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. 2005 Jan;79(1):225-33.
doi: 10.1128/JVI.79.1.225-233.2005.

The carboxyl-terminal region of human cytomegalovirus IE1491aa contains an acidic domain that plays a regulatory role and a chromatin-tethering domain that is dispensable during viral replication

Jens Reinhardt  1 , Geoffrey B Smith, Christopher T Himmelheber, Jane Azizkhan-Clifford, Edward S Mocarski
Affiliations

The carboxyl-terminal region of human cytomegalovirus IE1491aa contains an acidic domain that plays a regulatory role and a chromatin-tethering domain that is dispensable during viral replication

Jens Reinhardt et al. J Virol. 2005 Jan.

Abstract

The human cytomegalovirus major immediate-early (alpha) protein IE1(491aa) plays an important role in controlling viral gene expression at low multiplicities of infection. With a transient complementation assay, full-length IE1(491aa) enhanced the growth of ie1 mutant virus CR208 20-fold better than a deletion mutant lacking 71 carboxyl-terminal amino acids (IE1(1-420aa)). A 16-amino-acid domain between amino acids 476 and 491 was both necessary and sufficient for chromatin-tethering activity; however, this domain was completely dispensable for complementation of CR208 replication. The proximal 55-amino-acid acidic domain (amino acids 421 to 475) was found to be most important for function. A deletion mutant lacking only this domain retained chromatin-tethering activity but failed to complement mutant virus. Interestingly, serine phosphorylation (at amino acids 399, 402, 406, 423, 428, 431, 448, 451, and 455) was not required for complementation. These results show that IE1(491aa) is composed of at least two domains that support replication, a region located between amino acids 1 and 399 that complements ie1 mutant virus replication to low levels and an acidic domain between amino acids 421 and 479 that dramatically enhances complementation.

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Figures

FIG. 1.
FIG. 1.
Complementation of CR208 by transient transfection. (A) Titration of the ie1-deficient HCMV mutant CR208 by serial threefold dilution on complementing (ihfie1.3) and noncomplementing (HF) cells. TNTC, too numerous to count. (B) CR208 complementation by wild-type and mutant ie1 expression plasmids transfected into HFs. HFs were transfected with pCMV IE1full-lengthGFP, pCMV IE11-420GFP, or pCMV-GFP alone and infected with CR208 at an MOI of 0.005. The graph shows the mean plaque counts from three parallel assays, with the standard deviation of the mean indicated by bars. The input titer of CR208 was determined on ihfie1.3 cells (open bar). Expression constructs are named in the figure by the portion of IE1 that is expressed.
FIG. 2.
FIG. 2.
Localization of the chromatin-tethering domain in IE1491aa. (A) Sequence comparison of the acidic regions of primate CMV ie1 proteins. HCMV IE1491aa is depicted with amino acids 421 to 491 expanded and divided into a 55-amino-acid acidic domain (amino acids 421 to 475) and a 16-amino-acid chromatin-tethering domain (amino acids 476 to 491). Basic amino acids are underlined, and acidic amino acids are overlined. An alignment of the acidic region of HCMV IE1491aa and homologues from chimpanzee CMV (ChCMV), simian CMV (SCMV), and rhesus CMV (RhCMV) is displayed below the expanded sequence. Conserved amino acids are marked with asterisks; positions with conservative substitutions are indicated by a dot. Dashes indicate a gap in the alignment. (B) Metaphase chromatin tethering by wild-type and and mutant ie1 constructs. ihfie1.3 cells and HFs transfected with pCMV IE11-479, pCMV IE1Δ421-475GFP, or pCMV IE1476-491GFP were stained with anti-GFP and fluorescein isothiocyanate-labeled anti-rabbit immunoglobulin antibody (ihfie1.3 and IE1476-491GFP) to visualize GFP expression or IE1- and IE2-specific antibody and fluorescein isothiocyanate-labeled anti-mouse immunoglobulin (IE11-479 and IE1Δ421-475GFP) to visualize ie1 expression. Chromatin was stained with Hoechst 33342 or propidium iodide as indicated on the individual panels.
FIG. 3.
FIG. 3.
Role of the acidic domain (AD) and the chromatin-tethering domain (CTD) for complementation by IE1491aa. (A) Complementation of CR208 by different IE1491aa constructs at various MOIs. HFs transfected with pCMV IE1full-lengthGFP, pCMV IE11-479, pCMV IE11-420GFP, pCMV IE1Δ421-475GFP, or pCMV-GFP alone were infected with CR208 at the indicated MOIs. The black bars represent the mean plaque count of three replicate assays, with the standard deviation of the mean indicated by error bars. The input titer of CR208 was determined on ihfie1.3 cells (open bars). (B) MOI dependence of complementation of CR208 by ie1 constructs. The graph shows the percentage of plaques relative to the wild-type level achieved in HFs transfected with pCMV IE11-420GFP, pCMV IE11-479, pCMV IE1Δ421-475GFP, or pCMV-GFP alone and infected with CR208 at MOIs of 0.0004, 0.002, and 0.01. (C) Immunoblot detection of transfected IE1 constructs. Lysates of HFs transfected with pCMV IE1full-lengthGFP, pCMV IE11-420GFP, pCMV IE1Δ421-475GFP, and pCMV IE11-479. The IE11-479 migrates faster because it is not GFP tagged. The proteins were detected with IE1- and IE2-specific monoclonal antibody. β-Actin was detected on the same blot as a loading control. Expression constructs are named in the figure by the portion of IE1 that is expressed or deleted.
FIG. 4.
FIG. 4.
Impact of carboxyl-terminal mutations in IE1491aa on complementation of CR208. (A) Complementation of CR208 by IE1full-lengthGFP, pCMV IE11-442GFP, pCMV IE11-420GFP, pCMV IE11-414, pCMV IE11-399, pCMV IE1S→AGFP, pSG5 IE1S→A, pCMV IE1S→EGFP, pSG5 IE1S→E and pCMV-GFP alone. HFs transfected with pCMV were infected with CR208 at an MOI of 0.01. S→A and S→E mutants were assayed as GFP fusion proteins in the same expression plasmid as other mutants (pCMV-GFP) and as native proteins in the expression plasmid in which they were constructed (pSG5). The black bars represent mean plaque counts of three replicate assays of each construct, with the standard deviation of the mean indicated by error bars. (B) Summary of complementation results. A diagram of each construct with the number of acidic residues in the 55-amino-acid AD, the number of putative phosphorylation sites, and range of complementation as a percentage of that of the full-length construct. The positions of the acidic (AD) and the chromatin-tethering (CT) domains are indicated on the diagram of the full-length IE11-491 construct. Expression constructs are identified in the column by the portion of IE1 that is expressed or by the type of mutations introduced. The S→A mutant has alanines replacing serines at positions 399, 402, 406, 423, 428, 431, 448, 451, and 455 (positions denoted by gray circles); the mutant S→E has glutamates replacing these serines (position denoted by black circles).
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