Many viruses have evolved mechanisms to interact with the host’s immune system in order to promote virus survival within the host. Poxviruses and herpesviruses encode proteins that either mimic cytokines or their receptors as a method of evading or altering the host immune response (Alcami, 2003). Chemokines are chemoattractant cytokines that regulate the movement and function of leukocytes (Weinberg et al., 2002). The M3 gene of MHV-68 is a
viral chemokine binding protein, a class of protein that is secreted from the cell during infection and has the ability to neutralise chemokines in solution (Alcami, 2003). M3 encodes a 44kDa protein with a signal peptide which is
abundantly secreted by infected cells in vitro (van Berkel et al., 1999). M3 is
transcribed in vivo abundantly during lytic infection in the lung and during early
stages of latency; latently infected mice show expression of M3 in the spleen
(Simas et al., 1999; Virgin and Speck, 1999). The M3 protein has no clear homology with known cellular or viral gene products, other than a low level of homology with MHV-68 M1 (van Berkel et al., 1999). As a secreted protein, M3 is a candidate for interaction with the host immune response, and has been found to bind to a wide range of chemokines in vitro, including at least one
from each of the CC, CXC, C and CX3C subtypes (Table 1-3). Chemokine
binding by M3 is functional as shown by inhibition of chemokine-induced elevation of intracellular calcium levels and the inability of bound chemokines to bind with their receptors due to a higher affinity to M3 than their receptors (Parry et al., 2000; van Berkel et al., 2000). M3 was found not to bind CXCL12 /SDF-1 and evidence of binding of CXCL13/BCA-1 (also known as BLC) was variable between authors, which is of note, as these are both B cell chemokines (Parry et al., 2000; van Berkel et al., 2000). The binding of M3 to CCL2/MCP-1 and CXCL8/IL-8 has been found to be in a similar spatial arrangement to that of the receptors of these chemokines, despite the lack of sequence homology between these proteins (Alexander et al., 2002; Webb et al., 2003).
Intranasal infection of laboratory mice with M3-deficient MHV-68 reveals that
M3 is non-essential for lytic replication in the lung, nor for spread of the virus to the spleen (Bridgeman et al., 2001; van Berkel et al., 2002). However,
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amplification of latently infected B lymphocytes in the spleen fails to occur and leads to a decrease in splenic latent load compared to wild type MHV-68 infected mice, demonstrated by a decrease in vtRNA transcripts and viral DNA load and decreased reactivation of latent virus ex vivo (Bridgeman et al.,
2001). Depletion of CD8+ T cells abrogates the differences seen in M3-null
infections, which suggests that chemokine neutralisation by M3 blocks effective CD8+ T cell recruitment into lymphoid tissue during B lymphocyte proliferation (Bridgeman et al., 2001). Intracranial infection of mice with wild type and M3-null MHV-68 showed a greater difference when M3 was present; M3-null infected mice exhibit considerably lower viral titres in the brain and the
intracranial inflammatory response is altered from a neutrophilic to a lymphohistiocytic meningitis (van Berkel et al., 2002).
Systematic name
Alternative name(s)
Responding cell
types Notes Reference
CCL2 MCP-1
actT cells, MØ, EL, NK cells, BL, immDC
In vitro (van Berkel et al., 2000)
In vivo (GEM) (Martin et al., 2006)
CCL3 MIP-1α
actT cells, MØ, EL, NK cells, immDC
In vitro (van Berkel et al., 2000)
CCL5 RANTES
actT cells, MØ, EL, NK cells, immDC
In vitro (Parry et al., 2000; van Berkel et al., 2000) CCL19 MIP-3β T cells, actT cells, mDC In vitro (Jensen et al., 2003) CCL21 6Ckine T cells, actT cells,
mDC
In vitro
In vivo (GEM) (Jensen et al., 2003)
CXCL8 IL-8 NL In vitro (Parry et al., 2000; van Berkel et al., 2000) CXCL13 BCA-1
BLC B cells
In vitro
(weak binding) (Parry et al., 2000)
In vivo (GEM) (Martin et al., 2006)
CX3CL1 Fractalkine actT cells, MØ, NK cells In vitro (Parry et al., 2000; van Berkel et al., 2000) XCL1 Lymphotactin T cells In vitro (van Berkel et al., 2000) Table 1-3 Evidence for M3-binding of chemokines.
Key: actT cells = activated T cells; MØ = macrophages; EL = eosinophils; NL = neutrophils; BL = basophils; immDC = immature dendritic cells; mDC = mature dendritic cells; GEM = transgenic mice that expressed M3 and the chemokine of interest in the pancreas
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Expression of M3 following intranasal infection of wood mice is highest at days
12 and 14 pi, later than that seen in Mus musculus (Hughes, 2006). M3
transcripts are located in perivascular and peribronchiolar accumulations of B lymphocytes, and macrophages within granulomatous infiltrates at day 7 pi; at day 14 pi M3 is present in numerous lymphocytes in the iBALT, in addition
those in the perivascular and peribronchiolar infiltrates. Extrapulmonary M3
transcripts are present in the bronchial and submandibular lymph nodes from day 7 pi and within splenic follicles from day 10 pi onwards (Hughes, 2006). Infection of wood mice with a M3-deficient MHV-68 (M3.stop) leads to
alterations in the inflammatory response, including mixed T and B lymphocytic infiltrates in the interstitium, perivascular and peribronchiolar areas (opposed to B cell dominated in the wild type MHV-68). A decrease in vtRNA in perivascular lymphocytes occurs, indicating a decrease in latency, which is either secondary to the smaller infiltrates or a lower proportion of B cells and is attributed to the absence of M3. Additionally, no iBALT is present in the
M3.stop infected lungs, which suggests that iBALT formation is stimulated in the presence of M3 due to increased viral persistence and the promotion of B
cell proliferation (Hughes, 2006). In the spleens of M3.stop-infected wood mice, lymphoid follicles are expanded but lack germinal centres; this is similar to the response seen in MHV-68 infected Mus musculus. The number of cells
containing vtRNA transcripts is also decreased, and the reduction in latency can be confirmed by a reduction in infective centre assay in the spleen of M3.stop-infected wood mice (Hughes, 2006).
The presence or absence of M3 also alters the presence of chemokines in the lungs of MHV-68-infected wood mice. MIP-1α/CCL3, RANTES/CCL5 and MIP- 3β/CCL19 are increased in M3.stop infection, consistent with the in vitro
binding of M3 to these chemokines. These chemokines are involved in T cell recruitment and depletion of their effects in vivo decreases inflammation and
increases viral titre following viral infection; therefore binding of M3 to these chemokines may confer a survival advantage (Cook et al., 1995; Culley et al., 2006). Additionally, MIP-1β/CCL9, MIP-3α/CCL20, KC/CXCL1, MIP-2/CXCL2 and MIG/CXCL9, which either have not exhibited binding, or have been shown
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not to bind to M3, are also increased in M3.stop infection. Significantly, SDF- 1α/CXCL12, BLC/CXCL12 and CD30L are decreased in M3.stop infection, suggesting that the presence of M3 increased the levels of these chemokines, either in relative or absolute terms (Hughes, 2006). SDF-1α and BLC are B cell chemoattractants, so an increase in wild type MHV-68 infection corresponds with the higher numbers of B lymphocytes in the inflammatory response to wild type MHV-68 infection in the wood mouse. CD30L has a role of T and B cell segregation in the spleen and BLC/CXCL12 may be relevant to the formation of iBALT in the lung. The contradictory reports as to whether BLC is bound by M3 may be due to the location and timing of the increased levels of M3 in response to viral infection, or due to subtle differences in the coding of these chemokines between Mus musculus and Apodemus sylvaticus (Hughes,
2006).
Analysis of gene expression in the lung in response to M3.MR compared to M3.stop infected wood mice also reveals alteration in the expression of other genes. These include members of the Palate, lung and nasal epithelium clone (PLUNC) family of proteins, SPLUNC1 and LPLUNC1, which were expressed
at levels 17.6 and 7.3-fold higher, respectively, in M3.MR compared to M3.stop infected wood mice; Clara cell secretory protein (CCSP), which showed a 3.3-
fold increase in the presence of M3 and Anterior gradient homologue 3 (AGR3), which was also increased in the presence of M3 (Hughes, 2006).
Further details of these proteins are given below (1.5 Expression of proteins in the lung in relation to MHV-68 M3).