-wired undeviating fate but is reversible. These observations in rodents, if applicable as expected to humans, have important implications for clinical treatment of persistent infections. What mechanism(s) allows viruses to disrupt immune responses thereby allowing the virus to persist? Over the last two decades, it has become clear that viruses can disrupt the formation of immune responses by acting anywhere along the defined pathways known to generate, activate or expand T cells (reviewed in Plough, 1998; Whitton and Oldstone, 2001). Years before corresponding observations were made with HIV, mice infected with LCMV (Ahmed et al., 1987; Doyle and Oldstone, 1978) and also infection of human lymphocytes with viruses as diverse as CMV, measles and influenza developed disorders that prevented performance of their expected specialized functions including their capacity to act as killer cells or to manufacture immunoglobulins (Casali et al., 1984; McChesney et al., 1987, 1988; Schrier and Oldstone, 1986). Extension of such findings to multiple systems indicates that viruses can down-regulate the degradation of protein into peptides and inhibit transport of peptides from proteosomes to the ER or from ER to the cell surface where peptides complexed to MHC molecules are recognized by T cells. The result is diminished or aborted expression of MHC and costimulatory molecules on the cell surface (Hahm et al., 2005; Plough, 1998; Whitton and Oldstone, 2001). Proper processing of peptides and their binding to MHC molecules along with the presence and function of appropriate co-stimulatory molecules are necessary for T cell recognition (Zinkernagel and Doherty, 1974) (reviewed in Janeway et al., 2005). Additionally, viruses that cause persistent infections routinely infect and persist in lymphocytes, monocytes or macrophages and can alter their functions (reviewed in Whitton and Oldstone, 2001). What has come to the forefront recently is the knowledge that viruses with the capacity to cause persistence or immunosuppression often infect dendritic cells (DC) (reviewed in Curr. Topics Microbiol. Immunol., 2003). DCs bridge the innate and adoptive immune system and are sentinel cells in protecting the host against infectious organisms. DCs in the spleen and lymph nodes are the primary and most robust antigen-presenting cells of the host. They process viral antigens, incorporating them with MHC molecules to present to naive T cells and by that process activate

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lymphocytes to function as effectors. Hence, it is not surprising that viruses employ strategies to focus on preventing both the expansion and function of DCs. Several viruses can downregulate the expected surface expression of MHC and costimulatory molecules on DCs and thereby diminish the activation of T and B cells (for examples see Curr. Topics Microbiol. Immunol., 2003, for review). Examples of these dual strategies to prevent expansion and function of DC during LCMV and measles virus infections are shown in Figs. 1 and 2. Furthermore, when viral titers were lowered in persistently infected mice, the numbers of DCs infected by the virus decreased significantly, and T cell function was revived (Brooks et al., submitted for publication). Interestingly, the molecular mechanism whereby LCMV and measles virus disrupted DC function was the release of type I interferon that acted in a STAT-2-dependent but STAT-1-independent pathway (Hahm et al., 2005). These new observations indicate that: (1) viruses subverted interferon type I from its usual and expected antiviral, host protective role (see Biron and Sen, 2001, for review) to one that enhanced the viruses’ survival by suppressing immune responses; (2) the usual heterodimer of STAT-1/STAT-2 signaling was replaced by STAT-2 alone, suggesting the possibility of a novel, yet to be defined signaling pathway; and (3) the suspicion that viruses may selectively attack the STAT-2 pathway for their own purposes. Recent experiments performed by Matt Trifilo1 in our laboratory support the latter contention. Persisting virus can alter the differentiation function of a specialized cell in the absence of killing that cell Non-cytolytic LCMV infects DC and alters the migration of MHC molecules to their surface (Sevilla et al., 2000). This phenomenon of a persistently infecting virus altering a cell’s function without lysing it was initially recorded with LCMV infection of neuroblastoma cells (Oldstone et al., 1977) and has now been observed for a plethora of other viruses and cells (reviewed in de la Torre and Oldstone, 1996; Oldstone, 1988). After infecting neuroblastoma cells, the virus caused abnormalities in the synthesis and degradation of acetylcholine by interfering with the acetylase or esterase enzymes but none of the cells’ vital enzymes needed for its growth or survival. However, these persistently infected neuroblastoma cells were normal in morphology, growth rates, cloning efficiencies and levels of total RNA, DNA, protein and vital enzyme synthesis. These observations of alterations in cell function without destruction of the cell were subsequently extended to infected lymphocytes and monocytes (Ahmed et al., 1987; Casali et al., 1984; Doyle and Oldstone, 1978) and to infected Schwann cells in which myelin formation was adversely affected (Rambukkana et al., 2003). In vivo, infection of growth hormone (GH) cells in the anterior lobe of the pituitary led to deficiencies in growth and glucose metabolism (Oldstone et al., 1982, 1984); infection of selected neurons in the dentate 1 Trifilo, M. et al. Viruses target the STAT-2 pathway to establish persistence. Manuscript in preparation, 2005.

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Fig. 1. This figure displays how LCMV disrupts dendritic cell (DC) function and expansion in vivo. Virus selectively infects DCs because, among other immune cells, DCs preferentially express the viral receptor. Upper left panel reports that alpha-dystroglycan (a-DG) is the receptor for LCMV and a-DG is preferentially located on DC. Using three MOIs, LCMV Clone 13 is able to infect wild-type (wt) stem cells and wt cells in which DG has been knocked out only if DG is restored by reintroduction of DG either by transfection or use of an adenoviral vector encoding DG. However, virus does not infect stem cells in which DG is deleted. Biochemically, a-DG can be found on surfaces of DC (CD11c+ cells) but not on CD4 or CD8 T cells or B cells. The upper right panel shows that, during LCMV infection, quasispecies of viruses are generated. Within that group, several viral species bind at a 2- to 3-log higher affinity to a-DG than others. Sequence analysis determined that binding to the a-DG receptor can depend on a single amino acid change in the viral glycoprotein ligand (see Sevilla et al., 2000 for details). Since aDG is on the surface of DC, those viruses (e.g., LCMV Cl 13) that bind at a high affinity to a-DG preferentially infect DC, whereas those viruses with a low binding affinity, e.g., LCMV ARM 53b, do not. Infection of DC by non-lytic LCMV viruses like LCMV Cl 13 down-regulate MHC and co-stimulatory molecules on DC. Such molecules are required to present and stabilize antigen (peptides) to activate T cells. A failure to activate T cells leads to virus-induced immunosuppression and persistent infection. Furthermore, those LCMV strains or variants that cause immunosuppression also abort the host’s ability to expand DC. The lower left panel shows that a 10-day treatment with Flt-3 ligand, a cytokine that expands DC, enables both uninfected and LCMV ARM 53b-infected mice to produce over 20-fold more DC in vivo. In contrast, similar Flt-3 ligand treatment associated with LCMV Cl 13 infection fails to expand DC (expansion <1.5-fold). The lower right panel documents that this suppression in DC production requires the presence of interferon (IFN) type 1 since mice genetically deficient in type 1 IFN receptor (IFN-ahrec ko) expand DC in the face of LCMV Cl 13 infection as well as non-infected or LCMV ARM 53b infected mice do. For details, see Sevilla et al. (2004).

gyrus was associated with alterations in behavior and learning (Cao et al., 1997; de la Torre et al., 1996); and infection of DCs aborted their expansion and function (Hahm et al., 2005; Sevilla et al., 2000, 2004). In all instances, the anatomy of the persistently infected tissues and cells with specific function, i.e., production of GH, neurotransmitter, expression of MHC and co-stimulatory molecules, was normal by low and high resolution microscopy. Biochemical analysis revealed no general alteration or shut-down in RNA or protein synthesis. Thus, conceptually, the virus caused disease by altering a selective function of each specialized cell type without

destroying any cell. At the same time, the virus disordered the host immune system so that the foreign (viral) content of an infected cell was not recognized and the spread of infection was not curtailed. Selected examples of such virus-induced diseases in the absence of tissue or cell destruction are displayed in Fig. 3. Also shown in Fig. 3 is the puzzle of how a virus can selectively alter transcription of one differentiated product but not another, even within the same infected cell. For example, individual cloned PC (pituitary cells) make both GH and prolactin (PRL), but the virus preferentially turns off synthesis only of GH RNA and not PRL

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Fig. 2. Like LCMV, measles virus (MV) infection of DC provokes the release of type 1 IFN and causes the down-regulation of MHC (shown) and co-stimulatory (not shown) molecules. The signaling pathway by which IFN-type 1 works with both MV and LCMV Cl 13 infections is STAT-2-dependent but STAT-1-independent (for details, see Hahm et al., 2005). MV is a human pathogen and does not infect mice unless adapted by multiple passages. Use of vaccine or ‘‘wild-type’’ (wt) MV in the mouse becomes possible only when MV receptors SLAM (shown) or CD46 (not shown) are expressed. Panels on the left display the molecular construct used to express the MV receptor SLAM on DC. Also included are: results for immune cells obtained from such transgenic mice in terms of SLAM expression (middle panel: left) on immune cells and expression and infectibility of CD11c DC when expanded in vitro with GM-CSF (shown) or Flt-3 ligand (not shown). The upper left panel illustrates that MV infection of DC lowers MHC expression, which is restored in IFN-a-hrec knockout (IFNah / ) mice, indicating the essential role of type 1 IFN in the process of altering DC function. The right upper panel documents that, in STAT-1 ko or STAT-2 ko mice, the type 1 IFN effect with MV infection is via a STAT-2-dependent and STAT-1-independent signaling pathway (see Hahm et al., 2005 for details). In the lower right panel, similar to MV, LCMV Cl 13 also suppresses DC function through a STAT-2-dependent and STAT-1-independent pathway (see Hahm et al., 2005 for details).

RNA (Fig. 3). Similarly, in P12 phenochromocytoma cells, virus selectively turns off GAP-43 synthesis but does not affect synthesis of the amyloid precursor protein or c-fos. The molecular basis of this selectivity and at what advantage to the virus remain to be solved. Termination of persistent viral infection to cure related diseases Because persistently infected cells whose dysfunction is associated with disease are themselves not injured, then conceptually clearing the virus infection might be expected to restore normal cell function. If so, reinstating homeostasis could abort the related disease. To address this issue, we designed and successfully cured persistent LCMV infection by adaptively transferring LCMV immune memory cells (Oldstone et al.,

1986). As hoped, the transferred cells purged virus from GH cells and corrected the GH deficiency syndrome (Oldstone, 2002; Oldstone et al., 1984) and restored normal growth, glucose metabolism and life expectancy. Lastly and of greater importance is that these initial observations with clearance of persistent LCMV infection in mice (Oldstone et al., 1986) were extended to treatments for several human infections (reviewed Curr. Topics Microbiol. Immunol., 1994). While it is unlikely that this procedure will become a general and useful therapy due to the technical requirement of generating and expanding large numbers of autologous or syngeneic antiviral CD8 and CD4 T cells, the underlying principles are now established. The ease in manipulating the LCMV model of persistently infected mice enabled determination that the minimal number of virusspecific CD8 T cells required to clear virus from sera and

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Fig. 3. Viruses cause disease by aborting a specific product of a differentiated cell, thereby altering that cell’s function and unbalancing the host homeostasis. Two examples of this phenomenon are shown. In the upper panel, LCMV infected C3H/St mice are retarded in growth compared to non-infected littermates (left upper panel). In vivo, virus replicated preferentially in growth hormone (GH) synthesizing cells in the anterior lobe of the pituitary (red color: virus nucleoprotein [NP] antigens detected immunohistochemically with a monoclonal antibody to LCMV NP labeled with rhodamine), but no such replication was noted in the middle or posterior lobes of the pituitary gland nor in prolactin (PRL) or thyrotropin-producing cells. LCMV infected >99% of pituitary cells (PC) in vitro. The upper right panel shows that LCMV infection(+) selectively affected transcription of GH but not PRL (compared to non-infected [ ] cell). On a clonal level, each PC cell expressed both GH and PRL. Other studies mapped the affect to the GH promoter. Construction of recombinants between GH and PRL promoters indicated the sole involvement of a 69-base PIT-1 transcription factor in this process. Two LCMV genes were involved: the glycoprotein that enabled virus to enter the cell and the NP that bound to PIT-1. Details appear in de la Torre and Oldstone (1992), Oldstone et al. (1982) and Oldstone et al. (1984). Lower left panel depicts persistent LCMV infection of neurons in the dentate gyrus of the hippocampus (brown color: peroxidase stain using monoclonal antibody to LCMV NP). The adjacent panel shows a normal concentration GAP 43 in the dentate gyrus of non-infected mice, and the lower panel shows the marked 5- to 7-fold decrease in GAP 43 protein from a similar area in a littermate that was persistently infected with LCMV. Results from transcription studies in PC-12 pheochromocytoma cells infected or not with LCMV appear in the lower right panel. Note that LCMV infection (evident by expression of S RNA of LCMV and NP mRNA) aborted GAP 43 transcription even in the presence of nerve growth factor (NGF), a substance known to enhance transcription of GAP 43. Furthermore, although LCMV infection aborted GAP 43 transcription, it had no effect on either amyloid precursor protein (APP: shown) or c-fos (not shown) transcription. For details and mapping of the defect’s location, refer to Cao et al. (1997) and de la Torre et al. (1996).

tissues is 350,000, and for virus-specific CD4 T cells 7000, or approximately 5  107 CD8 and 1  106 CD4 T cells per square meter of body surface, a CD8:CD4 ratio of 50:1 (Berger et al., 2000). Knowing the numbers of CD8 and CD4 T cells required to clear the viral infection should help lead to effective strategies via vaccination or reversal of T cell tolerance non-responsiveness. Although CD8 CTL are the major effector cells for clearing virus in most acute infections, genetic knock-out and reconstitution studies have provided clear evidence that CD4 T cells are an absolute requirement to maintain CD8 T cell activity in clearing a persistent infection (Battegay et al., 1994; Matloubian et al., 1994; Tishon et al.,

1995). Furthermore, interferon-g is also essential (Guidotti and Chisari, 2001; Tishon et al., 1995). Conclusions Animal models, specifically the study of LCMV infection in its natural rodent host, has yielded insights into basic understanding of virus – immune interactions, persistent infections and associated diseases. A remarkable number of key concepts in virology and immunology over the last 50 years have come from multiple laboratories investigating LCMV infection and pathogenesis (reviewed Zinkernagel, 2002).

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Furthermore, most of these conclusions have been extended to include many DNA and RNA virus infections of humans and are helpful in understanding and manipulating the diseases they cause. Importantly, the complex multifaceted spectrum of events that define diverse outcomes of LCMV infections refocuses attention to the delicate balance governing virus – host interactions. Small differences in either viral or host genes seem to profoundly influence the course of infection and the resultant disease state. The future for viral pathogenesis will be to understand this complex interaction at the cellular and whole host level as well as uncode how RNA viruses with so few genes when compared to DNA viruses can cause such broad alterations in cell physiology, extent of injury and course of disease. Work will also be directed to uncovering what additional diseases currently of unknown etiology may originate with viral infections, especially those that persist. These diseases are likely to be manifested initially only by biochemical abnormalities associated with subtle or negligible tissue/cell injury. Acknowledgments This is Publication Number 17575-NP from the Molecular and Integrative Neurosciences Department and Department of Infectology, The Scripps Research Institute, La Jolla, CA. This work was supported in part by USPHS grants AI009484, AI045927, AI055540 and AI036222. References Ahmed, R., King, C.-C., Oldstone, M.B.A., 1987. Virus – lymphocyte interaction: T cells of the helper subset are infected with lymphocytic choriomeningitis virus during persistent infection in vivo. J. Virol. 61, 1571 – 1579. Battegay, M., Moskophidis, D., Rahemtulla, A., Hengartner, H., Mak, T.W., Zinkernagel, R.M., 1994. Enhanced establishment of a virus carrier state in adult CD4+ T-cell-deficient mice. J. Virol. 68, 4700 – 4708. Berger, D.P., Homann, D., Oldstone, M.B.A., 2000. Defining parameters for successful immunocytotherapy of persistent viral infection. Virology 266, 257 – 263. Biron, C., Sen, G., 2001. Interferons and other cytokines. In: Knipe, D., et al., (Eds.), Fields Virology, 4th edR Lippincott Williams and Wilkins, Philadelphia, pp. 321 – 352. Brooks, D.G., McGavern, D.B., Oldstone, M.B.A. Prevention of immune exhaustion and restoration of T cell function during persistent viral infection (Submitted for publication). Burnet, F., Fenner, F., 1949. The Production of Antibodies, vol. 2. MacMillan Co., New York. Cao, W., Oldstone, M.B.A., de la Torre, J.C., 1997. Viral persistent infection affects both transcriptional and post-transcriptional regulation of neuronspecific molecule GAP43. Virology 230, 147 – 154. Casali, P., Rice, G.P.A., Oldstone, M.B.A., 1984. Viruses disrupt functions of human lymphocytes: effects of measles virus and influenza virus in lymphocyte-mediated killing and antibody production. J. Exp. Med. 159, 1322 – 1337. Current Topics in Microbiology and Immunology, 1994. Cytotoxic Tlymphocytes in human viral and malaria infections, vol. 189. SpringerVerlag, Berlin-Heidelberg, pp. 1 – 203. Current Topics in Microbiology and Immunology, 2003. Dendritic cells, vol. 276. Springer-Verlag, Berlin-Heidelberg. de la Torre, J.C., Oldstone, M.B.A., 1992. Selective disruption of growth hormone transcription machinery by viral infection. Proc. Natl. Acad. Sci. U.S.A. 89, 9939 – 9943.

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