Molecular basis for differential activation of p101 and p84 complexes of PI3Kγ by Ras and GPCRs.

Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells and can form two distinct complexes (p110γ-p84 and p110γ-p101), which are differentially activated by G protein-coupled receptors (GPCRs) and Ras. Using a combination of X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS), electron microscopy, molecular modeling, single-molecule imaging, and activity assays, we identify molecular differences between p110γ-p84 and p110γ-p101 that explain their differential membrane recruitment and activation by Ras and GPCRs. The p110γ-p84 complex is dynamic compared with p110γ-p101. While p110γ-p101 is robustly recruited by Gßγ subunits, p110γ-p84 is weakly recruited to membranes by Gßγ subunits alone and requires recruitment by Ras to allow for Gßγ activation. We mapped two distinct Gßγ interfaces on p101 and the p110γ helical domain, with differences in the C-terminal domain of p84 and p101 conferring sensitivity of p110γ-p101 to Gßγ activation. Overall, our work provides key insight into the molecular basis for how PI3Kγ complexes are activated.

Neurovirulence of avian influenza virus is dependent on the interaction of viral NP protein with host factor FMRP in the murine brain.

Avian influenza viruses (AIVs) are zoonotic viruses that exhibit a range infectivity and severity in the human host. Severe human cases of AIVs infection are often accompanied by neurological symptoms, however, the factors involved in the infection of the central nervous system (CNS) are not well known. In this study, we discovered that avian-like sialic acid (SA)-α2, 3 Gal receptor is highly presented in mammalian (human and mouse) brains. In the generation of a mouse-adapted neurotropic H9N2 AIV (SD16-MA virus) in BALB/c mice, we identified key adaptive mutations in its hemagglutinin (HA) and polymerase basic protein 2 (PB2) genes that conferred viral replication ability in mice brain. The SD16-MA virus showed binding affinity for avian-like SA-α2, 3 Gal receptor, enhanced viral RNP polymerase activity, increased viral protein production and transport that culminated in elevated progeny virus production and severe pathogenicity. We further established that host Fragile X Mental Retardation Protein (FMRP), a highly expressed protein in the brain that physically associated with viral nucleocapsid protein (NP) to facilitate RNP assembly and export, was an essential host factor for the neuronal replication of neurotropic AIVs (H9N2, H5N1 and H10N7 viruses). Our study identified a mechanistic process for AIVs to acquire neurovirulence in mice.IMPORTANCE Infection of the CNS is a serious complication of human cases of AIVs infection. The viral and host factors associated with neurovirulence of AIVs infection are not well understood. We identified and functionally characterized specific changes in the viral HA and PB2 genes of a mouse-adapted neurotropic avian H9N2 virus responsible for enhanced virus replication in neuronal cells and pathogenicity in mice. Importantly, we showed that host FMRP was a crucial host factor that was necessary for neurotropic AIVs (H9N2, H5N1 and H10N7 viruses) to replicate in neuronal cells. Our findings have provided insights into the pathogenesis of neurovirulence of AIV infection.

An R195K Mutation in the PA-X Protein Increases the Virulence and Transmission of Influenza A Virus in Mammalian Hosts.

In the 21st century, the emergence of H7N9 and H1N1/2009 influenza viruses, originating from animals and causing severe human infections, has prompted investigations into the genetic alterations required for cross-species transmission. We previously found that replacement of the human-origin PA gene segment in avian influenza virus (AIV) could overcome barriers to cross-species transmission. Recently, it was reported that the PA gene segment encodes both the PA protein and a second protein, PA-X. Here, we investigated the role of PA-X. We found that an H9N2 avian influenza reassortant virus bearing a human-origin H1N1/2009 PA gene was attenuated in mice after the loss of PA-X. Reverse genetics analyses of PA-X substitutions conserved in human influenza viruses indicated that R195K, K206R, and P210L substitutions conferred significantly increased replication and pathogenicity on H9N2 virus in mice and ferrets. PA-X R195K was present in all human H7N9 and H1N1/2009 viruses and predominated in human H5N6 viruses. Compared with PA-X 195R, H7N9 influenza viruses bearing PA-X 195K showed increased replication and transmission in ferrets. We further showed that PA-X 195K enhanced lung inflammatory responses, potentially due to decreased host shutoff function. A competitive transmission study in ferrets indicated that 195K provides a replicative advantage over 195R in H1N1/2009 viruses. In contrast, PA-X 195K did not influence the virulence of H9N2 AIV in chickens, suggesting that the effects of the substitution were mammal specific. Therefore, future surveillance efforts should scrutinize this region of PA-X because of its potential impact on cross-species transmission of influenza viruses.IMPORTANCE Four influenza pandemics in humans (the Spanish flu of 1918 [H1N1], the Asian flu of 1957 [H2N2], the Hong Kong flu of 1968 [H3N2], and the swine origin flu of 2009 [H1N1]) are all proposed to have been caused by avian or swine influenza viruses that acquired virulence factors through adaptive mutation or reassortment with circulating human viruses. Currently, influenza viruses circulating in animals are repeatedly transmitted to humans, posing a significant threat to public health. However, the molecular properties accounting for interspecies transmission of influenza viruses remain unclear. In the present study, we demonstrated that PA-X plays an important role in cross-species transmission of influenza viruses. At least three human-specific amino acid substitutions in PA-X dramatically enhanced the adaptation of animal influenza viruses in mammals. In particular, PA-X 195K might have contributed to cross-species transmission of H7N9, H5N6, and H1N1/2009 viruses from animal reservoirs to humans.

The use of pyrosequencing for detection of hemagglutinin mutations associated with increased pathogenicity of H5N1 avian influenza viruses in mammals.

Hemagglutinin (HA) cleavage is critical for virulence of influenza viruses. The amino acid residue at the P6 position of the HA cleavage site (HACS) has been shown to be most variable and to have a direct correlation with the cleavage efficiency and pathogenicity of H5N1 avian influenza viruses (AIVs) in mammals. Among these amino acid variants, serine has been associated with the highest virulence in mammals, and its detection may serve as an indicator for H5N1 AIVs with high pathogenicity and potential public risk. We developed a rapid detection method based on reverse-transcription (RT)-PCR and pyrosequencing to detect a mutation at the HACS that is associated with increased pathogenicity of H5N1 AIVs in mammals. Herein, we provide a specific, sensitive, and reliable method for rapid detection of one of the virulence determinants associated with increased pathogenicity of H5N1 AIVs in mammals.

5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (PQR309), a Potent, Brain-Penetrant, Orally Bioavailable, Pan-Class I PI3K/mTOR Inhibitor as Clinical Candidate in Oncology.

Phosphoinositide 3-kinase (PI3K) is deregulated in a wide variety of human tumors and triggers activation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclinical characterization of compound 1 (PQR309, bimiralisib), a potent 4,6-dimorpholino-1,3,5-triazine-based pan-class I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concentrations. No off-target interactions were detected for 1 in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover, 1 did not bind tubulin, which was observed for the structurally related 4 (BKM120, buparlisib). Compound 1 is orally available, crosses the blood-brain barrier, and displayed favorable pharmacokinetic parameters in mice, rats, and dogs. Compound 1 demonstrated efficiency in inhibiting proliferation in tumor cell lines and a rat xenograft model. This, together with the compound's safety profile, identifies 1 as a clinical candidate with a broad application range in oncology, including treatment of brain tumors or CNS metastasis. Compound 1 is currently in phase II clinical trials for advanced solid tumors and refractory lymphoma.

Enhanced pathogenicity and neurotropism of mouse-adapted H10N7 influenza virus are mediated by novel PB2 and NA mutations.

The H10 subtype of avian influenza viruses (AIVs) circulates globally in wild birds and poultry, and this subtype has been shown to be increasingly prevalent in China. Among the various H10 viruses, H10N7 AIVs have caused repeated mammal and human infections. To investigate their genetic adaptation in mammals, we generated a mouse-adapted avian H10N7 variant (A/mallard/Beijing/27/2011-MA; BJ27-MA) which exhibited increased virulence in mice compared to wild-type virus and acquired neurotropism. Sequencing showed the absence of the widely recognized mammalian adaptation markers of E627K and D701N in PB2 in the mouse-adapted strain; instead, five amino acid mutations were identified: E158G and M631L in PB2; G218E in haemagglutinin (H3 numbering); and K110E and S453I in neuraminidase (NA). The neurovirulence of the BJ27-MA virus necessitated the combined presence of the PB2 and NA mutations. Mutations M631L and E158G of PB2 and K110E of NA were required to mediate increased virus replication and severity of infection in mice and mammalian cells. PB2-M631L was functionally the most dominant mutation in that it strongly upregulated viral polymerase activity and played a critical role in the enhancement of virus replication and disease severity in mice. K110E mutation in NA, on the other hand, significantly promoted NA enzymatic activity. These results indicate that the novel mutations in PB2 and NA genes are critical for the adaptation of H10N7 AIV in mice, and they could serve as molecular signatures of virus transmission to mammalian hosts, including humans.

PA-X protein contributes to virulence of triple-reassortant H1N2 influenza virus by suppressing early immune responses in swine.

Previous studies have identified a functional role of PA-X for influenza viruses in mice and avian species; however, its role in swine remains unknown. Toward this, we constructed PA-X deficient virus (Sw-FS) in the background of a Triple-reassortment (TR) H1N2 swine influenza virus (SIV) to assess the impact of PA-X in viral virulence in pigs. Expression of PA-X in TR H1N2 SIV enhanced viral replication and host protein synthesis shutoff, and inhibited the mRNA levels of type I IFNs and proinflammatory cytokines in porcine cells. A delay of proinflammatory responses was observed in lungs of pigs infected by wild type SIV (Sw-WT) compared to Sw-FS. Furthermore, Sw-WT virus replicated and transmitted more efficiently than Sw-FS in pigs. These results highlight the importance of PA-X in the moderation of virulence and immune responses of TR SIV in swine, which indicated that PA-X is a pro-virulence factor in TR SIV in pigs.

Prevailing PA Mutation K356R in Avian Influenza H9N2 Virus Increases Mammalian Replication and Pathogenicity.

UNLABELLED: Adaptation of the viral polymerase complex comprising PB1, PB2, and PA is necessary for efficient influenza A virus replication in new host species. We found that PA mutation K356R (PA-K356R) has become predominant since 2014 in avian H9N2 viruses in China as with seasonal human H1N1 viruses. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses whose six internal gene segments are derived from the H9N2 virus. We further demonstrated the mammalian adaptive functionality of the PA-K356R mutation. Avian H9N2 virus with the PA-K356R mutation in human A549 cells showed increased nuclear accumulation of PA and increased viral polymerase activity that resulted in elevated levels of viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutation of PA-K356R and PB2-E627K, a well-known mammalian adaptive marker, in the H9N2 virus showed further cooperative increases in virus production and severity of infection in vitro and in vivo In summary, PA-K356R behaves as a novel mammalian tropism mutation, which, along with other mutations such as PB2-E627K, might render avian H9N2 viruses adapted for human infection. IMPORTANCE: Mutations of the polymerase complex (PB1, PB2, and PA) of influenza A virus are necessary for viral adaptation to new hosts. This study reports a novel and predominant mammalian adaptive mutation, PA-K356R, in avian H9N2 viruses and human isolates of emergent H7N9 and H10N8 viruses. We found that PA-356R in H9N2 viruses causes significant increases in virus replication and severity of infection in human cells and mice and that PA-K356R cooperates with the PB2-E627K mutation, a well-characterized human adaptive marker, to exacerbate mammalian infection in vitro and in vivo Therefore, the PA-K356R mutation is a significant adaptation in H9N2 viruses and related H7N9 and H10N8 reassortants toward human infectivity.

Highly Pathogenic Avian Influenza H5N6 Viruses Exhibit Enhanced Affinity for Human Type Sialic Acid Receptor and In-Contact Transmission in Model Ferrets.

UNLABELLED: Since May 2014, highly pathogenic avian influenza H5N6 virus has been reported to cause six severe human infections three of which were fatal. The biological properties of this subtype, in particular its relative pathogenicity and transmissibility in mammals, are not known. We characterized the virus receptor-binding affinity, pathogenicity, and transmissibility in mice and ferrets of four H5N6 isolates derived from waterfowl in China from 2013-2014. All four H5N6 viruses have acquired a binding affinity for human-like SAα2,6Gal-linked receptor to be able to attach to human tracheal epithelial and alveolar cells. The emergent H5N6 viruses, which share high sequence similarity with the human isolate A/Guangzhou/39715/2014 (H5N6), were fully infective and highly transmissible by direct contact in ferrets but showed less-severe pathogenicity than the parental H5N1 virus. The present results highlight the threat of emergent H5N6 viruses to poultry and human health and the need to closely track their continual adaptation in humans. IMPORTANCE: Extended epizootics and panzootics of H5N1 viruses have led to the emergence of the novel 2.3.4.4 clade of H5 virus subtypes, including H5N2, H5N6, and H5N8 reassortants. Avian H5N6 viruses from this clade have caused three fatalities out of six severe human infections in China since the first case in 2014. However, the biological properties of this subtype, especially the pathogenicity and transmission in mammals, are not known. Here, we found that natural avian H5N6 viruses have acquired a high affinity for human-type virus receptor. Compared to the parental clade 2.3.4 H5N1 virus, emergent H5N6 isolates showed less severe pathogenicity in mice and ferrets but acquired efficient in-contact transmission in ferrets. These findings suggest that the threat of avian H5N6 viruses to humans should not be ignored.

Truncation of C-terminal 20 amino acids in PA-X contributes to adaptation of swine influenza virus in pigs.

The PA-X protein is a fusion protein incorporating the N-terminal 191 amino acids of the PA protein with a short C-terminal sequence encoded by an overlapping ORF (X-ORF) in segment 3 that is accessed by + 1 ribosomal frameshifting, and this X-ORF exists in either full length or a truncated form (either 61-or 41-condons). Genetic evolution analysis indicates that all swine influenza viruses (SIVs) possessed full-length PA-X prior to 1985, but since then SIVs with truncated PA-X have gradually increased and become dominant, implying that truncation of this protein may contribute to the adaptation of influenza virus in pigs. To verify this hypothesis, we constructed PA-X extended viruses in the background of a "triple-reassortment" H1N2 SIV with truncated PA-X, and evaluated their biological characteristics in vitro and in vivo. Compared with full-length PA-X, SIV with truncated PA-X had increased viral replication in porcine cells and swine respiratory tissues, along with enhanced pathogenicity, replication and transmissibility in pigs. Furthermore, we found that truncation of PA-X improved the inhibition of IFN-I mRNA expression. Hereby, our results imply that truncation of PA-X may contribute to the adaptation of SIV in pigs.

Serological survey of canine H3N2, pandemic H1N1/09, and human seasonal H3N2 influenza viruses in cats in northern China, 2010-2014.

BACKGROUND: The close contact between cats and humans poses a threat to public health because of the potential zoonotic transmission of influenza viruses to humans. Therefore, we examined the seroprevalence of pandemic H1N1/09, canine H3N2, and human H3N2 viruses in pet cats in northern China from 2010 to 2014. FINDING: Of 1794 serum samples, the seropositivity rates for H1N1/09, canine H3N2, and human H3N2 were 5.7%, 0.7%, and 0.4%, respectively. The seropositivity rate for H1N1/09 in cats was highest in 2010 (8.3%), and then declined continuously thereafter. Cats older than 10 years were most commonly seropositive for the H1N1/09 virus. CONCLUSIONS: Our findings emphasize the need for continuous surveillance of influenza viruses in cats in China.

A serological survey of canine H3N2, pandemic H1N1/09 and human seasonal H3N2 influenza viruses in dogs in China.

Influenza viruses have been isolated from dogs in China; however, the extent of influenza infection among dogs is not yet clear. Here, we examined the seroprevalence of avian-origin canine H3N2, pandemic H1N1/09 and human seasonal H3N2 influenza viruses in pet dogs in China during January 2012 to June 2013. The seropositivity rate of canine H3N2, H1N1/09 and human H3N2 were 3.5%, 1.5%, and 1.2%, respectively. Dogs aged 2-5 years were most commonly seropositive to canine H3N2 virus. It is worth noting that two serum samples were positive against both canine H3N2 and H1N1/09 viruses, suggesting the possibility of coinfection with both viruses. Our findings emphasize the necessity for continued surveillance of influenza viruses in dogs in China.

PKCß phosphorylates PI3Kγ to activate it and release it from GPCR control.

All class I phosphoinositide 3-kinases (PI3Ks) associate tightly with regulatory subunits through interactions that have been thought to be constitutive. PI3Kγ is key to the regulation of immune cell responses activated by G protein-coupled receptors (GPCRs). Remarkably we find that PKCß phosphorylates Ser582 in the helical domain of the PI3Kγ catalytic subunit p110γ in response to clustering of the high-affinity IgE receptor (FcεRI) and/or store-operated Ca²âº- influx in mast cells. Phosphorylation of p110γ correlates with the release of the p84 PI3Kγ adapter subunit from the p84-p110γ complex. Ser582 phospho-mimicking mutants show increased p110γ activity and a reduced binding to the p84 adapter subunit. As functional p84-p110γ is key to GPCR-mediated p110γ signaling, this suggests that PKCß-mediated p110γ phosphorylation disconnects PI3Kγ from its canonical inputs from trimeric G proteins, and enables p110γ to operate downstream of Ca²âº and PKCß. Hydrogen deuterium exchange mass spectrometry shows that the p84 adaptor subunit interacts with the p110γ helical domain, and reveals an unexpected mechanism of PI3Kγ regulation. Our data show that the interaction of p110γ with its adapter subunit is vulnerable to phosphorylation, and outline a novel level of PI3K control.

Proteomic analysis of colorectal cancer metastasis: stathmin-1 revealed as a player in cancer cell migration and prognostic marker.

Metastasis accounts largely for the high mortality rate of colorectal cancer (CRC) patients. In this study, we performed comparative proteome analysis of primary CRC cell lines HCT-116 and its metastatic derivative E1 using 2-D DIGE. We identified 74 differentially expressed proteins, many of which function in transcription, translation, angiogenesis signal transduction, or cytoskeletal remodeling pathways, which are indispensable cellular processes involved in the metastatic cascade. Among these proteins, stathmin-1 (STMN1) was found to be highly up-regulated in E1 as compared to HCT-116 and was thus selected for further functional studies. Our results showed that perturbations in STMN1 levels resulted in significant changes in cell migration, invasion, adhesion, and colony formation. We further showed that the differential expression of STMN1 correlated with the cells' metastatic potential in other paradigms of CRC models. Using immunohistochemistry, we also showed that STMN1 was highly expressed in colorectal primary tumors and metastatic tissues as compared to the adjacent normal colorectal tissues. Furthermore, we also showed via tissue microarray analyses of 324 CRC tissues and Kaplan-Meier survival plot that CRC patients with higher expression of STMN1 have poorer prognosis.

Structural basis for activation and inhibition of class I phosphoinositide 3-kinases.

Phosphoinositide 3-kinases (PI3Ks) are implicated in a broad spectrum of cellular activities, such as growth, proliferation, differentiation, migration, and metabolism. Activation of class I PI3Ks by mutation or overexpression correlates with the development and maintenance of various human cancers. These PI3Ks are heterodimers, and the activity of the catalytic subunits is tightly controlled by the associated regulatory subunits. Although the same p85 regulatory subunits associate with all class IA PI3Ks, the functional outcome depends on the isotype of the catalytic subunit. New PI3K partners that affect the signaling by the PI3K heterodimers have been uncovered, including phosphate and tensin homolog (PTEN), cyclic adenosine monophosphate-dependent protein kinase (PKA), and nonstructural protein 1. Interactions with PI3K regulators modulate the intrinsic membrane affinity and either the rate of phosphoryl transfer or product release. Crystal structures for the class I and class III PI3Ks in complexes with associated regulators and inhibitors have contributed to developing isoform-specific inhibitors and have shed light on the numerous regulatory mechanisms controlling PI3K activation and inhibition.

Structure of lipid kinase p110ß/p85ß elucidates an unusual SH2-domain-mediated inhibitory mechanism.

Phosphoinositide 3-kinases (PI3Ks) are essential for cell growth, migration, and survival. The structure of a p110ß/p85ß complex identifies an inhibitory function for the C-terminal SH2 domain (cSH2) of the p85 regulatory subunit. Mutagenesis of a cSH2 contact residue activates downstream signaling in cells. This inhibitory contact ties up the C-terminal region of the p110ß catalytic subunit, which is essential for lipid kinase activity. In vitro, p110ß basal activity is tightly restrained by contacts with three p85 domains: the cSH2, nSH2, and iSH2. RTK phosphopeptides relieve inhibition by nSH2 and cSH2 using completely different mechanisms. The binding site for the RTK's pYXXM motif is exposed on the cSH2, requiring an extended RTK motif to reach and disrupt the inhibitory contact with p110ß. This contrasts with the nSH2 where the pY-binding site itself forms the inhibitory contact. This establishes an unusual mechanism by which p85 SH2 domains contribute to RTK signaling specificities.

Form and flexibility in phosphoinositide 3-kinases.

PI3Ks (phosphoinositide 3-kinases) have important roles in a variety of cellular activities, including survival, proliferation, growth, shape, migration and intracellular sorting. Consistent with their function in cell survival and growth, the gene for the class Ialpha PI3K catalytic subunit is a common site of gain-of-function mutations in cancers. Ongoing structural studies of these enzymes and the complexes they make with their regulatory subunits have helped to clarify the mechanistic basis of this role in tumour development. The broad spectrum of biological activities associated with various isotypes of class I PI3Ks has led to an intense search for isotype-specific inhibitors as tools in mammalian cell biology and for therapeutic application. Structural studies of the class I PI3Ks suggest that flexibility may be a component of the catalytic cycle of the enzymes.