RESUMO
Over half of individuals infected with human immunodeficiency virus (HIV) suffer from HIV-associated neurocognitive disorders (HANDs), yet the molecular mechanisms leading to neuronal dysfunction are poorly understood. Feline immunodeficiency virus (FIV) naturally infects cats and shares its structure, cell tropism, and pathology with HIV, including wide-ranging neurological deficits. We employ FIV as a model to elucidate the molecular pathways underlying HIV-induced neuronal dysfunction, in particular, synaptic alteration. Among HIV-induced neuron-damaging products, HIV envelope glycoprotein gp120 triggers elevation of intracellular Ca2+ activity in neurons, stimulating various pathways to damage synaptic functions. We quantify neuronal Ca2+ activity using intracellular Ca2+ imaging in cultured hippocampal neurons and confirm that FIV envelope glycoprotein gp95 also elevates neuronal Ca2+ activity. In addition, we reveal that gp95 interacts with the chemokine receptor, CXCR4, and facilitates the release of intracellular Ca2+ by the activation of the endoplasmic reticulum (ER)-associated Ca2+ channels, inositol triphosphate receptors (IP3Rs), and synaptic NMDA receptors (NMDARs), similar to HIV gp120. This suggests that HIV gp120 and FIV gp95 share a core pathological process in neurons. Significantly, gp95's stimulation of NMDARs activates cGMP-dependent protein kinase II (cGKII) through the activation of the neuronal nitric oxide synthase (nNOS)-cGMP pathway, which increases Ca2+ release from the ER and promotes surface expression of AMPA receptors, leading to an increase in synaptic activity. Moreover, we culture feline hippocampal neurons and confirm that gp95-induced neuronal Ca2+ overactivation is mediated by CXCR4 and cGKII. Finally, cGKII activation is also required for HIV gp120-induced Ca2+ hyperactivation. These results thus provide a novel neurobiological mechanism of cGKII-mediated synaptic hyperexcitation in HAND.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo II/metabolismo , Síndrome de Imunodeficiência Adquirida Felina/virologia , HIV-1/fisiologia , Vírus da Imunodeficiência Felina/fisiologia , Sinapses/metabolismo , Animais , Cálcio/metabolismo , Gatos , Quimiocina CXCL12/farmacologia , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/metabolismo , Hipocampo/patologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Subunidades Proteicas/metabolismo , Receptores de AMPA/metabolismo , Proteínas Virais/metabolismoRESUMO
The mature HIV-1 reverse transcriptase is a heterodimer that comprises 66â¯kDa (p66) and 51â¯kDa (p51) subunits. The latter is formed by HIV-1 protease-catalyzed removal of a C-terminal ribonuclease H domain from a p66 subunit. This proteolytic processing is a critical step in virus maturation and essential for viral infectivity. Here, we report that tRNA significantly enhances in vitro processing even at a substoichiometric tRNA:p66/p66 ratio. Other double-stranded RNAs have considerably less pronounced effect. Our data support a model where interaction of p66/p66 with tRNA introduces conformational asymmetry in the two subunits, permitting specific proteolytic processing of one p66 to provide the mature RT p66/p51 heterodimer.
Assuntos
Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , RNA de Transferência/metabolismo , Transcriptase Reversa do HIV/química , HIV-1/genética , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Proteólise , RNA Viral/metabolismo , Ribonuclease H/metabolismoRESUMO
Secreted microvesicles (MVs) are potent inflammatory triggers that stimulate autoreactive B and T cells, causing Type 1 Diabetes in non-obese diabetic (NOD) mice. Proteomic analysis of purified MVs released from islet cells detected the presence of endogenous retrovirus (ERV) antigens, including Env and Gag sequences similar to the well-characterized murine leukemia retroviruses. This raises the possibility that ERV antigens may be expressed in the pancreatic islets via MV secretion. Using virus-like particles produced by co-expressing ERV Env and Gag antigens, and a recombinant gp70 Env protein, we demonstrated that NOD but not diabetes-resistant mice developed anti-Env autoantibodies that increase in titer as disease progresses. A lentiviral-based RNA interference knockdown of Gag revealed that Gag contributes to the MV-induced T-cell response, whose diabetogenic function can be demonstrated via cell-transfer into immune-deficient mice. Finally, we observed that Gag and Env are expressed in NOD islet-derived primary mesenchymal stem cells (MSCs). However, MSCs derived from the islets of diabetes-resistant mice do not express the antigens. Taken together, abnormal ERV activation and secretion of MVs may induce anti-retroviral responses to trigger autoimmunity.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Retrovirus Endógenos/imunologia , Produtos do Gene env/metabolismo , Produtos do Gene gag/metabolismo , Ilhotas Pancreáticas/imunologia , Células-Tronco Mesenquimais/metabolismo , Linfócitos T/imunologia , Transferência Adotiva , Animais , Autoanticorpos/sangue , Autoimunidade , Micropartículas Derivadas de Células/imunologia , Células Cultivadas , Feminino , Produtos do Gene env/genética , Produtos do Gene gag/genética , Humanos , Ilhotas Pancreáticas/metabolismo , Ativação Linfocitária , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , RNA Interferente Pequeno/genética , Linfócitos T/transplanteRESUMO
The purpose of this study was to assess humoral antibody responses as a function of disease progression (DP) in a well-defined HIV+ cohort. We quantified antibodies to HIV-1 gp120, Gag, and CD4 receptor by enzyme-linked immunosorbent assay in sera from a cohort of 97 HIV+ subjects at defined stages of DP. We also measured antibody-dependent cellular cytotoxicity (ADCC) as a function of the clinical status of the patients. We purified antibodies to CD4 and gp120 and assessed them for specificity, ability to block gp120 binding to target cells, ability to block virus infection, and ability to facilitate ADCC. All of the HIV+ patient samples were positive for antibodies to HIV gp120 and p24 and 80% showed evidence of hypergammaglobulinemia. Approximately 10% of cohort members were positive for antibodies to CD4, but we noted no significant correlation relevant to DP. There were statistically significant differences between the groups concerning the level of humoral response to gp120 and Gag. However, we observed no distinction in ability of anti-gp120 antibodies purified from each group to neutralize infection. In addition, there was a statistically significant difference in ADCC, with elite controllers exhibiting significantly lower levels of ADCC than the other five groups. We detected IgA anti-gp120 antibodies, but did not correlate their presence with either DP or ADCC levels. The results are consistent with the interpretation that the humoral antibody response to the antigens assessed here represents a signature of the level of viremia but does not correlate with clinical status of HIV infection.
Assuntos
Formação de Anticorpos , Autoanticorpos/sangue , Antígenos CD4/imunologia , Progressão da Doença , Anticorpos Anti-HIV/sangue , Infecções por HIV/imunologia , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Autoanticorpos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Anticorpos Anti-HIV/imunologia , Infecções por HIV/patologia , Humanos , Masculino , Estudos Prospectivos , Fatores de TempoRESUMO
Diagnosis of KSHV-infected individuals remains a challenge. KSHV prevalence is high in several populations with high prevalence of HIV, leading to increased risk of development of Kaposi's sarcoma (KS). While current assays are reliable for detecting antibodies to KSHV, none are routinely utilized to identify individuals with KSHV infection and thus at increased risk for KS due to assay complexity, lack of access to testing, and cost, particularly in resource-limited settings. Here we describe the addition of KSHV proteins LANA and K8.1 to a previously evaluated HIV/co-infection multiplexed fluorescence immunoassay system. This study demonstrates assay performance by measuring antibody reactivity for KSHV and HIV-1 in a collection of clinical specimens from patients with biopsy-proven KS and sourced negative controls. The KSHV assay correctly identified 155 of 164 plasma samples from patients with biopsy-proven KS and 85 of 93 KSHV antibody (Ab)-negative samples for a sensitivity of 95.1% and specificity of 91.4%. Assay performance for HIV-1 detection was also assessed with 100% agreement with independently verified HIV-1 Ab-positive and Ab-negative samples. These results demonstrate good sensitivity and specificity for detection of antibody to KSHV antigens, and demonstrate the potential for multiplexed co-infection testing in resource-limited settings to identify those at increased risk for HIV-1-related complications.
RESUMO
Heparan sulfate proteoglycans (HSPG) can act as binding receptors for certain laboratory-adapted (TCA) strains of feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV). Heparin, a soluble heparin sulfate (HS), can inhibit TCA HIV and FIV entry mediated by HSPG interaction in vitro. In the present study, we further determined the selective interaction of heparin with the V3 loop of TCA of FIV. Our current results indicate that heparin selectively inhibits infection by TCA strains, but not for field isolates (FS). Heparin also specifically interferes with TCA surface glycoprotein (SU) binding to CXCR4, by interactions with HSPG binding sites on the V3 loop of the FIV envelope protein. Peptides representing either the N- or C-terminal side of the V3 loop and containing HSPG binding sites were able to compete away the heparin block of TCA SU binding to CXCR4. Heparin does not interfere with the interaction of SU with anti-V3 antibodies that target the CXCR4 binding region or with the interaction between FS FIV and anti-V3 antibodies since FS SU has no HSPG binding sites within the HSPG binding region. Our data show that heparin blocks TCA FIV infection or entry not only through its competition of HSPG on the cell surface interaction with SU, but also by its interference with CXCR4 binding to SU. These studies aid in the design and development of heparin derivatives or analogues that can inhibit steps in virus infection and are informative regarding the HSPG/SU interaction.
Assuntos
Proteoglicanas de Heparan Sulfato/metabolismo , Heparina/farmacologia , Vírus da Imunodeficiência Felina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Gatos , Linhagem Celular , Proteoglicanas de Heparan Sulfato/química , Vírus da Imunodeficiência Felina/genética , Dados de Sequência Molecular , Ligação Proteica , Receptores CXCR4/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismoRESUMO
A library of 68 brominated fragments was screened against a new crystal form of inhibited HIV-1 protease in order to probe surface sites in soaking experiments. Often, fragments are weak binders with partial occupancy, resulting in weak, difficult-to-fit electron density. The use of a brominated fragment library addresses this challenge, as bromine can be located unequivocally via anomalous scattering. Data collection was carried out in an automated fashion using AutoDrug at SSRL. Novel hits were identified in the known surface sites: 3-bromo-2,6-dimethoxybenzoic acid (Br6) in the flap site and 1-bromo-2-naphthoic acid (Br27) in the exosite, expanding the chemistry of known fragments for development of higher affinity potential allosteric inhibitors. At the same time, mapping the binding sites of a number of weaker binding Br-fragments provides further insight into the nature of these surface pockets.
Assuntos
Bromo/química , Protease de HIV/química , HIV-1/enzimologia , Inibidores de Proteases/química , Bibliotecas de Moléculas Pequenas/química , Regulação Alostérica , Sítios de Ligação , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Protease de HIV/genética , Protease de HIV/metabolismo , Humanos , Éteres de Hidroxibenzoatos/química , Simulação de Acoplamento Molecular , Naftalenos/química , Inibidores de Proteases/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
Microsphere immunoassays (MIAs) allow rapid and accurate evaluation of multiple analytes simultaneously within a biological sample. Here we describe the development and validation of domestic cat-specific MIAs for a) the quantification of total IgG and IgA levels in plasma, and b) the detection of IgG and IgA antibodies to feline immunodeficiency virus (FIV) capsid (CA) and surface (SU) proteins, and feline CD134 in plasma. These assays were used to examine the temporal antibody response of domestic cats infected with apathogenic and pathogenic FIVs, and domestic cats infected with parental and chimeric FIVs of varying pathogenicity. The results from these studies demonstrated that a) total IgG antibodies increase over time after infection; b) α-CA and α-SU IgG antibodies are detectable between 9 and 28 days post-infection and increase over time, and these antibodies combined represent a fraction (1.8 to 21.8%) of the total IgG increase due to infection; c) measurable α-CD134 IgG antibody levels vary among individuals and over time, and are not strongly correlated with viral load; d) circulating IgA antibodies, in general, do not increase during the early stage of infection; and e) total IgG, and α-CA and α-SU IgG antibody kinetics and levels vary with FIV viral strain/pathogenicity. The MIAs described here could be used to screen domestic cats for FIV infection, and to evaluate the FIV-specific or total antibody response elicited by various FIV strains/other diseases.
Assuntos
Anticorpos Antivirais/sangue , Síndrome de Imunodeficiência Adquirida Felina/diagnóstico , Síndrome de Imunodeficiência Adquirida Felina/imunologia , Vírus da Imunodeficiência Felina/imunologia , Animais , Anticorpos Antivirais/imunologia , Antígenos de Superfície/imunologia , Proteínas do Capsídeo/imunologia , Gatos , Síndrome de Imunodeficiência Adquirida Felina/sangue , Imunoensaio/métodos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Microesferas , Receptores OX40/sangue , Receptores OX40/imunologia , Carga ViralRESUMO
Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between two FIV molecular clones with divergent disease potentials: a highly pathogenic isolate that replicates rapidly in vitro and is associated with significant immunopathology in vivo, FIV-C36 (referred to here as high-virulence FIV [HV-FIV]), and a less-pathogenic strain, FIV-PPR (referred to here as low-virulence FIV [LV-FIV]). Using PCR-driven overlap extension, we produced viruses in which vif, orfA, or both genes from virulent HV-FIV replaced equivalent genes in LV-FIV. The generation of these chimeras is more straightforward in FIV than in primate lentiviruses, since FIV accessory gene open reading frames have very little overlap with other genes. All three chimeric viruses exhibited increased replication kinetics in vitro compared to the replication kinetics of LV-FIV. Chimeras containing HV-Vif or Vif/OrfA had replication rates equivalent to those of the virulent HV-FIV parental virus. Furthermore, small interfering RNA knockdown of feline APOBEC3 genes resulted in equalization of replication rates between LV-FIV and LV-FIV encoding HV-FIV Vif. These findings demonstrate that Vif-APOBEC interactions play a key role in controlling the replication and pathogenicity of this immunodeficiency-inducing virus in its native host species and that accessory genes act as mediators of lentiviral strain-specific virulence.
Assuntos
Gatos/virologia , Citosina Desaminase/metabolismo , Produtos do Gene vif/metabolismo , Vírus da Imunodeficiência Felina/patogenicidade , Proteínas Virais Reguladoras e Acessórias/metabolismo , Replicação Viral/fisiologia , Análise de Variância , Animais , Linhagem Celular , Quimera/virologia , Primers do DNA/genética , Produtos do Gene vif/fisiologia , Células HEK293 , Humanos , Vírus da Imunodeficiência Felina/fisiologia , Reação em Cadeia da Polimerase , Interferência de RNA , Receptores OX40/metabolismo , Especificidade da Espécie , Proteínas Virais Reguladoras e Acessórias/fisiologia , VirulênciaRESUMO
An infectious chimeric feline immunodeficiency virus (FIV)/HIV strain carrying six HIV-like protease (PR) mutations (I37V/N55M/V59I/I98S/Q99V/P100N) was subjected to selection in culture against the PR inhibitor lopinavir (LPV), darunavir (DRV), or TL-3. LPV selection resulted in the sequential emergence of V99A (strain S-1X), I59V (strain S-2X), and I108V (strain S-3X) mutations, followed by V37I (strain S-4X). Mutant PRs were analyzed in vitro, and an isogenic virus producing each mutant PR was analyzed in culture for LPV sensitivity, yielding results consistent with the original selection. The 50% inhibitory concentrations (IC50s) for S-1X, S-2X, S-3X, and S-4X were 95, 643, 627, and 1,543 nM, respectively. The primary resistance mutations, V99(82)A, I59(50)V, and V37(32)I, are consistent with the resistance pattern developed by HIV-1 under similar selection conditions. While resistance to LPV emerged readily, similar PR mutations causing resistance to either DRV or TL-3 failed to emerge after passage for more than a year. However, a G37D mutation in the nucleocapsid (NC) was observed in both selections and an isogenic G37D mutant replicated in the presence of 100 nM DRV or TL-3, whereas parental chimeric FIV could not. An additional mutation, L92V, near the PR active site in the folded structure recently emerged during TL-3 selection. The L92V mutant PR exhibited an IC50 of 50 nM, compared to 35 nM for 6s-98S PR, and processed the NC-p2 junction more efficiently, consistent with increased viral fitness. These findings emphasize the role of mutations outside the active site of PR in increasing viral resistance to active-site inhibitors and suggest additional targets for inhibitor development.
Assuntos
Farmacorresistência Viral , Inibidores da Protease de HIV/farmacologia , Protease de HIV/metabolismo , HIV-1/enzimologia , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Mutação de Sentido Incorreto , Seleção Genética , Análise Mutacional de DNA , Protease de HIV/genética , HIV-1/genética , Vírus da Imunodeficiência Felina/genética , Vírus da Imunodeficiência Felina/crescimento & desenvolvimento , Vírus da Imunodeficiência Felina/isolamento & purificação , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Inoculações SeriadasRESUMO
The fragment indole-6-carboxylic acid (1F1), previously identified as a flap site binder in a fragment-based screen against HIV protease (PR), has been cocrystallized with pepstatin-inhibited PR and with apo-PR. Another fragment, 3-indolepropionic acid (1F1-N), predicted by AutoDock calculations and confirmed in a novel inhibition of nucleation crystallization assay, exploits the same interactions in the flap site in two crystal structures. Both 1F1 and 1F1-N bind to the closed form of apo-PR and to pepstatin:PR. In solution, 1F1 and 1F1-N raise the Tm of apo-PR by 3.5-5 °C as assayed by differential scanning fluorimetry (DSF) and show equivalent low-micromolar binding constants to both apo-PR and pepstatin:PR, assayed by backscattering interferometry (BSI). The observed signal intensities in BSI are greater for each fragment upon binding to apo-PR than to pepstatin-bound PR, consistent with greater conformational change in the former binding event. Together, these data indicate that fragment binding in the flap site favors a closed conformation of HIV PR.
Assuntos
Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , Protease de HIV/química , HIV-1/enzimologia , Conformação Proteica/efeitos dos fármacos , Cristalografia por Raios X , Infecções por HIV/tratamento farmacológico , Infecções por HIV/enzimologia , Infecções por HIV/virologia , Protease de HIV/metabolismo , HIV-1/efeitos dos fármacos , Humanos , Indóis/química , Indóis/farmacologia , Simulação de Acoplamento Molecular , Pepstatinas/química , Pepstatinas/farmacologia , Propionatos/química , Propionatos/farmacologiaRESUMO
Similar to HIV, FIV uses a two-receptor mechanism to infect CD4(+) T cells, the primary target cells in the cat. The T cell activation marker, CD134, serves as a primary binding receptor similar to the role of CD4 for HIV and facilitates interaction with the entry receptor, CXCR4. Heparan sulfate proteoglycans (HSPG) can also act as binding receptors for certain tissue culture adapted FIV and HIV isolates. In the present study, we employed site-directed mutagenesis to investigate the importance of specific residues on the FIV envelope for CD134 and HSPG interactions. We show that certain mutations that disrupt CD134 interactions facilitate HSPG binding by FIV-PPR. In particular, an E407K mutation at the base of the V3 loop knocks out CD134 binding; enhances HSPG binding; and in combination with additional Env mutations E656K and V817I increases entry into CD134(-), CXCR4(+) target cells by greater than 80-fold over wild type FIV-PPR. The CD134-independent mutant, termed FIV-PPRcr, exhibits a broadened host cell range, but also becomes readily susceptible to CD134-dependent neutralizing monoclonal antibodies. The findings are consistent with the notion that FIV-PPRcr Env has an "open" conformation that readily associates with CXCR4 directly, similar to wild type FIV-PPR Env after CD134 binding. The findings highlight the utility of a two-receptor mechanism that allows FIV V3 residues critical for CXCR4 binding to remain cryptic until reaction occurs with the primary binding receptor, thus thwarting immune surveillance.
RESUMO
Chimeric viruses constructed between a highly pathogenic Feline Immunodeficiency Virus isolate (FIV-C36) and a less pathogenic but neurotropic strain (FIV-PPR) have been used to map viral genetic determinants of in vivo pathogenicity. Chimeric virus FIV-PCenv, which contains FIV-C36 genome from the 3' region of pol to upstream of the 3'LTR on an FIV-PPR backbone, was previously shown to be replication-competent in vivo, inducing altered CD4(+) T-cell and neutrophil profiles intermediate between parental strains following a delay in viral replication during initial infection. Examination of FIV-PCenv proviral sequences recovered at week 11 post-infection revealed two changes compared to initial viral inoculum; the most significant being arginine to histidine in the integrase region of Pol at residue 813 (R813H). Pooled plasma from the initial in vivo study was used to inoculate a second cohort of cats to determine whether similar virulence and kinetics could be established following primary infection. Viral replication kinetics and immunocyte profiles were monitored in blood, bone marrow, and saliva over a one-year period. Passaged FIV-PCenv again displayed intermediate phenotype between parental strains, but unlike primary experiments, the onset of acute viremia was not delayed. CD4/8 alterations were noted in all groups of animals, though significant changes from controls were delayed in FIV-PPR infected animals compared to FIV-C36 and FIV-PCenv. In vivo passage of FIV-PCenv increased replication-competence relative to the initial molecularly-cloned chimera in association with one adaptive nucleotide change in the 5' end of the genome relative to primary tissue culture inoculum, while mutations in the 3' end of the genome were not detected. The results are consistent with the interpretation that 3' elements contribute to heightened virulence of FIV-C36, and that integrase residue 813 plays an important role in facilitating successful in vivo replication.
Assuntos
Região 3'-Flanqueadora/genética , Vírus da Imunodeficiência Felina/crescimento & desenvolvimento , Vírus da Imunodeficiência Felina/patogenicidade , Animais , Sangue/virologia , Medula Óssea/virologia , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Gatos , Genes Virais , Integrases , Cinética , Saliva/virologia , Replicação ViralRESUMO
HIV infection results in a highly prevalent syndrome of cognitive and motor disorders designated as HIV-associated dementia (HAD). Neurologic dysfunction resembling HAD has been documented in cats infected with strain PPR of the feline immunodeficiency virus (FIV), whereas another highly pathogenic strain (C36) has not been known to cause neurologic signs. Animals experimentally infected with equivalent doses of FIV-C36 or FIV-PPR, and uninfected controls were evaluated by magnetic resonance diffusion-weighted imaging (DW-MRI) and spectroscopy (MRS) at 17.5-18 weeks post-infection, as part of a study of viral clade pathogenesis in FIV-infected cats. The goals of the MR imaging portion of the project were to determine whether this methodology was capable of detecting early neuropathophysiology in the absence of outward manifestation of neurological signs and to compare the MR imaging results for the two viral strains expected to have differing degrees of neurologic effects. We hypothesized that there would be increased diffusion, evidenced by the apparent diffusion coefficient as measured by DW-MRI, and altered metabolite ratios measured by MRS, in the brains of FIV-PPR-infected cats relative to C36-infected cats and uninfected controls. Increased apparent diffusion coefficients were seen in the white matter, gray matter, and basal ganglia of both the PPR and C36-infected (asymptomatic) cats. Thalamic MRS metabolite ratios did not differ between groups. The equivalently increased diffusion by DW-MRI suggests similar indirect neurotoxicity mechanisms for the two viral genotypes. DW-MRI is a sensitive tool to detect neuropathophysiological changes in vivo that could be useful during longitudinal studies of FIV.
Assuntos
Complexo AIDS Demência/diagnóstico , Encéfalo/patologia , Imagem de Difusão por Ressonância Magnética/métodos , Síndrome de Imunodeficiência Adquirida Felina/diagnóstico , Vírus da Imunodeficiência Felina , Espectroscopia de Ressonância Magnética/métodos , Complexo AIDS Demência/sangue , Complexo AIDS Demência/etiologia , Complexo AIDS Demência/patologia , Complexo AIDS Demência/fisiopatologia , Complexo AIDS Demência/virologia , Animais , Doenças Assintomáticas , Peso Corporal , Encéfalo/fisiopatologia , Encéfalo/virologia , Gatos , Síndrome de Imunodeficiência Adquirida Felina/sangue , Síndrome de Imunodeficiência Adquirida Felina/complicações , Síndrome de Imunodeficiência Adquirida Felina/patologia , Síndrome de Imunodeficiência Adquirida Felina/fisiopatologia , Síndrome de Imunodeficiência Adquirida Felina/virologia , Vírus da Imunodeficiência Felina/fisiologia , Imuno-Histoquímica , Contagem de Linfócitos , Quinases de Proteína Quinase Ativadas por Mitógeno/análise , Especificidade da Espécie , Carga Viral/fisiologiaRESUMO
A chimeric feline immunodeficiency virus (FIV) protease (PR) has been engineered that supports infectivity but confers sensitivity to the human immunodeficiency virus (HIV) PR inhibitors darunavir (DRV) and lopinavir (LPV). The 6s-98S PR has five replacements mimicking homologous residues in HIV PR and a sixth which mutated from Pro to Ser during selection. Crystal structures of the 6s-98S FIV PR chimera with DRV and LPV bound have been determined at 1.7 and 1.8 Å resolution, respectively. The structures reveal the role of a flexible 90s loop and residue 98 in supporting Gag processing and infectivity and the roles of residue 37 in the active site and residues 55, 57 and 59 in the flap in conferring the ability to specifically recognize HIV PR drugs. Specifically, Ile37Val preserves tertiary structure but prevents steric clashes with DRV and LPV. Asn55Met and Val59Ile induce a distinct kink in the flap and a new hydrogen bond to DRV. Ile98ProâSer and Pro100Asn increase 90s loop flexibility, Gln99Val contributes hydrophobic contacts to DRV and LPV, and Pro100Asn forms compensatory hydrogen bonds. The chimeric PR exhibits a comparable number of hydrogen bonds, electrostatic interactions and hydrophobic contacts with DRV and LPV as in the corresponding HIV PR complexes, consistent with IC(50) values in the nanomolar range.
Assuntos
Ácido Aspártico Endopeptidases/química , Protease de HIV/química , HIV/enzimologia , Vírus da Imunodeficiência Felina/enzimologia , Ácido Aspártico Endopeptidases/genética , Cristalografia por Raios X , Protease de HIV/genética , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Especificidade por SubstratoRESUMO
Heparan sulfate proteoglycans (HSPGs) act as binding receptors or attachment factors for the viral envelope of many viruses, including strains of HIV and feline immunodeficiency virus (FIV). The FIV gp95 glycoprotein (SU) from laboratory-adapted strains (tissue culture adapted [TCA]) such as FIV-34TF10 can bind to HSPG, whereas SU from field strains (FS) such as FIV-PPR cannot. Previous studies indicate that SU-HSPG interactions occur within the V3 loop. We utilized a series of nested V3 peptides to further map the HSPG binding sites and found that both sides of the predicted V3 loop stem were critical for the binding but not the CXCR4 binding domain near the predicted tip of the V3 loop. Neutralization assays for TCA strain entry using the same set of V3 peptides showed that peptides targeting CXCR4 or HSPG binding sites can block infection, supporting the V3 loop as a critical neutralization target. Site-directed mutagenesis identified two highly conserved arginines, R379 and R389, on the N-terminal side of the V3 stem as critical for the contact between SU and HSPG. Residues K407, K409, K410, and K412 on the C-terminal side of the V3 stem form a second nonconserved domain necessary for HSPG binding, consistent with the observed specificity distinctions with FS FIV. Our findings discriminate structural determinants important for HSPG and CXCR4 binding by FIV SU and thus further define the importance of the V3 loop for virus entry and infection.
Assuntos
Aminoácidos/metabolismo , Glicoproteínas/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Vírus da Imunodeficiência Felina/metabolismo , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Animais , Sequência de Bases , Gatos , Linhagem Celular , Primers do DNA , Citometria de Fluxo , Glicoproteínas/química , Glicoproteínas/genética , Vírus da Imunodeficiência Felina/fisiologia , Fusão de Membrana , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Ligação Proteica , Receptores CXCR4/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
Feline immunodeficiency virus (FIV) OrfA is an accessory protein that is critical for productive viral replication and infection in T cells. Here, we show that OrfA acts to markedly reduce cell surface expression of the FIV primary binding receptor. Downregulation does not occur at the transcriptional or translational level in that the amounts of CD134 mRNA and protein in total cell lysates are not altered between parental 104-C1 T cells and the same cell line stably expressing OrfA (104-C1-OrfA). Analysis by confocal microscopy revealed significant accumulation of CD134 in the Golgi apparatus of 104-C1 cells expressing OrfA. OrfA does not cause a generalized disruption of membrane trafficking in that surface expression of CD9 is unaffected by OrfA overexpression. Consistent with the above observations, OrfA-negative FIV-34TF10 productively infects CrFK (CD134-negative) and 104-C1-OrfA (CD134 downregulated by OrfA) cells but fails to productively infect either 104-C1 (CD134-positive) cells or GFox (CrFK cells overexpressing CD134) cells. FIV-34TF10 in which the OrfA reading frame is open (OrfArep) productively infects CrFK, GFox, 104-C1, and 104-C1-OrfA cells. We hypothesize that reduced surface expression of the receptor, a hallmark of retrovirus infections, may facilitate an increase in virus release from the infected cell by minimizing receptor interactions with budding virus particles.
Assuntos
Vírus da Imunodeficiência Felina/fisiologia , Receptores OX40/metabolismo , Linfócitos T/virologia , Proteínas Virais/metabolismo , Animais , Gatos , Linhagem Celular , Membrana Celular/metabolismo , Separação Celular , Regulação para Baixo , Humanos , Vírus da Imunodeficiência Felina/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Receptores OX40/genética , Proteínas Virais/genética , Internalização do Vírus , Liberação de VírusRESUMO
The chemokine receptor CXCR4 is shared by primary and laboratory-adapted strains of feline immunodeficiency virus (FIV) for viral entry. Our previous studies implicated a contiguous nine-amino-acid region of the V3 loop of the FIV envelope surface as important in CXCR4 binding and virus entry. The binding is specific for CXCR4 since it can be inhibited by AMD3100, a selective CXCR4 inhibitor. Additional site-directed mutagenesis was used to further reveal the key residues. Binding studies indicated that basic residues R395, K397, R399 as well as N398 are critical for CXCR4 binding. The effect of other amino acid residues on receptor binding depends on the type of amino acid residue substituted. The binding study results were confirmed on human CXCR4-expressing SupT1 cells and correlated with entry efficiency using a virus entry assay. Amino acid residues critical for CXCR4 are not critical for interactions with the primary binding receptor CD134, which has an equivalent role as CD4 for HIV-1 binding. The ELISA results show that W394 and W400 are crucial for the recognition by neutralizing anti-V3 antibodies. Since certain strains of HIV-1 also use CXCR4 as the entry receptor, the findings make the feline model attractive for development of broad-based entry antagonists and for study of the molecular mechanism of receptor/virus interactions.
Assuntos
Glicoproteínas de Membrana/química , Receptores CXCR4/metabolismo , Proteínas Virais/química , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Gatos , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Vírus da Imunodeficiência Felina/classificação , Vírus da Imunodeficiência Felina/isolamento & purificação , Vírus da Imunodeficiência Felina/patogenicidade , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação/genética , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Receptores CXCR4/química , Receptores CXCR4/imunologia , Receptores OX40/metabolismo , Proteínas Virais/metabolismo , Internalização do VírusRESUMO
Clinically approved inhibitors of the HIV-1 protease function via a competitive mechanism. A particular vulnerability of competitive inhibitors is their sensitivity to increases in substrate concentration, as may occur during virion assembly, budding and processing into a mature infectious viral particle. Advances in chemical synthesis have led to the development of new high-diversity chemical libraries using rapid in-solution syntheses. These libraries have been shown previously to be effective at disrupting protein-protein and protein-nucleic acid interfaces. We have screened 44000 compounds from such a library to identify inhibitors of the HIV-1 protease. One compound was identified that inhibits wild-type protease, as well as a drug-resistant protease with six mutations. Moreover, analysis of this compound suggests an allosteric non-competitive mechanism of inhibition and may represent a starting point for an additional strategy for anti-retroviral therapy.
Assuntos
Técnicas de Química Combinatória , Inibidores da Protease de HIV/química , Protease de HIV/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV-1) proteases (PRs) share only 23% amino acid identity and exhibit distinct specificities yet have very similar 3-dimensional structures. Chimeric PRs in which HIV residues were substituted in structurally equivalent positions in FIV PR were prepared in order to study the molecular basis of PR specificity. Previous in vitro analyses showed that such substitutions dramatically altered the inhibitor specificity of mutant PRs but changed the rate and specificity of Gag cleavage so that chimeric FIVs were not infectious. Chimeric PRs encoding combinations of the I37V, N55M, M56I, V59I, L97T, I98P, Q99V, and P100N mutations were cloned into FIV Gag-Pol, and those constructs that best approximated the temporal cleavage pattern generated by wild-type FIV PR, while maintaining HIV-like inhibitor specificity, were selected. Two mutations, M56I and L97T, were intolerant to change and caused inefficient cleavage at NC-p2. However, a mutant PR with six substitutions (I37V, N55M, V59I, I98P, Q99V, and P100N) was selected and placed in the context of full-length FIV-34TF10. This virus, termed YCL6, had low-level infectivity ex vivo, and after passage, progeny that exhibited a higher growth rate emerged. The residue at the position of one of the six mutations, I98P, further mutated on passage to either P98H or P98S. Both PRs were sensitive to the HIV-1 PR inhibitors lopinavir (LPV) and darunavir (DRV), as well as to the broad-based inhibitor TL-3, with 50% inhibitory concentrations (IC(50)) of 30 to 40 nM, consistent with ex vivo results obtained using mutant FIVs. The chimeras offer an infectivity system with which to screen compounds for potential as broad-based PR inhibitors, define structural parameters that dictate specificity, and investigate pathways for drug resistance development.