RESUMEN
The infection and subsequent replication rates of human immunodeficiency virus type 1 (HIV-1) affect the pathogenicity. The initial stage of HIV-1 infection is largely regulated by viral envelope sequence. We previously reported that the defective doughnut-shaped particles produced from a persistently infected cell clone, named L-2, obtained from human CD4+ T-cell line MT-4 that was persistently infected with HIV-1 LAI strain, efficiently form particle-mediated syncytia with uninfected human CD4+ T-cell line, MOLT-4. Here, we prepared a molecular clone (pL2) containing the L-2 provirus to characterize the viral genetic region contributing to this activity to form particle-mediated syncytia. Several recombinants were constructed with pNL4-3 by replacing the pL2-derived region including full-length env. Characterization of the particles obtained by transfection with these recombinant clones confirmed that pL2-derived env carried the particle-mediated syncytia formation activity. It is noteworthy that the pL2-derived env region could also contribute to enhancement of infectivity in CD4+ T-cell lines as well as primary peripheral blood mononuclear cells (PBMCs). Thus, the HIV-1 particle-mediated syncytium formation activity could also contribute to the enhancement of HIV-1 infectivity.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Proteína gp120 de Envoltorio del VIH/fisiología , Infecciones por VIH/virología , VIH-1/patogenicidad , Linfocitos T CD4-Positivos/inmunología , ADN Viral/química , ADN Viral/genética , Técnica del Anticuerpo Fluorescente Indirecta , Células Gigantes/inmunología , Células Gigantes/ultraestructura , Células Gigantes/virología , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/genética , VIH-1/inmunología , Humanos , Microscopía Electrónica , Plásmidos , Provirus/genética , Proteínas Recombinantes , Transfección , Virión/inmunología , Virión/ultraestructura , Replicación ViralRESUMEN
An infectious molecular clone (named p95TNIH022) was constructed using long-range polymerase chain reaction products derived from a clinical isolate (95TNIH022) of HIV-1 CRF01_AE obtained from an asymptomatic Thai carrier in 1995. The virus in the supernatant from p95TNIH022-transfected 293T cells showed infectivity in peripheral blood mononuclear cells (PBMCs) as well as in MAGIC5 cells, which express CD4 and CCR5, but not in the original MAGI cells, indicating that p95TNIH022 is an infectious molecular clone with CCR5 tropism. Interestingly, p95TNIH022-derived virus induced profound cell killing in infected PBMCs, as in cells infected with the parental isolate.
Asunto(s)
Infecciones por VIH/virología , VIH-1/patogenicidad , Virulencia , Secuencia de Bases , Recuento de Linfocito CD4 , Línea Celular , Clonación Molecular , Cartilla de ADN , VIH-1/genética , HumanosRESUMEN
Borna disease virus (BDV) establishes a persistent infection in the central nervous system of vertebrate animal species as well as in tissue cultures. In an attempt to characterize the life cycle of BDV in persistently infected cultured cells, we developed 30 clones by single-cell cloning from a human oligodendroglioma (OL) cell line after infection with BDV. According to the percentage of cells expressing the BDV major proteins, p40 (nucleoprotein) and p24 (phosphoprotein), the clones were classified into two types: type I (>20%) and type II (<20%). mRNAs corresponding to both proteins were detected by in situ hybridization (ISH) in a percentage of cells consistent with that for the protein expression in the two types. Surprisingly, ISH for the detection of the genomic RNA, mainly in type II, revealed a significantly larger cell population harboring the genomic RNA than that with the protein as well as the mRNA expression. By recloning from type II primary cell clones, the same phenotype was confirmed in the secondary cell clones obtained: i.e., low percentage of protein-positive cells and higher percentage of cells harboring the genomic RNA. After nerve growth factor treatment, the two types of clones showed increases in the percentage of cells expressing BDV-specific proteins that reached 80% in type II clones, in addition to increased expression levels per cell. Such enhancement might have been mediated by the activation of the mitogen-activated protein kinase in the clones as revealed by the detection of activated ERK1/2. Thus, our findings show that BDV may have established a persistent infection at low levels of viral expression in OL cells with the possibility of a latent infection.