IE proteins

HHV-6 gene transcription follows a similar pattern that characterizes herpesvirus, with immediate-early (IE), early and late proteins expressed (Dockrell, 2003). IE proteins are the first proteins expressed following viral entry, independent of de novo protein synthesis and play a crucial role in the initiation of infection, and the establishing productive infections, regulating reactivation from latency and evading immune recognition.

HHV-6 immediate-early A locus (IE-A) locates in the position analogous to the human cytomegalovirus (HCMV) major IE (MIE) locus that is well-known to play critical roles in viral infection. Similarly to HCMV MIE, HHV-6 IE-A consists of two genetic units, IE1 and IE2, corresponding to ORFs U90-U89 and U90-U86/87, respectively (Papanikolaou et al., 2002). However, the HHV-6 IE-A locus exhibits limited sequence homology with the HCMV MIE locus. IE2 proteins derived from the U86/87 region with apparent molecular mass of 100, 85 and 55 kDa are detected in HHV-6-infected cells 3 days after infection, while IE1 proteins with apparent molecular mass greater than 170 kDa are detectable as early as 8h (Papanikolaou et al., 2002). Mapping of the IE2 proteins suggests differential splicing and alternative translation initiation in the IE2 genetic unit. The IE2 proteins show a mixed cytoplasmic and nuclear localization pattern. Thus, it is detected (8-48 h) as intranuclear granules, while at later time points (72-120 h) the IE2 protein coalesces into a few large immunoreactive patches. Transfection of cells with an IE2 expression vector alone does not reproduce the patch-like distribution, suggesting that other viral proteins are necessary for this process to occur (Papanikolaou et al., 2002). In addition, the 437 amino acid carboxyl-terminus domain of IE2 binds to a DNA fragment containing the putative IE-A promoter. Cotransfection experiments in T cells indicate that IE2 can induce the transcription of a complex promoter, such as the human immunodeficiency virus (HIV)-long terminal repeat (LTR), as well as simpler promoters, whose expression is driven by a unique set of responsive elements (CRE, NFAT and NF-kB) (Gravel et al., 2003). Moreover, minimal promoters having a single TATA box or no defined eukaryotic regulatory elements are significantly activated by IE2, suggesting that IE2 is likely to play an important role in initiating the expression of several HHV-6 genes.

Furthermore, the heterogeneous nuclear ribonucleoprotein K (hnRNP K) and the beta subunit of casein kinase 2 (CK2beta) interacts with HHV-6 IE2, indicating that these interactions may affect viral and cellular RNA transcription and translation in viral replication (Shimada et al., 2004).

Analyses of HHV-6 IE gene expression have revealed that the IE1 gene of the HHV-6A and HHV-6B variants exhibits a higher degree of sequence variation than other regions of the genome and no obvious similarity to its positional analogue in HCMV.

The IE1B (HHV-6B) transcript consists of five exons (3720 nucleotides), three of which are coding for the IE1 protein (Gravel et al., 2002). The 1078-amino acid-long IE1B protein is 62% identical and 75% similar to the 941-amino acid IE1 from HHV-6A. IE1B protein can be detected at 4h p.i.I, and it is distributed as small intranuclear structures. The maximal number of IE1 bodies (approximately 10-12/nucleus) is detected at 12 h p.i., after which the IE1 bodies condense into 1-3 larger entities by 24-48h p.i. (Gravel et al., 2002). During infection, the IE1B protein is phosphorylated on serine and threonine residues. HHV-6A IE1 can also be detected at 4h p.i. as small dots, and accumulates at later PI (Fig. 1).

IE1 proteins form a stable interaction with, PML-bodies [also known as ND10 or nuclear promyelocytic leukemia protein (PML) oncogenic domains (PODS)] (Stanton et al., 2002). Remarkably, PML bodies remain structurally intact and associate with the IE1 protein throughout lytic HHV-6 infection, unlike other herpesviruses, suggesting that HHV-6 appears to have no requirement or mechanism to induce PML-body dispersal during lytic replication. In addition, IE1 is

Fig. 1 Indirect immunofluorescence assay of HHV-6-infected cells. The HSB-2 cells infected with HHV-6A (strain GS) were stained with monoclonal antibody for IE1 (a) U27 (b) or gB (c) at 86 h p.i. (a) IE1 locates in nucleus with punctuated pattern. (b) U27 locates in nucleus like forming replication compartment. (c) gB locates in the cytoplasm. (for colour version: see colour section on page 349).

Fig. 1 Indirect immunofluorescence assay of HHV-6-infected cells. The HSB-2 cells infected with HHV-6A (strain GS) were stained with monoclonal antibody for IE1 (a) U27 (b) or gB (c) at 86 h p.i. (a) IE1 locates in nucleus with punctuated pattern. (b) U27 locates in nucleus like forming replication compartment. (c) gB locates in the cytoplasm. (for colour version: see colour section on page 349).

covalently modified by conjugation to the small ubiquitin-like protein, SUMO-1. Overexpression of SUMO-1 in cell lines results in substantially enhanced levels of IE1 expression; thus, sumoylation may bestow stability to the protein (Stanton et al., 2002). However, SUMOylation-deficient mutants of IE1 co-localize with PODs as efficiently as wild type IE1, indicating that POD targeting is independent of IE1 SUMOylation status (Gravel et al., 2004).

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