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 U L56 protein in vitro. The DNA sequences corresponding to the open read ing frame of the UL56 gene of HSV-1 strain F were amplified from genom ic viral DNA by PCR using primers corresponding to the translational s tart and termination regions of the UL56 ORF. The PCR product (705 bp) was inserted into the EcoRI/XbaI recognition sites of the bacterial e xpression vector pMal-c2. This procedure allowed the expression of the viral UL56 gene fused to the maltose-binding protein (MBP) of Escheri chia coli, and subsequent cleavage of the fusion protein with the spec ific protease factor Xa. The induced fusion protein was purified by af finity chromatography using amylose columns. The apparent molecular we ight of the fusion protein was about 70 kDa. Factor Xa cleaves the fus ion protein into two subfragments of 42 kDa (MBP) and 30 kDa (UL56). R abbit 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 immunofluorescenc e assays. A strong and increasing cytoplasmic fluorescence was observe d 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.