RESUMO
Adoptive T cell therapy using patient T cells redirected to recognize tumor-specific antigens by expressing genetically engineered high-affinity T-cell receptors (TCRs) has therapeutic potential for melanoma and other solid tumors. Clinical trials implementing genetically modified TCRs in melanoma patients have raised concerns regarding off-target toxicities resulting in lethal destruction of healthy tissue, highlighting the urgency of assessing which off-target peptides can be recognized by a TCR. As a model system we used the clinically efficacious NY-ESO-1-specific TCR C259, which recognizes the peptide epitope SLLMWITQC presented by HLA-A*02:01. We investigated which amino acids at each position enable a TCR interaction by sequentially replacing every amino acid position outside of anchor positions 2 and 9 with all 19 possible alternative amino acids, resulting in 134 peptides (133 altered peptides plus epitope peptide). Each peptide was individually evaluated using three different in vitro assays: binding of the NY-ESOc259 TCR to the peptide, peptide-dependent activation of TCR-expressing cells, and killing of peptide-presenting target cells. To represent the TCR recognition kernel, we defined Position Weight Matrices (PWMs) for each assay by assigning normalized measurements to each of the 20 amino acids in each position. To predict potential off-target peptides, we applied a novel algorithm projecting the PWM-defined kernel into the human proteome, scoring NY-ESOc259 TCR recognition of 336,921 predicted human HLA-A*02:01 binding 9-mer peptides. Of the 12 peptides with high predicted score, we confirmed 7 (including NY-ESO-1 antigen SLLMWITQC) strongly activate human primary NY-ESOc259-expressing T cells. These off-target peptides include peptides with up to 7 amino acid changes (of 9 possible), which could not be predicted using the recognition motif as determined by alanine scans. Thus, this replacement scan assay determines the "TCR fingerprint" and, when coupled with the algorithm applied to the database of human 9-mer peptides binding to HLA-A*02:01, enables the identification of potential off-target antigens and the tissues where they are expressed. This platform enables both screening of multiple TCRs to identify the best candidate for clinical development and identification of TCR-specific cross-reactive peptide recognition and constitutes an improved methodology for the identification of potential off-target peptides presented on MHC class I molecules.
Assuntos
Bioensaio , Epitopos de Linfócito T/análise , Ativação Linfocitária , Peptídeos/análise , Receptores de Antígenos/imunologia , Linfócitos T/imunologia , Linhagem Celular Tumoral , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Células HEK293 , Humanos , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Receptores de Antígenos/genética , Linfócitos T/citologiaRESUMO
Analyzing surface epitopes of single HIV particles holds great potential for the development of vaccine candidates. However, existing technologies do not allow corresponding screens at high throughput. We present here a single-virus droplet-based microfluidics platform enabling sorting of millions of HIV-1 particles with >99% efficiency, based on the expression of epitopes recognized by broadly neutralizing antibodies. We show that virus particles displaying these epitopes can be identified, sorted, and analyzed by next-generation sequencing: an approximately 1,900-fold enrichment of viral particles displaying neutralizing epitopes could be obtained in a single sort, thus opening the way for screening diverse virus libraries with optimal antigenic features for HIV vaccine candidates.
Assuntos
Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , HIV-1/imunologia , Ensaios de Triagem em Larga Escala/instrumentação , Dispositivos Lab-On-A-Chip , Vírion/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/químicaRESUMO
Droplet-based microfluidics enables assays to be carried out at very high throughput (up to thousands of samples per second) and enables researchers to work with very limited material, such as primary cells, patient's biopsies or expensive reagents. An additional strength of the technology is the possibility to perform large-scale genotypic or phenotypic screens at the single-cell level. Here we critically review the latest developments in antibody screening, drug discovery and highly multiplexed genomic applications such as targeted genetic workflows, single-cell RNAseq and single-cell ChIPseq. Starting with a comprehensive introduction for non-experts, we pinpoint current limitations, analyze how they might be overcome and give an outlook on exciting future applications.
Assuntos
Descoberta de Drogas/métodos , Perfilação da Expressão Gênica/métodos , Dispositivos Lab-On-A-Chip , Biologia Molecular/métodos , Animais , Descoberta de Drogas/instrumentação , Perfilação da Expressão Gênica/instrumentação , Humanos , Biologia Molecular/instrumentaçãoRESUMO
APOBEC3 proteins inhibit HIV-1 replication in experimental systems and induce hypermutation in infected patients; however, the relative contributions of several APOBEC3 proteins to restriction of HIV-1 replication in the absence of the viral Vif protein in human primary CD4(+) T cells and macrophages are unknown. We observed significant inhibition of HIV-1Δvif produced in 293T cells in the presence of APOBEC3DE (A3DE), APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H haplotype II (A3H HapII) but not APOBEC3B (A3B), APOBEC3C (A3C), or APOBEC3H haplotype I (A3H HapI). Our previous studies showed that Vif amino acids Y(40)RHHY(44) are important for inducing proteasomal degradation of A3G, whereas amino acids (14)DRMR(17) are important for degradation of A3F and A3DE. Here, we introduced substitution mutations of (40)YRHHY(44) and (14)DRMR(17) in replication-competent HIV-1 to generate vif mutants NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 to compare the antiviral activity of A3G to the combined antiviral activity of A3F and A3DE in activated CD4(+) T cells and macrophages. During the first 15 days (round 1), in which multiple cycles of viral replication occurred, both the NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 mutants replicated in activated CD4(+) T cells and macrophages, and only the NL4-3 YRHHY>A5 mutant showed a 2- to 4-day delay in replication compared to the wild type. During the subsequent 27 days (round 2) of cultures initiated with peak virus obtained from round 1, the NL4-3 YRHHY>A5 mutant exhibited a longer, 8- to 10-day delay and the NL4-3 DRMR>A4 mutant exhibited a 2- to 6-day delay in replication compared to the wild type. The NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 mutant proviruses displayed G-to-A hypermutations primarily in GG and GA dinucleotides as expected of A3G- and A3F- or A3DE-mediated deamination, respectively. We conclude that A3G exerts a greater restriction effect on HIV-1 than A3F and A3DE.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Citidina Desaminase/metabolismo , Citosina Desaminase/metabolismo , HIV-1/imunologia , Macrófagos/imunologia , Desaminase APOBEC-3G , Substituição de Aminoácidos , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , HIV-1/genética , HIV-1/fisiologia , Humanos , Macrófagos/virologia , Replicação Viral , Produtos do Gene vif do Vírus da Imunodeficiência Humana/deficiência , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genéticaRESUMO
Although xenotropic murine leukemia virus-related virus (XMRV) has been previously linked to prostate cancer and myalgic encephalomyelitis/chronic fatigue syndrome, recent data indicate that results interpreted as evidence of human XMRV infection reflect laboratory contamination rather than authentic in vivo infection. Nevertheless, XMRV is a retrovirus of undefined pathogenic potential that is able to replicate in human cells. Here we describe a comprehensive analysis of two male pigtailed macaques (Macaca nemestrina) experimentally infected with XMRV. Following intravenous inoculation with >10(10) RNA copy equivalents of XMRV, viral replication was limited and transient, peaking at ≤2,200 viral RNA (vRNA) copies/ml plasma and becoming undetectable by 4 weeks postinfection, though viral DNA (vDNA) in peripheral blood mononuclear cells remained detectable through 119 days of follow-up. Similarly, vRNA was not detectable in lymph nodes by in situ hybridization despite detectable vDNA. Sequencing of cell-associated vDNA revealed extensive G-to-A hypermutation, suggestive of APOBEC-mediated viral restriction. Consistent with limited viral replication, we found transient upregulation of type I interferon responses that returned to baseline by 2 weeks postinfection, no detectable cellular immune responses, and limited or no spread to prostate tissue. Antibody responses, including neutralizing antibodies, however, were detectable by 2 weeks postinfection and maintained throughout the study. Both animals were healthy for the duration of follow-up. These findings indicate that XMRV replication and spread were limited in pigtailed macaques, predominantly by APOBEC-mediated hypermutation. Given that human APOBEC proteins restrict XMRV infection in vitro, human XMRV infection, if it occurred, would be expected to be characterized by similarly limited viral replication and spread.
Assuntos
Modelos Animais de Doenças , Macaca nemestrina , Infecções por Retroviridae/virologia , Replicação Viral , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/fisiologia , Animais , Anticorpos Antivirais/imunologia , Humanos , Masculino , Filogenia , Infecções por Retroviridae/imunologia , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/classificação , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/genéticaRESUMO
Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation.
Assuntos
Peptídeo Hidrolases/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Fusão Celular , Linhagem Celular , Furina/genética , Furina/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Humanos , Leupeptinas , Mutação , Tripsina , Internalização do VírusRESUMO
Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus recently isolated from human prostate cancer and peripheral blood mononuclear cells (PBMCs) of patients with chronic fatigue syndrome (CFS). We and others have shown that host restriction factors APOBEC3G (A3G) and APOBEC3F (A3F), which are expressed in human PBMCs, inhibit XMRV in transient-transfection assays involving a single cycle of viral replication. However, the recovery of infectious XMRV from human PBMCs suggested that XMRV can replicate in these cells despite the expression of APOBEC3 proteins. To determine whether XMRV can replicate and spread in cultured PBMCs even though it can be inhibited by A3G/A3F, we infected phytohemagglutinin-activated human PBMCs and A3G/A3F-positive and -negative cell lines (CEM and CEM-SS, respectively) with different amounts of XMRV and monitored virus production by using quantitative real-time PCR. We found that XMRV efficiently replicated in CEM-SS cells and viral production increased by >4,000-fold, but there was only a modest increase in viral production from CEM cells (<14-fold) and a decrease in activated PBMCs, indicating little or no replication and spread of XMRV. However, infectious XMRV could be recovered from the infected PBMCs by cocultivation with a canine indicator cell line, and we observed hypermutation of XMRV genomes in PBMCs. Thus, PBMCs can potentially act as a source of infectious XMRV for spread to cells that express low levels of host restriction factors. Overall, these results suggest that hypermutation of XMRV in human PBMCs constitutes one of the blocks to replication and spread of XMRV. Furthermore, hypermutation of XMRV proviruses at GG dinucleotides may be a useful and reliable indicator of human PBMC infection.
Assuntos
Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Replicação Viral , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/imunologia , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/patogenicidade , Desaminase APOBEC-3G , Células Cultivadas , Citidina Desaminase/genética , Citidina Desaminase/imunologia , Citosina Desaminase/genética , Citosina Desaminase/imunologia , HumanosRESUMO
Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus reported to be associated with human prostate cancer and chronic fatigue syndrome. Since retroviruses cause various cancers, and XMRV replication might be facilitated by HIV-1 co-infection, we asked whether certain patients with HIV-associated lymphomas are infected with XMRV. Analysis of PMBCs and plasma from 26 patients failed to detect XMRV by PCR, ELISA, or Western blot, suggesting a lack of association between XMRV and AIDS-associated lymphomas.
RESUMO
Recent studies have shown that APOBEC3G (A3G), a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, is localized to cytoplasmic mRNA-processing bodies (P bodies). However, the functional relevance of A3G colocalization with P body marker proteins has not been established. To explore the relationship between HIV-1, A3G, and P bodies, we analyzed the effects of overexpression of P body marker proteins Mov10, DCP1a, and DCP2 on HIV-1 replication. Our results show that overexpression of Mov10, a putative RNA helicase that was previously reported to belong to the DExD superfamily and was recently reported to belong to the Upf1-like group of helicases, but not the decapping enzymes DCP1a and DCP2, leads to potent inhibition of HIV-1 replication at multiple stages. Mov10 overexpression in the virus producer cells resulted in reductions in the steady-state levels of the HIV-1 Gag protein and virus production; Mov10 was efficiently incorporated into virions and reduced virus infectivity, in part by inhibiting reverse transcription. In addition, A3G and Mov10 overexpression reduced proteolytic processing of HIV-1 Gag. The inhibitory effects of A3G and Mov10 were additive, implying a lack of functional interaction between the two inhibitors. Small interfering RNA (siRNA)-mediated knockdown of endogenous Mov10 by 80% resulted in a 2-fold reduction in virus production but no discernible impact on the infectivity of the viruses after normalization for the p24 input, suggesting that endogenous Mov10 was not required for viral infectivity. Overall, these results show that Mov10 can potently inhibit HIV-1 replication at multiple stages.
Assuntos
HIV-1/fisiologia , RNA Helicases/fisiologia , Replicação Viral/fisiologia , Desaminase APOBEC-3G , Linhagem Celular , Citidina Desaminase/fisiologia , Estruturas Citoplasmáticas/fisiologia , Estruturas Citoplasmáticas/virologia , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , HIV-1/genética , HIV-1/patogenicidade , Células HeLa , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Processamento de Proteína Pós-Traducional , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Viral/genética , RNA Viral/metabolismo , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/fisiologiaRESUMO
BACKGROUND: Platelets are associated with HIV in the blood of infected individuals and might modulate viral dissemination, particularly if the virus is directly transmitted into the bloodstream. The C-type lectin DC-SIGN and the novel HIV attachment factor CLEC-2 are expressed by platelets and facilitate HIV transmission from platelets to T-cells. Here, we studied the molecular mechanisms behind CLEC-2-mediated HIV-1 transmission. RESULTS: Binding studies with soluble proteins indicated that CLEC-2, in contrast to DC-SIGN, does not recognize the viral envelope protein, but a cellular factor expressed on kidney-derived 293T cells. Subsequent analyses revealed that the cellular mucin-like membranous glycoprotein podoplanin, a CLEC-2 ligand, was expressed on 293T cells and incorporated into virions released from these cells. Knock-down of podoplanin in 293T cells by shRNA showed that virion incorporation of podoplanin was required for efficient CLEC-2-dependent HIV-1 interactions with cell lines and platelets. Flow cytometry revealed no evidence for podoplanin expression on viable T-cells and peripheral blood mononuclear cells (PBMC). Podoplanin was also not detected on HIV-1 infected T-cells. However, apoptotic bystander cells in HIV-1 infected cultures reacted with anti-podoplanin antibodies, and similar results were obtained upon induction of apoptosis in a cell line and in PBMCs suggesting an unexpected link between apoptosis and podoplanin expression. Despite the absence of detectable podoplanin expression, HIV-1 produced in PBMC was transmitted to T-cells in a CLEC-2-dependent manner, indicating that T-cells might express an as yet unidentified CLEC-2 ligand. CONCLUSIONS: Virion incorporation of podoplanin mediates CLEC-2 interactions of HIV-1 derived from 293T cells, while incorporation of a different cellular factor seems to be responsible for CLEC-2-dependent capture of PBMC-derived viruses. Furthermore, evidence was obtained that podoplanin expression is connected to apoptosis, a finding that deserves further investigation.
Assuntos
Células Epiteliais/virologia , HIV-1/fisiologia , Lectinas Tipo C/metabolismo , Leucócitos Mononucleares/virologia , Glicoproteínas de Membrana/metabolismo , Ligação Viral , Células Cultivadas , HIV-1/química , Humanos , Vírion/químicaRESUMO
Xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus, has been isolated from human prostate cancer tissue and from activated CD4(+) T cells and B cells of patients with chronic fatigue syndrome, suggesting an association between XMRV infection and these two diseases. Since APOBEC3G (A3G) and APOBEC3F (A3F), which are potent inhibitors of murine leukemia virus and Vif-deficient human immunodeficiency virus type 1 (HIV-1), are expressed in human CD4(+) T cells and B cells, we sought to determine how XMRV evades suppression of replication by APOBEC3 proteins. We found that expression of A3G, A3F, or murine A3 in virus-producing cells resulted in their virion incorporation, inhibition of XMRV replication, and G-to-A hypermutation of the viral DNA with all three APOBEC3 proteins. Quantitation of A3G and A3F mRNAs indicated that, compared to the human T-cell lines CEM and H9, prostate cell lines LNCaP and DU145 exhibited 50% lower A3F mRNA levels, whereas A3G expression in 22Rv1, LNCaP, and DU145 cells was nearly undetectable. XMRV proviral genomes in LNCaP and DU145 cells were hypermutated at low frequency with mutation patterns consistent with A3F activity. XMRV proviral genomes were extensively hypermutated upon replication in A3G/A3F-positive T cells (CEM and H9), but not in A3G/A3F-negative cells (CEM-SS). We also observed that XMRV replication was susceptible to the nucleoside reverse transcriptase (RT) inhibitors zidovudine (AZT) and tenofovir and the integrase inhibitor raltegravir. In summary, the establishment of XMRV infection in patients may be dependent on infection of A3G/A3F-deficient cells, and cells expressing low levels of A3G/A3F, such as prostate cancer cells, may be ideal producers of infectious XMRV. Furthermore, the anti-HIV-1 drugs AZT, tenofovir, and raltegravir may be useful for treatment of XMRV infection.
Assuntos
Antivirais/farmacologia , Citidina Desaminase/imunologia , Gammaretrovirus/imunologia , Vírus da Leucemia Murina/imunologia , Neoplasias da Próstata/virologia , Infecções por Retroviridae/imunologia , Infecções Tumorais por Vírus/imunologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Linhagem Celular Tumoral , Citidina Desaminase/genética , Humanos , Masculino , Camundongos , Mutação , Organofosfonatos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Pirrolidinonas/farmacologia , Raltegravir Potássico , Infecções por Retroviridae/tratamento farmacológico , Tenofovir , Infecções Tumorais por Vírus/tratamento farmacológico , Zidovudina/farmacologiaRESUMO
Proteolytic activation of the hemagglutinin (HA) protein is indispensable for influenza virus infectivity, and the tissue expression of the responsible cellular proteases impacts viral tropism and pathogenicity. The HA protein critically contributes to the exceptionally high pathogenicity of the 1918 influenza virus, but the mechanisms underlying cleavage activation of the 1918 HA have not been characterized. The neuraminidase (NA) protein of the 1918 influenza virus allows trypsin-independent growth in canine kidney cells (MDCK). However, it is at present unknown if the 1918 NA, like the NA of the closely related strain A/WSN/33, facilitates HA cleavage activation by recruiting the proprotease plasminogen. Moreover, it is not known which pulmonary proteases activate the 1918 HA. We provide evidence that NA-dependent, trypsin-independent cleavage activation of the 1918 HA is cell line dependent and most likely plasminogen independent since the 1918 NA failed to recruit plasminogen and neither exogenous plasminogen nor the presence of the A/WSN/33 NA promoted efficient cleavage of the 1918 HA. The transmembrane serine protease TMPRSS4 was found to be expressed in lung tissue and was shown to cleave the 1918 HA. Accordingly, coexpression of the 1918 HA with TMPRSS4 or the previously identified HA-processing protease TMPRSS2 allowed trypsin-independent infection by pseudotypes bearing the 1918 HA, indicating that these proteases might support 1918 influenza virus spread in the lung. In summary, we show that the previously reported 1918 NA-dependent spread of the 1918 influenza virus is a cell line-dependent phenomenon and is not due to plasminogen recruitment by the 1918 NA. Moreover, we provide evidence that TMPRSS2 and TMPRSS4 activate the 1918 HA by cleavage and therefore may promote viral spread in lung tissue.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/fisiologia , Linhagem Celular , Humanos , Proteínas de Membrana/metabolismo , Neuraminidase/metabolismo , Plasminogênio/metabolismo , Serina Endopeptidases/metabolismoRESUMO
Plasmacytoid dendritic cells (PDC) are major producers of type I interferons (IFN) in response to human immunodeficiency virus type 1 (HIV-1) infection. To better define the underlying mechanisms, we studied the magnitude of alpha IFN (IFN-alpha) induction by recombinant viruses containing changes in the Env protein that impair or disrupt CD4 binding or expressing primary env alleles with differential coreceptor tropism. We found that the CD4 binding affinity but not the viral coreceptor usage is critical for the attachment of autofluorescing HIV-1 to PDC and for subsequent IFN-alpha induction. Our results illustrate the importance of the gp120-CD4 interaction in determining HIV-1-induced immune stimulation via IFN-alpha production.
Assuntos
Antígenos CD4/imunologia , Células Dendríticas/imunologia , HIV-1/imunologia , Interferon-alfa/biossíntese , Adolescente , Adulto , Antígenos CD4/metabolismo , Linhagem Celular , Células Cultivadas , Criança , HIV-1/isolamento & purificação , HIV-1/fisiologia , Humanos , Rim/citologia , Pessoa de Meia-Idade , Transfecção , Replicação ViralRESUMO
The C-type lectin DC-SIGN binds to oligosaccharides on the human and simian immunodeficiency virus (HIV, SIV) envelope glycoproteins and promotes infection of susceptible cells. Here, we show that DC-SIGN recognizes glycans involved in SIV sensitivity to neutralizing antibodies and that binding to DC-SIGN confers neutralization resistance to an otherwise sensitive SIV variant. Moreover, we provide evidence that mannose-binding lectin (MBL) can interfere with HIV-1 neutralization by the carbohydrate-specific antibody 2G12.
Assuntos
Anticorpos Antivirais/imunologia , Moléculas de Adesão Celular/metabolismo , Anticorpos Anti-HIV/imunologia , HIV/patogenicidade , Lectinas Tipo C/metabolismo , Lectina de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo , Vírus da Imunodeficiência Símia/patogenicidade , Animais , Linhagem Celular , Glicosilação , HIV/química , HIV/imunologia , HIV/metabolismo , Infecções por HIV/transmissão , Infecções por HIV/virologia , Humanos , Testes de Neutralização , Polissacarídeos/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/transmissão , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/química , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/metabolismoRESUMO
A variety of molecules in human blood have been implicated in the inhibition of HIV-1. However, it remained elusive which circulating natural compounds are most effective in controlling viral replication in vivo. To identify natural HIV-1 inhibitors we screened a comprehensive peptide library generated from human hemofiltrate. The most potent fraction contained a 20-residue peptide, designated VIRUS-INHIBITORY PEPTIDE (VIRIP), corresponding to the C-proximal region of alpha1-antitrypsin, the most abundant circulating serine protease inhibitor. We found that VIRIP inhibits a wide variety of HIV-1 strains including those resistant to current antiretroviral drugs. Further analysis demonstrated that VIRIP blocks HIV-1 entry by interacting with the gp41 fusion peptide and showed that a few amino acid changes increase its antiretroviral potency by two orders of magnitude. Thus, as a highly specific natural inhibitor of the HIV-1 gp41 fusion peptide, VIRIP may lead to the development of another class of antiretroviral drugs.
Assuntos
Proteína gp41 do Envelope de HIV/metabolismo , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Internalização do Vírus/efeitos dos fármacos , alfa 1-Antitripsina/farmacologia , Sequência de Aminoácidos , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Proteína gp41 do Envelope de HIV/química , Inibidores da Fusão de HIV/química , Inibidores da Fusão de HIV/metabolismo , HIV-1/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Replicação Viral , alfa 1-Antitripsina/química , alfa 1-Antitripsina/metabolismoRESUMO
Platelets can engulf human immunodeficiency virus type 1 (HIV-1), and a significant amount of HIV-1 in the blood of infected individuals is associated with these cells. However, it is unclear how platelets capture HIV-1 and whether platelet-associated virus remains infectious. DC-SIGN and other lectins contribute to capture of HIV-1 by dendritic cells (DCs) and facilitate HIV-1 spread in DC/T-cell cocultures. Here, we show that platelets express both the C-type lectin-like receptor 2 (CLEC-2) and low levels of DC-SIGN. CLEC-2 bound to HIV-1, irrespective of the presence of the viral envelope protein, and facilitated HIV-1 capture by platelets. However, a substantial fraction of the HIV-1 binding activity of platelets was dependent on DC-SIGN. A combination of DC-SIGN and CLEC-2 inhibitors strongly reduced HIV-1 association with platelets, indicating that these lectins are required for efficient HIV-1 binding to platelets. Captured HIV-1 was maintained in an infectious state over several days, suggesting that HIV-1 can escape degradation by platelets and might use these cells to promote its spread. Our results identify CLEC-2 as a novel HIV-1 attachment factor and provide evidence that platelets capture and transfer infectious HIV-1 via DC-SIGN and CLEC-2, thereby possibly facilitating HIV-1 dissemination in infected patients.
Assuntos
Plaquetas/virologia , Moléculas de Adesão Celular/fisiologia , HIV-1/metabolismo , Lectinas Tipo C/fisiologia , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Células Dendríticas/virologia , Infecções por HIV/sangue , Humanos , Células Jurkat , Leucócitos Mononucleares/virologia , Megacariócitos/virologiaRESUMO
We have recently demonstrated that the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor angiotensin converting enzyme 2 (ACE2) also mediates cellular entry of the newly discovered human coronavirus (hCoV) NL63. Here, we show that expression of DC-SIGN augments NL63 spike (S)-protein-driven infection of susceptible cells, while only expression of ACE2 but not DC-SIGN is sufficient for entry into nonpermissive cells, indicating that ACE2 fulfills the criteria of a bona fide hCoV-NL63 receptor. As for SARS-CoV, murine ACE2 is used less efficiently by NL63-S for entry than human ACE2. In contrast, several amino acid exchanges in human ACE2 which diminish SARS-S-driven entry do not interfere with NL63-S-mediated infection, suggesting that SARS-S and NL63-S might engage human ACE2 differentially. Moreover, we observed that NL63-S-driven entry was less dependent on a low-pH environment and activity of endosomal proteases compared to infection mediated by SARS-S, further suggesting differences in hCoV-NL63 and SARS-CoV cellular entry. NL63-S does not exhibit significant homology to SARS-S but is highly related to the S-protein of hCoV-229E, which enters target cells by engaging CD13. Employing mutagenic analyses, we found that the N-terminal unique domain in NL63-S, which is absent in 229E-S, does not confer binding to ACE2. In contrast, the highly homologous C-terminal parts of the NL63-S1 and 229E-S1 subunits in conjunction with distinct amino acids in the central regions of these proteins confer recognition of ACE2 and CD13, respectively. Therefore, despite the high homology of these sequences, they likely form sufficiently distinct surfaces, thus determining receptor specificity.
Assuntos
Coronavirus Humano 229E/patogenicidade , Coronavirus/patogenicidade , Glicoproteínas de Membrana/química , Peptidil Dipeptidase A/metabolismo , Receptores Virais/metabolismo , Proteínas do Envelope Viral/química , Enzima de Conversão de Angiotensina 2 , Animais , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Coronavirus/metabolismo , Coronavirus/fisiologia , Coronavirus Humano 229E/metabolismo , Coronavirus Humano 229E/fisiologia , Cricetinae , Humanos , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Receptores de Superfície Celular/metabolismo , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
The lectins DC-SIGN and DC-SIGNR augment infection by human immunodeficiency virus (HIV), Ebolavirus (EBOV) and other pathogens. The neck domain of these proteins drives multimerization, which is believed to be required for efficient recognition of multivalent ligands. The neck domain of DC-SIGN consists of seven sequence repeats with rare variations. In contrast, the DC-SIGNR neck domain is polymorphic and, in addition to the wild type (wt) allele with seven repeat units, allelic forms with five and six sequence repeats are frequently found. A potential association of the DC-SIGNR genotype and risk of HIV-1 infection is currently under debate. Therefore, we investigated if DC-SIGNR alleles with five and six repeat units exhibit defects in pathogen capture. Here, we show that wt DC-SIGNR and patient derived alleles with five and six repeats bind viral glycoproteins, augment viral infection and tetramerize with comparable efficiency. Moreover, coexpression of wt DC-SIGNR and alleles with five repeats did not decrease the interaction with pathogens compared to expression of each allele alone, suggesting that potential formation of hetero-oligomers does not appreciably reduce pathogen binding, at least under conditions of high expression. Thus, our results do not provide evidence for diminished pathogen capture by DC-SIGNR alleles with five and six repeat units. Albeit, we cannot exclude that subtle, but in vivo relevant differences remained undetected, our analysis suggests that indirect mechanisms could account for the association of polymorphisms in the DC-SIGNR neck region with reduced risk of HIV-1 infection.