We obtained 2D profiles of the protein expression in PBMCs of HIV-1/HCV mono- and co-infected patients and uninfected controls. Proteins related to protein synthesis, the ER, oxidative stress, the cytoskeleton, metabolism, and glycolysis are differentially and uniquely expressed in PBMCs of HIV-1/HCV mono- and co-infected patients. In general, several up-regulated proteins showed overlap between the HCV mono-infected and HIV-1/HCV co-infected samples, and only two up-regulated proteins, gelsolin and CBP2, were shared by HIV-1 mono-infected and HIV-1/HCV co-infected patient samples. In contrast to the results observed in control cells, a high level expression of CBP2 occurred in infected samples in agreement with previous studies showing that HIV and HCV promote expression of collagen
]. An anti-apoptotic signature through the up-regulation of GST, gelsolin and Ran was detected on HIV-infected cells. The down-regulation of Rho-GDI (known to lead to an apoptotic response) was observed on HCV infections. Gelsolin and Rho-GDI, key proteins that promote cell prosurvival mechanism, were up-regulated in co-infected PBMCs. These identified proteins have diverse cellular functions that will require further in-depth studies to understand the protective and apoptotic immune-mediated mechanisms triggered by HIV-1 and HCV. Figure
encompass the HIV-1, HCV and HIV-1/HCV induced differentially expressed proteins detected in this study. In the model, we propose that HIV and HIV-1/HCV induce a pro-survival anti-apoptotic mechanism, to protect the cell from the pathogenic interaction “cross-talk” of the two viruses, while HCV seem to trigger a more apoptotic protein signature.
Figure 6 Color reproduction on the Web. Proposed model highlighting specific protein signatures triggered by HIV-1 (A), HCV (B) and HIV-1/HCV (C) in peripheral blood mononuclear cells. ROS, reactive oxygen species; RS, reactive substances; GST, Glutathione S-transferase. (more ...) HIV up-regulated, CBP2, GST, Ran and Gelsolin.
CBP2 (also known as HSP47) is an ER resident protein involved in collagen synthesis. The observed up-regulation of CBP2 correlates with previous studies demonstrating significant increases in collagen deposition in HIV-1-infected tissue samples
]. GST constitutes the major intracellular antioxidant defense against reactive substances (RS) and oxidative stress. Moreover, the safe elimination of toxins via
GST pathways was shown to protect cellular DNA against reactive oxygen species (ROS)-induced damage
]. The up-regulation of GST in AIDS patients could have significant effect on HCV chronicity. Ran is a suppressor of Bcl-2-associated X protein (Bax), and a pro-apoptotic marker. HIV also induced the up-regulation of gelsolin that has been shown to inhibit HIV-induced T-cell apoptosis
HIV-1 down-regulated the expression of tropomyosin, profilin-1, and Rho-GDI.
Tropomyosins forms coiled-coil dimers that assemble end-to-end along an actin filament
] and provide an excellent example of spatial, temporal, and functional diversity
]. Their observed down-regulation is consistent with the fact that the actin network is involved in HIV-1-induced host cell apoptosis
]. Profilin-1 is an actin-binding protein thought to be a key regulator of actin polymerization in cells
]. Profilin-1 has been shown to have an impact on HIV-infected macrophages
]. Cytoskeletal proteins including profilin-1 have been shown to bind to HSP70 and to elicit resistance to simian immunodeficiency virus (SIV) infection of CD4+
]. Previously, an impairment in the production of reactive oxygen intermediates has been demonstrated during HIV infection
] and our finding of reduced Rho-GDI is in agreement with this observation. Rho-GDI has also been implicated in mediating HIV-infected cell migration through tight junctions
] and was shown to be under-expressed in HIV-1-resistant women
]. The mechanism by which these proteins are down-regulated remains to be investigated, but the up-regulation of mRNA (but not protein) for Rho-GDI and profilin-1 during HIV-1 infection could indicate impairment in protein translation induced by the virus. The possible interaction among these proteins during HIV-1 infection is represented in Figure
HCV up-regulated tropomyosin, profilin, ER ATPase, HSP90-beta, thioredoxin, a protein highly similar to CBP2, DPYSL3, DPYSL4 and EIF3K.
The up-regulation of a protein similar to CBP2 is in agreement with previous findings that HCV promotes expression of collagen
]. Since CBP2 was also up-regulated in HIV-infected patients, it highlights a possible common molecular mechanism between these two viral infections. DPYSL3 and DPYSL4 are involved in semaphorin signaling and in subsequent rearrangement of the cytoskeleton
]. EIF3K has been shown to be involved in apoptosis regulation by interacting with the subsequent liberation of caspase 3 into the cytosol
]. After HCV infection, PBMC generate the over-expression of thioredoxin, inducing high levels of ROS that results in the down-regulation of GST (Figure
B). In the cytoplasm, the liberated caspase 3 is known to cleave the Rho-GDI-Rac-GDP complex resulting in Rac activation, which is the first reaction for the cell apoptotic function
HCV down-regulated profilin-1 and Rho-GDI, as observed in HIV-1 infections.
GST and GTPase Ran were also down-regulated in HCV-infected samples. The down-regulation of Rho-GDI may represent a pro-survival mechanism for both HIV-1 and HCV by reducing the activation of NADPH. It seems that during HCV infection, the over expression of thioredoxin triggers oxidative stress inducing high levels of ROS that result in the down-regulation of pro-apoptotic GST. The up-regulated caspase 3 cleaves the Rho-GDI-Rac-GDP complex resulting in Rac activation, which is the first step that leads to apoptosis
]. The relevance of other down-regulated proteins with respect to HCV infection remains to be established. Figure
B represent the possible contribution of observed proteins in HCV pathogenesis.
HCV and HIV co-infection up-regulated CPB2 and the levels of expression of tropomyosin, profilin, and Rho-GDI as well as the pro-apoptotic proteins Annexin 5, NACHT, and TCP1. After the co-infection with HIV-1/HCV, oxidative stress could trigger the up-regulation of GST. In the cytoplasm, the up-regulated Rho-GDI is known to cause an anti-apoptotic signaling by inhibiting caspase 3 (Figure
HCV and HIV co-infection down-regulated tropomyosin alpha-4 isoform 1, pyruvate kinase isozyme M1/M2, an ER ATPase, GST, EIF3K, and GTPase Ran
of which the first three were down-regulated in HIV-1 but not in HCV-infected samples. Considering this, we postulate that their down-regulation is related to HIV-1 infection. Conversely, the down-regulation of GST and GTPase Ran might be due to the HCV infection. In addition, we observed that the protein, ER ATPase, a protein that is involved in ER-associated degradation pathway
], was down-regulated in HIV-1 mono- and HIV-1/HCV co-infected samples but up-regulated during HCV mono-infection, indicating an inflammatory immune response in HCV infection and immune suppression in HIV-1 infection. More importantly, it appears that there is a divergence between immune activation and immune suppression during co-infection, which would explain the diversity of liver disease in the co-infected patients. Overall, interplay of identified proteins with relevance to HIV-1/HCV co-infection is presented in Figure
The proteins unique to HIV-1/HCV co-infection included pro-inflammatory, apoptosis and immune response related proteins.
For example, NLRP13 (NACHT, LRR, and PYD domains-containing protein 13) or NALPs are implicated in the activation of pro-inflammatory caspases (e.g., CASP1; MIM 147678) via their involvement in multi-protein complexes called inflammasomes
]. Likewise, the NALP3 inflammasome is a crucial element in the adjuvant effect of aluminum and can direct a humoral adaptive immune response
]. Annexin A5 (ANXA5) has been shown to be an important modulator of the immune response by interfering with the immunosuppressive effects of apoptotic and necrotic cells and certain viruses by preferentially binding phosphatidylserine with high affinity and inhibiting the uptake of these particles by macrophages
], leading to defects in the clearance process that may cause chronic immune activity. Among the immune-related proteins identified only in the co-infected samples, TCP-1 (T-complex protein 1) and 60S acidic ribosomal protein P0 (RPL10) are worth mentioning. TCP-1, a molecular chaperone, assists the folding of proteins upon ATP hydrolysis and is known to play a role in the folding of actin and tubulin
]. Activity-based proteome profiling of hepatoma cells using protease substrate probes identified TCP-1 as one of the few proteins differentially expressed during HCV replication
], indicating the involvement of this chaperone in containing catalytic enzyme in HCV infection. Similarly, TCP-1 is differentially expressed in HCV polyprotein expressing cells
]. P0 (60S acidic ribosomal protein) is a protein that is involved in host-virus interactions and has been shown to be up-regulated in HBV- and HCV-associated HCC and fibrosis progression
]. These studies correlate with our findings and suggest that the co-infected PBMC protein profile is dominated by the proteins modulated primarily by HCV infection and that the similarity in the liver disease abnormalities in this patient population is due to the presence of HCV co-infection. The majority of the unique proteins identified were found in either HCV mono-infected or HIV-1/HCV co-infected samples. However, HSPA5, peptidyl-prolyl cis-trans isomerase A, and highly similar to phosphoglycerate kinase 1 proteins were not detected in infected PBMCs as compared to un-infected controls.
Overall, our findings revealed that the majority of the differentially modulated proteins overlap between HCV mono- and HIV-1/HCV co-infected PBMCs. It seems, that the liver disease progression is primarily HCV related and that HIV-1 co-infection may amplify the effects of stress and inflammation response prior to liver fibrosis, cirrhosis, and eventually carcinogenesis. In addition, the identified changes highlight protein signatures possibly involved in pro-inflammatory response and immune suppression that are highly implicated in progression of liver disease in chronic HCV infection and could potentially be used as drug targets. The specific pro and anti-apoptotic protein signatures revealed in this study could facilitate the understanding of protective immune-mediated mechanisms underlying HIV-1 and HCV co-infection. Accordingly, detailed analysis of the functional role of novel candidate molecular targets identified in this study would extend our understanding of the pathogenic effects of HIV, HCV and HIV/HCV co-infection and, in future, enable more specific and concurrent targeting of multiple key molecular pathways leading to better treatments.