Stem Cells, Vol. 14, No. 2, 177-184,
March 1996
© 1996 AlphaMed Press
Cytokines and Viral Anti-Immune Genes
Charles H. Evans
National Cancer Institute, Bethesda, Maryland, USA
Key Words. Anti-immune gene • Complement • Cytokine • Homolog • Interleukin • MHC gene presentation • Receptor decoy • Virus pirating of host defense genes
Dr. Charles H. Evans, Laboratory of Biology, National Cancer Institute, Building 37, Room 2A17, National Institutes of Health, Bethesda, MD 20892-4255, USA.
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Abstract
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Cytokines, the pleomorphic and pleiotropic director proteins of an increasingly defined diversity of differentiation and growth, are potent modulators of immune function and homeostasis. The antiviral and immunostimulatory actions of cytokines, like the interferons, have also been recognized for many years. In more recent developments, virus genomes were discovered in 1990 to contain anti-immune genes that control the synthesis of host cell proteins that disarm immune defenses. Many of the virus anti-immune genes are directed against cytokines. Recent discoveries of cytokine homologs and cytokine receptor homologs, and other viral gene products that disarm host immune defenses, provide a molecular basis for improved understanding of virus diseases and new targets for development of innovative therapeutic approaches against viral, cancer and perhaps a number of other diseases.
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Introduction
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The power of cytokines to act both as destructive and protective agents in virus infection is being rapidly revealed by increasing discoveries that virus genomes contain genes and produce products with significant structural and functional homology interfering with host cytokines and other defense molecules. Cytokine virus interactions of this nature provide important clues to the understanding, prevention and treatment of virus, cancer and other diseases where host defenses exert important modulatory actions. For nearly a century, viruses have been an important consideration in the etiology of cancer since Sanarelli [1] in 1898, Ellermann and Bang [2] in 1908 and Rous [3] in 1911 observed that rabbit myxomatosis and avian leukemia and sarcoma, respectively, are horizontally transmitted by cell-free infectious agents. Cytokines including lymphokines, monokines, interleukins, interferons and a variety of other growth factors produced by virtually every nucleated cell in the body, play pivotal roles in all of biology, including infectious disease and cancer, and are more recent molecular discoveries. The term cytokine was introduced in 1974 to recognize that lymphokines can be produced by other than lymphoid cells [4], with the first mediators identified as cytokines having been produced by nonlymphoid cells infected with virus [5, 6]. Twenty years later there are more than 16,000 biomedical articles with cytokine citations and greater than 75% of these have been published during the last two years.
Since 1990 a number of virus genes or gene products modulating cytokine and a variety of host noncytokine immunologic activities have been discovered. Some of the modulatory virus genes or their products are homologs of host cytokine, cytokine receptor and related host-defense genes and are aimed at disarming host defenses, a system dependent upon cytokines and related immunologic molecules for its activation, differentiation, expansion and protective function. Viral antihost defense genes are keys to understanding the means by which virus-infected or other abnormal cells escape host recognition and attack and survive in a foreign environment. Viral antihost defense genes and their products must also be viewed as points of potential therapeutic attack in diseased cells.
To appreciate the origin and extent of the disarming action of virus anti-immune genes and their potential as therapeutic targets, it is important to consider virus antihost defense genes and their products interacting with complement and antigen epitope presentation in concert with viral genes affecting the expanding group of bioregulatory molecules collectively classified as cytokines. If viruses can successfully pirate or otherwise steal mammalian cytokine and other host defense genes to thwart host antiviral defenses, it is likely that tumor and other diseased cells also do the same in achieving continued survival. Definition of selective antiviral and antitumor cytokine actions, therefore, will in addition facilitate further identification of viral and tumor anti-immune genes and their products. Understanding the variety of approaches viruses utilize to mimic, block or otherwise impede cytokine and related host antiviral defenses is important in the search for similar mechanisms in cancer cells and cells of other diseases. Discovery of antihost genes in nonviral diseased cells would markedly expand both our understanding of the pathobiology of viral-induced and related disorders and provide new points for therapeutic intervention.
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Disarming Host Defenses
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A variety of diverse strategies has been identified by which viruses disarm host defense mechanisms, strategies which in essence are largely encoded in viral anti-immune genes pirated from host genes. Currently recognized virus anti-immune genes are largely homologs of host cytokine, growth factor, complement and other immune-related protein or receptor genes, and have been the subject of several recent comprehensive reviews [7–9]. Molecular understanding of virus anti-immune genes began in 1990 with the discovery that the Shope fibroma virus genome contains a genetic sequence with extensive homology to the mammalian tumor necrosis factor (TNF) receptor [10]. The product of the gene is a soluble protein, T2, which has a 24% amino acid homology with the TNF receptor. T2 is a homolog of the binding portion, not the membrane anchor part, of the TNF receptor accounting for the 24% homology, and cleverly serves as a soluble TNF receptor decoy to bind TNF extracellularly and to prevent lysis of the virus-infected cell. Subsequently another T2-like protein coded by myxoma virus was discovered, and the functional importance of the T2 gene was demonstrated by showing that mutant myxoma virus, without the T2-like gene and product, was not lethal to rabbits [11]. Myxoma virus in addition codes for secretion of a homolog of the gamma interferon (IFN-
) receptor [12]. Poxviruses are now recognized as having extensive pirating of host defense genes [13].
A variety of DNA and RNA viruses code for homologs of a number of cytokine and antiviral host defense molecules or functions as illustrated in Table 1
. The attack on viruses and virus-infected cells by the mammalian immune system provides considerable selective pressure for viruses that have evolved vigorous countermeasures to preempt, neutralize or evade this host attack. These countermeasures are astonishingly diverse, and their study imparts fundamental information about immunology and the mechanisms enabling viruses to survive and cause disease [9]. Molecular analysis indicates that among all known viral protein sequences only a small number have significant sequence similarity to cellular proteins. Of these, the majority are proteins involved in regulating cell growth or in host defense. The former are found primarily among the oncogenic RNA retroviruses, whereas the latter are found primarily among the DNA pox viruses and herpes viruses. The sequence relationship of the host and viral homologs for the RNA viruses is very strong, generally more than 80% amino acid identity; yet for the DNA viruses it is usually weak, generally less than 40% amino acid identity [8]. The functions of many viral genes, however, are yet to be defined. It is very likely that some of these viral genes with undescribed function will be homologs of additional yet-to-be-discovered antiviral host defense mechanisms.
The diversity of known virus cytokine interactions can be grouped into several functional categories of virus anti-immune and antihost defense genes and gene products: A) cytokine, complement and other immunologic receptor decoys; B) modulators of cytokine signaling; C) inhibitors of cytokine processing and major histocompatability complex (MHC) antigen presentation, and D) regulators of cytokine synthesis (Table 1
). The distribution of viral defense disarming actions in the four groups also indicates that more virus gene and gene product-cytokine interactions are known that are directed by the virus to some aspect of cytokine synthesis and action or immune function than vice-versa—cytokine and other host defenses directed against virus function and/or persistence. The number of categories and the balance of the direction in terms of virus versus host-defense may change as more virus genomic sequence homologies and their functional interactions are discovered. Virus cytokine interactions must be viewed at this time as a snapshot of the very early development of this newly discovered and rapidly developing area of virus-host interaction. Although a number of specific host-defense molecule targets has been identified, many remain to be identified, and the precise action of a number of the identified virus anti-immune gene products and functional host-defense disarming categories remains to be elucidated.
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Receptor Homolog Decoys of Host Defenses
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The largest number of virus anti-immune genes and gene products as of the beginning of 1995 function as receptor decoys. Fifteen different genes have been discovered in pox, vaccinia, herpes family and hepatitis viruses that express antihost defense decoy gene products. Seven genes express homologs of five different cytokines, six genes express homologs of at least four components of the complement system, one gene expresses a homolog of a growth factor and another gene expresses a homolog of a subunit in the MHC antigen presentation complex (Table 1). The diversity of ways in which the virus anti-immune genes in the decoy category can immobilize host-defense mechanisms emphasizes the extraordinary ingenuity and capacity of virus-pirated homolog genes to disarm host antiviral defenses.
The first member of the receptor decoy group to be discovered was the Shope fibroma/myxoma T2 protein homolog of the TNF receptor. T2 is a secreted soluble protein mimicking the binding domain of the TNF receptor. Viruses have apparently pirated the binding, as opposed to the transmembrane anchoring sequences, of the TNF receptor gene allowing the virus to direct the infected cell to produce soluble receptor homolog molecules to bind TNF, prevent the cytokine from interacting with the cell and protect the virus-infected cell from TNF-induced lysis. Other genes coding cytokine receptor decoys are myxoma MT7 and swinepox C61 which bind to IFN-, swinepox K2R and ecrf3 and herpes saimiri ecrf3 whose products bind interleukin 8 (IL-8), vaccinia and cowpox B15R whose products bind IL-1ß, and hepatitis B virus PreS2 which binds IL-6.
Two genes have been identified in the human cytomegalovirus genome which affect inflammation and antigen presentation, two critical components in host defense to foreign microbes. US28 codes for a chemokine receptor homolog which binds several potent chemoattractants of lymphocytes and monocytes including macrophage inflammatory protein-1
(MIP-1), MIP-1ß, monocyte chemotactic protein-1 (MCP-1) and RANTES (an acronym for reduced on activation normal T-expressed and secreted, several characteristics of the gene and its product). The other human cytomegalovirus anti-immune gene UL18 codes for a competitive inhibitor of a key MHC Class I molecule binding to ß2-microglobulin, a subunit of Class I antigen molecules necessary for transport of the MHC to the cell surface. Binding of the UL18 product to the ß2-microglobulin subunit prevents the full assembly of the MHC necessary for antigen binding and presentation to cytotoxic lymphocytes, a critical host-defense function in the recognition and elimination of specific foreign microbes and altered host cells.
The remaining virus anti-immune gene group in the receptor decoy category consists of genes that block components in the complement system (C' and its components C1a, C3b, C4b, etc), another critical host antiviral defense mechanism that directly or in combination with antibodies and immune lymphocytes leads to lysis of virus-infected and other diseased or abnormal cells. Vaccinia B5R and herpes simplex virus (HSV) ORF4 genes lead to the production of complement homologs. The HSV gc1 gene product binds the C3b component of complement, and the herpes virus saimiri CCPH1 and vaccinia VCP gene products bind C4b, two critical components in the complement pathways leading to microbial and cellular lysis . The gE-gI gene product of HSV operates in a related yet different manner by binding to immunoglobulin G in or near the Fc region of the antibody molecule, resulting in inhibition of antibody-dependent complement-mediated lysis of virus-infected cells. It is likely that additional virus anti-immune genes will be identified that modulate this area of host defense due to the many components comprising the complement system.
The large number and diversity of virus anti-immune genes producing receptor decoys suggest that the cytokine and related immune components targeted by the receptor decoys represent either some of the easiest host defenses to counteract, or that these host-defense components are the most advantageous to disarm for the virus and other pathologic cells to insure their persistence. The known and future identified virus receptor decoy anti-immune genes thus provide extremely important clues for more effective antiviral, anticancer, anti-inflammatory and other therapeutic development.
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Interference with Cytokine Signaling
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Most cytokine and other ligands, once they have bound with their receptor on the cell surface, initiate a series of biochemical transmembrane signals critical to achieving their cellular effect. The second largest category of virus anti-immune genes are those virus genes which inhibit or otherwise modify cytokine signaling. Eleven genes that predominately affect TNF and IFN- are presently known in this category in adenoviruses, herpes viruses, poxviruses, reoviruses, retroviruses and vaccinia viruses.
Adenoviruses code for a number of proteins including E1A, E3–4.7K, E3–10.4K/14.5K and E1B–19K which influence the sensitivity of the virus-infected cell to the lytic action of TNF [14]. Although the precise points of interaction in the TNF signaling pathway are still being defined, the presence of the E1A product is known to increase cell sensitivity to TNF lysis whereas the other adenovirus gene products increase cellular resistance to lysis by this cytokine. Adenoviruses along with Eptsein Barr virus (EBV) and HIV-1 viruses also contain a variety of anti-immune genes coding for the production of abundant small RNA molecules that bind to the double-stranded binding site of PKR, a double-stranded RNA-dependent protein kinase, that is a critical part of the IFN signaling pathway leading to host synthesis of several proteins contributing to induction of an antiviral state. Reoviruses, swinepox and vaccinia viruses, on the other hand, block PKR through coding for proteins rather than small RNA molecules. The reovirus 
3 protein and vaccinia virus E3L protein bind double-stranded RNA and competitively inhibit PKR. The vaccinia and swinepox K3L genes encode a protein homolog of the amino terminus of eukaryotic initiation factor 2 (eif2), the substrate for PKR. The presence of K3L protein thus serves as a competitive inhibitor blocking PKR and establishment of the antiviral state. Retroviruses utilize still another approach that may contribute to immunosuppression by encoding for p15E, a protein that inhibits protein kinase C, an important enzyme in a number of cytokine signaling pathways.
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Disruption of Cytokine Processing and Antigen Presentation.
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Cowpox and vaccinia viruses express two very similar 38kDa proteins, crmA, with high sequence homology to serine-protease inhibitors (serpins) and which inhibit the pro-IL-1ß-converting enzyme that cleaves intracellular IL-1ß to its active secreted form. The crmA proteins may also function in other antihost defense modes by altering arachidonic acid metabolism resulting in reduced immune and inflammatory cell recruitment to the virus-infected area leading to a reduction in inflammation, and by inhibiting TNF-induced apoptosis [15]. crmA proteins may offer insights into additional new points of attack to interfere with or augment cytokine, inflammatory or programmed cell death.
A number of viruses suppress MHC class I expression, reducing the ability of host cytotoxic lymphocytes to destroy the virus-infected cells [16]. The adenovirus 12 E1A gene inhibits transcription of MHC class I genes and as mentioned above, the human cytomegalovirus UL18 protein binds to the ß2-microglobulin subunit of MHC class I molecules preventing assembly of a functional MHC antigen epitope presentation complex. The adenovirus E3gp19K protein has a C-terminal sequence that anchors in the endoplasmic reticulum and binds with high affinity to MHC antigen epitopes preventing their translocation to the cell surface. Cytomegalovirus expresses an early protein which has a similar action, and myxoma and poxviruses produce several late proteins which reduce expression of the cell surface MHC. The E5 protein of human papillomavirus (HPV) type 16 also produces a post-transcriptional loss of MHC Class I expression, apparently by causing a loss of transporter protein encoded by the TAP-1 gene [17]. Identification of viral and other disease cell subversion of processing and presentation of antigenic epitopes through the MHC offers new unparalleled opportunities for therapeutic intervention in a variety of disorders.
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Modulation of Cytokine and Viral Transcription
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Viruses affect the synthesis of cytokines in several diverse ways. EBV expresses a protein, BCRF1, a homolog of IL-10, and equine HSV induces secretion of a similar homolog. IL-10 was originally known as cytokine-synthesis inhibitory factor because it decreases IFN- and IL-2 expression by TH1 lymphocytes and polymorphonuclear leukocytes. IL-10 also increases the growth of B lymphocytes which are the host for EBV infection. The expression of IL-10 homologs favors the persistence of EBV by both decreasing cytokine synthesis and host-defense mechanisms, and independently by increasing host target cells for virus replication. Viruses also affect cytokine synthesis by the cells they infect. HTLV-1, possibly through the tat–1 gene, increases TNF- and TNF-ß secretion. Cytokines can influence not only virus expression but also virus host range and virus receptor expression as revealed by analysis of HIV-1 infection [18, 19]. The regulation of cytokine synthesis can be quite complex as EBV, herpes simplex type 1 and herpes simplex type 6 viruses differentially regulate IL-1, IL-6 and TNF synthesis in infected peripheral blood mononuclear leukocytes with the dominant effect being suppression of IL-6 and TNF synthesis by EBV and herpes simplex type 6 viruses [20].
Models of cytokine intervention in acute and chronic virus infection are useful for studying cytokine modulation of virus infection, virus gene expression, virus replication and the development of neoplasia. Leukoregulin (LR), a naturally occurring 50kDa cytokine secreted by lymphocytes and which increases membrane permeability and drug uptake in tumor but not in normal cells, is able to prevent secretion of infectious HSV-1 when applied shortly after infection. LR treatment of human WISH amnion cells 3 h after infection enhances 1–100 µM acyclovir (ACV) inhibition of HSV-1 up to 100-fold [21]. This indicates that combined immunotherapy and chemotherapy can produce a substantial inhibition of HSV-1 replication and provides a rationale for the application of this approach to the interventive treatment of virus infection. HPV, in particular type 16, are major factors in the etiology of cervical cancer. The HPV transforming genes E6 and E7 are retained and expressed in the majority of cervical cancers implying an important role for these proteins in maintenance of the malignant phenotype. LR, TGF-ß and interferon-, but not interferon-, selectively inhibit transcription of E6 and E7 in human epithelial cervical cells immortalized by HPV DNA [22]. Cytokines have the potential to selectively downregulate viral gene expression and associated neoplastic phenotypes, and to enhance therapy directed against viral infection and neoplastic cells (Fig 1).
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Fig 1. Cytokine and host-defense molecular target interactions and points of potential cellular interference or escape. Viral and potentially other disease-controlling genes have the potential (see text for details) to generate products: 1) decoying host ligand receptor interaction, 2, 3) interfering with ligand signaling metabolism or synthesis, 4) influencing drug uptake and action, and 5) modulating immunologic interactions resulting in disruption of the host's or interventive attempts to reduce new virus production or eliminate diseased cells.
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Persistent infection of human cervical cells with HPV type 16 is also associated with resistance to TNF lysis [23] and with increased responsiveness of the cells to LR enhancement of the cervical epithelial cell sensitivity to lymphocytotoxicity [24]. Cytokine interactions in virus infections are complex, sometimes seemingly contradictory, and offer unique opportunities to study cytokine signal transduction and modulation of gene expression, which will be helpful in further delineating the control of cytokine expression and pathways of molecular action. Molecular definition of the interactions will also identify new targets for developing potential therapeutic intervention to decrease abnormal cell growth and function and increase host defenses.
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Insights for Therapeutic Development
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The recent discoveries that a wide variety of DNA and RNA virus genomes contain genetic sequences with significant homology to human antidefense cytokine and other immunologic related genes, and that the virus antidefense genes express products that function to disarm host antiviral defenses, provide great opportunities to search for analogous changes in the genomes of cancer cells and cells of other diseases to provide new understanding of the neoplastic process, inflammation, autoimmunity and other pathologic processes, as well as unique new avenues for the prevention and control of these diseases. If, as current evidence suggests, the pirating of host antiviral gene sequences by viruses is an effective mechanism to permit virus persistence, then it is logical to anticipate that other microbial and otherwise altered host diseased cells likewise benefit from pirated or otherwise modified host gene sequences to also function in anti-immune capacities to facilitate persistence of the pathologic cells. The same anti-immune functions utilized by viruses to favor virus survival, such as: a) receptor decoys for cytotoxic, immunostimulatory and proinflammatory cytokines and other immune molecules, modulation of cytokine; b) signaling; and c) processing and MHC antigen presentation; and d) alteration of cytokine synthesis, can also serve to facilitate the permissive growth of cancer and other diseased cells. Just as viruses contain homologs of host-defense genes, it is expected that analogous homologs to host-defense genes useful in controlling the development and spread of neoplasia are present in cancer cells and cells of other diseases. Identification of these cancer cell homologs to host antineoplastic defense genes can provide new and exceptionally important gene targets for the development of innovative diagnostic, interventive and therapeutic approaches in the elimination of cells infected with nonviral microbes and in the cells of neoplastic, inflammatory, autoimmune and other disorders which interact with host defenses.
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accepted for publication September 15, 1995.
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Abstract
Introduction
