*1.1. PI3K-AKT Pathway*

Viruses activate metabolic pathways in order to meet their needs for production of appropriate macromolecules. Once such pathway is the multifunctional phosphatidylinositol 3-kinases (PI3K)-AKT pathway. Due to its central importance in metabolism but also other cellular functions, this pathway is a common target for viruses [1–4]. In this review, we describe the activation of the PI3K-AKT pathway by alphaviruses and the consequent cellular effects.

PI3Ks are a large family of kinases that is divided in three classes: class 1 (1A and 1B), class II, and class III. The PI3K-AKT pathway involves class 1A PI3Ks. These PI3Ks are heterodimers with a catalytic subunit (p110) and a regulatory subunit (p85). Class 1A PI3Ks are activated by receptor tyrosine kinases (RTK) or G protein-coupled receptors (GPCR) after binding of growth factors, either directly or indirectly via activation of the small GTPase RAS [5] (Figure 1). Once the regulatory action of p85 has been relieved, active class 1A PI3Ks phosphorylate phosphatidylinositol-4,5-biphosphate (PIP2, or PtdIns(4,5)P2) to phosphatidylinositol-3,4,5-triphosphate (PIP3, or PtdIns(3,4,5)P3) at the plasma membrane (PM). PIP3-enriched membranes are a docking site for the PI3K-dependent kinase-1 (PDK1) and mTORC2 (or PDK2). Furthermore, the serine/threonine kinase AKT (or protein kinase B, PKB) is relocated to membrane sites with PIP3, where it can be activated via phosphorylation by PDK1 and mTORC2 [6,7]. A number of downstream targets of AKT have been identified, several of which are multifunctional nodes, integrating AKT signalling with signalling through other pathways [7]. One of these is the serine/threonine kinase mammalian target of rapamycin (mTOR) in the multi-protein complex mTORC1. AKT signalling leads to the activation of mTORC1, which promotes cell growth by

inducing lipid biogenesis through activation of the transcription factors SREBP1 and PPARγ and by promoting protein synthesis by activating the S6 kinase (S6K) and by inactivating the translational inhibitor 4E-BP1. mTORC1 also inhibits autophagy by blocking ULK1 [6]. The serine/threonine kinase glycogen synthase kinase 3 (GSK3) is another multifunctional target of AKT. Through phosphorylation, active GSK3 inhibits most of its substrates. Upon phosphorylation by AKT, GSK3 itself is inhibited and thereby the downstream targets are positively regulated. These targets include the prosurvival BCL-2 family member MCL-1 and the transcription factor c-Myc, which is required for expression of many genes involved in proliferation. Other GSK3-targets, such as glycogen synthase, are involved in (regulation of) cellular metabolism [7]. The third multifunctional target of AKT are the forkhead box O (FoxO) transcription factors. Phosphorylation of FoxO transcription factors by AKT leads to acute translocation out of the nucleus. Thus, AKT signalling suppresses the expression of FoxO targets. These include targets involved in the induction of apoptosis, cell-cycle arrest, catabolic metabolism and growth inhibition. Thus, PI3K-AKT signalling via these multifunctional targets promotes cell survival, growth and proliferation and steers cellular metabolism towards anabolism.

**Figure 1.** Activation of PI3K-AKT pathway by external signals via G-protein coupled receptors (GPCR) or receptor tyrosine kinases (RTK) leads to dissociation of the p85 regulatory subunit from the active p110 PI3K subunit. The active subunit catalyses the conversion of PIP2 to PIP3 at the plasma membrane, leading to the recruitment and activation of the AKT kinase. Via multiple downstream effector pathways, cellular states of growth, proliferation, heightened metabolic activity and survival are promoted. For more details, see text.

The PI3K-AKT pathway is regulated in many ways. The RAF-MEK-ERK pathway also promotes cell survival and growth and the two pathways have co-regulated proteins and negatively regulate each other. For example, MEK promotes membrane localisation of the phosphatase and tensin homologue (PTEN), where it dephosphorylates PIP3 and inhibits AKT activation [6]. Also, post-translational modifications (including (de)phosphorylation and acetylation) of AKT play important roles in regulation. An important immediate negative feedback loop is provided by mTORC1. Through a variety of downstream targets of mTORC1, AKT signalling is inhibited [7].
