Allele-Specific H3K79 Di- versus Trimethylation Distinguishes Opposite Parental Alleles at Imprinted Regions

Imprinted gene expression corresponds to parental allele-specific DNA CpG methylation and chromatin composition. Histone tail covalent modifications have been extensively studied, but it is not known whether modifications in the histone globular domains can also discriminate between the parental alleles. Using multiplex chromatin immunoprecipitation-single nucleotide primer extension (ChIP-SNuPE) assays, we measured the allele-specific enrichment of H3K79 methylation and H4K91 acetylation along the H19/Igf2 imprinted domain. Whereas H3K79me1, H3K79me2, and H4K91ac displayed a paternal-specific enrichment at the paternally expressed Igf2 locus, H3K79me3 was paternally biased at the maternally expressed H19 locus, including the paternally methylated imprinting control region (ICR). We found that these allele-specific differences depended on CTCF binding in the maternal ICR allele. We analyzed an additional 11 differentially methylated regions (DMRs) and found that, in general, H3K79me3 was associated with the CpG-methylated alleles, whereas H3K79me1, H3K79me2, and H4K91ac enrichment was specific to the unmethylated alleles. Our data suggest that allele-specific differences in the globular histone domains may constitute a layer of the “histone code” at imprinted genes.Imprinted genes are defined by the characteristic monoallelic silencing of either the paternally or maternally inherited allele. Most imprinted genes exist in imprinted gene clusters (10), and these clusters are usually associated with one or more differentially methylated regions (DMRs) (27, 65). DNA methylation at DMRs is essential for the allele-specific expression of most imprinted genes (31). Maternal or paternal allele-specific DNA methylation of a subset of DMRs (germ line DMRs) is gamete specific (27, 39). These maternal or paternal methylation differences are established during oogenesis or spermatogenesis, respectively, by the de novo DNA methyltransferases Dnmt3a and Dnmt3b together with Dnmt3L (5, 26, 48). The gamete-specific methylation differences set the stage for the parental allele-specific action of germ line DMRs, some of which have been shown to control the monoallelic expression of the associated genes in the respective domains (11, 34, 36, 53, 66, 71-73, 77). These DMRs are called imprinting control regions (ICRs).Two recurring themes have been reported for ICR action. ICRs can function as DNA methylation-regulated promoters of a noncoding RNA or as methylation-regulated insulators. Recent evidence suggests that both of these mechanisms involve chromatin organization by either the noncoding RNA (45, 50) or the CTCF insulator protein (17, 32) along the respective imprinted domains. The CTCF insulator binds in the unmethylated maternal allele of the H19/Igf2 ICR and blocks the access of the Igf2 promoters to the shared downstream enhancers. CTCF cannot bind in the methylated paternal ICR allele; hence, here the Igf2 promoters have access to the enhancers (4, 18, 24, 25, 62). When CTCF binding is abolished in the ICR of the maternal allele, Igf2 expression becomes biallelic, and H19 expression is missing from both alleles (17, 52, 58, 63). Importantly, CTCF is the single major organizer of the allele-specific chromatin along the H19/Igf2 imprinted domain (17). Significantly, CTCF recruits, at a distance, Polycomb-mediated H3K27me3 repressive marks at the Igf2 promoter and at the Igf2 DMRs (17, 32).A role for chromatin composition is suggested in the parental allele-specific expression of imprinted genes. Repressive histone tail covalent modifications, such as H3K9me2 H3K9me3, H4K20me3, H3K27me3, and the symmetrically methylated H4R3me2 marks, are generally associated with the methylated DMR alleles, while activating histone tail covalent modifications, such as acetylated histone tails and also H3K4me2 and H3K4me3, are characteristic of the unmethylated alleles (7-9, 12-15, 17, 21, 33, 35, 43, 44, 51, 55, 56, 67, 69, 74, 75). Importantly, the maintenance of imprinted gene expression depends on the allele-specific chromatin differences. ICR-dependent H3K9me2 and H3K27me3 enrichment in the paternal allele (67) is required for paternal repression of a set of imprinted genes along the Kcnq1 imprinted domain in the placenta (30). Imprinted Cdkn1c and Cd81 expression depends on H3K27 methyltransferase Ezh2 activity in the extraembryonic ectoderm (64). Similarly, H3K9 methyltransferase Ehmt2 is required for parental allele-specific expression of a number of imprinted genes, including Osbpl5, Cd81, Ascl2, Tfpi2, and Slc22a3 in the placenta (44, 45, 70).There is increasing evidence that covalent modifications, not only in the histone tails but also in the histone globular domains, carry essential information for development and gene regulation. The H3K79 methyltransferase gene is essential for development in Drosophila (60) and in mice (22). H3K79 methylation is required for telomeric heterochromatin silencing in Drosophila (60), Saccharomyces cerevisiae (47, 68), and mice (22). The H4K91 residue regulates nucleosome assembly (76). Whereas mutations at single acetylation sites in the histone tails have only minor consequences, mutation of the H4K91 site in the histone H4 globular domain causes severe defects in silent chromatin formation and DNA repair in yeast (37, 42, 76).Contrary to the abundant information that exists regarding the allele-specific chromatin composition at DMRs of imprinted genes, no information is available about the parental allele-specific marking in the histone globular domains at the DMRs. We hypothesized that chromatin marks in the globular domains of histones also distinguish the parental alleles of germ line DMRs. In order to demonstrate this, we measured the allele-specific enrichment of H3K79me1, H3K79me2, H3K79me3, and H4K91ac at 11 mouse DMRs using quantitative multiplex chromatin immunoprecipitation-single nucleotide primer extension (ChIP-SNuPE) assays. In general, H3K79me3 was associated with the methylated allele at most DMRs, whereas the unmethylated allele showed enrichment for H3K79me1, H3K79me2, and H4K91ac. These results are consistent with the possibility that allele-specific differences in the globular domains of histones contribute to the “histone code” at DMRs.     

p85 Associates with Unphosphorylated PTEN and the PTEN-Associated Complex

The lipid phosphatase PTEN functions as a tumor suppressor by dephosphorylating the D3 position of phosphoinositide-3,4,5-trisphosphate, thereby negatively regulating the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. In mammalian cells, PTEN exists either as a monomer or as a part of a >600-kDa complex (the PTEN-associated complex [PAC]). Previous studies suggest that the antagonism of PI3K/AKT signaling by PTEN may be mediated by a nonphosphorylated form of the protein resident within the multiprotein complex. Here we show that PTEN associates with p85, the regulatory subunit of PI3K. Using newly generated antibodies, we demonstrate that this PTEN-p85 association involves the unphosphorylated form of PTEN engaged within the PAC and also includes the p110β isoform of PI3K. The PTEN-p85 association is enhanced by trastuzumab treatment and linked to a decline in AKT phosphorylation in some ERBB2-amplified breast cancer cell lines. Together, these results suggest that integration of p85 into the PAC may provide a novel means of downregulating the PI3K/AKT pathway.The phosphoinositide 3-kinase (PI3K)/AKT signaling pathway regulates glucose/nutrient homeostasis and cell survival and plays a central role in both normal metabolism and cancer. The PTEN tumor suppressor gene (29, 30, 54) negatively regulates the PI3K/AKT pathway by dephosphorylating the D3 hydroxyl subunit of phosphoinositide-3,4,5-trisphosphate, a key membrane phosphatidylinositol generated by PI3K (34). PTEN undergoes genetic or epigenetic inactivation in many malignancies, including glioblastoma, melanoma, and endometrial, prostate, and breast cancers, among others (6, 13, 22, 23, 47, 49-51, 55, 68). Similarly, germ line mutations of PTEN are associated with the development of hamartomatous neoplasias such as Cowden disease and Bannayan-Zonana syndrome (17, 21, 41).The tumor suppressor function of PTEN undergoes dynamic regulation involving both C-terminal phosphorylation and protein-protein interactions. Phosphorylation of serine and threonine residues at the PTEN C-terminal tail, mediated by kinases such as CK2 and glycogen synthase kinase 3β, alters its conformational structure and association with PDZ domain-containing proteins and attenuates PTEN enzymatic activity (1, 11, 20, 32, 45, 61-63, 66, 67, 71). Conversely, PTEN function is promoted in large part through its stabilization in unphosphorylated form by incorporation into a high-molecular-weight protein complex (the PTEN-associated complex [PAC]) (66). We first demonstrated the existence of the PAC through gel filtration studies of rat liver extracts, which identified PTEN within a high-molecular-mass peak (>600 kDa), as well as a low-molecular-mass peak (40 to 100 kDa) in which PTEN is monomeric and phosphorylated (66). Subsequently, several PDZ domain-containing proteins were shown to interact with PTEN, including MAGI-1b, MAGI-2, MAGI-3, ghDLG, hMAST205, MSP58/MCRS1, NHERF1, and NHERF2, which mediate indirect binding with platelet-derived growth factor (PDGF) receptor β (25, 36, 42, 57, 66). More recently, LKB1, a serine/threonine kinase tumor suppressor (7), was also found to interact with and phosphorylate PTEN in vitro (36). In aggregate, these data suggest that PTEN functional output is controlled by a complex interplay of protein interactions and regulation of C-terminal phosphorylation.Beyond these interactions, there is also evidence to support additional regulatory mechanisms by which the tumor suppressor function of PTEN is mediated. The herpesvirus-associated ubiquitin-specific protease was shown to interact directly with PTEN and promote its nuclear entry (53). Both ubiquitination and relocalization into the nucleus constitute important PTEN regulatory mechanisms (53, 64). In many tumors, PTEN nuclear exclusion has been associated with poor cancer prognosis and more aggressive cancer development (15, 44, 56). Moreover, successful treatment of acute promyelocytic leukemia was shown to be associated with an increase in monoubiquitinylation and relocation of PTEN into the nucleus (53).Like PTEN, the p85 regulatory subunit of PI3K serves as a prominent modulator of PI3K/AKT signaling. p85, which exists in three isoforms (α, β, and γ), targets the catalytic (110-kDa) PI3K subunit to the membrane, which brings it into proximity with membrane-associated phosphatidylinositol lipids. In the steady state, p85 forms a tight association with the catalytic PI3K subunit, usually p110α or p110β in nonhematopoietic cells, with p110δ predominating in leukocytes (19). Consistent with this notion, p85 and p110 exist in equimolar ratios in a wide variety of mammalian cell lines and tissues (19), although some studies have suggested a role for free p85 in cell signaling (33, 65).Several recent lines of evidence have begun to support a possible regulatory relationship between PTEN and p85 (reviewed in references 3 and 53). For example, liver-specific deletion of PIK3R1, which encodes the p85α regulatory subunit, reduces both the activation of PI3K and PTEN enzymatic activity in this context. As a result, p85α-deficient hepatic cells express elevated levels of phosphoinositide trisphosphate and exhibit prolonged AKT activation (60). In addition, both PTEN and p85 are regulated by small GTPase proteins such as RhoA, but PTEN coimmunoprecipitates with the RhoA effector Rock only in the presence of PI3K (18, 31, 37). Although only correlative in nature, these findings may suggest a possible role for PTEN in p85 regulation or vice versa, in addition to its known function as a direct antagonist of the PI3K/AKT pathway (3, 9, 52, 57, 60).In the present study, we demonstrate an endogenous association between p85 and PTEN. Using newly generated antibodies that selectively recognize the PTEN C-terminal tail in its unphosphorylated form, we demonstrate that this PTEN-p85 association preferentially involves the unphosphorylated form of PTEN. The specificity of this interaction was confirmed using multiple antibodies and through studies of both human cancer cells and murine embryonic fibroblasts (MEFs) deficient for specific p85 subunits. This association, which also engages p110β, is enhanced by trastuzumab treatment and correlates with diminished AKT phosphorylation. These results support a functional role for the PTEN-p85 association that may have important biological and therapeutic implications for PI3K/AKT pathway regulation.     

PDK1 Regulates Vascular Remodeling and Promotes Epithelial-Mesenchymal Transition in Cardiac Development

One essential downstream signaling pathway of receptor tyrosine kinases (RTKs), such as vascular endothelial growth factor receptor (VEGFR) and the Tie2 receptor, is the phosphoinositide-3 kinase (PI3K)-phosphoinositide-dependent protein kinase 1 (PDK1)-Akt/protein kinase B (PKB) cascade that plays a critical role in development and tumorigenesis. However, the role of PDK1 in cardiovascular development remains unknown. Here, we deleted PDK1 specifically in endothelial cells in mice. These mice displayed hemorrhage and hydropericardium and died at approximately embryonic day 11.5 (E11.5). Histological analysis revealed defective vascular remodeling and development and disrupted integrity between the endothelium and trabeculae/myocardium in the heart. The atrioventricular canal (AVC) cushion and valves failed to form, indicating a defect in epithelial-mesenchymal transition (EMT), together with increased endothelial apoptosis. Consistently, ex vivo AVC explant culture showed impeded mesenchymal outgrowth. Snail protein was reduced and was absent from the nucleus in AVC cells. Delivery of the Snail S6A mutant to the AVC explant effectively rescued EMT defects. Furthermore, adenoviral Akt delivery rescued EMT defects in AVC explant culture, and deletion of PTEN delayed embryonic lethality of PDK1 endothelial deletion mice by 1 day and rendered normal development of the AVC cushion in the PDK1-deficient heart. Taken together, these results have revealed an essential role of PDK1 in cardiovascular development through activation of Akt and Snail.Polypeptide growth factors, such as insulin, insulin-like growth factor 1 (IGF-I), vascular endothelial growth factor (VEGF), and angiopoietin 1 (Ang1), exert biological functions through binding to their transmembrane receptors that belong to a large family of receptor tyrosine kinases (RTKs) (4). Consequently, the receptor molecules form homo- or heterodimers, and the intracellular tyrosines at the carboxyl termini of the receptors become phosphorylated (37). Numerous distinct adaptor/regulatory proteins, through their Src homologous 2 (SH2) domains, bind to the phosphotyrosines and transduce the signal to downstream pathways, among which are two essential and well-characterized signaling cascades—the mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3K)-phosphoinositide-dependent protein kinase 1 (PDK1)-Akt signaling pathways (4, 13, 37).The regulatory subunit of PI3K, p85, possesses the SH2 domain and can, therefore, bind to phosphotyrosines on the RTKs and subsequently render activation of the catalytic subunit of PI3K, p110 (7, 8). Active p110 phosphorylates phosphoinositide biphosphate (PIP2), turning it into PIP3 that recruits PDK1 and Akt to the cellular membrane, where Akt is phosphorylated at threonine 308 (T308 for Akt1) by PDK (5, 23, 30). The serine 473 (S473) of Akt (Akt1) is phosphorylated by mTOR complex 2 (mTORC2) and other kinases (17, 36). Phosphorylation of Akt at these two amino acids brings it to full activation. In PDK1-deficient embryonic stem (ES) cells, T308 phosphorylation was abolished and most of the Akt activity was lost, although the S473 phosphorylation was intact (40).Akt plays an important role in multiple biological processes, such as cell survival, growth, glucose metabolism, and angiogenesis (2, 12, 14-16, 22, 23, 39, 41-43). In mammals, there are three Akt isoforms, termed Akt 1, -2, and -3. Previously, we generated Akt1- and Akt3-deficient mice and studied their roles in mouse development (2, 15, 39, 42, 43). We found that the Akt1 and -3 double knockout (KO) (DKO) mice were embryonically lethal at around embryonic day 12 (E12) and manifested developmental defects in multiple tissues, including the cardiovascular system (14, 15, 43). These studies suggest that the Akt signaling pathway is involved in cardiovascular development.Other than Akt isoforms, PDK1 also activates another group of AGC family kinases, such as p70 ribosomal S6 kinase (S6K) (32), serum, and glucocorticoid-induced protein kinase (SGK) (26), p90 ribosomal S6 kinase (RSK) (21), and atypical isoforms of protein kinase C (PKC) (31). Comprehensive and intensive mouse genetic studies performed mainly by Alessi and coworkers have confirmed the regulation of these AGC kinases by PDK1 (3, 9, 10, 27-29, 40).PDK1 knockout mice were severely growth retarded and died at around E9.0, indicating an essential role of PDK1 in development (27). However, its function and downstream targets in cardiovascular development are still elusive. To study this, we deleted PDK1 specifically in endothelial cells through Cre recombinase-mediated excision (25). The results have revealed an essential role of PDK1 in vascular remodeling and integrity and in cardiac development through activation of Akt and its downstream target of Snail.     

Regulation of IRSp53-Dependent Filopodial Dynamics by Antagonism between 14-3-3 Binding and SH3-Mediated Localization

Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia.The ability of a cell to rapidly respond to extracellular cues and direct cytoskeletal rearrangements is dependent on an array of signaling complexes that control actin assembly (58). The protrusive structures at the leading edges of motile cells are broadly defined as lamellipodia or filopodia (14). Lamellae are sheet-like protrusions composed of dendritic actin arrays that drive membrane expansion, with the “lamellipodium” representing a narrow region at the edge of the cell (in culture) characterized by rapid actin polymerization. This F-actin assembly is suggested to require Arp2/3 activity that nucleates new actin filaments from the sides of existing ones (58, 71) and capping proteins that limit the length of these new filaments and stabilize them (7). Arp2/3 activity in turn is regulated by the WASP/WAVE family of proteins, such as N-WASP and WAVE2 (68), whose regulation is a subject of intense interest (12, 29, 36, 41, 56, 76).Filopodia contain parallel bundles of actin filaments containing fascin (22). These are dynamic structures that emanate from the periphery of the cell and are retracted, with occasional attachment (to the dish in culture). Thus, they have been thought to have a sensory or exploratory role during cell migration (28). This is the case for neuronal growth cones, where filopodia sense attractant or repulsive cues and dictate direction in axonal path finding (9, 17, 25, 35). Filopodia have been shown to be important in the context of dendritic-spine development (64, 77), epithelial-sheet closure (26, 60, 79), and cell invasion/metastasis (80, 83).Lamellipodia have been well characterized since the pioneering work of Abercrombie et al. in the early 1970s (2, 3, 4). Filopodia require symmetry breaking at the leading edge (initiation), followed by elongation driven by a filopodial-tip protein complex (14, 28). A few proteins have been identified in this complex; Mena/Vasp serve to prevent capping at the barbed ends of bundled actin filaments (7, 53), and Dia2 promotes F-actin elongation (57, 85). Termination of filopodial elongation is not understood but nonetheless is likely to be tightly regulated. In the absence of F-actin elongation, retraction of the filopodium takes place by a rearward flow of F-actin and filament depolymerization (22).IRSp53 is in a position to play a pivotal role in generating filopodia; this brain-enriched protein was discovered as a substrate of the insulin receptor (87). Subsequently, IRSp53 was identified as an effector for Rac1 (50) and Cdc42 (27, 38), where it participates in filopodium and lamellipodium production (38, 51, 54, 86), neurite extension (27), dendritic-spine morphogenesis (1, 15, 66, 67), cell motility and invasiveness (24). The N terminus of IRSp53 contains a conserved helical domain that is found in five different gene products and is referred to as the IRSp53/MIM homology domain (IMD) (51, 70). This domain has been postulated to bind to Rac1 (50, 70) in a nucleotide-independent manner (52), but no convincing effector-like region has been identified. A Cdc42-specific CRIB-like sequence that does not bind Rac1 (27, 38) allows coupling of this and perhaps related Rho GTPases. The structure of the IMD reveals a zeppelin-shaped dimer that could bind “bent” membranes; thus, its potential as an F-actin-bundling domain (51, 82) could be an in vitro artifact often attributed to proteins with basic patches (46). Although there are reports of F-actin binding at physiological ionic strength (ca. 100 mM KCl) (82, 19), this region when expressed in isolation does not decorate F-actin in vivo.Two reports showed the IMD to be an “inverse-BAR” domain. BAR (Bin-amphipysin-Rvs) domains are found in proteins involved in endocytic trafficking, such as amphipysin and endophilin, and stabilize positively bent membranes, such as those on endocytic vesicles (31, 47). The IMD domains of both IRSp53 (70) and MIM-B (46) associate with lipids and can induce tubulations of PI(3,4,5)P₃ or PI(4,5)P₂-rich membranes, respectively. These tubulations are equivalent to membrane protrusions and are also referred to as negatively bent membranes. Ectopic expression of the IMD from IRSp53 (51, 70, 82, 86) or two other family members, MIM-B (11, 46) and IRTKS (52), can give rise to cells with many peripheral extensions. MIM-B is said to stimulate lamellipodia (11), while IRTKS generates “short actin clusters” at the cell periphery (52).In IRSp53 is a CRIB-like motif that mediates binding to Cdc42 (27, 38), but the function of this interaction in unclear. Cdc42 could relieve IRSp53 autoinhibition as described for N-Wasp (38), but there is little evidence for this. It has been suggested that Cdc42 controls IRSp53 localization and actin remodeling (27, 38), but another study indicated that these events are Cdc42 independent (19). IRSp53 contains a central SH3 domain that may bind proline-rich proteins, such as Dia1 (23), Mena (38), WAVE2 (49, 50, 69), and Eps8 (19, 24). However, it seems unlikely that all of these represent bona fide partners, and side-by-side comparison is provided in this study. Mena is involved in filopodium production (37), Dia1 in stress fiber formation (81), and WAVE2 in lamellipodium extension (72). Thus, Mena is a better candidate as a partner for IRSp53-mediated filopodia than Dia1 or WAVE2.There is good evidence for IRSp53 as a cellular partner for Eps8 (19). Eps8 is an adaptor protein containing an N-terminal PTB domain that can associate with receptor tyrosine kinases (65), and perhaps β integrins (13), and a C-terminal SH3 domain that can associate with Abi1 (30). Binding of the general adaptor Abi1 appears to positively regulate the actin-capping domain at the C terminus of Eps8 (18). It has been suggested that IRSp53 and Eps8 as a complex regulate cell motility, and perhaps Rac1 activation, via SOS (24); more recently, their roles in filopodium formation have been addressed (19). The involvement of IRSp53, but not MIM-B or IRTKS, in filopodium formation might be related to its role as a Cdc42 effector. We show here that, surprisingly, the CRIB motif is not essential for this activity, but rather, the ability of IRSp53 to associate via its SH3 domain is required, and that this domain is controlled by 14-3-3 binding.We have focused on the regulation of Cdc42 effectors that bind 14-3-3, including IRSp53 and PAK4, which are found as 14-3-3 targets in various proteomic projects (32, 44). In this study, we characterize the binding of 14-3-3 to IRSp53 and uncover how this activity regulates IRSp53 function. The phosphorylation-dependent 14-3-3 binding is GSK3β dependent, and 14-3-3 blocks the accessibility of both the CRIB and SH3 domains of IRSp53, thus indicating its primary function in controlling IRSp53 partners. This regulation of the SH3 domain by 14-3-3 is critical in the proper localization and termination of IRSp53 function to promote filopodium dynamics.     

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.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

mTORC1-Activated S6K1 Phosphorylates Rictor on Threonine 1135 and Regulates mTORC2 Signaling

The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. While mTOR complex 1 (mTORC1) regulates mRNA translation and ribosome biogenesis, mTORC2 plays an important role in the phosphorylation and subsequent activation of Akt. Interestingly, mTORC1 negatively regulates Akt activation, but whether mTORC1 signaling directly targets mTORC2 remains unknown. Here we show that growth factors promote the phosphorylation of Rictor (rapamycin-insensitive companion of mTOR), an essential subunit of mTORC2. We found that Rictor phosphorylation requires mTORC1 activity and, more specifically, the p70 ribosomal S6 kinase 1 (S6K1). We identified several phosphorylation sites in Rictor and found that Thr1135 is directly phosphorylated by S6K1 in vitro and in vivo, in a rapamycin-sensitive manner. Phosphorylation of Rictor on Thr1135 did not affect mTORC2 assembly, kinase activity, or cellular localization. However, cells expressing a Rictor T1135A mutant were found to have increased mTORC2-dependent phosphorylation of Akt. In addition, phosphorylation of the Akt substrates FoxO1/3a and glycogen synthase kinase 3α/β (GSK3α/β) was found to be increased in these cells, indicating that S6K1-mediated phosphorylation of Rictor inhibits mTORC2 and Akt signaling. Together, our results uncover a new regulatory link between the two mTOR complexes, whereby Rictor integrates mTORC1-dependent signaling.The mammalian target of rapamycin (mTOR) is an evolutionarily conserved phosphatidylinositol 3-kinase (PI3K)-related Ser/Thr kinase that integrates signals from nutrients, energy sufficiency, and growth factors to regulate cell growth as well as organ and body size in a variety of organisms (reviewed in references 4, 38, 49, and 77). mTOR was discovered as the molecular target of rapamycin, an antifungal agent used clinically as an immunosuppressant and more recently as an anticancer drug (5, 20). Recent evidence indicates that deregulation of the mTOR pathway occurs in a majority of human cancers (12, 18, 25, 46), suggesting that rapamycin analogs may be potent antineoplastic therapeutic agents.mTOR forms two distinct multiprotein complexes, the rapamycin-sensitive and -insensitive mTOR complexes 1 and 2 (mTORC1 and mTORC2), respectively (6, 47). In cells, rapamycin interacts with FKBP12 and targets the FKBP12-rapamycin binding (FRB) domain of mTORC1, thereby inhibiting some of its function (13, 40, 66). mTORC1 is comprised of the mTOR catalytic subunit and four associated proteins, Raptor (regulatory associated protein of mTOR), mLST8 (mammalian lethal with sec13 protein 8), PRAS40 (proline-rich Akt substrate of 40 kDa), and Deptor (28, 43, 44, 47, 59, 73, 74). The small GTPase Rheb (Ras homolog enriched in brain) is a key upstream activator of mTORC1 that is negatively regulated by the tuberous sclerosis complex 1 (TSC1)/TSC2 GTPase-activating protein complex (reviewed in reference 35). mTORC1 is activated by PI3K and Ras signaling through direct phosphorylation and inactivation of TSC2 by Akt, extracellular signal-regulated kinase (ERK), and p90 ribosomal protein S6 kinase (RSK) (11, 37, 48, 53, 63). mTORC1 activity is also regulated at the level of Raptor. Whereas low cellular energy levels negatively regulate mTORC1 activity through phosphorylation of Raptor by AMP-activated protein kinase (AMPK) (27), growth signaling pathways activating the Ras/ERK pathway positively regulate mTORC1 activity through direct phosphorylation of Raptor by RSK (10). More recent evidence has also shown that mTOR itself positively regulates mTORC1 activity by directly phosphorylating Raptor at proline-directed sites (20a, 75). Countertransport of amino acids (55) and amino acid signaling through the Rag GTPases were also shown to regulate mTORC1 activity (45, 65). When activated, mTORC1 phosphorylates two main regulators of mRNA translation and ribosome biogenesis, the AGC (protein kinase A, G, and C) family kinase p70 ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and thus stimulates protein synthesis and cellular growth (50, 60).The second mTOR complex, mTORC2, is comprised of mTOR, Rictor (rapamycin-insensitive companion of mTOR), mSin1 (mammalian stress-activated mitogen-activated protein kinase-interacting protein 1), mLST8, PRR5 (proline-rich region 5), and Deptor (21, 39, 58, 59, 66, 76, 79). Rapamycin does not directly target and inhibit mTORC2, but long-term treatment with this drug was shown to correlate with mTORC2 disassembly and cytoplasmic accumulation of Rictor (21, 39, 62, 79). Whereas mTORC1 regulates hydrophobic motif phosphorylation of S6K1, mTORC2 has been shown to phosphorylate other members of the AGC family of kinases. Biochemical and genetic evidence has demonstrated that mTORC2 phosphorylates Akt at Ser473 (26, 39, 68, 70), thereby contributing to growth factor-mediated Akt activation (6, 7, 52). Deletion or knockdown of the mTORC2 components mTOR, Rictor, mSin1, and mLST8 has a dramatic effect on mTORC2 assembly and Akt phosphorylation at Ser473 (26, 39, 79). mTORC2 was also shown to regulate protein kinase Cα (PKCα) (26, 66) and, more recently, serum- and glucocorticoid-induced protein kinase 1 (SGK1) (4, 22). Recent evidence implicates mTORC2 in the regulation of Akt and PKCα phosphorylation at their turn motifs (19, 36), but whether mTOR directly phosphorylates these sites remains a subject of debate (4).Activation of mTORC1 has been shown to negatively regulate Akt phosphorylation in response to insulin or insulin-like growth factor 1 (IGF1) (reviewed in references 30 and 51). This negative regulation is particularly evident in cell culture models with defects in the TSC1/TSC2 complex, where mTORC1 and S6K1 are constitutively activated. Phosphorylation of insulin receptor substrate-1 (IRS-1) by mTORC1 (72) and its downstream target S6K1 has been shown to decrease its stability and lead to an inability of insulin or IGF1 to activate PI3K and Akt (29, 69). Although the mechanism is unknown, platelet-derived growth factor receptor β (PDGF-Rβ) has been found to be downregulated in TSC1- and TSC2-deficient murine embryonic fibroblasts (MEFs), contributing to a reduction of PI3K signaling (80). Interestingly, inhibition of Akt phosphorylation by mTORC1 has also been observed in the presence of growth factors other than IGF-1, insulin, or PDGF, suggesting that there are other mechanisms by which mTORC1 activation restricts Akt activity in cells (reviewed in references 6 and 31). Recent evidence demonstrates that rapamycin treatment causes a significant increase in Rictor electrophoretic mobility (2, 62), suggesting that phosphorylation of the mTORC2 subunit Rictor may be regulated by mTORC1 or downstream protein kinases.Herein, we demonstrate that Rictor is phosphorylated by S6K1 in response to mTORC1 activation. We demonstrate that Thr1135 is directly phosphorylated by S6K1 and found that a Rictor mutant lacking this phosphorylation site increases Akt phosphorylation induced by growth factor stimulation. Cells expressing the Rictor T1135A mutant were found to have increased Akt signaling to its substrates compared to Rictor wild-type- and T1135D mutant-expressing cells. Together, our results suggest that Rictor integrates mTORC1 signaling via its phosphorylation by S6K1, resulting in the inhibition of mTORC2 and Akt signaling.     

Role of Phosphatidylinositol 3-Kinase in Friend Spleen Focus-Forming Virus-Induced Erythroid Disease

Infection of erythroid cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia in mice due to expression of its unique envelope glycoprotein, gp55. Erythroid cells expressing SFFV gp55 proliferate in the absence of their normal regulator, erythropoietin (Epo), because of interaction of the viral envelope protein with the erythropoietin receptor and a short form of the receptor tyrosine kinase Stk (sf-Stk), leading to constitutive activation of several signal transduction pathways. Our previous in vitro studies showed that phosphatidylinositol 3-kinase (PI3-kinase) is activated in SFFV-infected cells and is important in mediating the biological effects of the virus. To determine the role of PI3-kinase in SFFV-induced disease, mice deficient in the p85α regulatory subunit of class IA PI3-kinase were inoculated with different strains of SFFV. We observed that p85α status determined the extent of erythroid hyperplasia induced by the sf-Stk-dependent viruses SFFV-P (polycythemia-inducing strain of SFFV) and SFFV-A (anemia-inducing strain of SFFV) but not by the sf-Stk-independent SFFV variant BB6. Our data also indicate that p85α status determines the response of mice to stress erythropoiesis, consistent with a previous report showing that SFFV uses a stress erythropoiesis pathway to induce erythroleukemia. We further showed that sf-Stk interacts with p85α and that this interaction depends upon sf-Stk kinase activity and tyrosine 436 in the multifunctional docking site. Pharmacological inhibition of PI3-kinase blocked proliferation of primary erythroleukemia cells from SFFV-infected mice and the erythroleukemia cell lines derived from them. These results indicate that p85α may regulate sf-Stk-dependent erythroid proliferation induced by SFFV as well as stress-induced erythroid hyperplasia.The Friend spleen focus-forming virus (SFFV) is a highly pathogenic retrovirus that induces rapid erythroblastosis in susceptible strains of mice (for a review, see reference 42). Friend SFFV is a replication-defective virus with deletions in its env gene, giving rise to a unique glycoprotein, SFFV gp55. This unique glycoprotein confers pathogenicity to the virus; a vector encoding SFFV gp55 alone is sufficient to induce erythroblastosis in susceptible strains of mice (49). The Fv-2 gene encodes one of the key susceptibility factors for SFFV-induced erythroid disease (18, 37), as follows: the receptor tyrosine kinase Stk/RON, a member of the Met family of receptor tyrosine kinases (11-12). Susceptibility to SFFV-induced disease is associated with expression of a short form of the receptor tyrosine kinase Stk, termed sf-Stk, that is transcribed from an internal promoter within the Stk gene of Fv-2-susceptible (Fv-2^ss) mice but not Fv-2-resistant (Fv-2^rr) mice (37) and is abundantly expressed in erythroid cells (11). Infection of erythroid cells with the polycythemia-inducing strain of SFFV (SFFV-P) induces erythropoietin (Epo)-independent proliferation and differentiation, whereas erythroid cells infected with the anemia-inducing strain of SFFV (SFFV-A) proliferate in the absence of Epo but still require Epo for differentiation (42). Previous studies demonstrated that this Epo-independent erythroblastosis is due to the cell surface interaction of the SFFV envelope protein with the Epo receptor (EpoR) and sf-Stk (31). While interaction with the EpoR appears to be responsible mainly for the induction of Epo-independent differentiation (52), Epo-independent erythroid cell proliferation depends upon activation of sf-Stk. We recently demonstrated that sf-Stk covalently interacts with SFFV-P gp55 in hematopoietic cells that express the EpoR and that this interaction induces sf-Stk activation (31). Furthermore, exogenous expression of sf-Stk, but not a kinase-inactive mutant of sf-Stk, in bone marrow cells from sf-Stk null mice can restore Epo-independent erythroid colony formation in response to SFFV infection (5, 41). Thus, the SFFV envelope glycoprotein induces Epo-independent proliferation of erythroid cells mainly by activating sf-Stk. While sf-Stk is a key susceptibility factor for erythroblastosis induced by both SFFV-P and SFFV-A (18), it is not required for the induction of erythroblastosis by the SFFV mutant BB6, which encodes an envelope glycoprotein, gp42, that is deleted in the membrane-proximal extracellular domain (19) and does not induce sf-Stk activation (31). gp42 of SFFV-BB6 appears to exert its biological effects on erythroid cells by efficiently interacting with the EpoR (9). Compared with wild-type SFFV, SFFV-BB6 causes a relatively indolent and slowly developing disease in mice (19).A number of signaling pathways normally activated in erythroid cells after erythropoietin (Epo) binds to its cell surface receptor (40) are constitutively activated in erythroid cells infected with SFFV. These include JAK/STAT, Ras/Raf/mitogen-activated protein kinase (MAPK), Jun N-terminal kinase, and the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathways (24, 25, 28-30, 32). SFFV gp55 is thought to activate these pathways by interacting with either the EpoR or sf-Stk (17, 31, 43). In several in vitro systems, class IA PI3-kinase has been shown to be activated by Epo through the EpoR (8, 20, 21) or by SFFV through sf-Stk (5, 14). We and others have shown that the PI3-kinase pathway is important for the induction of Epo independence by SFFV (5, 29). The class IA subclass of PI3-kinase is a heterodimer comprising the p110 (α, β, δ) catalytic unit and one of five regulatory subunits (85α, p55α, p50α, 85β, and 55γ) (15). The first 3 regulatory subunits are all splice variants of the same gene (pik3r1). Deletion of pik3r1, which encodes p85α, p55α, and p50α, is lethal (6, 7), and these regulatory subunits of PI3-kinase are required for normal murine fetal erythropoiesis in mice (10).To determine the role of p85α in SFFV-induced erythroleukemia, we used a distinct nonlethal pik3r1 knockout mouse which lacks only the p85α regulatory subunit of PI3-kinase (45, 47), allowing the study of SFFV-induced erythroleukemia in adult mice. Our results indicate that p85α regulates SFFV-induced erythroid hyperplasia induced in vivo by sf-Stk-dependent, but not sf-Stk-independent, isolates of the virus as well as stress-induced erythropoiesis and suggest that this regulation may occur through the interaction of sf-Stk with p85α.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).