Asynchronous Expression of the Immediate-Early Protein of Herpesvirus Saimiri in Populations of Productively Infected Cells

Abstract

SUMMARYThe time course of virus replication in cultures of permissive cells infected with high multiplicities of herpesvirus saimiri (HVS), a gammaherpesvirus, is protracted relative to the replication of herpes simplex virus (HSV), an alphaherpesvirus, under similar conditions. The basis for this difference was investigated by quantitative immunofluorescence microscopy exploiting monoclonal antibodies specific to the HVS 52000 mol. wt. immediate-early polypeptide (IE 52K) and to delayed-early (DE 51K, DE 110K) and late (130K and capsid proteins) gene products to measure the timing of gene expression in individual cells of infected cultures. The timing of the transitions from IE to DE and from DE to late protein synthesis occurred at proportionately different intervals in the growth cycle of HVS, relative to that of HSV. In particular, the DE to late transition occurred relatively later in HVS infections. However, asynchrony in the events leading to the expression of the first class of virus proteins (IE 52K) was the main source of the extended course of HVS replication in populations of infected cells. This asynchrony was not modified significantly by infection at different stages of the host cell-cycle and was reduced, but not overcome, by very high applied multiplicities of infection. Double-antibody staining revealed a positive correlation between the accumulation of high concentrations of parental virus particles at perinuclear sites and early detection of HVS IE 52K gene expression. Both of these events remained sensitive to a microtubule poison (colcemid) for many hours after infection with HVS, whereas the rapid and synchronous expression of the IE 175K protein (ICP4) in HSV-1-infected cells was insensitive to post-infection exposures to this drug. We conclude that significant differences in early stages of virus entry and intracellular processing which precede immediate-early gene expression are largely responsible for differences between the replicative cycles of these representatives of gamma-and alphaherpesviruses in cultures of permissive cells.

Differential Expression of Two Immediate Early Genes of Herpesvirus Saimiri as Detected by in Situ Hybridization

“…This proportion changed from 10 to 20~ at 6 h in the presence of cycloheximide to 40 to 509/00 at 12 h. In the absence of cycloheximide only a very low level of hybridization was visualized with the EcoRI-I probe. These results of in situ hybridization to measure RNA accumulation resemble very closely the results from studies reported by Randall et al (1985) of the asynchronous accumulation of the 52K protein based on immunofluorescence microscopy.…”

“…Thus, in addition to cell-to-cell variations in entry and intracellular processing of virus particles which provides one component of asynchrony (Randall et al, 1985), it is now clear that the transcription of the IE 52K gene requires (or is prevented by) factors which do not affect the transcription of the IE gene in HindlII-G. Thus there is differential regulation of the expression of these two IE genes.…”

“…Synthesis of the IE 52K protein preceded the synthesis of the DE DNA-binding proteins (51K and 110K) in all cells, but there was a significant degree of cell-to-cell variation in the time from infection to detectable 52K expression. This asynchrony could be reduced, but not overcome, by the use of very high m.o.i, and was sufficient to account for the relatively protracted cycle of replication of HVS in tissue culture (Randall et al, 1985).…”

“…Thus there is differential regulation of the expression of these two IE genes. Our previous studies showed that expression of the 52K protein was followed by entry into the lyric cycle of DE and late gene expression (Randall et al, 1984a(Randall et al, , 1985. The nature of the transcription factors which differentiate between the HindlII-G and 52K IE genes of HVS seem likely to play a crucial role in determining the entry of this virus into a lytic cycle of virus gene expression.…”

Intranuclear Localization of Herpes Simplex Virus Immediate-Early and Delayed-Early Proteins: Evidence that ICP 4 Is Associated with Progeny Virus DNA

“…At high multiplicities of infection (10 post-infection (data not shown; Randall et al, 1985). The number of loci in which ICP 4 and ICP 8 accumulated per nucleus was dependent on the multiplicity of infection ( Fig.…”