Activation of the PI3K/Akt Pathway Early during Vaccinia and Cowpox Virus Infections Is Required for both Host Survival and Viral Replication

Viral manipulation of the transduction pathways associated with key cellular functions such as actin remodeling, microtubule stabilization, and survival may favor a productive viral infection. Here we show that consistent with the vaccinia virus (VACV) and cowpox virus (CPXV) requirement for cytoskeleton alterations early during the infection cycle, PBK/Akt was phosphorylated at S473 Akt(S473-P)], a modification associated with the mammalian target of rapamycin complex 2 (mTORC2), which was paralleled by phosphorylation at T308 Akt(T308-P)] by PI3K/PDK1, which is required for host survival. Notably, while VACV stimulated Akt(S473-P/T308-P) at early (1 h postinfection p.i.]) and late (24 h p.i.) times during the infective cycle, CPXV stimulated Akt at early times only. Pharmacological and genetic inhibition of PI3K ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) or Akt (Akt-X and a dominant-negative form of Akt-K179M) resulted in a significant decline in virus yield (from 80% to ≥90%). This decline was secondary to the inhibition of late viral gene expression, which in turn led to an arrest of virion morphogenesis at the immature-virion stage of the viral growth cycle. Furthermore, the cleavage of both caspase-3 and poly(ADP-ribose) polymerase and terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling assays confirmed that permissive, spontaneously immortalized cells such as A31 cells and mouse embryonic fibroblasts (MEFs) underwent apoptosis upon orthopoxvirus infection plus {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 treatment. Thus, in A31 cells and MEFs, early viral receptor-mediated signals transmitted via the PI3K/Akt pathway are required and precede the expression of viral antiapoptotic genes. Additionally, the inhibition of these signals resulted in the apoptosis of the infected cells and a significant decline in viral titers.The family Poxviridae is a family of large, linear, double-stranded DNA viruses that carry out their entire life cycle within the cytoplasmic compartment of infected cells. Vaccinia virus (VACV) is a prototypical member of the genus Orthopoxvirus, which also includes the closely related cowpox virus (CPXV) (12, 52). The genomes of these viruses are approximately 200 kbp in length, with a coding capacity of approximately 200 genes. The genes involved in virus-host interactions are situated at both ends of the genome and are associated with the evasion of host immune defenses (1). These evasion mechanisms operate mainly extracellularly. For example, the secretion of soluble cytokine and chemokine receptor homologues blocks the receptor recognition by intercepting the cognate cytokine/chemokine in the extracellular environment. This mechanism facilitates viral attachment and entry into cells (1, 70). Therefore, decoy receptors for alpha interferon (IFN-α), IFN-β, IFN-γ, and tumor necrosis factor alpha play an important immunomodulatory role by affecting both the host antiviral and apoptotic responses.To counteract the host proapoptotic response, poxviruses have developed a number of antiapoptotic strategies, including the inhibition of apoptotic signals triggered by the extrinsic pathway (those mediated by death receptors such as tumor necrosis factor and Fas ligand) or the intrinsic pathway (mediated by the mitochondria and triggered upon viral infection) (1, 25, 70, 74). Many studies previously identified viral inhibitors that block specific steps of the intrinsic pathway. These include the VACV-encoded E3L, F1L, and N1L genes and the myxoma virus (MYXV)-encoded M11L gene, which block cytochrome c release (14, 20, 34, 39, 45, 75, 90), and the CPXV-encoded cytokine response modifier gene (CrmA) as well as the VACV-encoded SPI-2 gene, which inhibits both caspase-1 and caspase-8 (25, 58, 61, 74).An emerging body of evidence has also highlighted the pivotal role played by intracellular signaling pathways in Orthopoxvirus biology (18, 48, 92). We and others have shown that poxvirus manipulation of signaling pathways can be virus specific. For example, while both VACV and CPXV stimulate the MEK/extracellular signal-regulated kinase (ERK)/EGR-1 pathway during a substantial length of time of their infective cycle, the pathway is required only for VACV replication, whereas its role in CPXV biology has yet to be identified (71). MYXV, a rabbit-specific poxvirus, also activates the MEK/ERK pathway in a mouse model of poxvirus-host interactions. However, this stimulation led to the expression of IFN-β, which consequently blocked virus replication and possibly explains why MYXV has such a restricted host range (87).Another signaling molecule associated with viral replication is Akt kinase (also known as protein kinase B). The MYXV host range factor M-T5 is able to reprogram the intracellular environment, thereby increasing human tumor cell permissiveness to viral replication, which is directly associated with levels of phosphorylated Akt (88). In addition, M-T5 is functionally replaced by the host phosphatidylinositol 3-kinase (PI3K) enhancer A protein (92).The transmission of intracellular signals mediated by the serine/threonine kinase Akt to downstream molecules in response to diverse stimuli such as growth factors, insulin, and hormones is dependent upon the phosphorylation of serine 473 (S473-P) and threonine 308 (T308-P). This phosphorylation is mediated by mammalian target of rapamycin complex 2 (mTORC2) and phosphoinositide-dependent protein kinase 1 (PDK1), which act as downstream effectors of the PI3K/Akt/mTORC1 pathway (2, 66). PI3Ks are a family of enzymes (classes I to III) that generate lipid second messengers by the phosphorylation of plasma membrane phosphoinositides. Class IA PI3Ks consist of a catalytic subunit (p110, comprising the three isoforms α, β, and δ) and an adaptor/regulatory subunit (p85, comprising the two isoforms α and β) (for a detailed review, see reference 80).The Akt family of proteins is comprised of the three isoforms α, β, and γ, which are composed of an N-terminal pleckstrin homology domain, a central catalytic domain, and a C-terminal hydrophobic domain. Akt is recruited to the plasma membrane through the binding of its pleckstrin homology domain to the phosphatidylinositol 3,4,5-triphosphate (PIP3), which is a product of PI3K that is anchored to the plasma membrane. PDK1 is also recruited to the plasma membrane through interactions with PIP3. As both PDK1 and Akt interact with PIP3, PDK1 colocalizes with Akt and activates it by phosphorylating threonine 308 (T308-P) (2, 66). Following its activation, Akt phosphorylates a number of downstream substrates such as caspase-9, BAD, glycogen synthase kinase 3β (GSK-3β), and FKHR. This leads to the suppression of apoptosis, cell growth, survival, and proliferation (11, 16, 56).Another downstream target of PI3K/Akt is mTOR, a serine/threonine kinase that plays a central role in the regulation of cell growth, proliferation, survival, and protein synthesis (26). mTOR kinase has recently been found to be associated with two functionally distinct complexes in mammalian cells, known as mTORC1 and mTORC2 (63, 66). Although these multiprotein complexes share molecules in common, distinct adaptor proteins are recruited into each complex: regulatory-associated protein of TOR (raptor) is recruited into mTORC1, while rapamycin-insensitive companion of TOR (rictor) is recruited into mTORC2 (33, 64). While mTORC1 controls cell growth and protein translation and has proven to be rapamycin sensitive, mTORC2 regulates the actin cytoskeleton and is assumed to be rapamycin insensitive, even though under conditions of prolonged exposure to the drug, it appears to inhibit mTORC2 assembly (29, 64, 65). Additionally, it has been demonstrated that mTORC2 regulates the activity of Akt through the phosphorylation of S473 (S473-P). S473-P appears to be required for the full activation of Akt, since S473-P has been shown to enhance the subsequent phosphorylation of T308 by PDK1 (66, 67, 94). Moreover, the phosphorylation of both S473 and T308 results in a four- to fivefold increase in Akt activity compared to T308-P by PDK1 alone (66).The PI3K/PDK1/Akt(T308)/mTORC1 pathway regulates vital cellular processes that are important for viral replication and propagation, including cell growth, proliferation, and protein translation. This pathway is particularly important for the replication of DNA viruses, as their replication is cap dependent. However, the Akt signaling pathway can also negatively affect viral replication. The stress response downstream of Akt signaling, including hypoxia and energy and amino acid depletion, inhibits mTORC1 (5, 9, 69). Therefore, DNA viruses must overcome these constraints to translate their mRNAs.Pharmacological disruption of the PI3K/Akt pathway with the specific PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (2-morpholino-8-phenyl-4H-1-benzopyran-4-one) (82) has been reported to not only increase the cleavage of downstream molecules associated with proapoptotic activity e.g., poly(ADP-ribose) polymerase (PARP) and the executioner caspase-3] (38, 41) but also promote microtubule stabilization, actin filament remodeling/cell migration, and bleb formation/viral infectivity (10, 35, 49, 54, 59).Because the PI3K/Akt and PI3K/Akt/mTOR pathways influence diverse cellular functions and possibly a healthy antiviral response, usurping these pathways could support an increase in viral replication. In support of this, a number of reports have demonstrated that either the PI3K/Akt or the PI3K/Akt/mTOR pathway plays a role in the replication of many viruses including flavivirus (38), hepatitis C virus (27), human immunodeficiency virus type 1 (93), human papillomavirus (44, 96), respiratory syncytial virus (77), coxsackievirus B3 (19), Epstein-Barr virus (17, 50, 73), human cytomegalovirus (36, 37, 72), herpes simplex virus type 1 (7, 83), varicella-zoster virus (60), Kaposi''s sarcoma-associated herpesvirus (89), adenovirus (55), and simian virus 40 (SV40) (95). With this in mind, we also investigated whether the PI3K/Akt pathway played a pivotal role in orthopoxvirus biology. In this study, we show that the VACV- and CPXV-stimulated PI3K/Akt pathway not only contributes to the prevention of host-cell death but also plays a beneficial role in the viral replication cycle.