Andes Virus Recognition of Human and Syrian Hamster β3 Integrins Is Determined by an L33P Substitution in the PSI Domain

Andes virus (ANDV) causes a fatal hantavirus pulmonary syndrome (HPS) in humans and Syrian hamsters. Human α_vβ₃ integrins are receptors for several pathogenic hantaviruses, and the function of α_vβ₃ integrins on endothelial cells suggests a role for α_vβ₃ in hantavirus directed vascular permeability. We determined here that ANDV infection of human endothelial cells or Syrian hamster-derived BHK-21 cells was selectively inhibited by the high-affinity α_vβ₃ integrin ligand vitronectin and by antibodies to α_vβ₃ integrins. Further, antibodies to the β₃ integrin PSI domain, as well as PSI domain polypeptides derived from human and Syrian hamster β₃ subunits, but not murine or bovine β₃, inhibited ANDV infection of both BHK-21 and human endothelial cells. These findings suggest that ANDV interacts with β₃ subunits through PSI domain residues conserved in both Syrian hamster and human β₃ integrins. Sequencing the Syrian hamster β₃ integrin PSI domain revealed eight differences between Syrian hamster and human β₃ integrins. Analysis of residues within the PSI domains of human, Syrian hamster, murine, and bovine β₃ integrins identified unique proline substitutions at residues 32 and 33 of murine and bovine PSI domains that could determine ANDV recognition. Mutagenizing the human β₃ PSI domain to contain the L33P substitution present in bovine β₃ integrin abolished the ability of the PSI domain to inhibit ANDV infectivity. Conversely, mutagenizing either the bovine PSI domain, P33L, or the murine PSI domain, S32P, to the residue present human β₃ permitted PSI mutants to inhibit ANDV infection. Similarly, CHO cells transfected with the full-length bovine β₃ integrin containing the P33L mutation permitted infection by ANDV. These findings indicate that human and Syrian hamster α_vβ₃ integrins are key receptors for ANDV and that specific residues within the β₃ integrin PSI domain are required for ANDV infection. Since L33P is a naturally occurring human β₃ polymorphism, these findings further suggest the importance of specific β₃ integrin residues in hantavirus infection. These findings rationalize determining the role of β₃ integrins in hantavirus pathogenesis in the Syrian hamster model.Hantaviruses persistently infect specific small mammal hosts and are spread to humans by the inhalation of aerosolized excreted virus (41, 42). Hantaviruses predominantly infect endothelial cells and cause one of two vascular leak-based diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (41). Hantavirus diseases are characterized by increased vascular permeability and acute thrombocytopenia in the absence of endothelial cell lysis (36, 41, 42, 54). In general, hantaviruses are not spread from person to person; however, the Andes hantavirus (ANDV) is an exception, since there are several reports of person-to-person transmission of ANDV infection (11, 37, 47, 52). ANDV is also unique in its ability to cause an HPS-like disease in Syrian hamsters and serves as the best-characterized hantavirus disease model with a long onset, symptoms, and pathogenesis nearly identical to that of HPS patients (20, 21, 50).Hantavirus infection of the endothelium alters endothelial cell barrier functions through direct and immunological responses (8, 14). Although the means by which hantaviruses cause pulmonary edema or hemorrhagic disease has been widely conjectured, the mechanisms by which hantaviruses elicit pathogenic human responses have yet to be defined. Hantaviruses coat the surface of infected VeroE6 cells days after infection (17), and this further suggests that dynamic hantavirus interactions with immune and endothelial cells are likely to contribute to viral pathogenesis. Hantavirus pathogenesis has been suggested to involve CD8⁺ T cells, tumor necrosis factor alpha or other cytokines, viremia, and the dysregulation of β₃ integrins (7, 8, 13-16, 25-28, 32, 34, 38, 44-46). However, these responses have not been demonstrated to contribute to hantavirus pathogenesis, and in some cases there are conflicting data on their involvement (18, 25-28, 34, 35, 44, 45, 48). Immune complex deposition clearly contributes to HFRS patient disease and renal sequelae (4, 7), but it is unclear what triggers vascular permeability in HPS and HFRS diseases or why hemorrhage occurs in HFRS patients but not in HPS patients (8, 36, 54). Acute thrombocytopenia is common to both diseases, and platelet dysfunction resulting from defective platelet aggregation is reported in HFRS patients (7, 8).Pathogenic hantaviruses have in common their ability to interact with α_IIbβ₃ and α_vβ₃ integrins present on platelets and endothelial cells (13, 16), and β₃ integrins have primary roles in regulating vascular integrity (1, 2, 6, 19, 22, 39, 40). Consistent with the presence of cell surface displayed virus (17), pathogenic hantaviruses uniquely block α_vβ₃ directed endothelial cell migration and enhance endothelial cell permeability for 3 to 5 days postinfection (14, 15). Pathogenic hantaviruses dysregulate β₃ integrin functions by binding domains present at the apex of inactive β₃ integrin conformers (38). α_vβ₃ forms a complex with vascular endothelial cell growth factor receptor 2 (VEGFR2) and normally regulates VEGF-directed endothelial cell permeability (2, 3, 10, 39, 40). However, both β₃ integrin knockouts and hantavirus-infected endothelial cells result in increased VEGF-induced permeability, presumably by disrupting VEGFR2-β₃ integrin complex formation (2, 14, 19, 39, 40). This suggests that at least one means for hantaviruses to increase vascular permeability occurs through interactions with β₃ integrins that are required for normal platelet and endothelial cell functions.α_vβ₃ and α_IIbβ₃ integrins exist in two conformations: an active extended conformation where the ligand binding head domain is present at the apex of the heterodimer and a basal, inactive bent conformation where the globular head of the integrin is folded toward the cell membrane (30, 53, 55). Pathogenic HTN and NY-1 hantaviruses bind to the N-terminal plexin-semaphorin-integrin (PSI) domain of β₃ integrin subunits and are selective for bent, inactive α_vβ₃ integrin conformers (38). Pathogenic hantavirus binding to inactive α_vβ₃ integrins is consistent with the selective inhibitory effect of hantaviruses on α_vβ₃ function and endothelial cell permeability (14, 15, 38). Although the mechanism of hantavirus induced vascular permeability has yet to be defined, there is a clear role for β₃ integrin dysfunction in vascular permeability deficits (5, 6, 22, 29, 39, 40, 51) which make an understanding of hantavirus interactions with β₃ subunits important for both entry and disease processes.The similarity between HPS disease in humans and Syrian hamsters (20, 21) suggests that pathogenic mechanisms of ANDV disease are likely to be coincident. Curiously, other hantaviruses (Sin Nombre virus [SNV] and Hantaan virus [HTNV]) are restricted in Syrian hamsters and fail to cause disease in this animal, even though they are prominent causes of human disease (50). Although the host range restriction for SNV and HTNV in Syrian hamsters has not been defined (33), the pathogenesis of ANDV in Syrian hamsters suggests that both human and Syrian hamster β₃ integrins may similarly be used by ANDV and contribute to pathogenesis.We demonstrate here that ANDV infection of the Syrian hamster BHK-21 cell line and human endothelial cells is dependent on α_vβ₃ and inhibited by α_vβ₃ specific ligands and antibodies. Further, polypeptides expressing the N-terminal 53 residues of human and Syrian hamster β₃ subunits block ANDV infection. This further indicates that ANDV interaction with the N-terminal 53 residues of both human and Syrian hamster β₃ integrins is required for viral entry. We also demonstrate that ANDV recognition of human and Syrian hamster β₃ integrins is determined by proline substitutions at residues 32/33 within the β₃ integrin PSI domain. These results define unique ANDV interactions with human and Syrian hamster β₃ integrins.     

Pathogenic Hantaviruses Andes Virus and Hantaan Virus Induce Adherens Junction Disassembly by Directing Vascular Endothelial Cadherin Internalization in Human Endothelial Cells

Hantaviruses infect endothelial cells and cause 2 vascular permeability-based diseases. Pathogenic hantaviruses enhance the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF). However, the mechanism by which hantaviruses hyperpermeabilize endothelial cells has not been defined. The paracellular permeability of endothelial cells is uniquely determined by the homophilic assembly of vascular endothelial cadherin (VE-cadherin) within adherens junctions, which is regulated by VEGF receptor-2 (VEGFR2) responses. Here, we investigated VEGFR2 phosphorylation and the internalization of VE-cadherin within endothelial cells infected by pathogenic Andes virus (ANDV) and Hantaan virus (HTNV) and nonpathogenic Tula virus (TULV) hantaviruses. We found that VEGF addition to ANDV- and HTNV-infected endothelial cells results in the hyperphosphorylation of VEGFR2, while TULV infection failed to increase VEGFR2 phosphorylation. Concomitant with the VEGFR2 hyperphosphorylation, VE-cadherin was internalized to intracellular vesicles within ANDV- or HTNV-, but not TULV-, infected endothelial cells. Addition of angiopoietin-1 (Ang-1) or sphingosine-1-phosphate (S1P) to ANDV- or HTNV-infected cells blocked VE-cadherin internalization in response to VEGF. These findings are consistent with the ability of Ang-1 and S1P to inhibit hantavirus-induced endothelial cell permeability. Our results suggest that pathogenic hantaviruses disrupt fluid barrier properties of endothelial cell adherens junctions by enhancing VEGFR2-VE-cadherin pathway responses which increase paracellular permeability. These results provide a pathway-specific mechanism for the enhanced permeability of hantavirus-infected endothelial cells and suggest that stabilizing VE-cadherin within adherens junctions is a primary target for regulating endothelial cell permeability during pathogenic hantavirus infection.Hantaviruses cause 2 human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (50). HPS and HFRS are multifactorial in nature and cause thrombocytopenia, immune and endothelial cell responses, and hypoxia, which contribute to disease (7, 11, 31, 42, 62). Although these syndromes sound quite different, they share common components which involve the ability of hantaviruses to infect endothelial cells and induce capillary permeability. Edema, which results from capillary leakage of fluid into tissues and organs, is a common finding in both HPS and HFRS patients (4, 7, 11, 31, 42, 62). In fact, both diseases can present with renal or pulmonary sequelae, and the renal or pulmonary focus of hantavirus diseases is likely to result from hantavirus infection of endothelial cells within vast glomerular and pulmonary capillary beds (4, 7, 11, 31, 42, 62). All hantaviruses predominantly infect endothelial cells which line capillaries (31, 42, 44, 61, 62), and endothelial cells have a primary role in maintaining fluid barrier functions of the vasculature (1, 12, 55). Although hantaviruses do not lyse endothelial cells (44, 61), this primary cellular target underlies hantavirus-induced changes in capillary integrity. As a result, understanding altered endothelial cell responses following hantavirus infection is fundamental to defining the mechanism of permeability induced by pathogenic hantaviruses (1, 12, 55).Pathogenic, but not nonpathogenic, hantaviruses use β₃ integrins on the surface of endothelial cells and platelets for attachment (19, 21, 23, 39, 46), and β₃ integrins play prominent roles in regulating vascular integrity (3, 6, 8, 24, 48). Pathogenic hantaviruses bind to basal, inactive conformations of β₃ integrins (35, 46, 53) and days after infection inhibit β₃ integrin-directed endothelial cell migration (20, 46). This may be the result of cell-associated virus (19, 20, 22) which keeps β₃ in an inactive state but could also occur through additional regulatory processes that have yet to be defined. Interestingly, the nonpathogenic hantaviruses Prospect Hill virus (PHV) and Tula virus (TULV) fail to alter β₃ integrin functions, and their entry is consistent with the use of discrete α₅β₁ integrins (21, 23, 36).On endothelial cells, α_vβ₃ integrins normally regulate permeabilizing effects of vascular endothelial growth factor receptor-2 (VEGFR2) (3, 24, 48, 51). VEGF was initially identified as an edema-causing vascular permeability factor (VPF) that is 50,000 times more potent than histamine in directing fluid across capillaries (12, 14). VEGF is responsible for disassembling adherens junctions between endothelial cells to permit cellular movement, wound repair, and angiogenesis (8, 10, 12, 13, 17, 26, 57). Extracellular domains of β₃ integrins and VEGFR2 reportedly form a coprecipitable complex (3), and knocking out β₃ causes capillary permeability that is augmented by VEGF addition (24, 47, 48). Pathogenic hantaviruses inhibit β₃ integrin functions days after infection and similarly enhance the permeability of endothelial cells in response to VEGF (22).Adherens junctions form the primary fluid barrier of endothelial cells, and VEGFR2 responses control adherens junction disassembly (10, 17, 34, 57, 63). Vascular endothelial cadherin (VE-cadherin) is an endothelial cell-specific adherens junction protein and the primary determinant of paracellular permeability within the vascular endothelium (30, 33, 34). Activation of VEGFR2, another endothelial cell-specific protein, triggers signaling responses resulting in VE-cadherin disassembly and endocytosis, which increases the permeability of endothelial cell junctions (10, 12, 17, 34). VEGF is induced by hypoxic conditions and released by endothelial cells, platelets, and immune cells (2, 15, 38, 52). VEGF acts locally on endothelial cells through the autocrine or paracrine activation of VEGFR2, and the disassembly of endothelial cell adherens junctions increases the availability of nutrients to tissues and facilitates leukocyte trafficking and diapedesis (10, 12, 17, 55). The importance of endothelial cell barrier integrity is often in conflict with requirements for endothelial cells to move in order to permit angiogenesis and repair or cell and fluid egress, and as a result, VEGF-induced VE-cadherin responses are tightly controlled (10, 17, 18, 32, 33, 59). This limits capillary permeability while dynamically responding to a variety of endothelial cell-specific factors and conditions. However, if unregulated, this process can result in localized capillary permeability and edema (2, 9, 10, 12, 14, 17, 29, 60).Interestingly, tissue edema and hypoxia are common findings in both HPS and HFRS patients (11, 31, 62), and the ability of pathogenic hantaviruses to infect human endothelial cells provides a means for hantaviruses to directly alter normal VEGF-VE-cadherin regulation. In fact, the permeability of endothelial cells infected by pathogenic Andes virus (ANDV) or Hantaan virus (HTNV) is dramatically enhanced in response to VEGF addition (22). This response is absent from endothelial cells comparably infected with the nonpathogenic TULV and suggests that enhanced VEGF-induced endothelial cell permeability is a common underlying response of both HPS- and HFRS-causing hantaviruses (22). In these studies, we comparatively investigate responses of human endothelial cells infected with pathogenic ANDV and HTNV, as well as nonpathogenic TULV.     

Serine Dephosphorylation of Receptor Protein Tyrosine Phosphatase α in Mitosis Induces Src Binding and Activation

Receptor protein tyrosine phosphatase α (RPTPα) is the mitotic activator of the protein tyrosine kinase Src. RPTPα serine hyperphosphorylation was proposed to mediate mitotic activation of Src. We raised phosphospecific antibodies to the two main serine phosphorylation sites, and we discovered that RPTPα Ser204 was almost completely dephosphorylated in mitotic NIH 3T3 and HeLa cells, whereas Ser180 and Tyr789 phosphorylation were only marginally reduced in mitosis. Concomitantly, Src pTyr527 and pTyr416 were dephosphorylated, resulting in 2.3-fold activation of Src in mitosis. Using inhibitors and knockdown experiments, we demonstrated that dephosphorylation of RPTPα pSer204 in mitosis was mediated by PP2A. Mutation of Ser204 to Ala did not activate RPTPα, and intrinsic catalytic activity of RPTPα was not affected in mitosis. Interestingly, binding of endogenous Src to RPTPα was induced in mitosis. GRB2 binding to RPTPα, which was proposed to compete with Src binding to RPTPα, was only modestly reduced in mitosis, which could not account for enhanced Src binding. Moreover, we demonstrate that Src bound to mutant RPTPα-Y789F, lacking the GRB2 binding site, and mutant Src with an impaired Src homology 2 (SH2) domain bound to RPTPα, illustrating that Src binding to RPTPα is not mediated by a pTyr-SH2 interaction. Mutation of RPTPα Ser204 to Asp, mimicking phosphorylation, reduced coimmunoprecipitation with Src, suggesting that phosphorylation of Ser204 prohibits binding to Src. Based on our results, we propose a new model for mitotic activation of Src in which PP2A-mediated dephosphorylation of RPTPα pSer204 facilitates Src binding, leading to RPTPα-mediated dephosphorylation of Src pTyr527 and pTyr416 and hence modest activation of Src.Protein tyrosine phosphatases (PTPs) are responsible for dephosphorylation of the phosphotyrosyl residues. The human genome contains approximately 100 genes that encode members of the four PTP families, and most of them have mouse orthologues (2, 48). According to their subcellular localization, the classical PTPs, encoded by less than half of the total PTP genes, are divided into two subfamilies: cytoplasmic and receptor protein tyrosine phosphatases (RPTPs). The majority of the RPTPs contain, besides a variable extracellular domain and a transmembrane domain, two highly homologous phosphatase domains (27), with the membrane-proximal domain comprising most of the catalytic activity (33).RPTPα is a typical RPTP with a small, highly glycosylated extracellular domain (13). RPTPα function is regulated by many mechanisms, including proteolysis (18), oxidation (55), dimerization (7, 23, 24, 47, 52), and phosphorylation of serine and tyrosine residues (16, 17, 49). RPTPα is broadly expressed in many cell types, and over the years, RPTPα has been shown to be involved in a number of signaling mechanisms, including neuronal (15) and skeletal muscle (34) cell differentiation, neurite elongation (8, 9, 56), insulin receptor signaling downregulation (3, 28, 30, 31, 35), insulin secretion (25), activation of voltage-gated potassium channel Kv1.2 (51), long-term potentiation in hippocampal neurons (32, 38), matrix-dependent force transduction (53), and cell spreading and migration (21, 45, 57).The majority of the roles played in these cellular processes involve RPTPα''s ability to activate the proto-oncogenes Src and Fyn by dephosphorylating their C-terminal inhibitory phosphotyrosine (5, 15, 39, 45, 61). Normally, this phosphotyrosine (pTyr527 in chicken Src) binds to the Src homology 2 (SH2) domain, keeping the protein in an inactive closed conformation. A displacement mechanism was proposed for RPTPα-mediated Src activation in which pTyr789 of RPTPα is required to bind the SH2 domain of Src before RPTPα dephosphorylates Tyr527 (58). This model is the subject of debate since other studies show that RPTPα lacking Tyr789 is still able to dephosphorylate and activate Src (12, 26, 29, 56). In normal cells, Src reaches its activation peak during mitosis (4, 11, 40, 42), and with the help of overexpressing cells, it was shown that this activation is triggered mainly by RPTPα. The model that emerged is that RPTPα is activated in mitosis due to serine hyperphosphorylation and detaches from the GRB2 scaffolding protein (59, 60) that normally binds most of the pTyr789 of RPTPα via its SH2 domain (14, 17, 46). Two serine phosphorylation sites were mapped in the juxtamembrane domain of RPTPα, Ser180 and Ser204 (49). The kinases that were found responsible for their phosphorylation were protein kinase C delta (PKCdelta) (10) and CaMKIIalpha (9), but there is no clear evidence that these kinases are activated in mitosis. We set out to investigate the role of serine phosphorylation of RPTPα in mitotic activation of Src.We generated phosphospecific antibodies and show that RPTPα pSer204, but not pSer180, is dephosphorylated in mitotic NIH 3T3 and HeLa cells, concomitantly with activation of Src. Selective inhibitors suggested that PP2A was the phosphatase that dephosphorylated pSer204. RNA interference (RNAi)-mediated knockdown of the catalytic subunit of PP2A demonstrated that indeed PP2A was responsible for mitotic dephosphorylation of RPTPα pSer204. It is noteworthy that PP2A is known to be activated in mitosis. Intrinsic PTP activities of RPTPα were similar in unsynchronized and mitotic cells, and mutation of Ser204 did not activate RPTPα in in vitro PTP assays. Yet, Src binding to RPTPα was induced in mitotic NIH 3T3 cells and RPTPα-S204D with a phosphomimicking mutation at Ser204 coimmunoprecipitated less efficiently with Src. Based on our results, we propose a mechanism for mitotic activation of Src that is triggered by dephosphorylation of RPTPα pSer204, resulting in enhanced affinity for Src and subsequent dephosphorylation and activation of Src.     

The γ134.5 Protein of Herpes Simplex Virus 1 Is Required To Interfere with Dendritic Cell Maturation during Productive Infection

The γ₁34.5 protein of herpes simplex virus 1 is an essential factor for viral virulence. In infected cells, this viral protein prevents the translation arrest mediated by double-stranded RNA-dependent protein kinase R. Additionally, it associates with and inhibits TANK-binding kinase 1, an essential component of Toll-like receptor-dependent and -independent pathways that activate interferon regulatory factor 3 and cytokine expression. Here, we show that γ₁34.5 is required to block the maturation of conventional dendritic cells (DCs) that initiate adaptive immune responses. Unlike wild-type virus, the γ₁34.5 null mutant stimulates the expression of CD86, major histocompatibility complex class II (MHC-II), and cytokines such as alpha/beta interferon in immature DCs. Viral replication in DCs inversely correlates with interferon production. These phenotypes are also mirrored in a mouse ocular infection model. Further, DCs infected with the γ₁34.5 null mutant effectively activate naïve T cells whereas DCs infected with wild-type virus fail to do so. Type I interferon-neutralizing antibodies partially reverse virus-induced upregulation of CD86 and MHC-II, suggesting that γ₁34.5 acts through interferon-dependent and -independent mechanisms. These data indicate that γ₁34.5 is involved in the impairment of innate immunity by inhibiting both type I interferon production and DC maturation, leading to defective T-cell activation.Herpes simplex virus 1 (HSV-1) is a human pathogen responsible for localized mucocutaneous lesions and encephalitis (51). Following primary infection, HSV-1 establishes a latent or lytic infection in which the virus interacts with host cells, which include dendritic cells (DCs), required to initiate adaptive immune responses (36). Immature DCs, which reside in almost all peripheral tissues, are able to capture and process viral antigens (15). In this process, DCs migrate to lymph nodes, where they mature and present antigens to T cells. Mature DCs display high levels of major histocompatibility complex class II (MHC-II) and costimulatory molecules such as CD40, CD80, and CD86. Additionally, DCs release proinflammatory cytokines such as interleukin-12 (IL-12), tumor necrosis factor alpha, alpha interferon (IFN-α), and IFN-β, which promote DC maturation and activation. An important feature of functional DCs is to activate naïve T cells, and myeloid submucosal and lymph node resident DCs are responsible for HSV-specific T-cell activation (2, 45, 52). Moreover, DCs play a direct role in innate antiviral immunity by secreting type I IFN.HSV-1 is capable of infecting both immature and mature DCs (20, 24, 34, 38, 42). A previous study suggested that HSV entry into DCs requires viral receptors HVEM and nectin-2 (42). Upon HSV infection, plasmacytoid DCs detect viral genome through Toll-like receptor 9 (TLR9) and produce high levels of IFN-α (16, 23, 30, 40). In contrast, myeloid DCs, which are major antigen-presenting cells, recognize viral components through distinct pathways, independently of TLR9 (16, 36, 40). It has been suggested previously that HSV proteins or RNA intermediates produced during viral replication trigger myeloid DCs (16, 40). Indeed, a protein complex that consists of HSV glycoproteins B, D, H, and L stimulates the expression of CD40, CD83, CD86, and cytokines in myeloid DCs (41). Hence, DCs sense HSV through TLR-dependent and -independent mechanisms (16, 40, 41). Nevertheless, HSV replication compromises DC functions and subsequent T-cell activation (3, 20, 24, 42). HSV-1 interaction with immature DCs results in the downregulation of costimulatory molecules and cytokines (20, 34, 38, 42). While HSV-2 induces rapid apoptosis, HSV-1 does so with a delayed kinetics in human DCs (4, 20, 38). HSV-1 is also reported to interfere with functions of mature DCs (24, 39). Upon infection, HSV-1 induces the degradation of CD83 but not CD80 or CD86 in mature DCs (24, 25). Additionally, HSV-1 reduces levels of the chemokine receptors CCR7 and CXCR4 on mature DCs and subsequently impairs DC migration to the respective chemokine ligands CCL19 and CXCL12 (39).Although HSV infection impairs DC functions, viral components responsible for this impairment have not been thoroughly investigated. It has been suggested previously that the virion host shut-off protein (vhs) of HSV-1 contributes partially to the viral block of DC activation (43). This activity is thought to stem from the ability of vhs to destabilize host mRNA. Emerging evidence suggests that ICP0 perturbs the function of mature DCs, where it mediates CD83 degradation via cellular proteasomes (25). Findings from related studies show that ICP0 inhibits the induction of IFN-stimulated genes mediated by IFN regulatory factor 3 (IRF3) or IRF7 in other cell types (11, 27, 32, 33). However, the link of ICP0 activities to DC maturation remains to be established. Recently, we found that γ₁34.5, an HSV virulence factor, associates with and inhibits TANK-binding kinase 1 (TBK1), an essential component of TLR-dependent and -independent pathways that activates IRF3 and cytokine expression (49). Interestingly, an HSV mutant lacking γ₁34.5 stimulates MHC-II surface expression in glioblastoma cells (47). These observations raise the hypothesis that γ₁34.5 may modulate DC maturation during HSV infection.In this study, we report that γ₁34.5 is required to perturb DC maturation during HSV infection, leading to impaired T-cell activation. Wild-type virus, but not the γ₁34.5 null mutant, suppresses the expression of costimulatory molecules as well as cytokines in DCs. We provide evidence that the viral block of DC maturation is associated with reduced IFN-α/β secretion. Furthermore, the expression of γ₁34.5 inhibits DC-mediated allogeneic T-cell activation and IFN-γ production. IFN-neutralizing antibodies partially reverse DC maturation induced by the γ₁34.5 null mutant. These results shed light on the role of γ₁34.5 relevant to DC maturation and T-cell responses in HSV infection.     

Lipid Phosphate Phosphatase 3 Stabilization of β-Catenin Induces Endothelial Cell Migration and Formation of Branching Point Structures

Endothelial cell (EC) migration, cell-cell adhesion, and the formation of branching point structures are considered hallmarks of angiogenesis; however, the underlying mechanisms of these processes are not well understood. Lipid phosphate phosphatase 3 (LPP3) is a recently described p120-catenin-associated integrin ligand localized in adherens junctions (AJs) of ECs. Here, we tested the hypothesis that LPP3 stimulates β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) to induce EC migration and formation of branching point structures. In subconfluent ECs, LPP3 induced expression of fibronectin via β-catenin/LEF-1 signaling in a phosphatase and tensin homologue (PTEN)-dependent manner. In confluent ECs, depletion of p120-catenin restored LPP3-mediated β-catenin/LEF-1 signaling. Depletion of LPP3 resulted in destabilization of β-catenin, which in turn reduced fibronectin synthesis and deposition, which resulted in inhibition of EC migration. Accordingly, reexpression of β-catenin but not p120-catenin in LPP3-depleted ECs restored de novo synthesis of fibronectin, which mediated EC migration and formation of branching point structures. In confluent ECs, however, a fraction of p120-catenin associated and colocalized with LPP3 at the plasma membrane, via the C-terminal cytoplasmic domain, thereby limiting the ability of LPP3 to stimulate β-catenin/LEF-1 signaling. Thus, our study identified a key role for LPP3 in orchestrating PTEN-mediated β-catenin/LEF-1 signaling in EC migration, cell-cell adhesion, and formation of branching point structures.Angiogenesis, the formation of new blood vessels, involves several well-coordinated cellular processes, including endothelial cell (EC) migration, synthesis and deposition of extracellular matrix proteins, such as fibronectin, cell-cell adhesion, and formation of branching point structures (1-3, 19, 33); however, less is known about the underlying mechanisms of these processes (6, 8, 12, 14, 16, 17). For example, adherens junctions (AJs), which mediate cell-cell adhesion between ECs, may be involved in limiting the extent of cell migration (2, 14, 38, 40). VE-cadherin, a protein found in AJs, is a single-pass transmembrane polypeptide responsible for calcium-dependent homophilic interactions through its extracellular domains (2, 38, 40). The VE-cadherin cytoplasmic domain interacts with the Armadillo domain-containing proteins, β-catenin, γ-catenin (plakoglobin), and p120-catenin (p120ctn) (2, 15, 38, 40, 43). Genetic and biochemical evidence documents a crucial role of β-catenin in regulating cell adhesion as well as proliferation secondary to the central position of β-catenin in the Wnt signaling pathway (13, 16, 25, 31, 44). In addition, the juxtamembrane protein p120ctn regulates AJ stability via binding to VE-cadherin (2, 7, 9, 15, 21, 28, 32, 43). The absence of regulation or inappropriate regulation of β-catenin and VE-cadherin functions is linked to cardiovascular disease and tumor progression (2, 6).We previously identified lipid phosphate phosphatase 3 (LPP3), also known as phosphatidic acid phosphatase 2b (PAP2b), in a functional assay of angiogenesis (18, 19, 41, 42). LPP3 not only exhibits lipid phosphatase activity but also functions as a cell-associated integrin ligand (18, 19, 35, 41, 42). The known LPPs (LPP1, LPP2, and LPP3) (20-23) are six transmembrane domain-containing plasma membrane-bound enzymes that dephosphorylate sphingosine-1-phosphate (S1P) and its structural homologues, and thus, these phosphatases generate lipid mediators (4, 5, 23, 35, 39). All LPPs, which contain a single N-glycosylation site and a putative lipid phosphatase motif, are situated such that their N and C termini are within the cell (4, 5, 22, 23, 35, 39). Only the LPP3 isoform contains an Arg-Gly-Asp (RGD) sequence in the second extracellular loop, and this RGD sequence enables LPP3 to bind integrins (18, 19, 22). Transfection experiments with green fluorescent protein (GFP)-tagged LPP1 and LPP3 showed that LPP1 is apically sorted, whereas LPP3 colocalized with E-cadherin at cell-cell contact sites with other Madin-Darby canine kidney (MDCK) cells (22). Mutagenesis and domain swapping experiments established that LPP1 contains an apical targeting signal sequence (FDKTRL) in its N-terminal segment. In contrast, LPP3 contains a dityrosine (109Y/110Y) basolateral sorting motif (22). Interestingly, conventional deletion of Lpp3 is embryonic lethal, since the Lpp3 gene plays a critical role in extraembryonic vasculogenesis independent of its lipid phosphatase activity (11). In addition, an LPP3-neutralizing antibody was shown to prevent cell-cell interactions (19, 42) and angiogenesis (42). Here, we addressed the hypothesis that LPP3 plays a key role in EC migration, cell-cell adhesion, and formation of branching point structures by stimulating β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) signaling.     

Identification of G Protein α Subunit-Palmitoylating Enzyme

The heterotrimeric G protein α subunit (Gα) is targeted to the cytoplasmic face of the plasma membrane through reversible lipid palmitoylation and relays signals from G-protein-coupled receptors (GPCRs) to its effectors. By screening 23 DHHC motif (Asp-His-His-Cys) palmitoyl acyl-transferases, we identified DHHC3 and DHHC7 as Gα palmitoylating enzymes. DHHC3 and DHHC7 robustly palmitoylated Gα_q, Gα_s, and Gα_i2 in HEK293T cells. Knockdown of DHHC3 and DHHC7 decreased Gα_q/11 palmitoylation and relocalized it from the plasma membrane into the cytoplasm. Photoconversion analysis revealed that Gα_q rapidly shuttles between the plasma membrane and the Golgi apparatus, where DHHC3 specifically localizes. Fluorescence recovery after photobleaching studies showed that DHHC3 and DHHC7 are necessary for this continuous Gα_q shuttling. Furthermore, DHHC3 and DHHC7 knockdown blocked the α_1A-adrenergic receptor/Gα_q/11-mediated signaling pathway. Together, our findings revealed that DHHC3 and DHHC7 regulate GPCR-mediated signal transduction by controlling Gα localization to the plasma membrane.G-protein-coupled receptors (GPCRs) form the largest family of cell surface receptors, consisting of more than 700 members in humans. GPCRs respond to a variety of extracellular signals, including hormones and neurotransmitters, and are involved in various physiologic processes, such as smooth muscle contraction and synaptic transmission (20, 25). Heterotrimeric G proteins, composed of α, β, and γ subunits, transduce signals from GPCRs to their effectors and play a central role in the GPCR signaling pathway (13, 21, 24, 32). Although the Gα subunit seems to localize stably at the cytosolic face of the plasma membrane (PM), a recent report suggested that Gα_o, a Gα isoform, shuttles rapidly between the PM and intracellular membranes (2). The PM targeting of Gα requires both interaction with the Gβγ complex and subsequent lipid palmitoylation of Gα (22). Thus, palmitoylation of Gα is a critical determinant of membrane targeting of the heterotrimer Gαβγ.Protein palmitoylation is a common posttranslational modification with lipid palmitate and regulates protein trafficking and function (7, 18). Gα is a classic and representative palmitoyl substrate (19, 38), and recent studies revealed that protein palmitoylation modifies virtually almost all the components of G-protein signaling, including GPCRs, Gα subunits, several members of the RGS (regulators of G-protein signaling) family of GTPase-activating proteins, GPCR kinase GRK6, and some small GTPases (7, 33). This common lipid modification plays an important role in compartmentalizing G-protein signaling to the specific microdomain, such as membrane caveolae and lipid raft (26). The palmitoyl thioester bond is relatively labile, and palmitates on substrates turn over rapidly, allowing proteins to shuttle between the cytoplasm/intracellular organelles and the PM (2, 3, 27). For example, binding of isoproterenol to the β-adrenergic receptor markedly accelerates the depalmitoylation of the associated Gα_s, shifting Gα_s to the cytoplasm (37). This receptor activation-induced depalmitoylation was also observed in a major postsynaptic PSD-95 scaffold, which anchors the AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid)-type glutamate receptor at the excitatory postsynapse through stargazin (6). On glutamate receptor activation, accelerated depalmitoylation of PSD-95 dissociates PSD-95 from postsynaptic sites and causes AMPA receptor endocytosis (6). Thus, palmitate turnover on Gα_s and PSD-95 is accelerated by receptor activation, contributing to downregulation of the signaling pathway. However, the enzymes that add palmitate to proteins (palmitoyl-acyl transferases [PATs]) and those that cleave the thioester bond (palmitoyl-protein thioesterases) were long elusive.Recent genetic studies in Saccharomyces cerevisiae identified Erf2/Erf4 (1, 40) and Akr1 (29) as PATs for yeast Ras and yeast casein kinase 2, respectively. Erf2 and Akr1 have four- to six-pass transmembrane domains and share a common domain, referred to as a DHHC domain, a cysteine-rich domain with a conserved Asp-His-His-Cys signature motif. Because the DHHC domain is essential for the PAT activity, we isolated 23 mammalian DHHC domain-containing proteins (DHHC proteins) and developed a systematic screening method to identify the specific enzyme-substrate pairs (11, 12): DHHC2, -3, -7, and -15 for PSD-95 (11); DHHC21 for endothelial NO synthase (10); and DHHC3 and -7 for GABA_A receptor γ2 subunit (9). Several other groups also reported that DHHC9 with GCP16 mediates palmitoylation toward H- and N-Ras (36) and that DHHC17, also known as HIP14, palmitoylates several neuronal proteins: huntingtin (14), SNAP-25, and CSP (14, 23, 35). However, the existence of PATs for Gα has been controversial because spontaneous palmitoylation of Gα could occur in vitro (4).In this study, we screened the 23 DHHC clones to examine which DHHC proteins can palmitoylate Gα. We found that DHHC3 and -7 specifically and robustly palmitoylate Gα at the Golgi apparatus. Inhibition of DHHC3 and -7 reduces Gα_q/11 palmitoylation levels and delocalizes it from the PM to the cytoplasm in HeLa cells and primary hippocampal neurons. Also, DHHC3 and -7 are necessary for the continuous Gα_q shuttling between the Golgi apparatus and the PM. Finally, blocking DHHC3 and -7 inhibits the α_1A-adrenergic receptor [α_1A-AR]/Gα_q-mediated signaling pathway, indicating that DHHC3 and -7 play an essential role in GPCR signaling by regulating Gα localization.     

Analysis of Human Immunodeficiency Virus Type 1 Matrix Binding to Membranes and Nucleic Acids

The human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein targets HIV-1 precursor Gag (PrGag) proteins to assembly sites at plasma membrane (PM) sites that are enriched in cholesterol and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂]. MA is myristoylated, which enhances membrane binding, and specifically binds PI(4,5)P₂ through headgroup and 2′ acyl chain contacts. MA also binds nucleic acids, although the significance of this association with regard to the viral life cycle is unclear. We have devised a novel MA binding assay and used it to examine MA interactions with membranes and nucleic acids. Our results indicate that cholesterol increases the selectivity of MA for PI(4,5)P₂-containing membranes, that PI(4,5)P₂ binding tolerates 2′ acyl chain variation, and that the MA myristate enhances membrane binding efficiency but not selectivity. We also observed that soluble PI(4,5)P₂ analogues do not compete effectively with PI(4,5)P₂-containing liposomes for MA binding but surprisingly do increase nonspecific binding to liposomes. Finally, we have demonstrated that PI(4,5)P₂-containing liposomes successfully outcompete nucleic acids for MA binding, whereas other liposomes do not. These results support a model in which RNA binding protects MA from associating with inappropriate cellular membranes prior to PrGag delivery to PM assembly sites.The matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) precursor Gag (PrGag) protein serves several functions in the viral replication cycle. One essential function is to target PrGag proteins to their assembly sites at the plasma membranes (PMs) of infected cells (4, 5, 11, 16, 25, 29, 30, 33, 35, 39, 43-45, 47, 50, 54, 56, 57). A second function is the recruitment of the viral surface/transmembrane (SU/TM; also referred to as gp120/gp41) envelope (Env) protein complex into virions (14, 15, 18, 19, 27, 51-53). In addition to these activities, numerous reports have attributed nucleic acid binding properties to retroviral MAs (24, 38, 47), and with some viruses MA appears to serve in an encapsidation capacity (24). While no encapsidation role has been assigned for HIV-1 MA, experiments have shown that MA can substitute for the HIV-1 nucleocapsid (NC) protein assembly function (38) under some circumstances, presumably by virtue of its facility to concentrate PrGag proteins by binding them to RNAs (38).A number of structural studies have been conducted on HIV-1 MA (1, 22, 41, 42, 49). The protein is N terminally myristoylated and composed of six α helices, capped by a three-strand β sheet (7, 22, 41, 42, 49). The protein trimerizes in solution and in crystals (22, 28, 49) and recently has been shown to organize as hexamers of trimers on lipid membranes (1). The membrane binding face of HIV-1 MA is basic, fostering its ability to associate with negatively charged phospholipid headgroups (1, 22, 30, 41, 42, 49). The importance of such an interaction has been underscored in molecular genetic experiments which demonstrated that depletion of PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] reduced the assembly efficiency of HIV-1 (9, 36). Consistent with these observations, HIV-1 MA preferentially binds to soluble PI(4,5)P₂ mimics through contacts with the headgroup and 2′ acyl chain, and binding promotes exposure of the MA myristate group and protein oligomerization (17, 21, 40-43, 46). However, PI(4,5)P₂ is not the only lipid to demonstrate an association with HIV-1. In particular, HIV-1 appears to assemble at cholesterol-rich PM sites, cholesterol is highly enriched in HIV-1 virions, and cholesterol depletion reduces viral infectivity (2, 6, 8, 20, 23, 26, 31, 34, 37). The HIV-1 lipidome shows additional differences from the PM lipids of infected cells (2, 5, 8), suggesting that other lipids could affect PrGag-membrane binding or virus assembly site selection.To gain a better understanding of the functions and interactions of HIV-1 MA, we have examined the liposome and nucleic acid binding properties of purified myristoylated MA. Using liposome flotation assays and a novel liposome bead binding assay, we have demonstrated that the PI(4,5)P₂ binding specificity of MA is enhanced by cholesterol, that protein myristoylation increases membrane binding efficiency but not specificity, and that 2′ acyl chain variation is compatible with PI(4,5)P₂ binding. We also examined whether soluble PI(4,5)P₂ mimics could compete with liposomes for MA binding. Surprisingly, we found that soluble mimics not only failed to compete with PI(4,5)P₂ liposomes but also increased MA binding to membranes that do not contain acidic phospholipids. Finally, we have observed that while MA does bind nucleic acids, nucleic acid binding is outcompeted by PI(4,5)P₂-containing liposomes. Our results suggest models for PrGag-membrane and RNA association and the HIV-1 assembly pathway.     

Interaction of αVβ3 and αVβ6 Integrins with Human Parechovirus 1

Human parechovirus (HPEV) infections are very common in early childhood and can be severe in neonates. It has been shown that integrins are important for cellular infectivity of HPEV1 through experiments using peptide blocking assays and function-blocking antibodies to α_V integrins. The interaction of HPEV1 with α_V integrins is presumably mediated by a C-terminal RGD motif in the capsid protein VP1. We characterized the binding of integrins α_Vβ₃ and α_Vβ₆ to HPEV1 by biochemical and structural studies. We showed that although HPEV1 bound efficiently to immobilized integrins, α_Vβ₆ bound more efficiently than α_Vβ₃ to immobilized HPEV1. Moreover, soluble α_Vβ₆, but not α_Vβ₃, blocked HPEV1 cellular infectivity, indicating that it is a high-affinity receptor for HPEV1. We also showed that HPEV1 binding to integrins in vitro could be partially blocked by RGD peptides. Using electron cryo-microscopy and image reconstruction, we showed that HPEV1 has the typical T=1 (pseudo T=3) organization of a picornavirus. Complexes of HPEV1 and integrins indicated that both integrin footprints reside between the 5-fold and 3-fold symmetry axes. This result does not match the RGD position predicted from the coxsackievirus A9 X-ray structure but is consistent with the predicted location of this motif in the shorter C terminus found in HPEV1. This first structural characterization of a parechovirus indicates that the differences in receptor binding are due to the amino acid differences in the integrins rather than to significantly different viral footprints.Picornaviruses consist of a positive-sense, single-stranded infectious RNA genome of approximately 7.3 kb enclosed in a capsid composed of 60 copies of each of the three or four capsid proteins (VP1 to VP4). Human parechovirus 1 (HPEV1) is a member of the Parechovirus genus of the Picornaviridae family (38, 70). There are currently eight completely sequenced human parechovirus types and 14 described types (4, 19, 24, 30, 38, 39, 51, 58, 78). In addition, the Parechovirus genus currently has four Ljungan virus members that infect rodents. HPEV1 exhibits several distinct molecular characteristics compared to other picornaviruses (38, 71). These include the lack of the maturation cleavage of the capsid proteins VP0 to VP4 (N-terminal) and VP2 (C-terminal), existence of an approximately 30-amino-acid-long extension to the N terminus of VP3, a unique nonstructural protein 2A, and a 5′ untranslated region that is more closely related to picornaviruses infecting animals than those infecting humans.HPEV infections are common during the first years of life and are often mild or asymptomatic (20, 28, 42, 73, 80). Recently, a number of new types have been identified, and their prevalence in stool samples, for example, highlights their clinical importance. Normally, they cause gastroenteritis and respiratory infections, but severe illnesses, such as infections of the central nervous system, generalized infections of neonates, and myocarditis, have also been associated with HPEV infections (1, 8, 10, 28, 80). Currently, the role of the unique molecular, structural, and antigenic characteristics of HPEVs in the pathogenesis of infection is unknown.HPEV types 1, 2, 4, 5, and 6 are known to possess an RGD motif near the C terminus of VP1 that is known to facilitate binding of cellular ligands (e.g., fibronectin) to α_v integrins. The motif is in an analogous position to motifs in coxsackievirus A9 (CAV9) and echovirus 9 (EV9; Barty strain) (Fig. ​(Fig.1).1). The role of the RGD sequence in cellular entry and subsequent replication of HPEV1 has been shown through blocking assays with RGD-containing peptides, mutation of the sequence, and function-blocking antibodies to α_v integrins (11, 43, 62, 71). These results strongly suggested that α_v integrins play a central role in the initiation of HPEV1 infection. Direct involvement of α_v integrins in the infectious entry of HPEV1 was further confirmed by overexpression of human α_vβ₁ and α_vβ₃ integrins in Chinese hamster ovary (CHO) cells, allowing successful virus infection (74). There are no reports yet on the identification of receptors for the HPEV types lacking the RGD motif (HPEV3, HPEV7, and HPEV8) (19, 39, 51).Open in a separate windowFIG. 1.Sequence alignments. Amino acid sequence alignment of the viral coat protein VP1 from different picornaviruses with the CAV9 secondary structure derived from the atomic model displayed above the alignment (34). The columns boxed in blue with red letters signify similarity, and the red column signifies identity. There is limited similarity between HPEV and other picornaviruses. C-terminal RGD motifs are boxed in red.Although the crystal structures of several picornaviruses have been determined (3, 26, 34, 35, 44, 57, 59, 65, 68, 72) and the receptor interactions have been studied in detail by X-ray crystallography, electron cryo-microscopy (cryo-EM), and three-dimensional (3D) image reconstruction (6, 9, 23, 31, 32, 47, 83), there is no structural information available for the parechoviruses or parechovirus-receptor complexes. Here, we compare the binding of α_Vβ₃ and α_Vβ₆ to HPEV1 in vitro by biochemical assays and determine the structures of HPEV1 and the corresponding HPEV1-integrin complexes.     

Utilization of Immunoglobulin G Fc Receptors by Human Immunodeficiency Virus Type 1: a Specific Role for Antibodies against the Membrane-Proximal External Region of gp41

Receptors (FcγRs) for the constant region of immunoglobulin G (IgG) are an important link between humoral immunity and cellular immunity. To help define the role of FcγRs in determining the fate of human immunodeficiency virus type 1 (HIV-1) immune complexes, cDNAs for the four major human Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa) were stably expressed by lentiviral transduction in a cell line (TZM-bl) commonly used for standardized assessments of HIV-1 neutralization. Individual cell lines, each expressing a different FcγR, bound human IgG, as evidence that the physical properties of the receptors were preserved. In assays with a HIV-1 multisubtype panel, the neutralizing activities of two monoclonal antibodies (2F5 and 4E10) that target the membrane-proximal external region (MPER) of gp41 were potentiated by FcγRI and, to a lesser extent, by FcγRIIb. Moreover, the neutralizing activity of an HIV-1-positive plasma sample known to contain gp41 MPER-specific antibodies was potentiated by FcγRI. The neutralizing activities of monoclonal antibodies b12 and 2G12 and other HIV-1-positive plasma samples were rarely affected by any of the four FcγRs. Effects with gp41 MPER-specific antibodies were moderately stronger for IgG1 than for IgG3 and were ineffective for Fab. We conclude that FcγRI and FcγRIIb facilitate antibody-mediated neutralization of HIV-1 by a mechanism that is dependent on the Fc region, IgG subclass, and epitope specificity of antibody. The FcγR effects seen here suggests that the MPER of gp41 could have greater value for vaccines than previously recognized.Fc receptors (FcRs) are differentially expressed on a variety of cells of hematopoietic lineage, where they bind the constant region of antibody (Ab) and provide a link between humoral and cellular immunity. Humans possess two classes of FcRs for the constant region of IgG (FcγRs) that, when cross-linked, are distinguished by their ability to either activate or inhibit cell signaling (69, 77, 79). The activating receptors FcγRI (CD64), FcγRIIa (CD32), and FcγRIII (CD16) signal through an immunoreceptor tyrosine-based activation motif (ITAM), whereas FcγRIIb (CD32) contains an inhibitory motif (ITIM) that counters ITAM signals and B-cell receptor signals. It has been suggested that a balance between activating and inhibitory FcγRs coexpressed on the same cells plays an important role in regulating adaptive immunity (23, 68). Moreover, the inhibitory FcγRIIb, being the sole FcγR on B cells, appears to play an important role in regulating self-tolerance (23, 68). The biologic role of FcγRs may be further influenced by differences in their affinity for immunoglobulin G (IgG); thus, FcγRI is a high-affinity receptor that binds monomeric IgG (mIgG) and IgG immune complexes (IC), whereas FcγRIIa, FcγRIIb, and FcγRIIIa are medium- to low-affinity receptors that preferentially bind IgG IC (10, 49, 78). FcγRs also exhibit differences in their relative affinity for the four IgG subclasses (10), which has been suggested to influence the balance between activating and inhibitory FcγRs (67).In addition to their participation in acquired immunity, FcγRs can mediate several innate immune functions, including phagocytosis of opsonized pathogens, Ab-dependent cell cytotoxicity (ADCC), antigen uptake by professional antigen-presenting cells, and the production of inflammatory cytokines and chemokines (26, 35, 41, 48, 69). In some cases, interaction of Ab-coated viruses with FcγRs may be exploited by viruses as a means to facilitate entry into FcγR-expressing cells (2, 33, 47, 84). Several groups have reported FcγR-mediated Ab-dependent enhancement (ADE) of HIV-1 infection in vitro (47, 51, 58, 63, 94, 96), whereas other reports have implicated FcγRs in efficient inhibition of the virus in vitro (19, 21, 29, 44-46, 62, 98) and possibly as having beneficial effects against HIV-1 in vivo (5, 27, 28, 42). These conflicting results are further complicated by the fact that HIV-1-susceptible cells, such as monocytes and macrophages, can coexpress more than one FcγR (66, 77, 79).HIV-1 entry requires sequential interactions between the viral surface glycoprotein, gp120, and its cellular receptor (CD4) and coreceptor (usually CCR5 or CXCR4), followed by membrane fusion that is mediated by the viral transmembrane glycoprotein gp41 (17, 106). Abs neutralize the virus by binding either gp120 or gp41 and blocking entry into cells. Several human monoclonal Abs that neutralize a broad spectrum of HIV-1 variants have attracted considerable interest for vaccine design. Epitopes for these monoclonal Abs include the receptor binding domain of gp120 in the case of b12 (71, 86), a glycan-specific epitope on gp120 in the case of 2G12 (13, 85, 86), and two adjacent epitopes in the membrane-proximal external region (MPER) of g41 in the cases of 2F5 and 4E10 (3, 11, 38, 93). At least three of these monoclonal Abs have been shown to interact with FcRs and to mediate ADCC (42, 43).A highly standardized and validated assay for neutralizing Abs against HIV-1 that quantifies reductions in luciferase (Luc) reporter gene expression after a single round of virus infection in TZM-bl cells has been developed (60, 104). TZM-bl (also called JC53BL-13) is a CXCR4-positive HeLa cell line that was engineered to express CD4 and CCR5 and to contain integrated reporter genes for firefly Luc and Escherichia coli β-galactosidase under the control of the HIV-1 Tat-regulated promoter in the long terminal repeat terminal repeat sequence (74, 103). TZM-bl cells are permissive to infection by a wide variety of HIV-1, simian immunodeficiency virus, and human-simian immunodeficiency virus strains, including molecularly cloned Env-pseudotyped viruses. Here we report the creation and characterization of four new TZM-bl cell lines, each expressing one of the major human FcγRs. These new cell lines were used to gain a better understanding of the individual roles that FcγRs play in determining the fate of HIV-1 IC. Two FcγRs that potentiated the neutralizing activity of gp41 MPER-specific Abs were identified.     

Cell Cycle Arrest by Transforming Growth Factor β1 Enhances Replication of Respiratory Syncytial Virus in Lung Epithelial Cells

Respiratory syncytial virus (RSV) is a common respiratory viral infection in children which is associated with immune dysregulation and subsequent induction and exacerbations of asthma. We recently reported that treatment of primary human epithelial cells (PHBE cells) with transforming growth factor β (TGF-β) enhanced RSV replication. Here, we report that the enhancement of RSV replication is mediated by induction of cell cycle arrest. These data were confirmed by using pharmacologic inhibitors of cell cycle progression, which significantly enhanced RSV replication. Our data also showed that RSV infection alone resulted in cell cycle arrest in A549 and PHBE cells. Interestingly, our data showed that RSV infection induced the expression of TGF-β in epithelial cells. Blocking of TGF-β with anti-TGF-β antibody or use of a specific TGF-β receptor signaling inhibitor resulted in rescue of the RSV-induced cell cycle arrest, suggesting an autocrine mechanism. Collectively, our data demonstrate that RSV regulates the cell cycle through TGF-β in order to enhance its replication. These findings identify a novel pathway for upregulation of virus replication and suggest a plausible mechanism for association of RSV with immune dysregulation and asthma.Respiratory syncytial virus (RSV) is a single-stranded RNA virus and is a common cause of severe respiratory infections in children. RSV predominantly infects lung epithelial cells, inducing bronchiolitis, and in high-risk individuals it can cause lung fibrosis, airway hyperresponsiveness, mucus secretion, and edema. Interestingly, there is substantial evidence to show that RSV infection induces a dysregulation of the immune response (13, 14, 24, 28, 49). However, the molecular underpinnings of this immune dysregulation are not yet completely understood.It has been established that through its interaction with the immune system, RSV is associated with development and exacerbations of asthma, which is a chronic inflammatory respiratory disease (17, 18, 36, 41). In comparison to healthy individuals, those with asthma have an exaggerated inflammatory response during respiratory virus infections. Despite many studies reporting the involvement of RSV with asthma development and exacerbations, the underlining mechanisms are not yet fully delineated.Previously, we reported that transforming growth factor β (TGF-β) treatment enhanced RSV replication (30). TGF-β is a pleiotropic cytokine with diverse effects on T-cell differentiation and immune regulation and potent anti-inflammatory functions (21, 27, 33, 45). In the lung microenvironment TGF-β inhibits cell proliferation, induces mucus secretion, and regulates airway fibrosis and remodeling (2, 5, 6, 20, 23, 34, 39, 46), all of which are hallmarks of chronic asthma. Specifically, it has been reported that TGF-β expression is elevated in bronchoalveolar lavage fluids and lung tissue of asthmatic patients (9, 32, 48).In addition, genetic studies have found an association between asthma phenotype and TGF-β (19, 26, 38, 43). These studies have identified several single-nucleotide polymorphisms (C509T, T869C, and G915C) in the promoter and coding region of TGF-β that contributed to the increase in gene expression and are significantly associated with childhood wheezing, asthma diagnosis, and asthma severity. Despite this correlation between TGF-β and asthma, the interaction between this key cytokine and respiratory viral infection is poorly understood.A well-known function of TGF-β is the regulation of cell cycle progression. Activation of TGF-β-induced signaling pathways promotes cell cycle arrest in both the G₀/G₁ and G₂/M phases of the cell cycle (7, 8, 25, 29, 40, 42, 44). In the current study, our data showed that TGF-β induction of cell cycle arrest was beneficial to RSV replication. The association of cell cycle arrest with RSV replication was determined by using three different pharmacological inhibitors of cell cycle progression, which enhanced RSV replication. Interestingly, RSV infection alone resulted in secretion of active TGF-β. Treatment of epithelial cells with anti-TGF-β or a specific inhibitor of TGF-β receptor (TGF-βR) signaling resulted in a reduction in RSV replication.In the current study, our data uncover a new pathway for virus regulation of the cell cycle. These findings support our hypothesis that RSV regulates and utilizes TGF-β in lung epithelium to enhance its replication, which may contribute to the physiological changes in the lung leading to immune dysregulation, asthma development, and exacerbations.     

A Basic Patch on α-Adaptin Is Required for Binding of Human Immunodeficiency Virus Type 1 Nef and Cooperative Assembly of a CD4-Nef-AP-2 Complex

A critical function of the human immunodeficiency virus type 1 Nef protein is the downregulation of CD4 from the surfaces of infected cells. Nef is believed to act by linking the cytosolic tail of CD4 to the endocytic machinery, thereby increasing the rate of CD4 internalization. In support of this model, weak binary interactions between CD4, Nef, and the endocytic adaptor complex, AP-2, have been reported. In particular, dileucine and diacidic motifs in the C-terminal flexible loop of Nef have been shown to mediate binding to a combination of the α and σ2 subunits of AP-2. Here, we report the identification of a potential binding site for the Nef diacidic motif on α-adaptin. This site comprises two basic residues, lysine-297 and arginine-340, on the α-adaptin trunk domain. The mutation of these residues specifically inhibits the ability of Nef to bind AP-2 and downregulate CD4. We also present evidence that the diacidic motif on Nef and the basic patch on α-adaptin are both required for the cooperative assembly of a CD4-Nef-AP-2 complex. This cooperativity explains how Nef is able to efficiently downregulate CD4 despite weak binary interactions between components of the tripartite complex.CD4, a type I transmembrane glycoprotein that serves as a coreceptor for major histocompatibility complex class II (MHC-II) molecules, is expressed on the surfaces of helper T lymphocytes and cells of the monocyte/macrophage lineage (8). Primate immunodeficiency viruses gain access to these cells by virtue of the interaction of the viral envelope glycoprotein (Env) with a combination of CD4 and a chemokine receptor (63). This interaction causes a conformational change within the Env protein that promotes the fusion of the viral envelope with the plasma membrane. Upon the delivery of the viral genetic material into the cytoplasm of the host cells, one of the first virally encoded proteins to be expressed is Nef, an accessory factor that modulates specific signal transduction and protein-trafficking pathways in a manner that optimizes the intracellular environment for viral replication (reviewed in references 21, 39, and 65). Perhaps the best characterized function of Nef is the downregulation of CD4 from the surfaces of the host cells (6, 22, 29, 45). CD4 downregulation prevents superinfection (6, 41) and enhances virion release (19, 38, 48, 66, 76), thereby contributing to the establishment of a robust infective state (24, 72).The mechanism used by the Nef protein of human immunodeficiency virus type 1 (HIV-1) to downregulate CD4 has been the subject of extensive study, but only recently have the molecular details of this process begun to be unraveled. It is generally acknowledged that HIV-1 Nef accelerates the internalization of CD4 from the plasma membrane by linking the cytosolic tail of the receptor to the clathrin-associated endocytic machinery (1, 12, 20, 34, 40, 64). In support of this model, a hydrophobic pocket comprising W57 and L58 on the folded core domain of Nef binds with millimolar affinity to the cytosolic tail of CD4 (28) (all residues and numbers correspond to the NL4-3 variant of HIV-1 Nef used in this study). In addition, a dileucine motif (ENTSLL, residues 160 to 165) (10, 16, 26) and a diacidic motif (D174 and D175) (2) on the C-terminal flexible loop of Nef mediate an interaction of micromolar affinity with the clathrin-associated, heterotetrameric (α-β2-μ2-σ2) adaptor protein 2 (AP-2) complex (12, 20, 40, 49). These interactions draw CD4 into clathrin-coated pits that eventually bud inwards as clathrin-coated vesicles (11, 27). Internalized CD4 is subsequently delivered to endosomes and then to lysosomes for degradation (3, 23, 59, 64).Despite progress in the understanding of the mechanism of Nef-induced CD4 downregulation, several important aspects remain to be elucidated. Previous studies have shown that the Nef dileucine and diacidic motifs interact with a combination of the α and σ2 subunits of AP-2 (referred to as the α-σ2 hemicomplex) (12, 20, 40, 49), but the precise location of the Nef binding sites is unknown. It also remains to be determined whether Nef can actually bind CD4 and AP-2 at the same time. Indeed, the formation of a tripartite CD4-Nef-AP-2 complex in which Nef links the cytosolic tail of CD4 to AP-2 has long been hypothesized but has never been demonstrated experimentally. Given the relatively weak affinity of Nef for the CD4 tail (28) and AP-2 (12, 40), it is unclear how such a complex could assemble and function in CD4 downregulation.In this study, we have addressed these issues by using a combination of yeast hybrid, in vitro binding, and in vivo CD4 downregulation assays. We report the identification of a candidate binding site for the Nef diacidic motif on the AP-2 complex. This site, a basic patch comprising K297 and R340 on α-adaptin, is specifically required for Nef binding and Nef-induced CD4 downregulation. We also show that the Nef diacidic motif and the α-adaptin basic patch are required for the cooperative assembly of a tripartite complex composed of the CD4 cytosolic tail, Nef, and the α-σ2 hemicomplex. The cooperative manner in which this complex is formed explains how Nef is able to efficiently downregulate CD4 from the plasma membrane despite weak binary interactions between the components of this complex.