Owen Schwartz

pH-Dependent Entry of Severe Acute Respiratory Syndrome Coronavirus Is Mediated by the Spike Glycoprotein and Enhanced by Dendritic Cell Transfer through DC-SIGN

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) synthesizes several putative viral envelope proteins, including the spike (S), membrane (M), and small envelope (E) glycoproteins. Although these proteins likely are essential for viral replication, their specific roles in SARS-CoV entry have not been defined. In this report, we show that the SARS-CoV S glycoprotein mediates viral entry through pH-dependent endocytosis. Further, we define its cellular tropism and demonstrate that virus transmission occurs through cell-mediated transfer by dendritic cells. The S glycoprotein was used successfully to pseudotype replication-defective retroviral and lentiviral vectors that readily infected Vero cells as well as primary pulmonary and renal epithelial cells from human, nonhuman primate, and, to a lesser extent, feline species. The tropism of this reporter virus was similar to that of wild-type, replication-competent SARS-CoV, and binding of purified S to susceptible target cells was demonstrated by flow cytometry. Although myeloid dendritic cells were able to interact with S and to bind virus, these cells could not be infected by SARS-CoV. However, these cells were able to transfer the virus to susceptible target cells through a synapse-like structure. Both cell-mediated infection and direct infection were inhibited by anti-S antisera, indicating that strategies directed toward this gene product are likely to confer a therapeutic benefit for antiviral drugs or the development of a SARS vaccine.

Abnormal display of PfEMP-1 on erythrocytes carrying haemoglobin C may protect against malaria.

Haemoglobin C, which carries a glutamate-to-lysine mutation in the β-globin chain, protects West African children against Plasmodium falciparum malaria. Mechanisms of protection are not established for the heterozygous (haemoglobin AC) or homozygous (haemoglobin CC) states. Here we report a marked effect of haemoglobin C on the cell-surface properties of P. falciparum-infected erythrocytes involved in pathogenesis. Relative to parasite-infected normal erythrocytes (haemoglobin AA), parasitized AC and CC erythrocytes show reduced adhesion to endothelial monolayers expressing CD36 and intercellular adhesion molecule-1 (ICAM-1). They also show impaired rosetting interactions with non-parasitized erythrocytes, and reduced agglutination in the presence of pooled sera from malaria-immune adults. Abnormal cell-surface display of the main variable cytoadherence ligand, PfEMP-1 (P. falciparum erythrocyte membrane protein-1), correlates with these findings. The abnormalities in PfEMP-1 display are associated with markers of erythrocyte senescence, and are greater in CC than in AC erythrocytes. Haemoglobin C might protect against malaria by reducing PfEMP-1-mediated adherence of parasitized erythrocytes, thereby mitigating the effects of their sequestration in the microvasculature.

Infection of Specific Dendritic Cells by CCR5-Tropic Human Immunodeficiency Virus Type 1 Promotes Cell-Mediated Transmission of Virus Resistant to Broadly Neutralizing Antibodies

ABSTRACT The tropism of human immunodeficiency virus type 1 for chemokine receptors plays an important role in the transmission of AIDS. Although CXCR4-tropic virus is more cytopathic for T cells, CCR5-tropic strains are transmitted more frequently in humans for reasons that are not understood. Phenotypically immature myeloid dendritic cells (mDCs) are preferentially infected by CCR5-tropic virus, in contrast to mature mDCs, which are not susceptible to infection but instead internalize virus into a protected intracellular compartment and enhance the infection of T cells. Here, we define a mechanism to explain preferential transmission of CCR5-tropic viruses based on their interaction with mDCs and sensitivity to neutralizing antibodies. Infected immature mDCs differentiated normally and were found to enhance CCR5-tropic but not CXCR4-tropic virus infection of T cells even in the continuous presence of neutralizing antibodies. Infectious synapses also formed normally in the presence of such antibodies. Infection of immature mDCs by CCR5-tropic virus can therefore establish a pool of infected cells that can efficiently transfer virus at the same time that they protect virus from antibody neutralization. This property of DCs may enhance infection, contribute to immune evasion, and could provide a selective advantage for CCR5-tropic virus transmission.

The aorta and heart differentially express RGS (regulators of G-protein signalling) proteins that selectively regulate sphingosine 1-phosphate, angiotensin II and endothelin-1 signalling

Normal cardiovascular development and physiology depend in part upon signalling through G-protein-coupled receptors (GPCRs), such as the angiotensin II type 1 (AT(1)) receptor, sphingosine 1-phosphate (S1P) receptors and endothelin-1 (ET-1) receptor. Since regulator of G-protein signalling (RGS) proteins function as GTPase-activating proteins for the G alpha subunit of heterotrimeric G-proteins, these proteins undoubtedly have functional roles in the cardiovascular system. In the present paper, we show that human aorta and heart differentially express RGS1, RGS2, RGS3S (short-form), RGS3L (long-form), PDZ-RGS3 (PDZ domain-containing) and RGS4. The aorta prominently expresses mRNAs for all these RGS proteins except PDZ-RGS3. Various stimuli that are critical for both cardiovascular development and function regulate dynamically the mRNA levels of several of these RGS proteins in primary human aortic smooth muscle cells. Both RGS1 and RGS3 inhibit signalling through the S1P(1) (formerly known as EDG-1), S1P(2) (formerly known as EDG-5) and S1P(3) (formerly known as EDG-3) receptors, whereas RGS2 and RGS4 selectively attenuate S1P(2)-and S1P(3)-receptor signalling respectively. All of the tested RGS proteins inhibit AT(1)-receptor signalling, whereas only RGS3 and, to a lesser extent, RGS4 inhibit ET(A)-receptor signalling. The conspicuous expression of RGS proteins in the cardiovascular system and their selective effects on relevant GPCR-signalling pathways provide additional evidence that they have functional roles in cardiovascular development and physiology.

Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein

Brutons tyrosine kinase (Btk) is required for human and mouse B cell development. Btk deficiency causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency in mice. Unlike Src proteins, Btk lacks a negative regulatory domain at the COOH terminus and may rely on cytoplasmic Btk-binding proteins to regulates its kinase activity by trans-inhibitor mechanisms. Consistent with this possibility, IBtk, which we identified as an inhibitor of Btk, bound to the PH domain of Btk. IBtk downregulated Btk kinase activity, Btk-mediated calcium mobilization and nuclear factor-κB–driven transcription. These results define a potential mechanism for the regulation of Btk function in B cells.

HIV-1 Assembly: Viral Glycoproteins Segregate Quantally to Lipid Rafts that Associate Individually with HIV-1 Capsids and Virions

Summary HIV-1 assembly depends on its structural protein, Gag, which after synthesis on ribosomes, traffics to the late endosome/plasma membrane, associates with HIV Env glycoprotein, and forms infectious virions. While Env and Gag migrate to lipid microdomains, their stoichiometry and specificity of interaction are unknown. Pseudotyped viral particles can be made with one viral core surrounded by heterologous envelope proteins. Taking advantage of this property, we analyzed the association of HIV Env and Ebola glycoprotein (GP), with HIV-1 Gag coexpressed in the same cell. Though both viral glycoproteins were expressed, each associated independently with Gag, giving rise to distinct virion populations, each with a single glycoprotein type. Confocal imaging demonstrated that Env and GP localized to distinct lipid raft microdomains within the same cell where they associated with different virions. Thus, a single Gag particle associates “quantally” with one lipid raft, containing homogeneous trimeric viral envelope proteins, to assemble functional virions.

Chemokines control naive CD8 + T cell selection of optimal lymph node antigen presenting cells

CCR5-binding chemokines produced in the draining lymph node after vaccinia virus infection guide naive CD8+ T cells toward DCs and away from the macrophage-rich zone, thereby facilitating optimal CD8+ T cell activation and cytokine production.

Disruption of the C-terminal region of EBA-175 in the Dd2:Nm clone of Plasmodium falciparum does not affect erythrocyte invasion

EBA-175 is a Plasmodium falciparum micronemal protein that binds to sialic acid in the context of the peptide backbone of glycophorin A and has been implicated in sialic acid-dependent invasion of erythrocytes. The existence of an alternative invasion pathway has been suggested by the finding that the P. falciparum clone Dd2/Nm can invade sialic acid-depleted erythrocytes. To study the role of EBA-175 in this alternative pathway, we have generated Dd2/Nm clones expressing a truncated form of EBA-175 that lacks region 6 and the cytoplasmic domain. The protein still appears to be localized to the apical end in the vicinity of the micronemes, suggesting that region 6 and the cytoplasmic domain are not involved in EBA-175 trafficking to the micronemes. In these genetically modified clones, the level of truncated EBA-175 protein expression was greatly reduced. EBA-175-disrupted clones displayed normal rates of invasion of untreated and enzyme-treated human and animal erythrocytes, suggesting a lack of involvement of EBA-175 in this alternative invasion pathway.

The influence of the thymic environment on the CD4-versus-CD8 T lineage decision

T cell receptor signaling is an essential factor regulating thymocyte selection, but the function of the thymic environment in this process is not clear. In mice transgenic for major histocompatibility complex class II–restricted T cell receptors, every thymocyte is potentially selectable for maturation in the CD4 lineage. To address whether selection frequency affects positive selection, we created hematopoietic chimeras with mixtures of selectable and nonselectable precursors. With increased proportions of nonselectable thymocytes, positive selection of MHC class II–specific precursors was enhanced, generating not only CD4 but also CD8 thymocytes. These results indicate that the CD4 versus CD8 fate of selectable precursors can be influenced by the selection potential of its neighbors.

Knockout of G protein β5 impairs brain development and causes multiple neurologic abnormalities in mice.

J. Neurochem. (2011) 119, 544–554.