VZV gC is a vCKBP

The results obtained in this study show that the ectodomain of VZV gC bound a broad range of chemokines with high affinity. These interactions enhanced chemokine activity in transwell migration experiments, independently of the cell type used. The domain involved in these interactions localized at the C-terminal region of the gC ectodomain where two immunoglobulin-like domains are predicted. In contrast, the N-terminal region of gC did not interact with chemokines tested. Partial binding to the R2D chip was observed when the chip was tested later on time. This aberrant behavior was not observed either with rSgC or IgD. Moreover, unpublished data from our lab in collaboration with Thomas Krey (Institute of Virology, Hannover Medical School) showed that the combination of the two immunoglobulin domains,

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but not each of them in isolation, is required for enhancement of chemokine activity.

Immunoglobuling-like structures have also been described in other viral cytokine and chemokine binding proteins. For example, the vaccinia virus Western Reserve gene B18R encodes for an IFN receptor with three immunoglobulin-like domains²⁶⁵. The same applies to the poxvirus fowlpox virus, whose IFN-γ binding protein contains also an immunoglobulin-like domain²⁶⁹. An immunoglobulin-like fold has been also described in the vCKBP R17 and M3, expressed by the gamma herpesviruses rodent herpesvirus Peru and MHV-68, respectively^193,270.

The broad range of chemokines that rSgC interacts with is probably an indicator of the need of VZV to modulate the immune system. For example, some chemokines found to interact with rSgC are related with inflammatory responses. CCL2, CCL8, CCL7 and CCL13 belong to the monocyte chemoattractant protein (MCP) family and induce the migration of monocyte, T and NK cells²⁷¹. CCL5 (also known as RANTES) recruits T-cells, macrophages, eosinophils and basophils, and in collaboration with IFN-γ, activates and regulates NK cell proliferation²⁷². The interaction of rSgC with these chemokines may indicate that VZV modulates the inflammatory response at the infection site. Also, although not tested in migration experiments, CXCL9, CXCL10 and CXCL11 are ligands of CXCR3, a receptor expressed by activated T-cells, memory T-cells and NK cells²⁷³. The expression of this group of chemokines is regulated by IFN-γ which is also produced by T-cells and NK cells²⁷⁴. This group of chemokines is involved in cell-mediated immunity so it would be interesting to determine whether rSgC can modulate this immune response by interacting with these chemokines. At the same time, is also important to remark the role of IFN-γ in the inflammatory response, a cytokine that also interacts with rSgC among other IFNs, interactions that have not been characterized yet.

145 Other chemokines that interact with rSgC are involved in homeostatic functions such as CXCL12α, a ligand of CXCR4. The signaling axis CXCL12α/CXCR4 has been intensively studied due to their implications in HIV and cancer. Physiologically, CXCL12α is involved in hematopoiesis, development of the central nervous system and the lymphoid tissue, through the induction of cell migration, survival and proliferation²⁷⁵.

When compared with other vCKBP discovered in herpesviruses, rSgC stands out by its broad range of chemokine ligands, in a similar way than M3¹⁹⁴. Actually, the only difference reported between this two vCKBP besides their selectivity in the chemokines they interact with, is that rSgC interacts with members from the CXC- and CC- chemokine family while M3 is able to interact with members from the four chemokine families although the affinity for CXC- chemokines is lower²³⁸.

Some differences and similarities are observed when we compare rSgC and gG.

Described in BoHV-1 and 5, EHV-1, ILTV, FeHV-1, PRV and HSV-1 and 2, gG interacts with members of the C-, CC- and CXC- chemokine families196,214,237, having in common with rSgC the CC- and CXC- chemokine families. However, the number of chemokines that rSgC can interact with is much higher than gG. Actually, in the case of gG, there is an specific selectivity that depends on the virus and host specie as for example, gG from HSV-1 and 2 can bind CCL18, CCL25, CCL26, CCL28, CXCL9, CXCL10, CXCL11, CXCL12α, CXCL12β, CXCL13 and CXCL14, but only gG from HSV-2 binds CCL22²³⁷. Another example of this selectivity can be found when we compared EHV-1 gG and BoHV gG. Both can interact with the CXC- and CC- family but however, BoHV gG binds with higher affinity CC- chemokines while in contrast, EHV-1 gG shows similar binding to both chemokine families¹⁹⁶. When

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comparing with rSgC, we can see that the VZV vCKBP interacts with CC- and CXC- chemokine families with similar affinities in a similar way than HSV or EHV-1 gG.

The other vCKBP discovered in herpesviruses are R17 expressed by RHVP and pUL21.5 expressed by human CMV. In the case of R17, the protein can interact with the C- and CC- chemokine family, sharing only this last one with rSgC. Among the chemokines tested for R17, is interesting to observe that only CCL3 and CCL4 are bound by R17 and not by rSgC¹⁹⁸. Finally, the case of pUL21.5 is difficult to compare with rSgC as this vCKBP has been only tested with one chemokine, CCL5²¹⁰. Therefore, it would be interesting to check how broad the binding range of pUL21.5 is by using an extensive chemokine library, and compare the results with rSgC.

Similarities and differences are observed when we compared rSgC with other vCKBP discovered in other herpesviruses. Among the differences we can see that depending on the virus and the host, these vCKBP have evolved to interact with different chemokine partners. However, even when the chemokine ligands are different, something very interesting to remark is their effect. While most of the vCKBP inhibit migration, rSgC enhances it, a feature only observed in gG from HSV-1 and 2. Very probably, the similarities and differences among these vCKBP respond to different immunomodulatory strategies related to each host and viral infection in particular.