Date Published: March 5, 2012
Publisher: Hindawi Publishing Corporation
Author(s): Lisa N. McKernan, David Momjian, Joseph Kulkosky.
An effective means to eradicate latent reservoirs in HIV-1-infected individuals remains elusive. Attempts to purge these reservoirs were undertaken over a decade ago without success. The subsequent lapse in further clinical attempts since may have been justified as our knowledge of the mechanisms which underpin the latent state still evolves. Although additional novel molecular antagonists of HIV-1 latency have subsequently been reported, these candidate agents have not been tested in human trials for reservoir ablation. This review provides an overview of the protein kinase C (PKC) pathway which can be modulated by small molecular agents to induce the expression of latent HIV-1 from within infected reservoir cells. Some of these agents have been tested against select cancers with seemingly tolerable side effects. As such, modulation of the PKC pathway may yet be a viable mechanism toward HIV-1 reservoir eradication.
Administration of highly active anti-retroviral therapy (HAART) to HIV-1-infected individuals results in effective suppression of viral replication in metabolically active cells bearing integrated viral DNA. However, a small population of infected cells is refractive to HAART treatment as a consequence of being quiescent and/or not actively expressing virus products [1–5]. This small population of cells, comprised largely of infected CD4+ resting T cells, constitutes the HAART-persistent latent reservoir. Most cells in this silent reservoir have long half lives [6, 7] and are hidden from immune surveillance which permits them to remain as a stable source for de novo viral production upon reactivation. One strategy for eradication of this reservoir rests upon the premise that cellular activation with concomitant upregulation of viral expression will hasten its elimination [8–11]. Cellular activation typically shortens the half-life of a cell relative to its quiescent counterpart, and a cell, actively expressing viral antigens, would be a more favorable target for immune clearance .
The protein kinase C (PKC) signal cascade is comprised of, and regulated by, several isoforms [31, 32]. Each isoform exhibits select characteristics as well as varying patterns of expression in specific cell types. The PKC cascade can affect receptor upregulation or downregulation, membrane and cytoskeleton remodeling, and positive or negative regulation of transcription to mediate specific processes within the cell. These activities can have global effects on cellular functions, in particular, growth, attachment, differentiation, maturation, and death [31, 32]. These varied functions are typically mediated by PKC phosphorylation of serine and threonine residues of downstream signaling factors [33, 34]. These phosphorylated factors then serve as intermediaries in the transduction of signals to various cellular locations in order to accomplish specific effector functions.
PKC pathway activation involves the participation of the phospholipase C (PLC) superfamily of proteins for most natural cellular processes. PLCs participate in phosphatidylinositol-4,5-bisphosphate (PIP2) metabolism and lipid signaling pathways in a calcium-dependent manner. Similar to the PKC pathway, the PLC superfamily consists of many isoforms which differ in their mode of activation, expression levels, catalytic regulation, cellular localization, and membrane binding affinity . All are capable of catalyzing the hydrolysis of PIP2 into two important second messenger molecules: diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). These two second messengers have differential cellular effects. IP3 molecules diffuse through the cytoplasm and bind to the endoplasmic reticulum (ER) resulting in the opening of calcium channels [31–34]. The released calcium from the ER into the cytoplasm is free to bind important regulatory proteins including but not restricted to calmodulin and calcineurin. The binding of calcium to calmodulin mediates critical organismal processes such as inflammation, metabolism, apoptosis, smooth muscle contraction, intracellular movement, short-term and long-term memory, nerve growth, and the immune response [35–37].
All PKC isoforms consist of a regulatory domain tethered to a catalytic domain [31, 32]. There are two primary classes of PKC protein isoforms. Calcium-dependent classical PKC isoforms (cPKCs), require calcium for their activity, while calcium-independent isoforms (nPKCs) do not. The cPKCs (α, βI, βII, and γ) are calcium and DAG-dependent, whereas the novel PKCs (δ, ε, η, and θ) are calcium-independent but DAG-responsive. A third class of PCKs is referred to as atypical includes the isoforms ξ and λ/l that uniquely lack responses to calcium and DAG [31, 32, 38].
The non-tumor-promoting deoxyphorbol esters which activate PKC emerged as candidates for HIV-1 latent reservoir eradication from studies initially performed at the National Cancer Institute (NCI). NCI investigated antiviral properties of several ethnobotanical compounds including the novel phorbol ester, prostratin. Prostratin had first been identified as a constituent of the poisonous New Zealand plant Pimelea prostrata . As observed by the ethnobotanist, Paul Cox, prostratin was later detected in bark extracts from the plant, Homalanthus nutans, used by native island tribesmen in Samoa as a remedy for jaundice . On this basis it was speculated to have antiviral properties. Initial investigations of the purified compound by NCI revealed that prostratin inhibited infectious viral spread but readily upregulated latent HIV-1 from the quiescently infected cell lines, ACH2 and U1 [44, 45]. Prior to the introduction of HAART, there was little interest in further investigating an agent that would produce additional virus from within the cells of HIV-1-infected patients.
There have been ongoing concerns regarding the clinical use of PKC modulators for HIV-1 reservoir eradication, particularly the phorbol ester family of compounds. As previously stated, phorbol esters activate PKC in multiple cell types raising the issue that systemic effects in treated patients would be broader than necessary given the limited cell types in the latent reservoir necessary to be targeted for eradication.
It has been observed that structural variants among the phorbol ester family of agents differ in their potency to upregulate latent viral expression which likely relate to differential PKC isoform affinities for effector targets [20, 40, 59]. In vitro concentrations of phorbol esters, required for the upregulation of latent virus in patient primary cells, are in the range of 0.1 μM to 10 μM as single treatment agents [20, 45, 46]. The mechanistic basis for the differences in effective concentrations of agents relates to variations in their chemical structure. As shown in Figure 4, both PMA and DPP have extended hydrophobic side groups which are not present in the base structure of prostratin. These hydrophobic entities exposed outward from the PKC/phorbol complex, likely associate with and retain the complexes at the plasma membrane resulting in sustained PKC action. Such sustained action can accentuate select processes including tumor promotion or apoptosis which are not typical of prostratin treatment relative to PMA and DPP at similar concentrations. On the basis of these observations, it was proposed that the ancillary toxicities of PKC activators could be minimized by altering select reactive groups to present the most favorable clinical index.
The ongoing efforts to formulate new lead compounds, to upregulate latent HIV-1 through PKC modulation which bear minimal clinical side effects, appears to be a rational approach toward the eradication of latent HIV reservoirs. Differential PKC isoform targeting may be important since those isotypes that modulate PKC downstream of PKC α, exhibit fewer deleterious cellular and tissue side effects. It is unlikely that one singular therapeutic approach will achieve complete reservoir ablation as multiple molecular mechanisms can elicit or maintain HIV-1 latency . The lessons from HAART usage indicate that some form of combination therapy with reservoir eradication agents, as well as multiple dosing, could be of utility. Nonetheless, treatments, which markedly accelerate the process of reservoir decay, should be regarded as a significant advancement toward the goal of curing HIV-1 infection, as well as compelling, given the considerable expense and notable side effects associated with the prolonged administration of HAART.