In vitro expression of UL56 gene of herpes simplex virus type 1; detection of UL56 gene product in infected cells and in virions.
In order to investigate the functional properties of the UL56 gene of herpes simplex virus type 1 (HSV-1), it was necessary to express the UL56 protein in vitro. The DNA sequences corresponding to the open reading frame of the UL56 gene of HSV-1 strain F were amplified from genomic viral DNA by PCR using primers corresponding to the translational start and termination regions of the UL56 ORF. The PCR product (705 bp) was inserted into the EcoRI/XbaI recognition sites of the bacterial expression vector pMal-c2. This procedure allowed the expression of the viral UL56 gene fused to the maltose-binding protein (MBP) of Escherichia coli, and subsequent cleavage of the fusion protein with the specific protease factor Xa. The induced fusion protein was purified by affinity chromatography using amylose columns. The apparent molecular weight of the fusion protein was about 70 kDa. Factor Xa cleaves the fusion protein into two subfragments of 42 kDa (MBP) and 30 kDa (UL56). Rabbit antisera induced against recombinant UL56 protein were used for detection of the UL56 gene product during the infection cycles of HSV-1. The presence of the UL56 protein was detected in infected cells and in HSV-1 virions by Western blot experiments and by immunofluorescence assays. A strong and increasing cytoplasmic fluorescence was observed in RC-37 cells infected with HSV-1 strain F between 6 and 16 h post-infection. In addition it was found that human HSV-1 IgM/IgG positive convalescent sera recognized the recombinant UL56 protein.
Kehm
R
R
Institut für Medizinische Virologie, Universität Heidelberg, Germany.
Lorentzen
E
E
Rösen-Wolff
A
A
Darai
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018076:DNA, Complementary; D005814:Genes, Viral; D000882:Haplorhini; D018259:Herpesvirus 1, Human / Q000235:genetics*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010957:Plasmids; D014760:Viral Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D014764:Viral Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D014771:Virion / Q000737:chemistry
D018076:DNA, Complementary; C089864:UL56 protein, Human herpesvirus 1; D014760:Viral Fusion Proteins; D014764:Viral Proteins