One of the hallmarks of HIV infection is the gradual elimination of the host's CD4+
T cells due to apoptosis. However, the mechanisms of HIV-induced apoptosis are complex and still controversial (1
). Several HIV-1 proteins have been attributed with apoptogenic properties, including Vpr (2
), Env, and Tat (1
). More recently, Vpu was reported to increase the sensitivity of HIV-infected cells to Fas killing (47
). However, the underlying mechanism remained unclear. In this study, we investigated in detail the apoptogenic properties of Vpu and we performed an in-depth analysis of the molecular mechanism. Our data suggest that Vpu, aside from Vpr, is one of the main inducers of apoptosis in HIV-infected cells and functions by inhibiting the NF-κB–dependent expression of antiapoptotic genes.
Both Vpr- and Vpu-induced apoptosis involve the activation of the caspase pathway (references 48
, and this study). Although the precise mechanism for Vpr-induced apoptosis is still unclear, recent observations suggest that it might be caused by a Vpr-induced permeabilization of mitochondrial membranes resulting in the release of apoptogenic proteins such as cytochrome c or apoptosis inducing factor and the subsequent activation of caspase (50
). While it was suggested that Vpu itself might have poreforming properties (51
) making a mechanism for induction of apoptosis similar to that of Vpr conceivable, our data suggest that Vpu instead functions by inhibiting the NF-κB–dependent expression of antiapoptotic genes. This is supported by the observation that mutation of the TrCP-binding motif (Ser52, 56
Asn), which in fact stabilized the pore-forming property of Vpu (52
), abolished its apoptogenic potential (). Based on the available experimental evidence, we therefore propose the following model for Vpu-induced apoptosis () : in unstimulated cells, NF-κB resides in the cytoplasm in an inactive complex with its inhibitor IκB (15
). Upon stimulation of cells by cytokines such as TNF-α ( no. 1), IκB is rapidly phosphorylated by an IκB-specific kinase ( no. 2), which results in the rapid degradation of IκB via a TrCP-dependent pathway ( no. 3). Infection of cells by HIV-1 results in the gradual intracellular accumulation of Vpu. Because of its constitutively active TrCP-binding motif and the fact that it is not sensitive to TrCP-mediated proteolysis, Vpu functions as a competitive inhibitor of TrCP. This results in the gradual accumulation of IκB and the progressive impairment of the cell's ability to activate NF-κB ( no. 4). The inhibition of NF-κB blocks the synthesis of antiapoptotic proteins such as the Bcl-2 family proteins (e.g., Bcl-xL and A1/Bfl-1) or TNFR complex proteins (e.g., TRAF1; no. 5). TRAF1 is induced by TNF-α treatment and normally inhibits activation of caspase-8 ( no. 6). In Vpu-expressing cells, the levels of TRAF1, in response to TNF stimulation, are reduced and no longer sufficient to inhibit the cytokine-induced activation of caspase-8 ( no. 6). Activated caspase-8 in turn induces the release of cytochrome c from the mitochondria ( no. 7). Release of cytochrome c is normally inhibited by the Bcl-2 family of proteins. However, in Vpu-expressing cells the levels of Bcl-2 proteins are limiting and no longer sufficient to block cytochrome c release ( no. 8). After its release from the mitochondria, cytochrome c forms ternary complexes with Apaf-1 and caspase-9 ( no. 9), resulting in the activation of caspase-3 ( no. 10). Active caspase-3 finally triggers a reaction that results in the cleavage of a number of target proteins including Bcl-2 family proteins ( no. 11) and leads to cell death ( no. 12).
Model for Vpu-induced apoptosis through activation of the caspase pathway. Details of the model are explained in the Discussion. Broken arrows symbolize inhibitory effects. Steps inhibited by Vpu are marked in red.
While our data clearly demonstrate the ability of Vpu to induce apoptosis in HIV-infected cells, its role in promoting apoptosis of uninfected bystander cells, which has been observed for CD4+
as well as CD8+
), remains to be addressed. The latter phenomenon is presumably a consequence of a continuous immune activation and could be due to exposure of these cells to secreted HIV proteins or to the disturbance of cytokine regulatory networks (55
). Most cytokines important for cellular and humoral immune response, including IL-2, IL-4, IL-10, IL-12, as well as TNF-α are transcriptionally regulated by NF-κB (18
) and it is therefore possible that Vpu expression during the course of HIV infection could affect their expression. Thus, even though Vpu is not a secretory protein and is unlikely to directly promote apoptosis of bystander cells, its expression in HIV-infected cells could nevertheless indirectly affect uninfected bystander cells through its possible effect on cytokine production. While it is tempting to speculate on a possible role of Vpu in restricting the cellular immune response to HIV infection through its ability to inhibit NF-κB–dependent gene expression, regulation of cytokine production in vivo is complex and influenced by a multitude of factors, which will make it difficult to assess the contribution of individual viral factors such as Vpu in vivo. Nevertheless, the noted reversion of a Vpu mutant in a monkey model and its correlation with disease progression (59
) attests to the importance of Vpu for virus replication in vivo.
Chemokines are another family of cellular proteins that is regulated by NF-κB and whose expression could thus be affected by Vpu. These include: regulated on activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, and MIP-1β (18
), which are secreted from CD4+
as well as CD8+
cells and act through their specific surface receptor CCR5 (60
). Endogenous expression of these chemokines was found to suppress HIV-1 replication in vitro (61
) and inhibited HIV replication, presumably through competition for the HIV coreceptor (62
). In fact, there appears to be a correlation between increased production of RANTES and resistance to HIV infection (63
) and, conversely, decreased production of RANTES and MIP-1α with disease progression (64
) in vivo. Thus, suppression of chemokine production by Vpu could provide a selective advantage to the virus and thus have a severe impact on disease progression.
HIV-2 infection is generally associated with a reduced rate of disease development as compared with HIV-1 (65
) and is characterized by an extended asymptomatic phase. Interestingly, lymphocytes from HIV-2–infected patients were found to be less susceptible to apoptosis than those derived from HIV-1–infected cells during the asymptomatic phase (66
). Therefore, it is tempting to speculate that the apoptogenic property of Vpu, for which there is no functional complement in HIV-2, contributes to the increased pathogenicity of HIV-1. In fact, there is some evidence from the macaque monkey model supporting the importance of vpu
in vivo. For example, when monkeys were infected with a vpu
-defective chimeric SHIV variant carrying an ATG to ACG mutation in the vpu
initiation codon, the vpu
gene was found to revert back to a functional open reading frame during the course of infection (59
), demonstrating the in vivo selective pressure for maintaining a functional vpu
gene. In addition, reversion of the vpu
open reading frame was correlated with disease progression in infected animals (59
) and expression of Vpu was associated with increased viremia (68
), demonstrating the importance of Vpu for viral replication and/or persistence in vivo and suggesting a role for Vpu in viral pathogenesis.
Despite the fact that HIV-1 encodes at least four proteins that promote apoptosis, it is difficult to envision a scenario in which the induction of apoptosis per se could provide a selective advantage for HIV-1. It appears that, in this respect, other primate lentiviruses have much better adapted to their hosts. In particular, simian IVs, which are endemic in their natural hosts, do not generally induce disease (69
). It seems therefore more plausible that the apoptogenic properties of HIV-1 proteins are unfortunate side effects of other important functions of these viral proteins. In the case of Vpu, it could be argued that its ability to induce rapid degradation of CD4 provides a selective advantage to HIV-1 by preventing the intracellular retention of Env in CD4/Env complexes (25
). Such complexes can form between de novo synthesized Env and CD4 proteins in the endoplasmic reticulum (70
). They are highly stable and unable to traffic to the cell surface (70
). The benefits of Vpu-mediated degradation of CD4 for HIV-1 are therefore twofold: (i) it releases Env from its intracellular trap and ensures its expression at the cell surface, and (ii) at the same time, Vpu prevents surface expression of CD4, which would interfere both with virus release (73
) as well as with the infectivity of the particles produced (74
). These functions of Vpu are particularly important for HIV-1 due to the affinity of its Env protein to CD4, which is significantly higher than HIV-2 Env (76
). The evolution of Vpu thus provides an intriguing example of how viruses redirect existing cellular mechanisms to their own advantage even if it is at the expense of their host.