RESUMEN
GPR15 is a G protein-coupled receptor (GPCR) proposed to play a role in mucosal immunity that also serves as a major entry cofactor for HIV-2 and simian immunodeficiency virus (SIV). To discover novel endogenous GPR15 ligands, we screened a hemofiltrate (HF)-derived peptide library for inhibitors of GPR15-mediated SIV infection. Our approach identified a C-terminal fragment of cystatin C (CysC95-146) that specifically inhibits GPR15-dependent HIV-1, HIV-2, and SIV infection. In contrast, GPR15L, the chemokine ligand of GPR15, failed to inhibit virus infection. We found that cystatin C fragments preventing GPR15-mediated viral entry do not interfere with GPR15L signaling and are generated by proteases activated at sites of inflammation. The antiretroviral activity of CysC95-146 was confirmed in primary CD4+ T cells and is conserved in simian hosts of SIV infection. Thus, we identified a potent endogenous inhibitor of GPR15-mediated HIV and SIV infection that does not interfere with the physiological function of this GPCR.
Asunto(s)
Cistatina C/genética , Infecciones por VIH/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Péptidos/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Animales , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/patogenicidad , Humanos , Receptores Virales/genética , Transducción de Señal/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/patología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Linfocitos T/metabolismo , Linfocitos T/virología , Internalización del VirusRESUMEN
BACKGROUND: Platelets, anucleate cell fragments abundant in human blood, can capture HIV-1 and platelet counts have been associated with viral load and disease progression. However, the impact of platelets on HIV-1 infection of T cells is unclear. RESULTS: We found that platelets suppress HIV-1 spread in co-cultured T cells in a concentration-dependent manner. Platelets containing granules inhibited HIV-1 spread in T cells more efficiently than degranulated platelets, indicating that the granule content might exert antiviral activity. Indeed, supernatants from activated and thus degranulated platelets suppressed HIV-1 infection. Infection was inhibited at the stage of host cell entry and inhibition was independent of the viral strain or coreceptor tropism. In contrast, blockade of HIV-2 and SIV entry was less efficient. The chemokine CXCL4, a major component of platelet granules, blocked HIV-1 entry and neutralization of CXCL4 in platelet supernatants largely abrogated their anti-HIV-1 activity. CONCLUSIONS: Release of CXCL4 by activated platelets inhibits HIV-1 infection of adjacent T cells at the stage of virus entry. The inhibitory activity of platelet-derived CXCL4 suggests a role of platelets in the defense against infection by HIV-1 and potentially other pathogens.
Asunto(s)
Linfocitos T CD4-Positivos/virología , VIH-1/inmunología , VIH-1/fisiología , Activación Plaquetaria , Factor Plaquetario 4/inmunología , Internalización del Virus/efectos de los fármacos , Células Cultivadas , HumanosRESUMEN
Access of four new tumor specific folic acid/ansamitocin conjugates is reported that relies on a synthetic strategy based on the combination of mutasynthesis and semisynthesis. Two bromo-ansamitocin derivatives were prepared by mutasynthesis or by a modified fermentation protocol, respectively, that served as starting point for the semisynthetic introduction of an allyl amine linker under Stille conditions. A sequence of standard coupling steps introduced the pteroic acid/glutamic acid/cysteine unit to the modified ansamitocins. All new derivatives, including those that are expected to be generated after internalization of the folic acid/ansamitocin conjugates into the cancer cell and reductive cleavage of the disulfide linkage showed good to strong antiproliferative activity (IC(50) <10â nM) for different cancer cell lines. Finally, the four conjugates were exposed to two cancer cell lines [cervix carcinoma, KB-3-1 (FR+) and lung carcinoma, A-459 (FR-)], the latter devoid of the membrane-bound folic acid receptor (FR-). All four conjugates showed strong antiproliferative activity for the FR+ cancer cell line but were inactive against the FR- cell line. The synthetic strategy pursued is based on the combination of mutasynthesis and semisynthesis and proved to be powerful for accessing new ansamitocin derivatives that are difficult to prepare by total synthesis.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Maitansina/análogos & derivados , Actinobacteria/metabolismo , Animales , Antineoplásicos/química , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Receptores de Folato Anclados a GPI/metabolismo , Ácido Fólico/análogos & derivados , Ácido Fólico/metabolismo , Células HCT116 , Humanos , Concentración 50 Inhibidora , Células KB , Masculino , Maitansina/síntesis química , Maitansina/química , Maitansina/farmacología , Ratones , Resonancia Magnética Nuclear Biomolecular , Sintasas Poliquetidas/metabolismoRESUMEN
Infection with Ebola virus (EBOV) causes hemorrhagic fever in humans with high case-fatality rates. The EBOV-glycoprotein (EBOV-GP) facilitates viral entry and promotes viral release from human cells. African fruit bats are believed not to develop disease upon EBOV infection and have been proposed as a natural reservoir of EBOV. We compared EBOV-GP interactions with human cells and cells from African fruit bats. We found that susceptibility to EBOV-GP-dependent infection was not limited to bat cells from potential reservoir species, and we observed that GP displayed similar biological properties in human and bat cells. The only exception was GP localization, which was to a greater extent intracellular in bat cells as compared to human cells. Collectively, our results suggest that GP interactions with fruit bat and human cells are similar and do not limit EBOV tropism for certain bat species.
Asunto(s)
Quirópteros , Ebolavirus/metabolismo , Glicoproteínas/metabolismo , Proteínas Virales/metabolismo , Animales , Células Cultivadas , Cricetinae , Reservorios de Enfermedades , Regulación Viral de la Expresión Génica/fisiología , Humanos , Especificidad de la Especie , Replicación ViralRESUMEN
BACKGROUND: Many HIV-2 and SIV isolates, as well as some HIV-1 strains, can use the orphan 7-transmembrane receptor GPR15 as co-receptor for efficient entry into host cells. GPR15 is expressed on central memory and effector memory CD4(+) T cells in healthy individuals and a subset of these cells is susceptible to HIV-1 and SIV infection. However, it has not been determined whether GPR15 expression is altered in the context of HIV-1 infection. RESULTS: Here, we show that GPR15 expression in CD4(+) T cells is markedly up-regulated in some HIV-1 infected individuals compared to the rest of the infected patients and to healthy controls. Infection of the PM1 T cell line with primary HIV-1 isolates was found to up-regulate GPR15 expression on the infected cells, indicating that viral components can induce GPR15 expression. Up-regulation of GPR15 expression on CD4(+) T cells was induced by activation of Toll-like receptor 3 signalling via TIR-domain-containing adapter-inducing interferon-ß (TRIF) and was more prominent on gut-homing compared to lymph node-homing CD4(+) T cells. CONCLUSION: These results suggest that infection-induced up-regulation of GPR15 expression could increase susceptibility of CD4(+) T cells to HIV infection and target cell availability in the gut in some infected individuals.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Infecciones por VIH/inmunología , VIH-1/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Receptor Toll-Like 3/metabolismo , Adulto , Antígenos CD19/metabolismo , Biopsia , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/citología , Separación Celular , Colon/metabolismo , Colon/patología , Femenino , Citometría de Flujo , Humanos , Memoria Inmunológica , Leucocitos Mononucleares/citología , Masculino , Persona de Mediana Edad , Transducción de Señal , Regulación hacia ArribaRESUMEN
Many SIV isolates can employ the orphan receptor GPR15 as coreceptor for efficient entry into transfected cell lines, but the role of endogenously expressed GPR15 in SIV cell tropism is largely unclear. Here, we show that several human B and T cell lines express GPR15 on the cell surface, including the T/B cell hybrid cell line CEMx174, and that GPR15 expression is essential for SIV infection of CEMx174 cells. In addition, GPR15 expression was detected on subsets of primary human CD4(+), CD8(+) and CD19(+) peripheral blood mononuclear cells (PBMCs), respectively. However, GPR15(+) PBMCs were not efficiently infected by HIV and SIV, including cells from individuals homozygous for the defective Δ32 ccr5 allele. These results suggest that GPR15 is coexpressed with CD4 on PBMCs but that infection of CD4(+), GPR15(+) cells is not responsible for the well documented ability of SIV to infect CCR5(-) blood cells.
Asunto(s)
Linfocitos B/virología , Receptores CCR5/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Péptidos/genética , Receptores Virales/genética , Virus de la Inmunodeficiencia de los Simios/fisiología , Linfocitos T/virología , Antígenos CD/metabolismo , Linfocitos B/metabolismo , Antagonistas de los Receptores CCR5 , Línea Celular , Humanos , ARN Interferente Pequeño/genética , Receptores CCR5/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/antagonistas & inhibidores , Receptores de Péptidos/metabolismo , Receptores Virales/antagonistas & inhibidores , Receptores Virales/metabolismo , Linfocitos T/metabolismo , Transfección , Tropismo Viral/fisiología , Internalización del Virus , Replicación Viral/fisiologíaRESUMEN
The interferon-induced host cell factor tetherin inhibits release of human immunodeficiency virus (HIV) from the plasma membrane of infected cells and is counteracted by the HIV-1 protein Vpu. Influenza A virus (FLUAV) also buds from the plasma membrane and is not inhibited by tetherin. Here, we investigated if FLUAV encodes a functional equivalent of Vpu for tetherin antagonism. We found that expression of the FLUAV protein NS1, which antagonizes the interferon (IFN) response, did not block the tetherin-mediated restriction of HIV release, which was rescued by Vpu. Similarly, tetherin-mediated inhibition of HIV release was not rescued by FLUAV infection. In contrast, FLUAV infection induced tetherin expression on target cells in an IFN-dependent manner. These results suggest that FLUAV escapes the antiviral effects of tetherin without encoding a tetherin antagonist with Vpu-like activity.