obtained from patients with HCV also have greater TRAIL expression than hepatocytes from uninfected controls [21,22]. We have reported that X4 HIV gp120 envelope protein binding to CXCR4 on the surface of human hepatocytes activates JNK II, causing a transcriptional up regulation of the TRAIL receptor, TRAIL-R2 (DR5/CD262), which also is associated with an acquired state of TRAIL sensitivity [14]. More recently this observation has been extended to JFH1 HCV infected Huh 7.5.1 cells, where inactivated HIV causes upregulation of TRAIL-R2, and initiates a TRAIL dependent apoptosis [23] Altogether, such data support a model wherein HIV enhances TRAIL-R2 expression and TRAIL sensitivity, while an unknown factor associated with HCV infection promotes TRAIL expression and function. The purpose of the current report is to discover which factor associated with HCV causes hepatocyte expression of TRAIL, and to determine whether these changes result in the autocrine or paracrine death of HCV expressing, or HIV treated cells, and to determine whether the changes which are occur in vitro are also seen in vivo. Results X4 HIV Gp120 Increases TRAIL-R2 but not TRAIL on Huh7 cells It is established that HIV increases TRAIL-R2 expression on hepatocytes, renders the cell sensitive to TRAIL, and indepen- dently induces a low degree of hepatocyte apoptosis [14,24]. Therefore, we questioned whether HIV gp120 also increases TRAIL expression, which then might cause the observed hepatocyte apoptosis. As we have previously demonstrated, there was greater TRAIL-R2 expression, but not TRAIL-R1, -R3 nor – R4 when Huh7 cells were incubated with increasing concentra- tions of HIV gp120 (Fig. 1A and B)[14]. This is confirmed by real time PCR (Fig. 1C and D). However, increasing concentrations of HIV gp120 did not alter TRAIL expression on Huh7 cells by flow HCV Hepatocytes Kill HIV Hepatocytes through TRAIL cytometry (Fig. 2A) or Western blot (Fig. 2C). Therefore, HIV gp120 increases TRAIL-R2 expression on Huh7 cells, but does not alter TRAIL expression. Although the Huh7 cell is TRAIL sensitive after HIV gp120 treatment, there is little spontaneous death of the hepatocyte without the addition of exogenous TRAIL [14]. HCV Core Protein Increases TRAIL but not TRAIL-R2 on Huh7 cells It has been previously observed that HCV infection in the JFH1 HCV model using Huh7.5.1 cells results in enhanced TRAIL expression [23], and a similar effect has been reported to occur after HCV infection of livers in vivo [21,22,25]. Since it remains unknown what factor (s) associated with HCV are necessary or sufficient for this effect, we questioned whether individual HCV protein(s) could induce TRAIL expression in hepatocytes. Ultimately we focused on the HCV core protein since it is highly conserved and has been associated with several immunomodula- tory functions [26,27], including modulation of apoptosis signaling [28,29]. Huh7 cells were transfected with HCV genotype 1b core protein, as this represents the most common HCV genotype in North America and Western Europe, and patients with this virus have accelerated liver disease [30]. Cells expressing HCV core had increased TRAIL surface expression compared to control, as determined by flow cytometry (Fig. 3A and B) and western blot (Fig. 3D and E). A similar increase in TRAIL was seen following HIV infection using JFH1 HCV in Huh7.5.1 cells (Fig. 3C). The HCV core protein expression system is doxycycline responsive, however, doxycycline alone did not change TRAIL levels (Fig. 3F). The increase in TRAIL only occurred when HCV core protein was intracellularly expressed and not by extracellular soluble HCV core protein (Fig. 3G). Conversely, HCV core expression in Huh7 cells did not increase TRAIL-R2 expression (Fig. 3H), demon- strating that HIV and HCV exert complementary effects on the hepatocyte; HCV core protein increases TRAIL, but not TRAIL- Figure 1. HIV gp120 Increases TRAIL-R2. (A) Huh7 cells were incubated with increasing concentrations of HIV gp120 and analyzed for TRAIL- Receptor expression by Flow cytometry for TRAIL-R1, -R2, -R3, -R4 (BSA = black, HIV gp120 = Grey), (B and D) Western blot (C) or real time PCR. doi:10.1371/journal.pone.0027171.g001 PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27171