Our proteomic analysis demonstrates that infection of the T-cell line PM1 by HIV-1 gives rise to coordinated changes in protein expression patterns, together representing reprogramming of the T cell. In absolute amounts, these changes are less strong than one might have naively expected. However, this result is in agreement with microarray analyses and a recent proteomic analysis of HIV T-cell interactions (9
). Such relatively subtle changes in protein levels can be reliably identified by our approach using multiple independent replicates, and this analysis becomes even more convincing given the concerted changes of many proteins within certain metabolic pathways. Metabolic rerouting as shown in Fig. 5
is apparent, as well as a “tug of war” between host cell and virus with regard to apoptotic signals.
We observed upregulation of proteins involved in fatty acid oxidation and amino acid catabolism, the TCA cycle, and oxidative phosphorylation. Thus, enzymes involved in the generation of ATP from fatty acids (e.g., the beta oxidation trifunctional enzyme alpha and beta subunits) and amino acids are strongly upregulated, which would lead to increased energy production. In striking contrast, all the differentially regulated proteins that catalyze steps in glycolysis are downregulated: glucose-6-phosphate isomerase, aldolase A, phosphoglycerate mutase 1, and triosephosphate isomerase. HIV apparently induces a metabolic change in the infected cells to decrease energy production from glucose but increases the energy production from amino acids and fatty acids (Tables 1
). That this rerouting represents a highly specific metabolic program is clear not only from the downregulation of glycolytic enzymes but also from the downregulation of E-FABP, the immune cell-specific fatty acid transport protein that is consistently upregulated in activated immune cells (20
). Apart from the specific downregulation of the glycolytic enzymes, our results agree with the very recent proteomic studies of Chan et al. (9
). They describe one enzyme that is (also) involved in glycolysis (pyruvate kinase), which is upregulated. Although they used a totally different technique, similar effects with regard to oxidative phosphorylation, the TCA cycle, and valine, leucine, and isoleucine degradation were observed. However, their findings appear less consistent: indications for a “dysregulated” TCA cycle are presented, and some proteins involved in respiration (such as cytochrome c
oxidase II) are downregulated.
Several studies have shown effects of HIV on cell metabolism. An effect of HIV on glucose metabolism of human intestinal epithelial cells was measured (32
). HIV infection resulted in a disturbance of glycolytic and oxidative activities. The results indicated an increase in intracellular glucose concentration due to either increased uptake or decreased glycolysis. Glycolytic impairment was detected in neuronal cells upon incubation with HIV gp120 protein only (53
). How could “metabolic rerouting” by the virus be induced at the molecular level? It is known that the retroviral Vpr protein interferes with the suppressive effects of insulin on FOXO transcription factors, which normally stimulate gluconeogenesis by upregulating glucose-6-phosphatase and phosphoenolpyruvate carboxykinase (26
). FOXO transcription factors are negatively regulated through phosphorylation by the protein kinase Akt in response to insulin and growth factors and subsequent relocalization from the nucleus into the cytoplasm via interaction with 14-3-3 proteins (25
). Vpr is known to interact with 14-3-3 proteins and thus influence their binding specificities. FOXO factors could thus be positively regulated by Vpr, which binds 14-3-3 proteins, allowing FOXO to go to the nucleus. Another way to interfere with nuclear relocalization of certain transcription factors would be downregulation of the 14-3-3 proteins themselves. We indeed find the 14-3-3 gamma, epsilon, tau, and zeta proteins to be significantly (all about 1.6-fold) downregulated. The 14-3-3 pathway is also found to be strongly influenced upon HIV-1 infection (9
). Besides their role in glucose metabolism, FOXO transcription factors are involved in cell cycle arrest (as are 14-3-3 proteins, mediating Vpr's cell cycle-arresting activity, e.g., by enhanced binding of Cdc25C [25
]), DNA repair, apoptosis, and stress resistance. For instance, FOXO factors stimulate Mn superoxide dismutase transcription, and we indeed find it to be upregulated after HIV infection (as would be appropriate given the increase in mt oxidation). In muscle cells, repression of FOXO activity by Akt signaling induces a decrease in protein degradation. Thus, a Vpr-mediated indirect increase in FOXO activity could lead to enhanced protein degradation, generating substrates for the increased amino acid catabolism that we seem to observe upon HIV infection.
The role of FOXO transcription factors in mediating cell cycle arrest, DNA repair, apoptosis, and stress resistance mimics the functions of the tumor suppressor protein p53, and FOXO and p53 seem to be part of a complex regulatory network (19
). HIV infection indeed activates p53 (15
). Recently, p53 has also been shown to regulate glucose metabolism and autophagy via TIGAR (4
). An important role of TIGAR is to redirect glucose from catabolism (energy production) to anabolism (including synthesis of nucleotides) by blocking glycolysis and activating the pentose phosphate shunt (18
). The observed downregulation of glycolysis, leading to an increase in glucose available for ribose precursors, should induce the pentose phosphate pathway. This would lead to a concomitant increase in NADPH, which can also be used for the synthesis of new viral particles and which is needed for the stress response. Indeed, we find a twofold upregulation of the purine nucleoside phosphorylase (also called inosine phosphorylase), a central player in nucleoside metabolism of blood cells, inactivation of which results in severe combined immunodeficiency (37
). In this context, the slight upregulation of UDPglucose pyrophosphorylase, which plays a central role as a glucosyl donor, should also be mentioned. This is in agreement with studies of HIV infection of human intestinal epithelial cells (32
), where fructose-1,6-phosphate was found to be decreased. Apart from these possibly indirect p53 effects on metabolism, p53 also downregulates stathmin (the central regulator of proliferative activation of microtubules), which is strongly reduced (2.3-fold) upon HIV infection, in agreement with p53-mediated cell cycle arrest.
The activation of p53 might again be mediated by Vpr. p53 has been shown to be activated by Vpr (16
). This is possibly a consequence of Vpr-induced DNA double-strand breaks activating ataxia telangiectasia mutated protein-dependent signals such as p53 (36
). HIV-1-induced p53 signaling is also in agreement with our observations concerning RuvBL1 and RuvBL2, which are part of a large nuclear protein complex containing TIP60. This multifunctional complex can act as a transcriptional coregulator for several factors, including p53 (44
). It can be recruited to double-strand breaks and activate ataxia telangiectasia mutated protein by acetylation and, like FOXO and p53, is also involved in cell cycle regulation and apoptosis (44
). The HIV Tat protein, however, is known to suppress TIP60's apoptotic function, either via inhibition of its acetylation activity or by TIP60 degradation via polyubiquitination (44
). The upregulated RuvBL1 and downregulated RuvBL2 proteins (ATP-dependent DNA helicases with 3′-to-5′ and 5′-to-3′ activity, respectively) indeed play antagonistic roles, thus underlining the coordinated changes that we observe (24
Many HIV-1 effects could be mediated by Vpr through FOXO, p53, and TIP60. These three all share regulation by the p300/CBP nuclear coactivator (19
), which contains acetyltransferase and ubiquitin ligase activity (41
). p300/CBP is targeted by several viruses, and both HIV Vpr and Tat can bind and influence this coactivator (7
hnRNPs and stress responses.
The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of proteins sharing common structural domains, which seem to have roles in DNA repair, telomere biogenesis, cell signaling, RNA export and splicing, and regulation of gene expression. We find hnRNPs K and A1 to be downregulated. The hnRNP A1 protein regulates HIV-1 Tat mRNA splicing and seems to be involved in HIV-1 mRNA export from the nucleus (49
). However, hnRNP H, which is also involved in HIV-1 mRNA splicing (6
), is upregulated. Another “anomaly” is obtained with the regulation of protein breakdown, in which we find the general ubiquitin-activating enzyme E1 to be downregulated, whereas the proteasome beta type 4 subunit is upregulated nearly fourfold. Its regulatory subunit 13 (PSMD13) is also increased. The latter two proteins are involved in the HIV-1 Vpu-mediated degradation of CD4 (33
Proteins involved in stress responses are also strongly influenced by HIV infection and virus replication. The first one detected, in the “single” 42-h infection, is the cytoplasmic beta subunit of HSP90. Not surprisingly, many chaperonins (mostly ER located) are upregulated in the peak infection, including GRP 78 (BIP), ERp44, GRP 94 (endoplasmin), ERp 29 and mt HSP60. Many ER protein disulfide isomerases (such as A3, A4, and A6) are also upregulated, as are the alpha subunit of glucosidase II, heme oxygenase 2, and the 150-kDa hypoxia-upregulated protein 1 (HYOU1). The last two could be upregulated because of the increased mt oxidation of fatty acids and amino acids described above. This could explain the upregulation of the mt [Mn] superoxide dismutase as well. Interestingly, hypoxia can signal via Akt (already mentioned above as the protein kinase that can negatively regulate FOXO transcription factors; see also Fig. 5
), and indications for the activation of this pathway have been found as well (9
). Surprisingly, AHSA1, the 38-kDa activator of the ER-located 90-kDa heat shock protein, is slightly downregulated. It is known to interact with the cytoplasmic tail of the vesicular stomatitis virus glycoprotein (45
), but interactions with HIV proteins have not been found so far. However, such interactions have been documented for two of our upregulated ER stress proteins, endoplasmin and cyclophilin B (a peptidyl-prolyl cis-trans
-isomerase). Endoplasmin, which can also be induced by hypoxia, seems to be bound by the virion (10
), although the specific interaction is still unknown. Cyclophilins interact with Vpr; although this has been shown much more convincingly for cyclophilin A (21
). These interactions probably are important for virus routing and packaging, as are the interactions of the virus with cytoskeletal (associated) proteins.
Our observations confirm known interactions with HIV-1 proteins in the case of p21-activated kinase 2, which is bound by Nef to inhibit Bad-mediated apoptotic death and increase virus production (30
), and of eIF-5A, the eukaryotic translation initiation factor bound by Rev, which is involved in the nuclear export of Rev and HIV-1 replication (5
). Both p21-activated kinase 2 and eIF-5A are downregulated.
More than 30 mt proteins are differentially expressed upon HIV-1 infection, and all of these are upregulated. We do not think this stems from a systematic artifact, as we find an upregulation not of all mt proteins but of only a small subclass and because the magnitude of upregulation observed varies among proteins. Of these upregulated mt proteins, two remain to be mentioned: the glycoprotein gC1qBP/P33, of unknown function, which can interact with rubella virus capsid protein, and SSB, a single-stranded DNA binding protein (which is known to interact with viral proteins). The production of HIV proteins is seen more strongly (CA-p24 is more upregulated and Nef can also be detected) after 42 h (see Table S2 in the supplemental material). The six upregulated cellular proteins at 42 h are all also detected at peak infection, but the downregulated DDX3X was detected only in this experiment. Recent studies showed that DEAD box protein RNA helicases, such as DDX3 and DDX1, are important for HIV infection by facilitating the export of singly spliced or even unspliced HIV RNAs from the nucleus via the CRM1-Rev pathway. Of the DEAD box protein family, DDX3 showed the strongest mRNA downregulation upon HIV-1 replication (at 24 h after induction of replication), in agreement with our observation at the protein level (29
To study the T-cell-virus interaction, we used transformed PM1 cells cultured in vitro, which likely differ in several ways from primary T cells found in vivo. It is currently not clear how the observed PM1 changes compare to cellular changes in vivo or whether direct cytopathicity of HIV-1 is enhanced in vitro, although large-scale apoptosis was not observed. How the observed changes relate to pathogenicity for the infected individual is also unclear. Though superficially the cellular proteome seems relatively stable, PM1 cells are reprogrammed upon HIV-1 infection. This reprogramming begins early on in infection and has functions, among others, at the level of general metabolism, cytoskeletal organization, and primary suppression of apoptotic responses. Many of these processes are known to be affected by the viral Vpr protein, subverting cellular pathways involved in their regulation at many different levels (Fig. 5
). Our DIGE analysis is, for the most part, in agreement with the recent findings (9
) in a HIV-T-cell interaction study using LC-MS/MS. There are even many instances in which the findings perfectly complement each other (e.g., upregulation of components of the TCA cycle or the pyruvate dehydrogenase complex in both studies, but with different sets of proteins). There are three notable differences. We observed downregulation of glycolytic enzymes, upregulation of proteins involved in fatty acid breakdown (whereas the previous study [9
], e.g., found a strong increase in fatty acid synthase), and a much more consistent increase in TCA cycle enzymes. Possibly, these variations result from differences in the cell types used, infection procedures, and harvest times. More likely the differences are due to the different methods used, with LC-MS/MS yielding more candidate proteins but at the cost of more false positives. This first DIGE analysis of changes in the T-cell proteome upon HIV-1 infection not only uncovered specific reprogramming of T cells but also allowed us to identify (and verify) many proteins potentially involved in the virus-host interactions. Upon verification of these new cofactors of HIV-1 replication, they may become future drug targets.