Characterization of Regulatory Functions of the HSV-1 Immediate-Early Protein ICP22

Varicella-zoster virus (VZV) open reading frame 63 (ORF63)protein is expressed during latency in human sensory ganglia. Deletion of ORF63 impairs virus replication in cell culture and establishment of latency in cotton rats. We found that cells infected with a VZV ORF63 deletion mutant yielded low titers of cell-free virus and produced very few enveloped virions detectable by electron microscopy compared with those infected with parental virus. Microarray analysis of cells infected with a recombinant adenovirus expressing ORF63 showed that transcription of few human genes was affected by ORF63; a heat shock 70-kDa protein gene was downregulated, and several histone genes were upregulated. In experiments using VZV transcription arrays, deletion of ORF63 from VZV resulted in a fourfold increase in expression of ORF62, the major viral tran- Varicella-zoster virus (VZV) causes chicken pox upon primary infection and then establishes latency in cranial nerve and dorsal root ganglia and can reactivate to cause shingles (herpes zoster). The lifetime risk of shingles is 10 to 20% for adults in the United States who are seropositive for VZV (14). Transcripts corresponding to VZV open reading frames (ORFs) 4, 21, 29, 62, 63, and 66 have been detected in latently infected human ganglia (8,10,24,25,36). ORF63 is the most frequently detected and most abundant of these transcripts (10, 25). ORF63 protein has also been detected in latently infected human (25,31,33) and rodent (11, 22) ganglia.ORF63 encodes a virion tegument protein (28, 38), but the function of this protein is unclear. ORF63 protein enhances the activation of the minimal gI promoter by VZV ORF62 protein (32) and represses other viral genes (3,20). In transient-transfection assays, ORF63 protein represses promoters containing typical TATA boxes (12), including VZV ORF4 and ORF28, heterologous viral promoters, and the human interleukin-8 promoter. The VZV gI promoter, which contains an atypical TATA box, is only slightly repressed. In contrast, ORF63 protein activates the cellular elongation factor 1 (EF-1) alpha promoter in some cell types (53).A recombinant VZV in which 90% of both ORF63 and its duplicate gene, ORF70, are deleted is viable but impaired for growth in cell culture and latency in cotton rats (5). Analysis of additional ORF63 mutants showed that viruses that are impaired for replication in cell culture are also impaired for latency, while mutants that are not impaired for replication establish latencies at frequencies and copy numbers similar to those of parental virus (6).In this study, we examined the effects of VZV ORF63 on virion production and regulation of viral and cellular gene expression. We found that ORF63 downregulates transcription of ORF62 and that cells infected with VZV mutants impaired for replication and latency show increased transcription of ORF62, while cells infected with a mutant that is not impaired express wild-type levels of ORF62. MATERIALS AND METHODS Cells and viruses.Human diploid fibroblasts (MRC-5) were obtained from...

Section: Discussionmentioning

confidence: 71%

Downregulation of Varicella-Zoster Virus (VZV) Immediate-Early ORF62 Transcription by VZV ORF63 Correlates with Virus Replication In Vitro and with Latency

Hoover

Cohrs²,

Rangel

et al. 2006

“…Since previous studies have suggested that ICP22 can repress transcription from a large number of promoters in transient re- porter assays, we wanted to determine if ICP22 can repress expression from its own promoter (13). To this end, Vero cells were transfected with the ICP22 promoter-luciferase construct and increasing amounts of pCDNA3:22ORF.…”

Section: Resultsmentioning

confidence: 99%

“…Initial studies by Prod'hon et al (13) examined the effect of expression of ICP22 on HSV promoter activity. In contrast to its role in upregulating late viral gene expression during infection, cotransfection of ICP22 alone with a panel of HSV-1 promoters repressed transcription of reporter genes.…”

Section: Replication Of Icp22mentioning

confidence: 99%

Transient Expression of Herpes Simplex Virus Type 1 ICP22 Represses Viral Promoter Activity and Complements the Replication of an ICP22 Null Virus

Bowman

Orlando

Davido

et al. 2009

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.

“…To a degree, all kinetic classes of viral genes are affected during infection with ICP22 Ϫ viruses. Although ICP22 is required to achieve wild-type levels of viral proteins in infected cells, it has been reported that ICP22 negatively regulates expression from viral promoters in transient-transfection assays (22,33). Because ICP22 is able to both up-and downregulate gene expression under different conditions, it is possible that its role may be to control the regulation of viral gene expression.…”

Section: Icp0mentioning

confidence: 99%

ICP22 Is Required for Wild-Type Composition and Infectivity of Herpes Simplex Virus Type 1 Virions

Orlando

Balliet

Kushnir

et al. 2006

The immediate-early regulatory protein ICP22 is required for efficient replication of herpes simplex virus type 1 in some cell types (permissive) but not in others (restrictive). In mice infected via the ocular route, the pathogenesis of an ICP22؊ virus, 22/n199, was altered relative to that of wild-type virus. Specifically, tear film titers of 22/n199-infected mice were significantly reduced at 3 h postinfection relative to those of mice infected with wild-type virus. Further, 22/n199 virus titers were below the level of detection in trigeminal ganglia (TG) during the first 9 days postinfection. On day 30 postinfection, TG from 22/n199-infected mice contained reduced viral genome loads and exhibited reduced expression of latency-associated transcripts and reduced reactivation efficiency relative to TG from wild-type virus-infected mice. Notably, the first detectable alteration in the pathogenesis of 22/n199 in these tests occurred in the eye prior to the onset of nascent virus production. Thus, ICP22؊ virions appeared to be degraded, cleared, or adsorbed more rapidly than wild-type virions, implying potential differences in the composition of the two virion types. Analysis of the protein composition of purified extracellular virions indicated that ICP22 is not a virion component and that 22/n199 virions sediment at a reduced density relative to wild-type virions. Although similar to wild-type virions morphologically, 22/n199 virions contain reduced amounts of two ␥ 2 late proteins, U S 11 and gC, and increased amounts of two immediate-early proteins, ICP0 and ICP4, as well as protein species not detected in wild-type virions. Although ICP22؊ viruses replicate to near-wild-type levels in permissive cells, the virions produced in these cells are biochemically and physically different from wild-type virions. These virion-specific differences in ICP22 ؊ viruses add a new level of complexity to the functional analysis of this immediate-early viral regulatory protein.