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. 2018 May 24;10(6):282.
doi: 10.3390/v10060282.

K15 Protein of Kaposi's Sarcoma Herpesviruses Increases Endothelial Cell Proliferation and Migration through Store-Operated Calcium Entry

Wei Chen  1 , Changqing Xu  2 , Liuqing Wang, Bing Shen  3 , Linding Wang  4
Affiliations

K15 Protein of Kaposi's Sarcoma Herpesviruses Increases Endothelial Cell Proliferation and Migration through Store-Operated Calcium Entry

Wei Chen et al. Viruses. .

Abstract

Kaposi's sarcoma (KS) is a tumor of the vascular endothelium that is caused by Kaposi's sarcoma-associated herpesvirus (KSHV). K15 of KSHV is a specific gene encoding a transmembrane protein. Two highly different forms of K15, the predominant (K15P) and minor (K15M) have been identified in different KSHV strains. In genomic locations and protein topology, two K15 alleles resemble the latent membrane protein (LMP) 1 and LMP2A of Epstein⁻Barr virus. Both K15 proteins have motifs similar to those found in LMP1 and LMP2A. K15 therefore seems to be a hybrid of a distant evolutionary relative of LMP1 and LMP2A. Ca2+ is a second messenger and participates in numerous activities in cells, like proliferation, migration and metastasis. It has been found previously that LMP1 increased Ca2+ influx through store-operated calcium channels and blockade of LMP1 reduced store-operated Ca2+ entry (SOCE). LMP2A has similar activity. So we sought to determine whether K15 had similar activity. We showed that K15P induced Ca2+ influx and enhanced expression of Orail1, which is a vital protein in SOCE, and overexpression of K15P improved cell motility. Mutant K15P did not show these activities in HEK-293T and EA.hy 926 cells. Our results showed that K15P increased cell proliferation and migration though SOCE and established a novel mechanism for the development of KS and KSHV-associated diseases.

Keywords: K15; Kaposi’s sarcoma-associated herpesvirus; Orail1; cell migration; cell proliferation; store-operated calcium entry.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Store-operated calcium entry (SOCE) in HEK-293T and EA.hy926 cells. (A) Changes in cytosolic Ca2+ concentration ([Ca2+]cyto) were measured in HEK-293T cells. Representative traces showing Ca2+ release in Ca2+-free saline solution by Ca2+-indicator fura-8 and 2 mM Ca2+ application-induced [Ca2+]I rise. (B) Histogram in the right panel shows the average peak from the baseline after thapsigargin-induced depletion of ER Ca2+ in HEK-293T cells. (C) Summary of data showing increase in [Ca2+]I in response to extracellular Ca2+ application after thapsigargin in HEK-293T cells. (D) Changes in [Ca2+]cyto were measured in EA.hy926 cells. (E) Histogram in the right panel shows the average peak from the baseline after thapsigargin-induced depletion of ER Ca2+ in EA.hy926 cells (F) Summary of data showing changes in [Ca2+]I in response to extracellular Ca2+ application after thapsigargin in EA.hy 926 cells. n = 5 per group (the n refers to technical replicates). Data are presented as the mean ± standard error. ** p < 0.01.
Figure 2
Figure 2
Expression of STIM1 and Orail1 in the two cell lines. (A) Western blots of K15P, STIM1, and Orail1 expression in HEK-293T cells. The right panel shows expression of STIM1 and Orail1 quantified by densitometric analyses. (B) Western blots of K15P, STIM1, and Orail1 expression in EA.hy926 cells. The right panel shows expression of STIM1 and Orail1.GAPDH was used as a control. Data are presented as the mean ± standard error. n = 5 per group. ns, not significant. ** p < 0.01, * p < 0.05.
Figure 3
Figure 3
Cell proliferation and migration determined by CCK-8 assays and wound scratch assays. (A) Percentage cell viability with different treatments in HEK-293T cells. (B) Percentage cell viability with different treatments in EA.hy926 cells. (C) Representative photographs of migratory cells were captured at three times point after scratching (100×). (D) The right panel shows that cell migration was quantified by measuring the maximum migration distance with different treatments at indicated times in EA.hy926 cells. Data are presented as the mean ± standard error. n = 3 per group (the n refers to technical replicates). ns, not significant. ** p < 0.01, * p < 0.05.
Figure 4
Figure 4
Proposed mechanism of K15-induced disturbance of Ca2+ influx. (A) In the control group with vector infection, [Ca2+]cyt was in equilibrium between Ca2+ entry by SOCE and Ca2+ exit by plasma membrane Ca2+ ATPases (PMCA) and the Na+/Ca2+ exchanger (NCX). (B) In K15P-infected cells, K15P was expressed, inducing an increase of SOCE and an increase of Ca2+ influx. The residual [Ca2+]cyt levels increased compared with the control and K15P (YF) groups. (C) In K15P (YF)-infected cells, the mutant protein induced an decrease of SOCE and Ca2+ influx, and the residual [Ca2+]cyt levels did not increase compared with other group.
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