RESUMEN
We defined the effects of the cytoplasmic domain (CT) of the Env glycoprotein on co-receptor usage of HIV-1 by reciprocal exchanges of regions containing V3-V5 loops between CD4-dependent and CD4-independent isolates. Primary HIV-1 isolate Env clones CD8 CXCR4-tropic 92UG046 CT84 with an 84-aa truncated CT domain, CD4 CXCR4-tropic 92UG046, and CD4 CCR5-tropic SF162 with full-length (FL) CT domains were used for comparison. The parental 92UG046 Env with CT84 was not fusogenic, but a chimeric SF162 V3-V5-CT84 with an 84-aa truncated CT domain, which demonstrated a switched co-receptor specificity, exhibited syncytium-formation activity with 3T3T4X4 cells. The wild-type (WT) SF162 Env with CT84 or full-length CT was fusogenic in 3T3T4R5 cells. By exchange of V3-V5 loops, we were able to alter WT SF162 to switch its co-receptor preference, which was not dependent on CT domain length. These results provide evidence that CT domains can induce conformational changes in functional regions of gp120 and determine receptor tropism but do not modulate HIV-1 co-receptor specificity.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/fisiología , Receptores del VIH/metabolismo , Tropismo Viral , Acoplamiento Viral , Sitios de Unión , Línea Celular , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/genética , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica , Conformación Proteica , Dominios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
We investigated the effects on assembly and antigenic properties of specific modifications of the transmembrane spanning (TMS) and cytoplasmic tail (CT) domains of HIV-1 Env from a transmitted/founder (T/F) ZM53 Env glycoprotein. A construct containing a short version of the TMS domain derived from the mouse mammary tumor virus (MMTV) Env with or without a GCN4 trimerization sequence in the CT exhibited the highest levels of incorporation into VLPs and induced the highest titers of anti-Env IgG immune responses in a VLP context. Sera from guinea pigs immunized by VLPs with high Env content, and containing the CT trimerization sequence, had increased neutralization activity and antibody avidity. A cross-clade prime-boost regimen with clade B SF162 or clade C ZM53 Env DNA priming and boosting with VLPs containing modified ZM53 Env further enhanced these immune responses. The modified VLPs demonstrate improved potential as HIV-1 vaccine antigens.
Asunto(s)
Infecciones por VIH/prevención & control , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/química , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Secuencias de Aminoácidos , Animales , Femenino , Cobayas , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/química , VIH-1/genética , Humanos , Inmunización , Ratones , Pruebas de Neutralización , Productos del Gen env del Virus de la Inmunodeficiencia Humana/administración & dosificación , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
We investigated the effects of introducing specific sequences that are predicted to affect trimer stability into the CT domain of the SIV Env protein. Two constructs, 3HBai and 3HBaa, with additional GCN4-related sequences in the CT domain (45 aa) had enhanced infectivity, and differed in their fusion activity and trimer stability. Another construct, 3HBii, exhibited a very stable trimeric structure. Pseudotyped virions containing 3HBii retained infectivity despite the lack of syncytia formation. In contrast, 3HBai and 3HBaa, which caused extensive syncytia formation, had a less stable trimeric structure. We observed an inverse correlation between trimer stability and fusion activity but no correlation between syncytia formation activity and infectivity. Quantitative cell-cell fusion assays, analysis of Env incorporation, measurement of ectodomain conformation by CD4 binding, and CCR5 blocking assays indicated differential effects on fusion activity and infectivity of the viruses with Env CT modifications. Differences in interaction with CD4 were not affected by trimer stability and were not related to fusion activity or infectivity. The results indicate that changes in the stability of the CT domain can have significant effects on functional activities of the Env external domain and can impact viral biological properties.
Asunto(s)
Fusión Celular , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/metabolismo , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Secuencia de Aminoácidos , Animales , Antígenos CD4/metabolismo , Línea Celular , Genes env , Proteína gp41 de Envoltorio del VIH/genética , Humanos , Fusión de Membrana , Ratones , Mutación , Células 3T3 NIH , Estructura Terciaria de Proteína , Receptores CCR5/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Virión/metabolismoRESUMEN
Previously, a modified HIV Env protein with a heterologous membrane anchor was found to be incorporated into HIV virus-like particles (VLPs) at 10-fold-higher levels than those of unmodified Env. To further improve the immunogenicity of such VLPs, membrane-anchored forms of bacterial flagellin (FliC) or a flagellin with a truncated variable region (tFliC) were constructed to be incorporated into the VLPs as adjuvants. HIV-specific immune responses induced by the resulting VLPs were determined in a guinea pig model. The VLPs induce enhanced systemic antibody responses by either systemic or mucosal vaccination and enhanced mucosal immunity by a mucosal immunization route, as demonstrated by high levels of HIV-specific serum IgG and mucosal IgG and IgA. The quality of the antibody responses was also improved, as shown by enhanced neutralization capacity. VLPs incorporating FliC were more effective in inducing systemic responses, while VLPs containing tFliC were more effective in inducing mucosal IgA responses. The IgG titers in sera were found to last for at least 5 months without a significant drop. These results indicate that HIV VLPs incorporating high levels of Env and a molecular adjuvant have excellent potential for further development as a prophylactic HIV vaccine.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Flagelina/inmunología , Infecciones por VIH/inmunología , VIH/inmunología , Inmunidad Mucosa , Virión/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Adyuvantes Inmunológicos/genética , Animales , Anticuerpos Antivirales/inmunología , Femenino , Flagelina/administración & dosificación , Flagelina/genética , Cobayas , VIH/genética , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , Humanos , Salmonella typhimurium/genética , Salmonella typhimurium/inmunología , Virión/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/administración & dosificación , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
BACKGROUND: The Env glycoproteins of retroviruses play an important role in the initial steps of infection involving the binding to cell surface receptors and entry by membrane fusion. The Env glycoprotein also plays an important role in viral assembly at a late step of infection. Although the Env glycoprotein interacts with viral matrix proteins and cellular proteins associated with lipid rafts, its possible role during the early replication events remains unclear. Truncation of the cytoplasmic tail (CT) of the Env glycoprotein is acquired by SIV in the course of adaptation to human cells, and is known to be a determinant of SIV pathogenicity. RESULTS: We compared SIV viruses with full length or truncated (T) Env glycoproteins to analyze possible differences in entry and post-entry events, and assembly of virions. We observed that early steps in replication of SIV with full length or T Env were similar in dividing and non-dividing cells. However, the proviral DNA of the pathogenic virus clone SIVmac239 with full length Env was imported to the nucleus about 20-fold more efficiently than proviral DNA of SIVmac239T with T Env, and 100-fold more efficiently than an SIVmac18T variant with a single mutation A239T in the SU subunit and with a truncated cytoplasmic tail (CT). In contrast, proviral DNA of SIVmac18 with a full length CT and with a single mutation A239T in the SU subunit was imported to the nucleus about 50-fold more efficiently than SIVmac18T. SIV particles with full length Env were released from rhesus monkey PBMC, whereas a restriction of release of virus particles was observed from human 293T, CEMx174, HUT78 or macrophages. In contrast, SIV with T Envs were able to overcome the inhibition of release in human HUT78, CEMx174, 293T or growth-arrested CEMx174 cells and macrophages resulting in production of infectious particles. We found that the long CT of the Env glycoprotein was required for association of Env with lipid rafts. An Env mutant C787S which eliminated palmitoylation did not abolish Env incorporation into lipid rafts, but prevented virus assembly. CONCLUSION: The results indicate that the long cytoplasmic tail of the SIV Env glycoprotein may govern post-entry replication events and plays a role in the assembly process.
Asunto(s)
Productos del Gen env/fisiología , Estructura Terciaria de Proteína/fisiología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Adaptación Fisiológica , Animales , Línea Celular , Citoplasma/virología , Productos del Gen env/química , Humanos , Virus de la Inmunodeficiencia de los Simios/química , Acoplamiento Viral , Replicación ViralRESUMEN
Sulfonated porphyrins and phthalocyanines have been shown to have anti-HIV activity and are under consideration as microbicides. Both categories of compounds are small negatively charged molecules and both were previously shown to inhibit cell fusion induced by the HIV Env protein and to block binding of gp120 to the CD4 receptor. In the present study we show that these compounds inhibit transmission of cell-associated HIV, inactivate a broad range of HIV-1 primary isolates and are active against DS polyanion-resistant virus. The compounds tested are active over a range of pH values, and possess no detectable activity against normal bacterial flora. These results support the conclusion that anionic tetrapyrroles are promising candidates as microbicides for HIV prevention.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Indoles/farmacología , Porfirinas/farmacología , Aniones/química , Aniones/farmacología , Línea Celular , Células Cultivadas , Femenino , Infecciones por VIH , VIH-1/clasificación , Humanos , Indoles/química , Isoindoles , Lactobacillus/efectos de los fármacos , Linfocitos/virología , Pruebas de Sensibilidad Microbiana/métodos , Porfirinas/química , Relación Estructura-Actividad , Vagina/microbiologíaRESUMEN
Sulfonated 5,10,15,20-tetra(1-naphthyl)porphyrin (T1NapS) and 5,10,15,20-tetra(2-naphthyl)porphyrin (T2NapS) and their copper and iron chelates show activity against the human immunodeficiency virus (HIV-1). The porphyrins were prepared by sulfonation of the parent structures with sulfuric acid. More highly sulfonated structures were prepared by sulfonation for longer times. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry showed species with as many as eight sulfonates. Some of the mass spectral peaks for the copper chelates were consistent with loss of water, apparently from intramolecular sulfone formation between two adjacent naphthalene rings that took place during copper insertion. The compounds could be separated using capillary electrophoresis; addition of beta- or gamma-cyclodextrin gave substantially better separation of the components. Activity against HIV was evaluated using an epithelial HeLa-CD4-CCR5 cell line; EC50 values for HIV-1 IIIB and HIV-1 JR-FL ranged from 1 to 15 microg/ml. The compounds exhibit low toxicity for human epithelial cells and have potential as microbicides which might be used to provide protection against sexual transmission of HIV.
Asunto(s)
Alcanosulfonatos/química , Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Porfirinas/farmacología , Fármacos Anti-VIH/química , Quelantes/química , Cobre/química , Ciclodextrinas/química , Electroforesis Capilar , Células HeLa , Humanos , Hierro/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Porfirinas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-Actividad , Factores de TiempoRESUMEN
SIVmac239 and SIVmac1A11 are wild-type viruses encoding Env proteins with full-length or truncated cytoplasmic tails (CTs), respectively. A mutant designated SIVmac239T has a site-specific mutation which introduces a stop codon in the env gene resulting a truncated protein of similar length to SIVmac1A11 Env. To investigate the role of specific sequence differences in these Env proteins, we constructed SIV mutants encoding 1A11 or 239 Env proteins with reciprocal exchanges of the CT or exchanges of both the surface unit (SU) and CT sequences. A truncated CT in the context of the 1A11 SU subunit was found to significantly enhance replication in CEMx174 (human T-cell line) and rhesus PBMCs. However, similar Env CT truncation did not enhance replication of SIVmac239 in human or monkey cells. SIVmac1A11 with a full-length SIVmac239 CT did not replicate in human T-cell lines, but truncation of the CT by a stop codon resulted in replication. We also observed that these viruses differed significantly in sensitivity to neutralization by antibody. Taken together, the results indicated that the length of the CT domain as well as specific sequence differences in the SU domain affect viral replication capacity as well as sensitivity to neutralization.
Asunto(s)
Virus de la Inmunodeficiencia de los Simios/patogenicidad , Proteínas del Envoltorio Viral/fisiología , Replicación Viral/fisiología , Animales , Anticuerpos Antivirales , Especificidad de Anticuerpos , Línea Celular , Humanos , Macaca mulatta , Pruebas de Neutralización , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/fisiología , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
The ability of selected phthalocyanines and metallophthalocyanines to block HIV infection has been evaluated in an epithelial HeLa-CD4 cell line with an integrated LTR-beta-galactosidase gene. Sulfonated phthalocyanine itself (PcS), as well as its copper, nickel, and vanadyl chelates, were the most effective in blocking viral infection. These compounds were also very effective in blocking the fusion activity of the viral Env proteins. All of these compounds are expected to bind axial ligands weakly or not at all. In contrast, sulfonated phthalocyanines bearing metals expected to bind axial ligands more tightly (aluminum, cobalt, chromium, iron, silicon, and zinc) were less effective in blocking HIV infection and also less effective at inhibiting fusion. A number of active compounds were found to block binding of gp120 to CD4. Selected cationic and carboxy phthalocyanines, as well as porphyrazines, were also evaluated. Our results indicate that at least some of the compounds render the virus noninfectious, i.e. that they are virucidal. These compounds have potential as microbicides that might be used to provide protection against sexually transmitted HIV.
Asunto(s)
Fármacos Anti-VIH/farmacología , Infecciones por VIH/prevención & control , Indoles/farmacología , Animales , Fármacos Anti-VIH/química , Secuencia de Bases , Antígenos CD4/metabolismo , Línea Celular , ADN Viral/genética , Evaluación Preclínica de Medicamentos , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/genética , VIH-1/patogenicidad , VIH-1/fisiología , Haplorrinos , Células HeLa , Humanos , Indoles/química , Isoindoles , Fusión de Membrana/efectos de los fármacos , Ratones , Relación Estructura-ActividadRESUMEN
BACKGROUND: Prevention of poxvirus infection is a topic of great current interest. We report inhibition of vaccinia virus in cell culture by porphyrins and phthalocyanines. Most previous work on the inhibition of viruses with tetrapyrroles has involved photodynamic mechanisms. The current study, however, investigates light-independent inhibition activity. METHODS: The Western Reserve (WR) and International Health Department-J (IHD-J) strains of vaccinia virus were used. Virucidal and antiviral activities as well as the cytotoxicity of test compounds were determined. RESULTS: Examples of active compounds include zinc protoporphyrin, copper hematoporphyrin, meso(2,6-dihydroxyphenyl)porphyrin, the sulfonated tetra-1-naphthyl and tetra-1-anthracenylporphyrins, selected sulfonated derivatives of halogenated tetraphenyl porphyrins and the copper chelate of tetrasulfonated phthalocyanine. EC50 values for the most active compounds are as low as 0.05 microg/mL (40 nM). One of the most active compounds was the neutral meso(2,6-dihydroxyphenyl)porphyrin, indicating that the compounds do not have to be negatively charged to be active. CONCLUSIONS: Porphyrins and phthalocyanines have been found to be potent inhibitors of infection by vaccinia virus in cell culture. These tetrapyrroles were found to be active against two different virus strains, and against both enveloped and non-enveloped forms of the virus, indicating that these compounds may be broadly effective in their ability to inhibit poxvirus infection.
Asunto(s)
Pirroles/farmacología , Virus Vaccinia/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Células HeLa , Humanos , Luz , Porfirinas/farmacología , Porfirinas/uso terapéutico , Infecciones por Poxviridae/prevención & control , Protoporfirinas/farmacología , Protoporfirinas/uso terapéutico , Pirroles/uso terapéutico , Virus Vaccinia/clasificación , Virus Vaccinia/fisiologíaRESUMEN
We have evaluated the anti-human immunodeficiency virus (HIV) activity of a series of natural and synthetic porphyrins to identify compounds that could potentially be used as microbicides to provide a defense against infection by sexually transmitted virus. For assays we used an epithelial HeLa-CD4 cell line with an integrated long terminal repeat-beta-galactosidase gene. For structure-activity analysis, we divided the porphyrins tested into three classes: (i) natural porphyrins, (ii) metallo-tetraphenylporphyrin tetrasulfonate (metallo-TPPS4) derivatives, and (iii) sulfonated tetra-arylporphyrin derivatives. None of the natural porphyrins studied reduced infection by more than 80% at a concentration of 5 micro g/ml in these assays. Some metal chelates of TPPS4 were more active, and a number of sulfonated tetra-aryl derivatives showed significantly higher activity. Some of the most active compounds were the sulfonated tetranaphthyl porphyrin (TNapPS), sulfonated tetra-anthracenyl porphyrin (TAnthPS), and sulfonated 2,6-difluoro-meso-tetraphenylporphine [TPP(2,6-F2)S] and its copper chelate [TPP(2,6-F2)S,Cu], which reduced infection by 99, 96, 94, and 96%, respectively. Our observations indicate that at least some of these compounds are virucidal, i.e., that they render the virus noninfectious. The active compounds were found to inhibit binding of the HIV type 1 gp120 to CD4 and also to completely inhibit the ability of Env proteins expressed from recombinant vectors to induce cell fusion with receptor-bearing target cells. These results support the conclusion that modified porphyrins exhibit substantial activity against HIV and that their target is the HIV Env protein.