Transient expression of herpes simplex virus type 1 ICP22 represses viral promoter activity and complements the replication of an ICP22 null virus

Abstract

Of the five herpes simplex virus type 1 immediate early (IE) proteins, the least is known about the function of ICP22 during productive infection and latency. Research characterizing the physical and functional properties of the protein has been limited because ICP22 has proven to be difficult to express in transient assays. In addition, genetic analysis of ICP22 has been complicated by the fact that the C terminus of ICP22 is expressed as a discrete protein product. In order to characterize properties of mutant and wild-type ICP22, we developed a transient expression system. We found that ICP22 can be expressed at detectable levels when placed under the control of the cytomegalovirus IE promoter, confirming recent observations by K. A. Fraser and S. A. Rice (J. Virol. 81:5091-5101, 2007). We extended this analysis to show that ICP22 can also be expressed from its own promoter in the presence of other viral factors, either by coexpression with ICP0 or by infection with an ICP22 null virus. Notably, infection of cells transfected with an ICP22 expression vector yielded ICP22 protein that was modified in a manner similar to that of ICP22 protein detected in wild-type-infected cells. We go on to demonstrate that the failure of ICP22 protein to be expressed in transiently transfected cells was not due to inactivity of the ICP22 promoter, but rather to the ability of ICP22 to inhibit expression of reporter gene activity, including its own, in transient assays. Of special note was the observation that expression of ICP22 was sufficient to prevent transactivation of reporter genes by ICP0. Finally, transient expression of ICP22 was sufficient to complement replication of an ICP22 null virus, demonstrating that this system can be used to study functional properties of ICP22. Collectively, this transient expression system facilitates tests of the physical and functional properties of ICP22 and ICP22 mutants prior to introduction of mutant genes into the viral genome.

FIG. 1.

Construction and characterization of d22:GFP. (A) Diagram of the KOS genome. The positions of the unique (lines) and repeat (boxes) regions of the genome are indicated. The position of the U_S1 gene is noted. TR_L, terminal repeat long; TR_S, terminal repeat short; IR_L, internal repeat long; IR_S, internal repeat short. (B) The EcoRI-EcoRI fragment of the KOS genome containing U_S1 is shown. The positions of the U_S1 and U_S2 genes are represented by the gray boxes. The positions of the ICP22-specific probe is marked by the hatched bar. (C) Map of the d22:GFP genome. The ICP22 ORF was deleted (section in parentheses) and replaced with an eGFP cassette. Introduction of GFP introduced a novel fragment containing a NotI site, which is shown. The probe specific to eGFP is represented by the hatched bar. (D) Southern blot of KOS and d22:GFP. KOS and d22:GFP DNAs were digested with EcoRI and NotI and probed for ICP22- and eGFP-specific sequences by Southern blotting. (E) Replication of d22:GFP in Rab-9 cells. Replicate cultures of Rab-9 cells were infected with 2.5 PFU/cell KOS, 22/n199 (N199), or d22:GFP. At 24 hours postinfection, total virus was harvested and assayed on Vero cells by standard plaque assays. The experiment was repeated three times, and the error bars indicate the standard deviations. (F) Viral gene expression in d22:GFP-infected Rab-9 cells. Replicate cultures of Rab-9 cells were mock infected or infected with 2.5 PFU/cell KOS, 22/n199, or d22:GFP. At 6 hours postinfection, total cell lysates were prepared, and levels of ICP22, ICP4, and gC were determined by Western blotting.

FIG. 2.

Transient expression of ICP22. (A) Maps of ICP22 expression vectors. HSV-1-specific sequences are denoted by solid lines. Dotted lines indicate vector sequences. The promoter and polyadenylation sequences utilized in each vector are noted. (B) Expression of ICP22 from plasmid vectors in transfected mock-infected and transfected d22:GFP-infected Vero and Rab-9 cells. The position of the ICP22-specific bands is marked with a bracket. A nonspecific band routinely detected with the anti-ICP22 Ab is marked with an arrow.

FIG. 3.

Cotransfection of ICP0 and ICP22. (A) Western blots of Vero and Rab-9 cells cotransfected with plasmids expressing ICP22 and ICP0. The ICP22-specific band is marked with an arrow. (B) ICP0 and ICP22 RNA levels in transfected Rab-9 cells as determined by Northern blotting. (C) ICP22 protein expression in Rab-9 cells transfected with increasing amounts of an ICP0 expression vector.

FIG. 4.

ICP22 promoter activity in Rab-9 cells. Vero cells were transfected with the luciferase reporter constructs indicated. Data presented are the averages of the results for three experiments. Error bars indicate the standard deviations.

FIG. 5.

Repression of promoter activity by ICP22. (A) ICP22 represses its own promoter. Vero cells were transfected with the ICP22 luciferase reporter construct and increasing amounts of the ICP22 expression vector. Firefly luciferase activity was measured and normalized to the “no pCDNA3:22ORF” control. (B) Western blots of ICP22 expression. MT represents mock-transfected cells, and the plus sign indicates a positive cell lysate control. (C) ICP22 expression represses expression from the CMV IE promoter. (D) ICP22 prevents transactivation by ICP0. The ICP22 promoter construct was cotransfected with the ICP0 expression vector, pSH, and increasing concentrations of pCDNA3:22ORF as indicated. The level of luciferase activity was measured 24 hours posttransfection. Data presented are the averages of the results for three experiments. Error bars indicate the standard deviations.

FIG. 6.

Complementation of d22:GFP replication in Rab-9 cells. Rab-9 cells were transfected with the amounts of plasmid indicated. Total virus replication was scored by a plaque assay on Vero cells and reported as an n-fold increase over that on pCMV:GFP-transfected cells. Bars represent the averages of the results for three experiments (except for at 0.25 μg, for which there were two experiments), and error bars represent the standard deviation.