Epstein-Barr virus (EBV) is a human lymphocryptovirus that is associated with several malignancies. Elevated EBV DNA in the blood is observed in transplant recipients prior to, and at the time of post-transplant lymphoproliferative disease; thus, a vaccine that either prevents EBV infection or lowers the viral load might reduce certain EBV malignancies. Two major approaches have been suggested for an EBV vaccine- immunization with either EBV glycoprotein 350 (gp350) or EBV latency proteins (e.g. EBV nuclear antigens [EBNAs]). No comparative trials, however, have been performed. Rhesus lymphocryptovirus (LCV) encodes a homolog for each gene in EBV and infection of monkeys reproduces the clinical, immunologic, and virologic features of both acute and latent EBV infection. We vaccinated rhesus monkeys at 0, 4 and 12 weeks with (a) soluble rhesus LCV gp350, (b) virus-like replicon particles (VRPs) expressing rhesus LCV gp350, (c) VRPs expressing rhesus LCV gp350, EBNA-3A, and EBNA-3B, or (d) PBS. Animals vaccinated with soluble gp350 produced higher levels of antibody to the glycoprotein than those vaccinated with VRPs expressing gp350. Animals vaccinated with VRPs expressing EBNA-3A and EBNA-3B developed LCV-specific CD4 and CD8 T cell immunity to these proteins, while VRPs expressing gp350 did not induce detectable T cell immunity to gp350. After challenge with rhesus LCV, animals vaccinated with soluble rhesus LCV gp350 had the best level of protection against infection based on seroconversion, viral DNA, and viral RNA in the blood after challenge. Surprisingly, animals vaccinated with gp350 that became infected had the lowest LCV DNA loads in the blood at 23 months after challenge. These studies indicate that gp350 is critical for both protection against infection with rhesus LCV and for reducing the viral load in animals that become infected after challenge. Our results suggest that additional trials with soluble EBV gp350 alone, or in combination with other EBV proteins, should be considered to reduce EBV infection or virus-associated malignancies in humans.

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-typeinfected 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.

ICP22, an immediate-early protein of herpes simplex virus type 1 (HSV-1), is required for viral replication in nonpermissive cell types and for expression of a class of late viral proteins which includes glycoprotein C. An understanding of the mechanism of ICP22 function has been complicated by the coexpression of the full-length protein with an in-frame, C-terminus-specific protein, U S 1.5. In this report, we confirm that the U S 1.5 protein is a bona fide translation product since it is detected during infections with three laboratory strains and two low-passage clinical isolates of HSV-1. To clarify the expression patterns of the ICP22 and U S 1.5 proteins, we examined their synthesis from plasmids in transient expression assays. Because previous studies had identified two different U S 1.5 translational start sites, we attempted to determine which is correct by studying the effects of a series of deletion, nonsense, and methionine substitutions on U S 1.5 expression. First, amino acids 90 to 420 encoded by the ICP22 open reading frame (ORF) migrated at the mobility of U S 1.5 in sodium dodecyl sulfate-polyacrylamide gels. Second, introduction of a stop codon downstream of M90 ablated expression of both ICP22 and U S 1.5. Finally, mutation of M90 to alanine (M90A) allowed expression of full-length ICP22 while dramatically reducing expression of U S 1.5. Levels of U S 1.5 but not ICP22 protein expression were also reduced in cells infected with an M90A mutant virus. Thus, we conclude that expression of IC22 and that of U S 1.5 can occur independently of each other and that U S 1.5 translation initiates at M90 of the ICP22 ORF.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, U S 1.5. The purpose of this study was to obtain information sufficient to develop techniques adequate for the genetic and functional analysis of ICP22 and U S 1.5 as separate entities.Although information regarding the functions of ICP22 and/or U S