An important function of the immune system is to confer protection from invading pathogenic microorganisms such as bacteria, viruses and fungi. When a pathogen enters the host, it is confronted by the innate immune response. The adaptive immune response is also prepared to act accordingly to eliminate the invading pathogen at this time. The adaptive immune response is broadly grouped into 2 categories: the humoral response which is antibody mediated to target extracellular pathogens and the cell-mediated response which can eliminate intracellular pathogens such as viruses and some bacteria such as Salmonella. The innate immune system responds to a viral infection by producing cytokines such as interferons (IFNs) which can induce antiviral states in cells and limit viral proliferation. Cytokines can also enhance antigen presentation by the major histocompatibility (MHC) class 1 molecules. Killing of virus infected cells by natural killer (NK) or cytotoxic CD8+ T cells can also be mediated by cytokines. If a virus is to establish a successful infection in a host, it must overcome the barrier presented by cytokines as well as being able to successfully avoid elimination by NK and CD8+ T cells. Latency is one of the most successful immune evasion techniques employed by viruses. It is often employed by members of the herpesviridae family, such as Murine Gammaherpesvirus-68 (MHV-68) which is often used as an infection model for human herpesviruses. It also encodes a chemokine binding protein, M3.
Structure of the M3 gene product
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In 2002, Alexander et al. elucidated the crystal structure of the M3 protein (Alexander et al., 2002). The M3 protein forms an antiparallel homodimer with itself with the N-Terminal domain (NTD) of one subunit adjacent to the C-Terminal domain (CTD) of the second subunit. Each M3 monomer has a 2-lobed structure. The NTD and the CTD are each composed of an extended β-sandwich with large loops and helical regions. Residues 1 - 210 comprise the NTD while residues 211 - 382 comprise the CTD. The NTD is composed of 5-stranded and 8-stranded β-sheets. The CTD exhibits a structure which resembles the V-type Ig-fold. Alexander et al. also elucidated the crystal structure of M3 complexed with the CC chemokine monocyte chemoattractant protein 1 (MCP-1), also known as CCL2. It has been shown to be chemotactic for both T-lymphocytes and monocytes (Carr et al., 1994). It is interesting to note that the interaction mimics that of CCL2 to its receptor, CCR2 (Alcami, 2003). One molecule of MCP-1 can bind at either end of the M3 homodimer. This means that M3 binds MCP-1 in a 2:2 stoichiometry. There are deep clefts between the NTD and CTD interface in the homodimer which act as the chemokine binding sites. 26 residues from MCP-1 and 29 residues of M3 participate in the interaction. Both the NTD and CTD contribute equally to the chemokine-binding cleft. The NTD region of the cleft is made up of highly acidic residues. This complements MCP-1 which is highly basic. Hydrophobic contacts are also observed between M3 and MCP-1. MCP-1 makes contact with six side chains of the CTD. The A-B, A''-A' and E-F loops of the CTD act to sequester the MCP-1 residue Y13 which has been shown to be critical for GPCR binding (Jarnagin et al., 1999). M3 undergoes a conformational change upon chemokine binding. The binding sites in the uncomplexed M3 homodimer are asymmetric. One binding site resembles the open chemokine-complexed conformation while the other is more tightly shut due to loops of the opposing NTD and CTD of each monomer interacting. Alexander et al. also performed size exclusion chromatography (SEC) on both M3 alone and M3 bound to MCP-1 to examine whether or not it exists as a dimer in solution. Their result indicates that it exists as a dimer in solution whether or not it is complexed with MCP-1. Mutation studies on M3 performed by Alexander et al. show that only 2 substitutions had a measurable effect on M3 binding affinity; Y13 (10-fold reduction) and K19 (2-fold reduction). The NTD and the CTD each bind seven side chains of MCP-1. The surface areas of MCP-1 which are bound by M3 have been identified as being the same residues which are involved in receptor contact (Hemmerich et al., 1999).
Activities of M3 in vitro
There are four chemokine classes; C, CC, CXC and CX3C (the letter C represents the amino acid cysteine while the letter X represents any amine acid). Their function is to attract various cells of the immune system to sites of infection. M3 is a broad spectrum chemokine binding protein which can bind chemokines from each of these classes (Parry et al., 2000). M3 can bind both mouse and human chemokines with high affinity which blocks chemokine signalling (van Berkel et al., 2000). They found that M3 bound the human chemokines Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES, also known as CCL5), interleukin-8 (also known as CXCL8) and the murine chemokines monocyte chemoattractant protein 1α (MIP-1α), lymphotactin, a C chemokine and the chemokine fractalkine, a CX3C chemokine. All of these chemokines were bound with high affinity. Chemokine-mediated Calcium flux, which is an early signalling event in chemokine-recptor signalling was found to be lower in human neutrophils, murine neutrophils and murine macrophages. Van Berkel and colleagues also found that binding was selective as seven CX3C chemokines were not bound by M3. M3 has been identified as being the only chemokine binding protein encoded by MHV-68 (Parry et al., 2000) Parry and colleagues also showed that M3 does not bind human B cell-specific or murine neutrophil-specific CXC chemokines. Another study by Jensen et al. showed that M3 also inhibits chemotaxis in vitro and in vivo by CCL19 and CCL21 (Jensen et al., 2003). These chemokines are highly expressed in secondary lymphoid tissue. It's possible that the expression of M3 by infected cells helps to prolong the infection by altering the blocking the chemotaxis of lymphocytes and dendritic cells. In addition to binding their respective receptors, chemokines are also known to bind to glycosoaminoglycans (GAGs) in the extracellular matrix (ECM). These interactions are thought to be important in the establishment of stable chemokine concentration gradients in tissue (Witt and Lander, 1994, Lalani et al., 1997, Lalani and McFadden, 1997). M3 has been shown to inhibit this interaction as well as the chemokine-receptor interaction (Webb et al., 2004). It seems clear from these results that M3's function is to delay activation of the adaptive immune response by preventing recruitment of cells such as antigen presenting cells (APCs), natural killer (NK) cells and CD8+ T cells.
Role of M3 in pathogenesis of MHV-68 infection
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An important characteristic of the herpesviruses is their ability to establish a latent infection in the host. Like its relatives, Epstein-Barr virus (EBV) and Kaposi's Sarcoma Associated Herpesvirus (KSHV), MHV-68 latently infects B cells (Stevenson et al., 2002). Tumours are rare in mice infection models, though this could be due to the short lifespan of laboratory mice. MHV-68 shares many characteristics with EBV and KSHV such as primary lytic replication, ability to establish a latent infection and viral reactivation. In 2001, Bridgeman et al. showed that the M3 protein is essential for the virus to successfully establish a latent infection in the mouse. They disrupted the M3 gene and found that the lytic cycle and progression to the lymphoid tissues was normal but the amplification of B cells which were infected latently failed to occur (Bridgeman et al., 2001). Further evidence for the model that M3's function is to protect infected cells from CD8+ T cell-mediated cell death can be found in a study by Rice et al. They introduced the M3 gene into tumour cells before exposing them to CD8+ T cells which had been primed by a DNA vaccine for a specific tumour epitope (Rice et al., 2002). The cells were protected from the Cytotoxic T lymphocyte (CTL) response by M3. In 1999, Stevenson et al. conducted a study examining the effects of vaccinating murine lung cells with a CD8+ T cell epitope. They found that lytic replication in the inital stage of infection was reduced but that the CD8+ T cell epitope had no effect on the establishment of a latent infection (Stevenson et al., 1999). This result, along with the result of the Bridgeman et al. paper suggests that the role of M3 in lytic infection is minimal and that is has a crucial role in establishing and maintaining a latent infection. Van Berkel et. al conducted another study investigating the role of M3 in gammaherpesvirus-induced lethal meningitis. They found that an M3-deficient mutant virus was 100-fold less virulent than the wild types virus following intracerebral inoculation. CC chemokine expression was induced by MHV-68 infection in the CNS. The wild type infection model induced a predominantly neutrophilic chemotactic infiltrate. Lymphocytes and macrophages predominated the infiltrate in the M3 defective infection model (van Berkel et al., 2002). Another study by May et al. in 2004, the authors deregulated ORF50, the major lytic transactivation gene of MHV-68. They found that the replication-competent virus which had a severe deficit in establishing a latent infection was cleared 10 days after infection. This result suggests that both latency and M3 are critical in the establishment of a long term infection by MHV-68.
Potential therapeutic use of M3
As stated above, MHV-68 is closely related to the human gammaherpesvirus pathogens EBV and KSHV. This means that MHV-68 infection of mice can yield useful insights into the pathogenesis of EBV and KSHV. EBV is associated with various diseases such as infectious mononucleosis, Burkitt's lymphoma, Hodgkin's Disease and nasopharyngeal carcinoma. KSHV is associated with Kaposi's Sarcoma (Flano et al., 2002). The main problem with vaccination against herpesviruses is latency. Studies have shown that M3 is essential for MHV-68 to establish a latent infection. Structural and biophysical properties have been elucidated in studies by Alexander et al. and Alexander-Brett et al. (Alexander et al., 2002, Alexander-Brett and Fremont, 2007). In 2004, Obar et al. attempted to DNA vaccinate mice with the M3 ORF. The idea behind DNA vaccination is that a segment of a pathogen's genetic material is injected into cells. Here, it is expressed as a protein which is subsequently recognised as foreign. It is then degraded and presented via Major Histocompatibility Class (MHC) molecules. They found that, despite M3 being expressed in both the lytic and latent stages of the viral replication cycle the immune response to the epitope followed the kinetics as lytic cycle antigens. This shows that M3 can also be targeted by the CTL response.
M3 has also shown considerable therapeutic value. In one study by Liu et al. intimal hyperplasia was reduced in rat models of aortic allograft transplant vasculopathy after the administration of M3 or another chemokine binding protein. Early T cell and macrophage invasion inhibition was linked to a decrease in late stage development of vasculopathy (Liu et al., 2004). Another study by Pyo et al. showed that M3 was effective in reducing the levels of intimal hyperplasia in transgenic mice. They found that M3 expression reduced intimal area and a reduction of 68% in intimal/medial ration following femoral arterial injury (Pyo et al., 2004). It is suggested from these results that chemokines have a role in intimal hyperplasia and M3 may provide a potential therapeutic option. Martin et al. also showed that M3 prevented lymphocytic insulitis and diabetes induced by multiple low doses of Streptozotocin in mice (Martin et al., 2007). They found that Streptozotocin caused up-regulation of several chemokines and that transgenic mice expressing M3 were resistant to induced diabetes. They had fewer inflammatory cells and lower chemokine levels.
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Chemokines have been implicated in a wide variety of diseases (Luster, 1998). As a result their blockade may provide a novel therapeutic strategy for treatment of these diseases. However, more work is needed to investigate whether M3 may have therapeutic potential for other disorders such as autoimmunity. M3 may also represent a novel vaccination strategy as one study demonstrated that a latency-incompetent MHV-68 protected mice from infection with the wild type virus (Rickabaugh et al., 2004). It is conceivable that a similar strategy could be adopted regarding EBV and KSHV but more research is needed to fully elucidate the mechanisms underlying the role of M3 in pathogenesis.