An interaction with a host cell surface receptor(s) is the first essential step in any viral infection of target cells. Though binding to the target cells could occur at low temperatures, entry of a virus or viral genome into the cells and subsequent movement in the cytoplasm are energy-dependent phenomena. Virus interactions with specific target cell receptors have probably evolved to accommodate these needs, and the manipulation of preexisting host cell signal pathways via cell surface receptors is probably one of the best strategies to fulfill these requirements. Even though several host cell surface receptors have been identified for human and animal herpesviruses, how these interactions with cell surfaces facilitate infection is largely unexplored. KSHV is the first herpesvirus shown to interact with integrin molecules (4
). Within minutes of its binding to target cells, KSHV induces a variety of preexisting host cell signal pathway molecules, such as FAK, Src, PI-3K, RhoGTPases, PKC-χ, MEK, and ERK1/2 (32
). Chief among them is the integrin-dependent protein component FAK, and our studies here demonstrate that FAK plays a critical role in KSHV entry.
Since FAK-null human cells were not available, FAK-null mutant MEF (FAK−/−
) and their wild-type counterpart Du17 cells were used (21
). The present study demonstrates that KSHV entered the mouse cells with an efficiency similar to that of primary human fibroblasts or endothelial cells, and the kinetics of viral gene expression was remarkably similar to that reported in the human primary cells (23
). Our previous study demonstrated reduced infectivity in the absence of FAK (32
). Since infection was monitored by counting the number of GFP-expressing cells 48 h after GFP-HHV-8 infection, it was not clear whether the reduced infectivity was due to reduced viral entry, slow movement of virus particles to the nucleus, or downregulation of viral gene expression. The assays carried out here were designed to differentiate the role of FAK in the various steps of KSHV infection. If FAK phosphorylation and the subsequent activation of signal cascades played no role in viral entry but played a role in nuclear delivery of internalized virus particles, we should have observed no reduction in entry into Du3 cells but reduced nuclear entry and gene expression. Similarly, if FAK activation played no role in viral entry and in nuclear delivery of internalized virus particles but played a role in viral gene expression, we should have seen no reduction in entry and nuclear delivery of viral DNA, and we should have observed reduced viral gene expression. Instead, we observed a 70% reduction in KSHV entry into Du3 cells. However, the ratio of delivery of viral DNA into the nuclei and viral gene expression to the internalized viral DNA in Du3 cells was comparable to that in Du17 cells. This demonstrated that the reduced infectivity in Du3 cells was certainly due to reduced viral entry. The role played by FAK at the entry stage of KSHV infection was also confirmed with the introduction of Wt-FAK into Du3 cells and the expression of FRNK in Du17 cells.
Even though integrins are used by several diverse groups of viruses, the mechanism by which the integrin-virus interactions facilitate the infection is not well studied. Our demonstration of the role of FAK in KSHV entry is the first for these viruses. Adenovirus types 2 and 5 (group C) bind to a primary receptor, the coxsackie virus B Ad receptor (CAR). The CAR-docked particles activate integrin coreceptors, and this triggers a variety of cell responses, including endocytosis (25
). Ad2/Ad5 endocytosis is clathrin mediated and involves the large GTPase dynamin and the adaptor protein 2. A second endocytic process, macropinocytosis, is induced simultaneously with viral uptake. Macropinocytosis requires integrins, F-actin, PKC, and the Rho family of small G-proteins but not dynamin. A major question arising from studies of adenovirus entry is whether integrins promote virus entry via specific cell signaling events. Adenovirus induces the ERK1/ERK2 mitogen-activated protein kinases via FAK and consequently cell motility. However, FAK activation appears to have little role in integrin-mediated adenovirus endocytosis (25
). FAK is phosphorylated and activated upon adenovirus entry, but this kinase is not required for virus uptake into cells or infection, since FAK-null Du3 cells were infected with an efficiency similar to that of parental FAK+/+
DU17 cells (25
). Expression of FRNK also failed to inhibit adenovirus entry into the wild-type cells. Adenovirus internalization requires PI-3K activation-dependent p130Cas
, and entry was blocked by the pharmacologic inhibitors of PI-3K (25
). Our studies show that the presence of FAK is critical for KSHV entry, which is remarkably different from adenovirus infection. FAK has been shown to play a significant role in human cytomegalovirus-induced smooth muscle cell migration (48
). FAK was also phosphorylated during the infection of CaSki cells with herpes simplex virus types 1 and 2 (HSV-1 and -2) (12
), and a recent study showed the role of FAK in the infection of target cells by HSV-2 (13
). Further studies are essential to determine the role of FAK and the associated signal pathways in KSHV entry and infection.
As has been shown for FAK, Pyk2 has also been shown to participate in various signaling events. However, unlike that of FAK, Pyk2 signaling could also be nonintegrin related. Pyk2 was not expressed in Du17 cells (44
), and it was expressed in MEF only in the absence of FAK. The mechanism by which FAK expression regulates the expression of Pyk2 is not known. Our results show that Pyk2 plays a role in the KSHV entry process in Du3 cells. During KSHV infection of Du3 cells, but not Du17 cells, Pyk2 was phosphorylated at Tyr402
, which is functionally equivalent to Tyr397
of FAK. The role of Pyk2 was further confirmed by the introduction of F402 Pyk2 into Du3 cells. Expression of F402 Pyk2 has previously been shown to reduce the tyrosine phosphorylation of wild-type Pyk2, resulting in the inhibition of downstream signaling events such as ERK phosphorylation (44
). Overexpression of F402 Pyk2 in osteoblast-like cells inhibited bone resorption, like a deletion in Src in these cells, indicating that phosphorylation at Tyr402
was important in the interaction with Src (30
). The expression of this autophosphorylation site mutant form of Pyk2 in Du3 cells resulted in reduced phosphorylation of Pyk2 during KSHV infection, indicating that Pyk2 had a role at this stage and that phosphorylation of Pyk2 was directly correlated with viral entry into Du3 cells. Studies of Pyk2 inhibition by the Ca2+
chelator EGTA further confirmed these results. The effect of expression of Pyk2 along with FAK is not known, and it can be speculated that Pyk2 may compete for the target molecules, thus resulting in reduced viral entry.
The present study did not address the mechanism by which KSHV entry is facilitated by FAK and Pyk2. KSHV enters human fibroblast cells (2
), B cells (5
), and epithelial cells (22
) via endocytosis. We hypothesized that KSHV binding- and entry-activated signaling pathways may play roles at the following stages of viral infection: (i) to aid in virus entry through the formation of either clathrin-coated vesicles, caveolin coated vesicles, or macropinocytic vesicles, and/or an ill-defined route of non-clathrin, non-caveola-dependent vesicles, and their movement; (ii) to aid in the release of viral capsids with viral DNA from these vesicles into the cytoplasmic environment; (iii) to aid in the movement of viral capsids to the nuclear membrane boundary; (iv) to aid in the release and delivery of viral DNA into the nucleus; and (v) to aid in viral gene expression.
Based on the endocytic mode of KSHV entry into the target cell, certain reasonable scenarios can be hypothesized (Fig. ). Numerous recent reports show that endocytosis and signal pathways are highly interlinked (1
). Protein components of signal transduction cascades assemble at clathrin-coated pits and remain associated with endocytic vesicles following their dynamin-dependent release from the plasma membrane (9
). Following ligand interaction with receptors, associated tyrosine kinases are activated by autophosphorylation on tyrosine residues and recruit signaling complexes to the plasma membrane, which then rapidly translocate to clathrin, caveolae, and other vesicles (50
). Src-mediated tyrosine phosphorylation of clathrin regulates clathrin translocation to the plasma membrane (1
). Clathrin subsequently interacts with a number of other essential proteins, as well as dynamin (1
). Src-dependent phosphorylation also regulates dynamin self-assembly and ligand-induced endocytosis by releasing the internalized endocytic vesicles from the plasma membrane (1
). Through interactions with growth factor receptor bound protein 2 (Grb2), murine son of sevenless protein (mSOS) is recruited to the plasma membrane to activate Ras. Src-dependent phosphorylation initiates the assembly of a plasma membrane-associated Ras activation complex. Rho and Rab-GTPases activated by PI-3K and Ras are critical for the formation of various types of endocytic vesicles and their movement, and for microtubule and microfilament reorganization (9
FIG. 9. Schematic diagram depicting the overlapping dynamic phases of early events of KSHV infection of target cells, KSHV-induced signal pathways, and their role in infection. (Phase 1) KSHV binds to the cell surface HS molecules via its envelope glycoproteins (more ...)
The interplay between virus-induced signaling with endocytosis and the movement of capsids in the cytoplasm has not been thoroughly analyzed. In recent years, it has become increasingly clear that integrins and the associated signaling pathways are the converging point for the signals induced by other receptor tyrosine kinases, including growth factor receptors, and cross talk initiated by integrin-ligand interactions plays vital roles in various cellular processes (43
). The first step in any integrin-ligand interaction is the induction of two major overlapping signaling pathways, namely, (i) the FAK/Src signaling pathways and (ii) the caveolin-1/Shc signaling pathways (19
). The converging point for both pathways is the activation of Ras via Grb2 and mSOS (19
). FAK activation also results in the induction of other signaling pathways via the induction of Src kinase (Fig. ). FAK interacts with a number of signaling and cytoskeletal proteins, including Src, paxillin, PI3-K, p130Cas
, and Grb2. These interactions link FAK to signaling pathways that modify the cytoskeleton, aid in the formation, movement, and recycling of endocytic vesicles, and aid in the activation of other cascades (7
). FAK activation has been shown to be essential for invasion by certain bacteria (7
), and for endocytosis of integrin-bound photoreceptors (17
). Activation of FAK by KSHV thus may aid in virus entry by virtue of its critical role in endocytosis (Fig. ). The immediate converging point of FAK and Pyk2 induction is the activation of Src (Fig. ) (7
). KSHV infection and incubation with purified soluble KSHV gB induced comparable levels of Src phosphorylation in both Du17 and Du3 cells (42
). Since Src activation is the initial critical step that leads to the phosphorylation of clathrin, dynamin, Ras, and other associated molecules necessary for the formation of endocytic vesicles, as well as for the activation of PI-3K and RhoGTPases involved in the movement and recycling of endocytic vesicles, the role of FAK and Pyk2 in KSHV could be due to their ability to activate Src. Further studies are in progress to decipher the role of Src in KSHV infection and in the formation of endocytic vesicles and their movement.