Background
Viruses from a range of virus families exert direct influence on host cytokine responses. Such influence can be mediated through expression of viral homologs of host cytokines or cytokine receptors, or through expression of viral factors that alter expression of host cytokines [
1‐
3]. Several herpesviruses such as Epstein-Barr virus (EBV) [
4], equid herpesvirus 2 [
5], ovine herpesvirus 2 [
6], and primate cytomegaloviruses [
7‐
9] encode a homolog to the IL-10 gene. Infection of B cells by EBV results in expression of virus-encoded IL-10 (vIL-10, encoded by the
BCRF1 gene) as well as induction of the endogenous cellular IL-10 (cIL-10) gene. vIL10 mRNA is detectable within six hours of EBV infection
in vitro[
10] and thus is expressed early in infection [
11]. In fact, EBV virions carry a number of viral mRNAs including those encoding vIL-10, which may be translated immediately upon infection [
12]. Subsequently, expression of the cIL-10 gene is induced twenty to forty hours post-EBV infection [
10,
13,
14] and is upregulated by the EBV latency-associated LMP-1 and small non-coding RNAs (EBERs) [
15,
16]. The differential timing of expression of these two IL-10 forms may reflect differences in their respective roles in viral infection.
IL-10 is a highly pleotropic, regulatory cytokine with differential effects in T-cell populations that generally reduce inflammation and cytotoxic responses, while favoring a humoral immune response [
17]. The EBV vIL-10 [
4], like its cellular counterpart, inhibits cytokine synthesis [
18]. EBV vIL-10 is 84% homologous to human IL-10, with most divergence occurring at the N terminus [
4] resulting in an altered N-terminal structure [
19]. cIL-10 and vIL-10 enhance B-cell viability, whereas only cIL-10 upregulates MHC II on B cells [
20]. vIL-10 also lacks cIL-10’s ability to stimulate mast cells [
21] and to induce proliferation of mature and immature thymocytes [
22]. EBV vIL-10 has 1000-fold lower affinity for the IL-10 receptor, perhaps explaining its greatly reduced ability to inhibit IL-2 production by helper T cells [
23]. Thus, the viral and cellular homologs share many immunosuppressive activities, while vIL-10 generally lacks cIL-10’s immunostimulatory functions. These differences are attributed primarily to a single amino acid substitution [
24].
In earlier studies involving an EBV mutant in which the vIL-10-encoding gene was deleted, vIL-10 was concluded to have no effect on replication, immortalization and establishment of latency within B cells
in vitro, and to have no effect on tumorigenicity when the resulting EBV-infected B lymphoblastoid cell lines were injected into SCID mice [
11]. More recent
in vitro studies with vIL-10-deficient EBV virus have demonstrated that vIL-10’s early expression protects infected B cells by altering the cytokine response, reducing NK cell killing, and inhibiting CD4+ T cell activity [
12].
In vivo, expression of the vIL-10 gene of the betaherpesvirus rhesus cytomegalovirus (RhCMV), significantly limits innate immune responses to primary infection, which in turn reduces both T- and B-cell responses [
25]. However, the applicability of the latter study to understanding EBV vIL-10 function is questionable given the low (27%) homology of RhCMV vIL-10 with cIL-10 [
9] and the high binding affinity of RhCMV vIL-10 relative to EBV vIL-10 for the IL-10 receptor [
26]. To date, studies probing the nuances of EBV vIL-10 in gammaherpesvirus infection have not been presented in an
in vivo model of viral pathogenicity.
Murine gammaherpesvirus 68 (MHV-68, γHV68, murid herpesvirus 4) infection of laboratory mice serves as a tractable animal model for gammaherpesvirus pathogenesis [
27]. Upon intranasal (i.n.) inoculation of mice, MHV-68 rapidly establishes an acute, productive infection of alveolar epithelial cells which is essentially cleared about 10 days post-infection (p.i.) [
28]. As the acute phase resolves, a syndrome similar to EBV-induced infectious mononucleosis ensues. This phase is characterized by splenomegaly [
29], non-antigen-specific B-cell activation [
30], and peripheral blood lymphocytosis primarily reflecting the expansion of CD8+ T cells expressing a Vβ4 T-cell receptor [
31]. This syndrome peaks at day 14 p.i. and resolves by about day 21 p.i. Latent virus has been detected in peritoneal macrophages [
32], splenic macrophages and dendritic cells [
33], and B cells [
33‐
36]. B cells are likely to be the means for trafficking MHV-68 from the lung to the spleen [
37] and expression of vtRNAs, a marker for latency, has been localized to the germinal centers in the spleen [
38‐
40]. CD4+ T cells are required for MHV-68-induced splenomegaly [
29,
41], while CD8+ T cells are critical for limiting productive pulmonary infection and for the resolution of splenomegaly [
35,
41‐
43].
MHV-76, a variant of MHV-68, contains a deletion of 9538 bp within the left end of the unique-sequence domain of the MHV-68 prototype genome – the region which includes the MHV-68 genes
M1-
M4 as well as eight vtRNA genes [
44,
45]. In comparison to MHV-68, MHV-76 is cleared more rapidly from the lungs and induces less pronounced splenomegaly and fewer numbers of latently infected cells in the spleen, although replication of the viruses in culture does not differ [
44,
45]. The
M2 gene encodes a latency-associated protein that serves as a target for cell-mediated immunity [
46]. The M2 protein binds Vav signaling proteins and promotes cell proliferation and survival [
47]. MHV-68 strains lacking only M2 expression show the same reduction in latency and reactivation as MHV-76; however, they do not exhibit MHV-76’s reduction of splenomegaly [
48‐
50]. While MHV-68 does not carry a native vIL-10 gene, M2 stimulates cIL-10 expression [
51,
52]. It has been proposed that M2’s role in stimulating cIL-10 and its resulting effects may represent a conserved gammaherpesvirus strategy that is also represented by the EBV vIL-10 gene [
51], although, as noted above, the timing of vIL-10 expression differs from that of cIL-10 induction. Therefore, we sought to determine the effects of vIL-10 expression on viral pathogenesis in a murine gammaherpesvirus strain lacking
M2 (MHV-76). Here we show that vIL-10 expression by MHV-76 enhances spleen cell proliferation and viral titers in the lung during acute infection, but does not affect splenic latency or reactivation of virus replication from latently infected cells.
Discussion
These results provide the first evidence of vIL-10’s role in gammaherpesvirus infection in an
in vivo model of viral pathogenicity. vIL-10 expression in MHV-76-infected mice increases acute-phase pathogenicity, but does not increase the percentage of latently infected splenocytes or the level of reactivation of latent virus. In accordance with these findings, in EBV, in which vIL-10 is expressed in its native environment, several studies have associated the gene’s expression with the acute phase of infection [
13,
21,
55].
Nevertheless, IL-10 and its ability to drive B cell proliferation and differentiation into plasma cells are considered significant factors in the process of gammaherpesvirus reactivation from latency. The MHV-68 M2 gene product stimulates cIL-10 expression and subsequent B cell proliferation and differentiation [
51]. The M2 protein exerts this effect by activating the NFAT signal transduction pathway which induces expression of interferon regulatory factor-4 (IRF-4), in turn inducing cIL-10 expression [
52]. cIL-10 expression is also induced in EBV-infected cells by latency-associated LMP-1 and small non-coding RNAs (EBERs) [
15,
16], and in Kaposi’s sarcoma-associated herpesvirus (KSHV) by viral-encoded miRNA [
56]. Whether vIL-10 plays a role in EBV infection in the human similar to that of cIL-10 induction by MHV-68’s M2 protein in the mouse remains to be shown. However, with EBV’s other means of inducing cIL-10 during latency, it is possible that EBV expression of vIL-10 serves a different function. Results presented in this paper suggest that vIL-10’s role may be in enhancing infection during the acute (lytic) phase.
Early expression of vIL-10 homologues by herpesviruses appears to increase the local pool of host cells permissive for infection, thus increasing the chance for trafficking of infected cells to other sites. For example, human cytomegalovirus (HCMV) and RhCMV encode IL-10 homologs that induce the differentiation of macrophages [
25], a cell type shown to be permissive for CMV infection [
57,
58]. Our results have shown that EBV vIL-10 expression by MHV-76 increases splenomegaly, where MHV-induced splenomegaly results, in part, from an increase in B cells [
29], and infected B cells are likely vehicles for trafficking MHV-68 from the lung epithelium to the spleen [
37].
In addition to increasing the pool of host cells to expand and disseminate primary infection, early expression of viral IL-10 homologues reduces virus-specific effector responses, helping to ensure the survival of infected cells into the latent phase. Such inhibition can occur upstream of effector cell activation by inhibiting innate responses critical to the transition to adaptive immunity. RhCMV IL-10 reduces dendritic cell populations in draining lymph nodes, resulting in a lower frequency of virus-specific T cells [
25]. EBV vIL-10 modulates cytokine responses [
12], reduces MHC I expression [
59], avoids increasing MHC II expression [
20], and inhibits monocytes [
60]. vIL-10 can also inhibit effector cell responses directly. For example, vIL-10 limits NK cell killing of infected B cells and inhibits CD4+ T cell activity [
12]. Studies are planned that will assess effector responses in mice infected with 76.vIL-10.
The timing and level of expression of vIL-10 are likely to relate significantly to vIL-10’s influence on pathogenicity. Expression of vIL-10 by the recombinant virus used in this study was quantified by an ELISA that distinguished vIL-10 from any cIL-10 that might have been produced by host cells. However, it is difficult to know how this level of expression compares to expression of vIL-10 in EBV-infected cells. Published studies have reported vIL-10 concentrations in functional units [
14] or fluorescence units [
61] rather than in units of mass. Furthermore, the difference in culture conditions for recombinant MHV and EBV would make such comparisons difficult to interpret. Finally, it would be of interest to determine if recombinant viruses package vIL-10 mRNA in the virion and express the product immediately upon infection as has been shown for EBV [
12] as well as to ascertain the kinetics of expression of vIL-10 by recombinant MHV in the host animal.
Details are emerging to clarify our understanding of viral modulation of immune responses via cIL-10 and vIL-10. Such understanding may expand our ability to intervene in diseases such as EBV-associated lymphoproliferative disease [
62]. Several studies have exploited the immunomodulatory properties of EBV vIL-10 for increasing the survival of allografts [
63‐
70]. The establishment of this model murine gammaherpesvirus expressing vIL-10 may contribute further to such work.
Acknowledgments
For technical assistance, we thank Drew Burk, Kristen Campbell, Joiceann Compton, Chris Davis, Meghan Davis, Carol Dickerson, Jeff Freyder, Lauren Jackson, Kristina Lynch, Audrey Marsidi, Megan McKenna, Sandra Obreza, and Desiree Steimer. For advice, reagents and/or sponsorship, we thank Laurie Krug and Peter Doherty. This work was funded by the National Institutes of Health [5 R01 CA90208 (JTS); 1 R15 AI068680 (GJL)] and Rhodes College Faculty Development Endowment.
Competing interests
The authors declare that they have no competing of interest.
Authors’ contributions
GL conceived of the study; GL, JPS and JTS designed the study. KG helped generate recombinant herpesviruses and conducted growth curve experiments and animal studies. GL coordinated all and conducted many of the experiments and wrote the manuscript. All authors have approved the final manuscript.