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Short Communication |
1 Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Kyonggido 440-746, Korea
2 School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea
Correspondence
Jin-Hyun Ahn
jahn{at}med.skku.ac.kr
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ABSTRACT |
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Arg substitutions at positions 175 and 180, the two major sumoylation sites. When human fibroblast (HF) cells were infected with the reconstituted mutant virus, (i) viral growth kinetics, (ii) the accumulation of IE1 (UL123), IE2 (UL122), p52 (UL44) and pp65 (UL83) proteins and (iii) the relocalization of the cellular small ubiquitin-like modifier (SUMO)-1, p53 and proliferating cell nuclear antigen proteins into viral DNA replication compartments were comparable with those of the wild-type and the revertant virus. The data demonstrate that sumoylation of IE2 is not essential for virus growth in cultured HF cells. |
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, and references therein). A study using a recombinant viral genome revealed that IE2 is essential for the expression of all lytic cycle viral genes in tissue culture (Marchini et al., 2001). IE2 was also found to act as a repressor of its own major IE (MIE) promoter by directly binding to the MIE cis repression signal near the 5' cap site (Liu et al., 1991; Pizzorno & Hayward, 1990; Pizzorno et al., 1988). Studies on the intranuclear targeting of IE2 have shown that IE2 initially targets adjacent to premyelocytic leukaemia (PML) protein-associated nuclear bodies, known as PML oncogenic domains (PODs) or nuclear domain 10 (ND10), to form immediate transcription domains, and accumulates in viral DNA replication compartments at the later stages of infection (Ahn et al., 1999; Ishov et al., 1997).
IE2 has been shown to be covalently modified by small ubiquitin-like modifiers (SUMOs) at two Lys residues at positions 175 and 180 (Ahn et al., 2001; Hofmann et al., 2000). Several studies have suggested that sumoylation of IE2 is associated with the increased transactivation activity of IE2 in target reporter gene assays in DNA-transfected cells. Hofmann et al. (2000) reported that a sumoylation-deficient K175/180R mutant IE2 from the AD169 strain of HCMV showed significantly reduced transcriptional activation of the two viral promoters UL84 and UL112–113. In a study using IE2 from the Towne strain, it was shown that transactivation of the polymerase (UL54) promoter by the K175/180R mutant IE2 was only slightly reduced compared with the wild-type. However, IE2-mediated transactivation of the cellular cyclin E promoter was significantly increased in cells co-transfected with SUMO-1 and Ubc9, a SUMO E2 conjugation enzyme (Ahn et al., 2001). In a previous study, we found that PIAS1, known as a SUMO E3 ligase, enhanced the sumoylation level of IE2 and thereby increased the IE2-mediated transactivation of both UL54 and cyclin E promoters (Lee et al., 2003). Recently, a correlation between the transactivation activity of IE2 and its sumoylation level was suggested by an analysis of IE2 variations in different HCMV strains (Barrasa et al., 2003). In this study, a ThrAla substitution at position 541 significantly increased both the sumoylation level and the transactivation activity of IE2. This appears to be the primary reason why IE2 of the AD169 strain has a higher transactivation activity than that of the Towne strain. However, all of these functional assays were carried out in cells transiently co-transfected with DNA. Therefore, it is not clear whether the sumoylation of IE2 does in fact play a role in viral infection.
To examine whether sumoylation of IE2 is required for virus growth, we generated a recombinant HCMV genome encoding the K175/180R mutant IE2. A bacterial artificial chromosome (BAC) was used to create a Towne strain HCMV–BAC clone (T-BAC) (Marchini et al., 2001), which was used as a template for mutagenesis (Fig. 1a). This clone has a 9 kb deletion from a dispensable part of the US region (from US1 to US12) and contains both F plasmid sequences and a GFP expression cassette in the deleted region. To create a transfer vector for IE2 mutagenesis, a 2·4 kb BglII–StuI fragment containing the IE2(K175/180R) allele from the Towne strain was cloned into pGS284, a derivative of the positive suicide selection vector pCV442 (Marchini et al., 2001). To transfer the DNA sequences in pGS284 to T-BAC, Escherichia coli S17-
pir containing the GS284 donor plasmid was conjugated with a RecA+ derivative of E. coli DH10B harbouring the T-BAC DNA (Smith & Enquist, 1999). Co-integrates and exoconjugates were selected sequentially with antibiotics and sucrose (Fig. 1b). To isolate recombinant T-BAC clones containing mutations, DNA fragments containing the mutated allele were amplified by PCR and sequenced directly (Fig. 1c). In addition, the lack of any apparent alteration of the viral genome was checked by comparing the restriction endonuclease digestion patterns of the wild-type and mutant T-BAC clones (data not shown). The revertant T-BAC clone was generated by the allelic exchange of the mutant with the 4·1 kb PvuII–SalI wild-type fragment cloned in pGS284. The correct conversion of the mutant allele into the wild-type allele was confirmed by direct sequencing (data not shown).
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exon5, which lacks exon 5 of IE2 and is not infectious (Marchini et al., 2001), was used as a negative control. For each electroporation, 5x106 HF cells suspended in 250 µl medium supplemented with 10 % serum were mixed with 4 µg BAC DNA, 1 µg plasmid pCMV71 encoding pp71 (UL82) and 1 µg plasmid pEGFP-N1 (Clontech) in a 0·4 cm cuvette. Following electroporation at 250 V and 960 µF, cells were plated in 10 cm tissue culture plates. When the cells became confluent, they were split 1 : 3. Cells were cultured at 37 °C and the spread of the GFP signal was monitored. The electroporation experiments were repeated at least three times for each T-BAC. Based on the initial transient GFP signals of the transfected cells, the transfection efficiency was 10–15 % in all experiments. These transient GFP signals disappeared within 1 week of electroporation. When HF cells received the T-BAC-wt, the GFP signals (from the GFP cassette in the viral genome) began to reappear 3–4 weeks after electroporation and subsequently spread into surrounding cells. When cells received T-BAC-IE2(K175/180R), the reappearance of GFP signals was slightly delayed by 1 or 2 weeks but eventually spread to all surrounding cells (data not shown). These results suggested that sumoylation of IE2 is not essential for virus replication, but that it may facilitate the full infectivity of the transfected viral genome. In the control, the revertant T-BAC produced a wild-type T-BAC phenotype.
We next investigated the growth kinetics of the mutant virus. HF cells were infected with wild-type virus, the K175/180R mutant or its revertant at an m.o.i. of 0·2 or 0·01. Supernatants were collected at various times after infection and the titre of the infectious progeny virus was determined by plaque assays. Our results showed that the growth kinetics of the mutant virus were slightly delayed relative to the wild-type virus, but were similar to those of the revertant (Fig. 2a). To confirm that the inocula contained the same amount of input virus, inocula were harvested after adsorption and assayed for virus titre. The inoculum titres are presented as virus yields on day 0 and showed that similar amounts of virus were used (Fig. 2a). This was also confirmed by an indirect immunofluorescence assay (IFA) of the HF cells using anti-IE1 monoclonal antibody (mAb) 6E1 (Vancouver Biotech) (Fig. 2b). To circumvent contamination problems, K175/180R and the revertant alleles were confirmed by directly sequencing viral DNAs obtained from cells infected with the virus stocks used in these experiments (data not shown). We also examined the accumulation of viral IE proteins IE1 and IE2, an early protein (p52, a DNA polymerase processivity factor) and a late protein (pp65, a tegument protein) in virus-infected cells. HF cells were infected with recombinant virus encoding wild-type or the K175/180R mutant or the revertant at an m.o.i. of 0·5. Total cell extracts were prepared at 48, 96 and 144 h after infection and analysed by Western blotting using specific antibodies. The result showed that the accumulation of viral proteins in wild-type, mutant and the revertant virus-infected cells was comparable (Fig. 2c). The sumoylated form of IE2 was not detectable in this experiment due to the low level of IE2. However, the lack of IE2 sumoylation by the mutant virus and its restoration by the revertant were confirmed by immunoblot analysis using cell extracts that were prepared 72 h after a high m.o.i. (5·0) (Fig. 2d). Considering the slightly delayed growth of both the mutant virus and the revertant relative to the wild-type in our multistep growth curve analysis, we could not exclude the possibility that the viral genome may have been altered at another site during the construction of the initial mutant virus. However, our overall results indicated that sumoylation of IE2 was not essential for the progression of infection in cultured fibroblast cells.
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). This result suggested that sumoylation of IE2 is not required for initial POD targeting to generate immediate transcription domains or for its incorporation into virus RCs in virus-infected HF cells. It has been shown that the cellular SUMO signals, possibly in the form of sumoylated cellular proteins, accumulate in virus RCs at late times in HCMV-infected cells (Ahn et al., 1999). This suggests that the sumoylated cellular proteins may participate in or affect virus RCs. The two cellular proteins p53 and proliferating cell nuclear antigen (PCNA), identified as being involved in cellular DNA replication or repair, have been shown to be sumoylated (Gostissa et al., 1999; Hoege et al., 2002; Rodriguez et al., 1999). Interestingly, p53 has been shown to accumulate in virus RCs in both herpes simplex virus type-1 (HSV-1) and HCMV-infected cells, whereas PCNA has been detected in virus RCs in HSV-1-infected cells (Fortunato & Spector, 1998; Wilcock & Lane, 1991). IE2 was found to interact physically with SUMO-1 in vitro and some IE2-interacting partners were observed to interact with SUMO-1 in yeast, suggesting that IE2 may be associated with several cellular proteins by binding to covalently conjugated SUMO moieties (Ahn et al., 1999). We thus investigated whether the lack of IE2 sumoylation affected the accumulation of cellular sumoylated proteins (including p53 and PCNA) in virus RCs. HF cells were mock-infected or infected with either the wild-type or the mutant IE2(K175/180R) virus. At 48 h post-infection, cells were fixed in methanol and subjected to a double-labelled IFA. Mouse mAb against p52 and rabbit polyclonal antibody (pAb) against the UL112–113 viral proteins were used to detect the virus RCs (Ahn et al., 1999). Antibodies against SUMO-1 (FL-101), p53 (DO-1) and PCNA (PC10) cellular proteins were purchased from Santa Cruz Biotech. The results obtained showed that IFA signals for SUMO-1, p53 and PCNA were detected in virus RCs in wild-type and in IE2(K175/180R) mutant virus-infected cells. This suggested that the lack of IE2 sumoylation did not affect the accumulation of IE2 in virus RCs and that it did not interfere with the recruitment of other virus replication proteins (p52 and UL112–113) or sumoylated cellular proteins into the virus RCs.
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). However, when the reconstituted mutant virus was used, it grew in HF cells as efficiently as the wild-type and revertant viruses, suggesting that sumoylation of IE2 is not required for virus growth in HF cells. The absence of significant virus growth defects in the sumoylation-deficient mutant IE2 virus may be explained by the complementary role of virion proteins present in the tegument and matrix. Alternatively, cell signalling elicited upon virus entry may compensate for the modest loss of transactivation activity shown by the mutant IE2. Recently, a recombinant HCMV encoding a mutant IE2 with a deletion of aa 136–290 was reported to exhibit delayed virus growth and severe reductions in the steady-state levels of some viral late proteins such as pp65 (UL83) and pp28 (UL99) at a low m.o.i. (Sanchez et al., 2002). The two sumoylation sites mutated in our study lie within this deleted region, suggesting that the defect shown by the 136–290 mutant virus may be related to a lack of IE2 sumoylation. However, our results with the K175/180R mutant virus indicated that the observed defect of the 136–290 mutant virus was not due to the absence of IE2 sumoylation. Although we did not observe the effect of the absence of IE2 sumoylation in HF cells infected with the mutant virus in tissue culture, the possibility that the sumoylation of IE2 affects viral gene expression and replication only under the certain conditions that enhance cellular sumoylation activity cannot be excluded.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Ahn, J. H., Xu, Y., Jang, W. J., Matunis, M. J. & Hayward, G. S. (2001). Evaluation of interactions of human cytomegalovirus immediate-early IE2 regulatory protein with small ubiquitin-like modifiers and their conjugation enzyme Ubc9. J Virol 75, 3859–3872.
Barrasa, M. I., Harel, N., Yu, Y. & Alwine, J. C. (2003). Strain variations in single amino acids of the 86-kilodalton human cytomegalovirus major immediate-early protein (IE2) affect its functional and biochemical properties: implications of dynamic protein conformation. J Virol 77, 4760–4772.
Castillo, J. P. & Kowalik, T. F. (2002). Human cytomegalovirus immediate early proteins and cell growth control. Gene 290, 19–34.[CrossRef][Medline]
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Gostissa, M., Hengstermann, A., Fogal, V., Sandy, P., Schwarz, S. E., Scheffner, M. & Del Sal, G. (1999). Activation of p53 by conjugation to the ubiquitin-like protein SUMO-1. EMBO J 18, 6462–6471.[CrossRef][Medline]
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Pizzorno, M. C., O'Hare, P., Sha, L., LaFemina, R. L. & Hayward, G. S. (1988). trans-Activation and autoregulation of gene expression by the immediate-early region 2 gene products of human cytomegalovirus. J Virol 62, 1167–1179.
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Received 6 January 2004;
accepted 6 April 2004.
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), the IE2(K175/180R) mutant (
) or its revertant (
) at an input multiplicity of 0·2 or 0·01. The results shown represent the time courses of total p.f.u. of infectious virus in 4 ml culture supernatant at the indicated sampling times, as measured by plaque formation on HF cells. Titres are the mean results of two parallel titrations. The inoculum titre was also determined by plaque assay and is shown as virus yield on day 0. (b) Detection of the immediate-early IE1 (UL123) protein by IFA. HF cells in chamber slides were infected with the same viral stocks as described in (a) at an m.o.i. of 0·2. At 6 h post-infection, cells were fixed in methanol and stained with IE1-specific mouse mAb 6E1. The nuclei of all cells were stained with DAPI, which was included in the mounting solution. (c) Accumulation of viral IE protein, early protein and late protein during infection. HF cells in six-well plates were infected as described in (a) at an m.o.i. of 0·5. At 48, 96 and 144 h post-infection, total cell extracts were prepared in RIPA buffer, subjected to 8 % SDS-PAGE and immunoblotted using a mixture of anti-IE1 (UL123)/IE2 (UL122) mouse mAb 8131 (Chemicon) and anti-p52 (UL44) mouse mAbs (Advanced Biotech) (top panel), or anti-pp65 (UL83) mouse mAb (bottom panel). The alternative versions of p52, which are specifically expressed at the late stages of infection, are indicated as p52*. (d) Lack of sumoylation of IE2 by the mutant virus. HF cells in 60 mm dishes were infected with viruses as described in (a) at an m.o.i. of 5·0. At 72 h post-infection, total cell extracts were prepared in RIPA buffer containing 10 mM N-ethylmaleimide, a protease inhibitor, and subjected to 8 % SDS-PAGE followed by immunoblot analysis using mAb 8131. The results of two independent experiments are shown. The sumoylated forms of IE1 and IE2 are indicated as IE1-S and IE2-S, respectively.