Immediate-early expression of the herpes simplex virus type 1 ICP27 transcript is not critical for efficient replication in vitro or in vivo

Abstract

We constructed a promoter mutation altering the immediate-early expression of the herpes simplex virus type 1 (HSV-1) ICP27 transcript and its cognate wild-type rescue viruses in order to assess the role of the ICP27 protein in the earliest stages of viral infection by global transcriptional analysis with a DNA microarray. This mutant, ICP27/VP16, replaces the whole ICP27 promoter/enhancer with the VP16 promoter. It demonstrates loss of immediate-early expression of ICP27 according to the criteria expression in the absence of de novo protein synthesis and earliest expression in the kinetic cascade. Significant differences in relative transcript abundances between the mutant and wild-type rescue viruses were limited at the earliest times measured and not evident at all by 4 h after infection. Consistent with this observation, levels of some critical proteins were reduced in the mutant as compared to rescue virus infections at the earliest times tested, but were equivalent by 8 h postinfection. Further, both single and multistep levels of virus replication were equivalent with both mutant and rescue viruses. Thus, altering the immediate-early kinetics of ICP27 leads to a suboptimal quantitative lag phase in gene expression but without consequence for replication fitness in vitro. Infections in vivo also revealed equivalent ability of mutant and rescue viruses to invade the central nervous system of mice following footpad injections. Limitations to an immediate-early role of ICP27 in the biology of HSV are discussed in light of these observations.

FIG. 1.

Western blot analysis of HSV protein synthesis following infection with ICP27 mutants. Extracts from cells with the indicated viruses were fractionated by SDS-PAGE, the proteins were transferred to nitrocellulose, and the blots were probed with antibodies specific for ICP4, ICP0, ICP27, gB, and gC. Infections were performed at an MOI of 1 for the indicated times.

FIG. 2.

Replication of the ICP27/VP16 kinetic mutant in vitro and in vivo. (A) Cultures of confluent MEFs were infected with 1,000 PFU of virus as described in the text, and the titer of the virus yield was determined 12 and 24 h p.i. The experiment is described in the Results section. (B) Multistep replication of the ICP27/VP16 kinetic mutant in confluent MEFs. Circles represent the rescue values, and squares represent the mutant values. The vertical size of the symbols is a measure of the standard deviation of each experimental determination (see Results and Materials and Methods for details). (C) Recovery of the ICP27/VP16 kinetic mutant in mouse DRG at 5 days following footpad infection (see Results and Materials and Methods for details).