RESUMO
Antimicrobial peptides are effectors of innate immunity, providing their hosts with rapid non-specific defence against parasitic invaders. In this report, the effects are assessed of two well-characterized antimicrobial amphipathic peptides (melittin and cecropin) on human immunodeficiency virus 1 (HIV-1) replication and gene expression in acutely infected cells at subtoxic concentrations. Production of infectious, cell-free virus was inhibited in a dose-dependent manner, with ID50 values in the range 0.9-1.5 microM for melittin and 2-3 microM for cecropin. Analysis of the effect of melittin on cell-associated virus production revealed decreased levels of Gag antigen and HIV-1 mRNAs. Transient transfection assays with HIV long terminal repeat (LTR)-driven reporter gene plasmids indicated that melittin has a direct suppressive effect on activity of the HIV LTR. HIV LTR activity was also reduced in human cells stably transfected with retroviral expression plasmids for the melittin or cecropin gene. It is concluded that antimicrobial peptides such as melittin and cecropin are capable of inhibiting cell-associated production of HIV-1 by suppressing HIV-1 gene expression.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos , HIV-1/efeitos dos fármacos , Meliteno/farmacologia , Peptídeos/farmacologia , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Expressão Gênica/efeitos dos fármacos , Genes Virais/efeitos dos fármacos , Repetição Terminal Longa de HIV/efeitos dos fármacos , HIV-1/genética , HIV-1/fisiologia , Humanos , Meliteno/genética , Peptídeos/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Transfecção , Proteínas Virais/biossíntese , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
The objective of this study was to assess the inhibitory effect of potential negative regulatory elements on human immunodeficiency virus (HIV)-1 long terminal repeat (LTR) activity. This was carried out by pairwise comparisons of reporter gene activities of HIV-LTR-CAT constructs differing in the presence and absence of nef sequences in transient transfection assays. Parallel transfections were performed in two persistently HIV-infected cell lines and the uninfected parental cell lines. The negative regulatory element (NRE) of the LTR did not suppress HIV LTR activity in any of the cell lines examined. However, a non-LTR-derived fragment of the nef gene had a distinct suppressive effect on activity of the full-length LTR in chronically infected astrocytoma cells. A weaker negative effect of this nef partial sequence (nps) was detected in the other cell lines with constructs lacking the NRE. The nps was capable of suppressing LTR activity in trans in chronically infected astrocytoma cells in a concentration-dependent manner. These results stress a negative role of a non-LTR nef partial sequence in a cell-specific manner. In addition, our data indicate that nps functions in trans with promoters unrelated to HIV LTR such as SV40 early and CMV immediate-early promoters.
Assuntos
Regulação Viral da Expressão Gênica , Genes nef/genética , Repetição Terminal Longa de HIV/genética , HIV-1/genética , Linhagem Celular , Regulação para Baixo , HumanosRESUMO
Comparative sequence analyses were performed on 14 genes encoding bacterial elongation factors EF-Tu and 7 genes encoding the beta-subunit of bacterial F1F0 type ATP-synthases. The corresponding predicted amino acid sequences were compared with published primary structures of homologous molecules. Phylogenetic trees were reconstructed from both data sets of aligned protein sequences and from an equivalent selection of 16S rRNA sequences by applying distance matrix and maximum parsimony methods. The EF-Tu data were in very good agreement with the rRNA data, although the resolution within the EF-Tu tree was reduced at certain phylogenetic levels. The resolution power of the ATPase beta-subunit sequence data were more reduced than those of the EF-Tu data. In comparison with the 16S rRNA tree there are minor differences in the order of adjacent branchings within the ATPase beta-subunit tree.
Assuntos
Bactérias/genética , Bactérias/metabolismo , Genes Bacterianos , Fator Tu de Elongação de Peptídeos/genética , Filogenia , ATPases Translocadoras de Prótons/genética , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Substâncias Macromoleculares , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
OBJECTIVE: To study expression of HIV-1 in human glial cell lines. DESIGN: Chronically HIV-1-infected glial cell lines were established to evade potential artefacts resulting from unphysiological viral entry (i.e., transfection). These cell lines were used to study viral expression and regulation. METHODS: Chronically infected glial cell lines were established by terminal dilution cloning of human glioma cells exposed to HIV-1. Virus production and expression were assayed by measuring reverse transcriptase activity, p24-antigen levels and syncytia-inducing capacity in C8166 target cells (extracellular), or by indirect immunoperoxidase staining, immunoblot analysis, and p24- and Nef-antigen-capture enzyme-linked immunosorbent assays (intracellular). HIV-long terminal repeat (LTR)-dependent expression of the chloramphenicol acetyltransferase reporter gene was determined in transient transfection assays. RESULTS: Culture supernatant from chronically HIV-1-infected glial cells contained only low levels of virus compared with chronically HIV-infected fibroblasts and T-lymphoma cells. Detailed study of HIV-antigen expression in representative glial cell line TH4-7-5 indicated the presence of all major structural proteins, albeit at low levels, and of Vif, Tat, Rev and Nef. Intracellular levels of Nef exceeded p24-antigen levels by approximately 10-fold. Virus was recovered from TH4-7-5 cells by cocultivation with blood-derived target cells, indicating that low-level virus production is not due to defective provirus. Prominent negative regulatory element (NRE)-mediated suppression of exogenous HIV-LTR activity was observed in TH4-7-5 cells and was unequalled by chronically HIV-producing fibroblast cells or by uninfected fibroblast and glial cells. CONCLUSIONS: Our results suggest that restricted virus production by chronically infected glial cells involves LTR-mediated regulation of virus expression.
Assuntos
Regulação Viral da Expressão Gênica/genética , Glioma/microbiologia , Repetição Terminal Longa de HIV/genética , HIV-1/crescimento & desenvolvimento , Neuroglia/microbiologia , Replicação Viral/genética , Sequência de Bases , Southern Blotting , Western Blotting , Genes nef/genética , Proteína do Núcleo p24 do HIV/análise , Transcriptase Reversa do HIV , HIV-1/genética , Humanos , Dados de Sequência Molecular , Provírus/genética , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Células Tumorais CultivadasRESUMO
The nucleotide sequences of cloned genes coding for the elongation factor Tu of seven eubacteria have been determined. These genes were from Anacystis nidulans, Bacillus subtilis, Bacteroides fragilis, "Deinonema" spec., Pseudomonas cepacia, Shewanella putrefaciens and Streptococcus oralis. The primary structures of the genes were compared to the available sequences of prokaryotic elongation factors Tu and eukaryotic elongation factors 1 alpha. A conservation profile was determined for homologous amino acid residues. Sites of known or putative functions are usually located at highly conserved positions or within highly conserved sequence stretches. The aligned 24 amino acid sequences were used as basis for a phylogenetic analysis. The phylogenetic tree corroborates the kingdom as well as phylum concept deduced from 16S rRNA data.