Co-localization of HSV-1 DNA and ICP35 protein by in situ hybridization and immunocytochemistry.
In an effort to obtain a better signal-to-noise ratio and ultrastructural preservation, we sought to improve electron microscopic in situ hybridization technique. In our method, protease treatment was omitted and visualization of the digoxigenin (DIG)-labelled deoxyribonucleic acid (DNA)-probe was enhanced using a three-step procedure. These improvements allowed us to localize viral DNA with good signal-to-noise ratio. DNA specific to herpes simplex virus 1 (HSV-1) was localized by this method to HSV-1 infected cultured cells; DNA was not observed in the empty-cored HSV-1. Using this method and the immunogold cytochemical method, we co-localized viral DNA and capsid protein ICP35 on Lowicryl-embedded sections of HSV-1 infected cells. Interestingly, labelling for both DNA and ICP was observed on some HSV-1 particles in cell nucleus. This finding is consistent with the notion that ICP35 is necessary for assembly of viral DNA. Combination of in situ hybridization and immunocytochemical techniques is a powerful tool for examination of the functional relationship between viral DNA and proteins and help us to study protein function in viral multiplication.
Morioka
H
H
Laboratory of Electron Microscopy, Kyoto Prefectural University of Medicine, Japan.
Kobayashi
K
K
Tachibana
M
M
Imanishi
J
J
D000818:Animals; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000032:analysis*; D018259:Herpesvirus 1, Human / Q000737:chemistry* / Q000235:genetics* / Q000254:growth & development; D007150:Immunohistochemistry / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D008854:Microscopy, Electron; D014709:Vero Cells; D014764:Viral Proteins / Q000032:analysis*
D004279:DNA, Viral; C054582:ICP35 protein, Human herpesvirus 1; D014764:Viral Proteins