RESUMEN
The heterocomplex of glycoproteins H (gH) and L (gL) of herpes simplex virus type 1 (HSV-1) is essential for viral infectivity and is involved in viral penetration, cell-to-cell spread, and syncytium formation. We constructed an HSV-1 mutant expressing a gH-EGFP (enhanced green fluorescent protein) fusion protein under the control of the gH true late promoter. The EGFP coding sequence was cloned after the gH signal peptide into the HSV-1 genome. Superinfection of transfected, gH-nontranscomplementing cells with gH-negative HSV-1 resulted in a replication-competent recombinant virus. Cells infected with the recombinant virus exhibited strong and stable EGFP-specific fluorescence late in infection, and autofluorescence was detected in purified virions. The recombinant genotype of the mutant was confirmed by PCR. The 140-kD gH-EGFP fusion protein showed an N-linked glycosylation pattern similar to gH-1, was recognized by the conformation-dependent gH-specific monoclonal antibodies 52S and LP11 and formed a heterocomplex with gL which was transported to the cell surface and integrated into the viral envelope. Infectivity of the gH-EGFP mutant was neutralized by antibodies 52S and LP11. To our knowledge, this is the first replication-competent HSV-1 mutant expressing an autofluorescent essential glycoprotein which will be a versatile tool for studies of penetration, late gene expression, transport and tissue spread.
Asunto(s)
Herpes Simple/virología , Simplexvirus/metabolismo , Proteínas del Envoltorio Viral/genética , Animales , Células COS , Chlorocebus aethiops , Clonación Molecular , Fluorescencia , Expresión Génica , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Mutación Puntual , Proteínas Recombinantes de Fusión/biosíntesis , Simplexvirus/genética , Factores de Tiempo , Transfección , Células Vero , Proteínas del Envoltorio Viral/biosíntesis , Replicación ViralRESUMEN
Using the method of Haseloff and Gerlach, we constructed a ribozyme specifically targeted against the virion infectivity factor (vif) of HIV-1. Both, the vif gene and an oligonucleotide representing the catalytic RNA sequence were cloned into pSPT19 downstream of the T7 promoter and transcribed with T7 RNA polymerase. Efficient cleavage of vif RNA by the synthetic ribozyme occurred at pH 7.5 and 37 degrees C in the presence of magnesium ions in vitro. No measurable activity was observed with a vif antisense RNA. A deletion in the hybridizing region of the ribozyme decreased the cleavage rate, while a mutation in the consensus cleavage domain abolished its catalytic activity. Thus, we could demonstrate an in-vitro activity of a specifically designed ribozyme against HIV-1 vif RNA.
Asunto(s)
Genes vif , VIH-1/genética , ARN Catalítico/metabolismo , ARN Viral/metabolismo , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos , ARN Catalítico/genética , ARN Viral/genética , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
To investigate the interaction of herpes simplex virus type 1 (HSV-1) with the cell surface, we studied the formation of complexes by HSV-1 virion proteins with biotinylated cell membrane components. HSV-1 virion proteins reactive with surface components of HEp-2 and other cells were identified as gC, gB, and gD. Results from competition experiments suggested that binding of gC, gB, and gD occurred in a noncooperative way. The observed complex formation could be specifically blocked by monospecific rabbit antisera against gB and gD. The interaction of gD with the cell surface was also inhibited by monoclonal antibody IV3.4., whereas other gD-specific monoclonal antibodies, despite their high neutralizing activity, were not able to inhibit this interaction. Taken together, these data provide direct evidence that at least three of the seven known HSV-1 glycoproteins are able to form complexes with cellular surface structures.