Comprehensive Characterization of Interaction Complexes of Herpes Simplex Virus Type 1 ICP22, UL3, UL4, and UL20.5

Abstract

It has been reported that herpes simplex virus type 1 UL3, UL4, and UL20.5 proteins are localized to small, dense nuclear bodies together with ICP22 in infected cells. In the present study, we comprehensively characterized these interactions by subcellular colocalization, coimmunoprecipitation, and bimolecular fluorescence complementation assays. For the first time, it was demonstrated that both UL3 and UL20.5 are targeted to small, dense nuclear bodies by a direct interaction with ICP22, whereas UL4 colocalizes with ICP22 through its interaction with UL3 but not UL20.5 or ICP22. There was no detectable interaction between UL3 and UL20.5.Of the 84 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) known to be expressed, more than half are functionally poorly understood and identified as encoding dispensable proteins for viral replication at least in some cell lines (21,22). However, these ORFs appear to be essential for viral survival in nature, since viruses lacking these genes have not been isolated. Because mutant viruses lacking these genes have no obvious phenotype in infected cells, it remains difficult to functionally characterize these genes.As an immediate-early protein, ICP22 appears to be essential for viral replication (5, 19). It localizes in the nucleus of infected cells and functions as a transcriptional repressor for both viral and cellular genes (5). Furthermore, ICP22 associates with transcriptional complexes containing the viral transactivator protein ICP4, RNA polymerase II (4, 9, 14, 16), and other host factors, including cdc25C (25, 26) and cdk9 (7). Previous studies have reported that both UL3 and UL4 are expressed late in infection and are dispensable for viral replication in cultured cells in vitro (2,8,12). As a novel identified ORF situated between genes encoding UL20 and UL21 of the HSV-1 genome, the UL20.5 gene is not essential for growth and belongs to the ␥2 kinetic class (27). Nevertheless, although the proteins encoded by these genes are dispensable for growth in cell culture, their functions are still unknown.ICP22 localizes to small, dense nuclear structures early in infection, followed by the transition to the more diffuse replication complexes with nascent DNA and RNA polymerase II, ICP4, and other proteins associated with late gene transcription after the onset of viral DNA synthesis (11,14). During later infection, ICP22 aggregates with UL3, UL4, and UL20.5 proteins as discrete, dense spots in the nucleus (11,18,27). However, deletion of ICP22 resulted in a decreased accumulation of UL3 and UL4, and neither UL3 nor UL4 localizes to

Herpes Simplex Virus 1 UL24 Abrogates the DNA Sensing Signal Pathway by Inhibiting NF-κB Activation

“…HEK293T cells, Vero cells, and HFF cells were grown in Dulbecco's modified Eagle medium (DMEM) (Gibco-BRL) supplemented with 10% fetal bovine serum (FBS) (Gibco-BRL) and 100 U/ml of penicillin and streptomycin. HeLa cells were maintained in Eagle's minimum essential medium (MEM) (Gibco-BRL) supplemented with 10% FBS (58,59). The WT HSV-1 KOS strain and the UL24 mutant virus (UL24X) were originally from Don Coen's laboratory (39).…”

Herpes Simplex Virus 1 Ubiquitin-Specific Protease UL36 Abrogates NF-κB Activation in DNA Sensing Signal Pathway

“…HEK293T cells, Vero cells, and HFF cells were grown in Dulbecco modified Eagle medium (Gibco-BRL) supplemented with 10% fetal bovine serum and 100 U of penicillin and streptomycin/ml. The WT HSV-1 F strain and its derivative UL36USP mutant HSV-1 strain (C40A mutant HSV-1) were propagated in Vero cells and titrated as described previously (52). Two-step Red-mediated recombination was performed to construct C40A rescue HSV-1 (C40AR) as previously described (24).…”

“…Coimmunoprecipitation (Co-IP) assays and Western blot (WB) analysis were performed as previously described (52). Briefly, cells were transfected or infected with WT HSV-1 or C40A mutant HSV-1 as indicated in the figure legends.…”

Molecular cloning and characterization of the pseudorabies virus US1 gene

“…It appears to be a universal phenomenon that almost every protein is marked with a localization signal (Feng, 2002). Subcellular localization research of HSV-1 ICP22 suggests that in an HSV-1-infected cell, ICP22 predominantly locates in the nucleus, particularly in the small, dense nuclear bodies early in infection and in the diffuse replicative complexes after onset of DNA synthesis (Jahedi et al, 1999;Xing et al, 2011). PICP22 was predicted to be localized in the cytoplasm where it might function as a cytoplasmic-targeted protein, although it does contain a putative nuclear localization signal (NLS, 47PPKRGRY53).…”