Origin of Expression of the Herpes Simplex Virus Type 1 Protein US1.5

The first genes of herpes simplex virus type 1 (HSV-1) to be expressed, the immediate-early (IE) genes, encode infected cell protein 0 (ICP0), ICP4, ICP22, ICP27, and ICP47, which prime the cell to promote efficient viral transcription and DNA replication. While four IE proteins (ICP0, -4, -27, and -47) have been studied extensively, the functional roles of ICP22, a 420-amino-acid (aa) phosphoprotein, in both productive infection and latency have not been determined. Elucidation of the functions of ICP22 has been hindered by several factors. First, ICP22 was originally described as nonessential for viral replication in cell culture and as such was largely ignored (22). Second, transient expression of ICP22 in the absence of other viral factors has proven to be highly inefficient and has been reported for only a few isolated studies (9, 23). Third, genetic analysis of ICP22 has been complicated by coexpression of the in-frame, C-terminal variant of ICP22, US1.5. The purpose of this study was to obtain information sufficient to develop techniques adequate for the genetic and functional analysis of ICP22 and US1.5 as separate entities. Although information regarding the functions of ICP22 and/or US1.5 is limited, several physical properties have been described for the two overlapping proteins during productive infection. ICP22 is expressed from the Us1 transcript, which initiates within the short internal repeat sequence of the HSV-1 genome (Fig. ​(Fig.1).1). The ICP22 open reading frame (ORF) is located entirely within the unique short (US) region of the genome. The US1.5 protein has been reported to be expressed from its own transcript, which initiates within the ICP22 ORF at codon 146 (6). Both ICP22 and US1.5 localize to the nuclei of infected cells, and ICP22 localizes to dense foci which costain for ICP4 and RNA polymerase II (9, 14). ICP22 migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as multiple bands ranging in size from 65 to 90 kDa, much larger than the size predicted for the 420-aa protein (24). Similarly, US1.5 is also detected in multiple forms ranging in size from 40 to 50 kDa, once again larger than the 30 kDa predicted for the 249-aa protein (16). The multiple forms of both proteins and their reduced mobilities in SDS-PAGE are due in large part to extensive posttranslational modification by both viral and cellular enzymes. Infection of cells with viral mutants lacking the two viral Ser/Thr protein kinases (Us3 and Ul13) results in a loss of most of the modified forms of ICP22 and US1.5, suggesting that both proteins are extensively phosphorylated by the two viral kinases (16, 24). Consistent with this hypothesis, direct phosphorylation of ICP22 by Us3 (11) and UL13 (1) has been demonstrated in vitro. In addition to being phosphorylated by viral kinases, ICP22 is phosphorylated on Tyr116 by an unknown cellular enzyme (5). It has also been reported that ICP22 is nucleotydilated by casein kinase II (3). FIG. 1. (A) Map of the HSV-1 genome showing the location of ICP22 and Us1.5. The positions of unique and repeat sequences are indicated by lines and boxes, respectively. The Us1 transcript initiates within the internal repeat short (IRs) segment of the genome ... Although little is known about the functions of ICP22 and US1.5, functional analysis of mutant viruses that fail to express both proteins has provided some insight into their roles in productive infection. The absence of both ICP22 and US1.5 in mutant viruses results in the failure of these viruses to replicate efficiently in a number of commonly used cell lines, including Rab-9, human embryonic lung cells, human foreskin fibroblasts, and rabbit skin cells but not Vero, Hep-2, or HeLa cells (17, 20, 26). From a molecular perspective, this cell type-specific defect in replication is coincident with a decrease in the rate of synthesis of viral transcripts, the failure to express wild-type (WT) levels of a subset of late viral proteins, and changes in the composition and density of mature extracellular virions (18, 24, 25). In addition, ICP22/US1.5 null viruses replicate poorly in the mouse ocular model during the acute phase of infection, establish latency with reduced efficiency, and exhibit reduced virulence (18, 19, 26). While these multiple and various phenotypes are intriguing, the mechanism by which ICP22 facilitates WT replication is unclear. In addition, the individual contributions of ICP22 and/or US1.5 to each of the phenotypes are unknown. To begin to address these questions genetically and at the molecular level, expression and characterization of each protein independently of the other is necessary. Although previous studies have succeeded in expressing US1.5 in the absence of ICP22, all the ICP22-negative, US1.5-positive mutant viruses generated to date have been characterized only for the efficiency of expression of a subset of late viral genes and for the mobility of US1.5 in SDS-PAGE gels and not for any of the other properties ascribed to ICP22 mentioned above (16). In addition, the ICP22-negative, US1.5-positive mutant viruses were generated using cosmid libraries, and the integrity of the complete mutant viral genomes was not analyzed. Finally, mutant viruses that express ICP22 but not US1.5 were not generated or compared to mutants expressing only US1.5. These studies have been further complicated by the fact that the start site originally reported by Ogle and Roizman for US1.5, M147 (16) was later reported by the same group to be incorrect, and they reported that US1.5 actually initiates at M171 (21). In order to resolve these issues and begin to elucidate the roles of ICP22 and US1.5 individually during infection, we utilized a recently described transient expression system (4) for ICP22 to study mutants lacking one protein or the other. Using this system, we have attempted to identify the bona fide start site of US1.5. Specifically, we introduced a panel of deletion, nonsense, and site-directed point mutations into the ICP22 ORF. We found that while US1.5 expression was not dependent on expression of full-length ICP22, mutation of M171 to alanine failed to alter US1.5 expression. More-extensive mutational analysis of the ICP22 ORF suggests that translation of US1.5 initiates at M90, much further upstream than indicated by all previous reports. Initiation at M90 would also preclude expression of US1.5 from the previously described US1.5 transcript, raising questions concerning the mechanism that is responsible for US1.5 translation.