Kuniharu Ohnishi

SUMO-1 modification of the major immediate-early (IE) 1 and 2 proteins of human cytomegalovirus is regulated by different mechanisms and modulates the intracellular localization of the IE1, but not IE2, protein

Summary.We have previously shown that two proteins with apparent molecular masses of 91- and 102-kDa (p91 and p102, respectively) in human cytomegalovirus (HCMV)-infected cells are antigenically and structurally related to the major immediate-early (IE) 1 and 2 proteins (IE1p68 and IE2p80, respectively) of HCMV, respectively. In this study, we extended the characterization of p91 and p102 and our results were as follows; (i) Lysine at amino acid position 450 in IE1p68 and at amino acid position 175 or 180 in IE2p80, to which SUMO-1 has been shown to be covalently linked, are required for production of p91 and p102, respectively. (ii) A reversal of cycloheximide (CH) block in the presence of actinomycin D imposed at the time of infection inhibited production of p91, but not p102. (iii) The steady-state levels of p91, but not p102, were remarkably decreased by treatment with proteasome inhibitor MG132, but coincubation with CH inhibited this decrease of p91. (iv) Cell fractionation by differential detergent extraction demonstrated that p91 is preferentially found in detergent-insoluble fraction, although p102 as well as IE1p68 and IE2p80 distributes into all fractions. These results demonstrate that p91 and p102 correspond to SUMO-1-modified IE1p68 and IE2p80, respectively, that the production and degradation of SUMO-1-modified IE1p68 is regulated by mechanisms different from those of SUMO-1-modified IE2p80, and that SUMO-1 modification modulates the intracellular localization of IE1p68, but not IE2p80.

STRUCTURE OF CHROMOSOMAL DNA CODING FOR PSEUDOMONAS PUTIDA S-1 SALICYLATE HYDROXYLASE

A gene coding for the salicylate hydroxylase has been isolated from chromosomal DNA of Pseudomonas putida S-1 and sequenced. The DNA fragment contained an open reading frame of 1266 bp encoding a polypeptide of 421 amino acid residues. The predicted amino acid sequence of the protein gave a good agreement with the sequences determined with the peptides isolated from the enzyme but methionine residue in the amino terminal was deleted in the N-terminal sequence of the enzyme protein. The nucleotide and amino acid sequences of the salicylate hydroxylase shared several common characteristics with those of the enzyme encoded on the plasmid DNA of P. putida PpG7; homology of nucleotide sequence is 58% and that of amino acid sequence is 56%. We could find two large conserved regions of the amino acid sequence at or near FAD- and NADH-binding regions. The FAD-binding site locates on the amino terminal and a lysine residue, functioning as an NADH-binding site (K. Suzuki, M. Mizuguchi, T. Gomi, and E. Itagaki, 1995, J. Biochem. 117,579-585), locates as Lys163.

The major immediate-early genes of human cytomegalovirus induce two novel proteins with molecular weights of 91 and 102 kilodaltons

Summary. Mouse monoclonal antibody MAB810 is known to recognize the major immediate-early (IE) proteins, 68 kDa IE1 (IE1p68) and 80 kDa IE2 (IE2p80), of human cytomegalovirus (HCMV). Using this antibody we found that two additional proteins with higher molecular weights of approximately 91 (p91) and 102 kDa (p102) are also synthesized in HCMV-infected cells. p91 and p102 were produced in cells stably transfected with plasmid expressing IE1p68 and IE2p80, respectively, and shown to be related with IE1p68 and IE2p80, respectively, in primary amino acid sequence. Taken together, these results indicate that p91 and p102 are expressed from the IE1 and IE2 genes, respectively.

The effect of cyclic AMP on expression of the major immediate-early genes and replication of human cytomegalovirus in human central nervous system cell lines

SummaryThe effect of dibutyryl cyclic AMP (dbcAMP) on interaction of human cytomegalovirus (HCMV) with human central nervous system cell lines was examined. Activation of the major immediate-early (IE) promoter (MIEP) of HCMV by dbcAMP was observed in human neuroblastoma IMR-32 cells, but not in glioma 118MGC and astrocytoma U373-MG cells. The 19 bp repeat element in the enhancer of the MIEP was most likely to be the cis-element that responded to dbcAMP in IMR-32 cells as we expected. In IMR-32 cells activation of the MIEP led to enhanced synthesis of the major IE proteins and infectious HCMV.