Ultrastructural localization of herpes simplex virus RNA by in situ hybridization.
We studied the subcellular localization of virally encoded RNA by pre-embedding in situ hybridization, using colloidal gold as an electron-dense marker. Fibroblasts infected with Herpes simplex virus (HSV) were fixed, permeabilized, then hybridized with a biotinylated HSV DNA probe under conditions favoring DNA-RNA hybrid formation. HSV probe was localized with 5-nm streptavidin-gold conjugates. Transmission electron microscopy revealed 5-nm gold in clusters and singlets within HSV-infected cells. Formalin-fixed cells contained a mean of 4.6 clusters per cytoplasmic profile and 13.2 clusters per nuclear profile. Combined formalin-glutaraldehyde fixation increased the mean number of clusters per cytoplasmic and nuclear profile to 7.2 (57% increase) and 17.5 (33% increase), respectively. Gold clusters were frequently located in regions adjacent to the nuclear envelope but were not bound to viral nucleocapsids or endoplasmic reticulum. Labeling was unaffected by pre-hybridization DNAse treatment of cells. RNAse eliminated 87% of cytoplasmic and 97% of nuclear clusters. These findings indicate that clustered gold particles labeled viral RNA, with probable binding of multiple DNA probe molecules and/or gold particles to RNA strands. This novel pre-embedding technique may be a useful tool for ultrastructural evaluation of virus-host cell interactions.
Wolber
R A
RA
Department of Pathology, University of Michigan Medical School, Ann Arbor.
Beals
T F
TF
Maassab
H F
HF
D002467:Cell Nucleus / Q000032:analysis; D002478:Cells, Cultured; D003102:Colloids; D003593:Cytoplasm / Q000032:analysis; D015342:DNA Probes; D005347:Fibroblasts / Q000032:analysis / Q000648:ultrastructure*; D005404:Fixatives; D005557:Formaldehyde; D005976:Glutaral; D006046:Gold; D006801:Humans; D008854:Microscopy, Electron; D009693:Nucleic Acid Hybridization; D012367:RNA, Viral / Q000032:analysis*; D018139:Simplexvirus / Q000235:genetics*
D003102:Colloids; D015342:DNA Probes; D005404:Fixatives; D012367:RNA, Viral; D005557:Formaldehyde; D006046:Gold; D005976:Glutaral