The studies presented here demonstrate for the first time that during primary infection of endothelial cells, KSHV-induced ROS promote KSHV entry and the amplification of the initial host signal cascade, including EphA2, FAK, and Src. This amplification loop is probably essential for sustained EphA2, FAK, and Src phosphorylation, which in turn contributes to the downstream signal cascades that are essential for the translocation of the virus to LRs, actin cytoskeleton rearrangement, bleb formation, macropinocytic entry of KSHV, and the establishment of infection (). EphA2 has been shown to be associated with integrin-associated signaling during KSHV infection (14
), and the involvement of ROS in modulating integrin translocation into LRs, EphA2 activation, FAK and Src signaling, and KSHV entry reveals new insights into the amplification loop necessary for virus entry.
Fig 10 Schematic depicting the induction of ROS by KSHV early during primary infection of endothelial cells to promote virus entry. KSHV initially binds and interacts with heparan sulfate, integrins (α3β1, αVβ3, αVβ5), (more ...)
Accumulating evidence has suggested an important role for ROS and oxidative stress during viral infection (56
). It is known that virus binding and entry activate several secondary messengers and signaling pathways. We observed a 1.5-fold increase in ROS production at early time points following KSHV infection and a 2.5-fold increase at 24 h p.i. The kinetics and fold increases were comparable to those observed in other viral infections. It has been shown that during adenovirus 5 (Ad5) infection, ROS production increases rapidly following lysosomal rupture (57
). Within 15 min of infection, Ad5 induced ROS production that was maintained at a ca. 2-fold increase for more than 2 h p.i (58
). In addition, a recent study demonstrated that enterovirus 71 (EV71) increased ROS production as early as 30 min p.i. (59
). This study also indicated that EV71 increased ROS production following its binding to integrin β1, Rac1, and NADPH oxidase (59
). In results similar to ours, ROS were also found to be induced by several herpesviruses. Herpes simplex virus 1 (HSV-1) entry and replication induce ROS production, lasting for a prolonged period, as early as 1 h p.i. (60
), and glycoprotein gJ has been shown to induce ROS production (61
). HSV-1 infection of microglia induced a rapid 1.5-fold increase in ROS production as early as 3 h p.i., and ROS production was more robust at 24 h p.i., with a 2-fold increase (62
). ROS production after HSV-1 infection was dependent on NADPH oxidase activity. Herpes simplex virus 2 (HSV-2) induced a 1.5- to 2-fold increase in ROS production 1 h after the infection of RAW 264.7 cells (64
). Similarly, the gamma-1 herpesvirus Epstein-Barr virus (EBV) induced oxidative stress during the early stages of primary infections in B lymphocytes and epithelial and lymphoblastic cell lines (65
). In this study, a 2-fold increase in malondialdehyde activity, indicative of lipid peroxidation, was measured.
We observed that ROS production was sustained after the establishment of latent KSHV infection in HMVEC-d cells. This result is in accordance with studies showing the production of high levels of ROS in latent EBV-positive Burkitt's lymphomas (66
). Several mechanisms are proposed to be involved in ROS production in EBV-infected cells, depending on the type of latency. ROS production is dependent on paracrine secretion of interleukin 10 (IL-10) in latency I, whereas expression of EBV nuclear antigen 2 (EBNA-2) and latent membrane protein 1 (LMP1) is proposed in latency III (66
). EBNA-1 could also be involved in ROS production (67
). Very recently, Cao et al. have shown that long-term expression of EBNA-1 in nasopharyngeal carcinoma resulted in an increase in ROS production (69
). Taken together, these studies indicated that ROS production induced by EBV could play a role in genome instability as well as in metastasis. ROS can also function in KSHV-induced oncogenesis, a notion supported by several studies. Guilluy et al. have shown that ROS production leads to endothelial junction dysregulation and increased vascular permeability (7
). Moreover, the antioxidant NAC has been shown to efficiently reduce KSHV-induced oncogenesis in animal models (5
In addition to a role in tumorigenesis, ROS can play an important role in facilitating viral infection. Studies suggest that RNA and DNA viruses use oxidative stress to regulate their life cycles. Antioxidants have been shown to block the replication of RNA viruses, including influenza virus, EV71, and HIV-1 (59
). In the herpesvirus family, the oxidative stress induced by HSV-1 is required for efficient viral replication (60
). Indeed, the antioxidant compound Ebselen inhibited the replication of HSV-1. In contrast, H2
is instrumental in the maintenance of EBV latency, since it inhibits the expression of EBV immediate-early lytic genes (74
). Interestingly, it has been shown previously that oxidative stress can lead to KSHV reactivation in PEL cells and endothelial cells (4
In contrast to the earlier studies demonstrating the role of KSHV latency and lytic reactivation, our studies demonstrate for the first time that ROS induction promotes the early stage of KSHV infection and entry into HMVEC-d cells. KSHV has probably evolved to bind to integrin molecules to modulate several downstream signaling events that facilitate entry and infection (10
). Interestingly, several reports indicate an increase in ROS production following integrin engagement (33
). KSHV gB also interacts with integrins α3β1, αVβ3, and αVβ5 (10
), and this interaction is followed by several downstream signaling events, such as the phosphorylation of FAK, Src, PI-3K, and Rho GTPases (RhoA, Cdc42, and Rac1) (21
). Since these signal pathways play vital roles in host cell endocytosis and the movement of particulate materials in the cytoplasm, the early stages of KSHV interaction with host cells may provide an environment very conducive to the successful infection of target cells. Interestingly, we observed that the ROS scavenger NAC efficiently reduced FAK activation. Further investigations are needed to determine the identity of the KSHV envelope glycoprotein(s) involved in the ROS induction observed during the early time points of primary infection.
Previous studies have demonstrated an important role for integrin molecules α3β1, αVβ3, and αVβ5 in KSHV entry (10
). However, our attempts to block viral infection by preincubating the cells with anti-integrin antibodies before viral infection did not block ROS production (data not shown). This could be due to cross-linking of integrins by these antibodies and, consequently, induction of downstream signaling, including the production of ROS, which has been reported previously (29
). In addition, since KSHV interacts with a multitude of integrin subunits, simultaneous knockdown of all the integrin subunits (α3, αV, β1, and β3) by RNA interference is challenging.
An intriguing observation from our studies is that KSHV-induced ROS activate the FAK, Src, and EphA2 signal pathways. However, further detailed studies are essential to decipher the mechanism by which ROS activate these signal pathways. From the available literature, we speculate the following. ROS are essential cellular messengers, since they modulate several kinase and phosphatase activities through their transient and reversible oxidation. Especially, protein tyrosine phosphatases (PTP) contain cysteine residues in their active sites that could be targeted by oxidative stress, leading to their inactivation. One of these phosphatases is the low-molecular-weight protein tyrosine phosphatase (LMW-PTP). Indeed, H2
has been shown to inactivate LMW-PTP through the oxidation of cysteine residues (76
). Interestingly, LMW-PTP has been shown to dephosphorylate FAK (30
). Another substrate of LMW-PTP is EphA2 (78
). Therefore, LMW-PTP could be an attractive link between integrin, ROS, FAK, and EphA2 signaling. We speculate that the increased ROS production after integrin engagement in KSHV-infected cells could shift LMW-PTP to an oxidized/inactive status and consequently allow the amplification of FAK and EphA2 phosphorylation. On the other hand, the inhibition of ROS production during NAC treatment could increase the quantity of reduced/active LMW-PTP and consequently decrease FAK and EphA2 phosphorylation early during KSHV infection. Src kinase has also been shown to be directly and indirectly regulated by ROS (80
). Directly, oxidation of cysteine residues in the Src homology 2 (SH2) domain and/or kinase domain causes hyperphosphorylation and activation of Src. Indirectly, it is proposed that oxidation of the Csk kinase keeps Src active longer (by blocking the phosphorylation of Src Tyr527). In addition, Src is activated through the inhibition of phosphatases that dephosphorylate Src Try418 (80
). Additional studies examining the possibilities discussed above could shed light on the mechanism by which KSHV-induced ROS activate signal pathways.
In summary, our studies show that induction of ROS very early during infection by KSHV is essential for viral entry into HMVEC-d cells. Since the antioxidant NAC blocked viral entry by blocking the recruitment of integrin to the LRs, the phosphorylation of EphA2, the actin remodeling observed during macropinocytosis, and the activation of integrin-associated signaling molecules such as FAK, Src, and Rac1, antioxidants that have already been shown to be attractive drugs against KSHV oncogenesis are also attractive therapeutic drugs for controlling primary infection of endothelial cells with KSHV.