Kinetics of expression of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1

Abstract

The 65-kilodalton DNA-binding protein (65KDBp) of herpes simplex virus type 1, encoded by gene UL42, is required for herpes simplex virus origin-dependent DNA replication (C. A. Wu, N. J. Nelson, D. J. McGeoch, and M. D. Challberg, J. Virol. 62:435-443, 1988). We found by indirect immunofluorescence with monoclonal antibody to 65KDBP that the protein was first detectable at 3 h postinfection. It localized first to the inner periphery of the nucleus, but accumulated in large globular compartments within the nucleus by 6 h postinfection in a pattern similar to that displayed by the major DNA-binding protein ICP8. Immune electron microscopy revealed that 65KDBP was associated with the marginated heterochromatin at the early times, but migrated further into the nucleus at late times when the only discernible areas devoid of 65KDBP were the nucleoli and heterochromatin. The 65KDBP gene is a member of the 0i kinetic class as determined by the ability of the mRNA to be expressed at significant levels even in the absence of viral DNA synthesis. Furthermore, in the presence or absence of the DNA polymerase inhibitor phosphonoacetic acid, the patterns of accumulation of protein as well as mRNA were virtually indistinguishable from those displayed by the model 0 genes encoding ICP8 and thymidine kinase. Nuclear run-on experiments demonstrated that maximum rates of

Identification and characterization of the herpes simplex virus type 1 protein encoded by the UL37 open reading frame

“…As internal controls for the individual time points, the protein extracts were probed with antisera directed against UL42, a 65-kDa DNA-binding protein (14,22), and ICP8, the major HSV-1 DNA-binding protein (3,27,30). The kinetics of appearance in time course experiments for both of these proteins have been reported previously (15,26). Both the UL42 and the ICP8 proteins appeared early, being detected by 6 HPI (Fig.…”

Topoisomerase II cleavage of herpes simplex virus type 1 DNA in vivo is replication dependent

“…Another possibility is that viral genomes undergo subnuclear relocalization during replication, which makes their DNA accessible to topoisomerase II. Support for the latter possibility comes from several reports, which suggest that nuclear relocalization of the viral DNA correlates with HSV-1 DNA replication (14,21,38). In any case, the interaction between topoisomerase II and DNA only becomes apparent when infected cells are exposed to agents that stabilize the cleavage intermediate.…”

Localization of the herpes simplex virus type 1 65-kilodalton DNA-binding protein and DNA polymerase in the presence and absence of viral DNA synthesis

“…Cells were fixed in 3.7% formaldehyde and permeabilized for 2 min with -20°C acetone as described previously (10). In single-fluorochrome labeling experiments, the MAb or monospecific antibody was first added, the cells were washed with phosphate-buffered saline, and the secondary species-specific IgG (Cappel Laboratories, West Chester, Pa.) conjugated to either fluorescein isothiocyanate (FITC) or rhodamine isothiocyanate (RITC) was added (10). In double-labeling experiments, a second set of primary antibody-secondary antibody incubations was performed before the addition of mounting medium (90% glycerol, 10% phosphate-buffered saline, 150 mM propyl gallate [Eastman Kodak Co., Rochester, N.Y.]).…”