RESUMEN
The Portland alpha1-antitrypsin variant (alpha1-PDX) inhibits gp160 cleavage into gp120 and gp41 by different prohormone convertases (PCs) including furin, PC5 and PC7. Jurkat cells stably transfected with this inhibitor (J-PDX cells) and, as controls, Jurkat cells transfected with the empty vector (J-pcDNA3) were tested for their susceptibility to HIV-1 infection. We found that HIV-1 replication was significantly impaired in J-PDX cells. However, the analysis of the infectivity of HIV-1 viruses produced in J-PDX cells on different days during the infection indicated that they recovered infectivity starting from 13 days post-infection. The sequencing of viruses collected earlier and later from J-PDX cells revealed no mutations in envelope-glycoprotein precursor (Env) maturation sites or in the N-terminal sequence of gp41 fusion peptide, which plays a key role in membrane fusion. Although conserved mutations were detected at the C-terminus of the gp41 fusion peptide and ectodomain, the replication of mutant HIV-1 viruses produced on day 20 in J-PDX cells was inhibited at a similar level to wild-type viruses after a second passage in J-PDX cells. We then investigated the expression of the alpha1-PDX protein, and found that HIV-1 replication activated its proteolysis since the 54 kDa cleaved form became predominant later on in the infection. In contrast, the expression of PC7, a protein that is transported through the secretory pathway, was unaltered in HIV-1 infected cells. We conclude that recovered HIV-1 infectivity in J-PDX cells was due to a loss of alpha1-PDX activity via its extensive processing by intracellular proteases that cleave it through the substrate pathway.
Asunto(s)
VIH-1/fisiología , Replicación Viral , alfa 1-Antitripsina/farmacología , Western Blotting , Linfocitos T CD4-Positivos , Secuencia Conservada , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteínas gp160 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Humanos , Células Jurkat , Mutación , Péptidos/química , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , TransfecciónRESUMEN
We investigated the effects of alpha1-antitrypsine Portland variant (alpha1-PDX) and decanoylRVKRchloromethylketone (decRVKRcmk) on HIV-2(ROD) replication in the Jurkat lymphoblastoid cell line. To this end, cells were stably transfected with the alpha1-PDX (J-PDX) and used as targets for HIV-2(ROD) infection. Controls were prepared with the empty vector (J-pcDNA3). HIV-2(ROD) and HIV-1(LAI) replications were significantly inhibited and delayed in the presence of the alpha1-PDX protein. When decRVKRcmk was used at 35 microM, inhibition rates were 70-80% for HIV-2(ROD) and HIV-1(LAI), while total inhibition occurred at 70 microM. Control peptides consisting of decanoylRVKR and acetylYVADcmk had no effect. In the presence of the alpha1-PDX or the decRVKRcmk at 35 microM, the infectivity of HIV-2(ROD) and HIV-1(LAI) produced was 3-4-fold lower. Both molecules inhibited syncytium formation by HIV-2(ROD) and HIV-1(LAI) to a considerable extent. Finally, the inhibition of viral replication was correlated with the ability of the decRVKRcmk at 35 and 70 microM and of the alpha1-PDX, to inhibit the processing of envelope glycoprotein precursors. The alpha1-PDX protein and the decRVKRcmk peptide at 35 microM inhibited HIV-2 and HIV-1 to a similar level suggesting that identical or closely related endoproteases are involved in the maturation of their envelope glycoprotein precursors into surface and transmembrane glycoproteins. The significant inhibition observed with alpha1-PDX indicates that furin or furin-like endoproteases appear to play a major role in the maturation process.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , VIH-2/efectos de los fármacos , VIH-2/fisiología , Péptidos/farmacología , Replicación Viral/efectos de los fármacos , alfa 1-Antitripsina/farmacología , Clorometilcetonas de Aminoácidos , Secuencia de Aminoácidos , Relación Dosis-Respuesta a Droga , Productos del Gen env/metabolismo , Células Gigantes/efectos de los fármacos , Células Gigantes/virología , Proteínas gp160 de Envoltorio del VIH/metabolismo , VIH-1/fisiología , Humanos , Células Jurkat , Péptidos/química , Precursores de Proteínas/metabolismo , Ensamble de Virus/efectos de los fármacos , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
We have analyzed the calcium requirement for HIV-1 gp160 processing in cultured nonlymphoid (CV-1 and HeLa-CD4) and human-lymphoid [Jurkat, Molt-4 and peripheral blood lymphocytes (PBMCs)] cells. The processing of gp160 in these cells, infected with recombinant vaccinia virus encoding the gp160 gene, was only partially affected by intracellular calcium depletion induced by the calcium ionophore A23187 and calcium chelator EGTA. These observations prompted us to purify the Ca(2+)-independent gp160 processing enzyme from natural targets of HIV-1 PBMCs. The endoprotease was purified to homogeneity by the use of four chromatography fractionation steps and the constant detection of the Ca(2+)-independent activity at each one of them. The enzyme was believed to be a membrane-associated heteromeric 120-kDa protein composed of three subunits of 66, 32, and 24 kDa. It was found to be specifically inhibited by substrate analogues, decanoyl-RVKR-chloromethyl ketone, and serine protease inhibitors including diisopropyl fluorophosphate (DFP) and TLCK. In contrast, no effect was observed with reducing agents including 2-beta-mercaptoethanol, N-ethylmaleimide, L-cysteine, and dithiothreitol. There were significant similarities between inhibition profiles of the purified enzyme in vitro and those of the endogenous endoprotease(s) in cell culture experiments. Therefore, the selectivity of purified endoprotease for the gp160 cleavage site, its requirement for additional residues around this consensus sequence, and its isolation from natural targets of HIV-1, made it a good candidate in the gp160 maturation process. We provide more direct and supporting evidence that HIV-1 gp160 maturation may involve at least two families of divergent endoproteases according to calcium dependence.
Asunto(s)
Calcio/fisiología , Endopeptidasas/química , Endopeptidasas/aislamiento & purificación , Proteínas gp160 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Linfocitos/enzimología , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Línea Celular , Células Cultivadas , Fenómenos Químicos , Química Física , Chlorocebus aethiops , Endopeptidasas/sangre , Células Gigantes/enzimología , Células Gigantes/virología , Células HeLa , Humanos , Células Jurkat , Linfocitos/virología , Datos de Secuencia Molecular , Células Tumorales CultivadasRESUMEN
The present work investigated the potential role of alpha-1 antitrypsin Portland variant (alpha 1-PDX), a bioengineered serine proteinase inhibitor (serpin), in the interference with the viral replication of HIV-1, induction of syncytia and maturation of envelope glycoprotein gp160 to gp120 and gp41. A Jurkat lymphoid cell line transfected with a plasmid containing the alpha 1-PDX cDNA (J-PDX) and expressing the protein in a stable manner was infected with HIV-1(Lai). Controls were Jurkat cells transfected with the same vector pcDNA3 without the cDNA insert (J-pcDNA3). The results showed that viral replication of HIV-1 was significantly inhibited with a delay in replication kinetics in J-PDX cells as compared with J-pcDNA3 cells. In addition, a comparison of the infectious capacity of viruses produced in the presence and absence of alpha 1-PDX revealed that this capacity differed. It was found that alpha 1-PDX exerts its effect by interfering with the formation of syncytia between J-PDX cells infected with gp160 recombinant vaccinia virus, or after infection by HIV-1 and co-culture with uninfected Molt-4 cells. In contrast, when the same experiments were performed with J-pcDNA3 cells, a large number of syncytia was obtained. Analysis of viral proteins by Western blotting and densitometry showed that the inhibition of the cytopathic effect of HIV-1 and viral replication was correlated with the capacity of alpha 1-PDX to interfere with the maturation of gp160 to gp120 and gp41.
Asunto(s)
VIH-1/metabolismo , Replicación Viral/efectos de los fármacos , alfa 1-Antitripsina/farmacología , Western Blotting , Línea Celular , ADN Complementario/metabolismo , Densitometría , Células Gigantes/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteínas gp160 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Humanos , Células Jurkat , Plásmidos/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Activación Transcripcional , Transfección , Virus VacciniaRESUMEN
This study clearly shows that hepatitis E virus (HEV) was the major aetiological virus in an outbreak in the south of Morocco, in 1994. Acute hepatitis E was diagnosed using recombinant antigen-based enzyme immunoassays and reverse transcription polymerase chain reaction in 77.3% of patients. In the west of Morocco, 6.1% of controls were positive for anti-HEV IgG. The anti-HEV prevalence in patients was significantly higher than that of controls (84.0% vs. 6.1%) (P < 0.001). In healthy contacts residing in southern Morocco, 10.4% had anti-HEV IgG, indicating past HEV infection. Furthermore, HEV-specific IgM was associated with subclinical HEV infection in 9 contacts and was noted in 10 others who were convalescent. Faecal contamination of drinking water samples collected from the epidemic city was observed. It also appeared that primary infection with HEV accounted for more than 86% of the cases. A longitudinal study showed waning of anti-HEV antibodies in patients and healthy contacts six months after the initial testing. Subclinical HEV infection was significantly prevalent in a paediatric population younger than 10 years (P < 0.05). Our results also showed that anti-HEV IgG in healthy contacts decreased significantly after 30 years of age (P < 0.01), whereas the clinical acute HEV infection incidence increased significantly with age (P < 0.01). From this study, it appears that HEV is present in both the west and the south of Morocco.
Asunto(s)
Anticuerpos Antihepatitis/sangre , Virus de la Hepatitis E/inmunología , Hepatitis E/epidemiología , Enfermedad Aguda , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Brotes de Enfermedades , Ensayo de Inmunoadsorción Enzimática/métodos , Heces/virología , Femenino , Hepatitis E/inmunología , Hepatitis E/transmisión , Humanos , Lactante , Recién Nacido , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Marruecos/epidemiología , ARN Viral/análisis , ARN Viral/sangre , Estudios Seroepidemiológicos , Abastecimiento de AguaRESUMEN
Hepatitis C virus (HCV) seroprevalence and transmission routes were investigated in several groups of the Moroccan population. This study showed a low HCV seroprevalence in the Moroccan general population. However, haemodialysis patients and haemophiliacs were at higher risk of having HCV infection, since the prevalences were, respectively, 35.1 and 42.4% in comparison with the blood donors' prevalence (1.1%). These results indicated that parenteral exposure is the transmission pathway of HCV. To investigate the possibility of vertical HCV transmission, a cohort of healthy, unselected pregnant women were included in the study. A prevalence of 1% was found among them. Seven newborns were anti-HCV-positive, although, when RT-PCR was used to search for HCV RNA in their sera, none of them was viraemic. These data indicated that anti-HCV antibodies were passively acquired in these cases. We concluded that vertical transmission is absent when mothers are at low risk of contracting other parenterally or sexually transmitted diseases. Three percent of a group of patients of a centre for sexually transmitted diseases were repeatedly anti-HCV-positive, suggesting the possible sexual transmission of HCV. When screening 116 sera of anti-HIV-positive subjects, 19.8% were anti-HCV-positive. Furthermore, 17.9% of the sixty-seven patients who were proven to have sexually contracted HIV were also anti-HCV-positive. These data might reflect a likely cotransmission of these two viruses, hence suggesting HIV is a cofacter for HCV sexual transmission, as previously reported.