Isolation and characterization of a low-abundance splice variant from the human cytomegalovirus major immediate-early gene region.

The major immediate-early (IE) gene region of human cytomegalovirus (HCMV) encodes several proteins as a result of differential RNA splicing events. By expression vector cloning of HCMV IE mRNA, we isolated and characterized a cDNA for a novel splice variant from the major IE gene region. The RNA product is a derivative of the IE55 mRNA and contains an additional splice from nucleotides 170,635 to 170,307 in the IE2 gene region (UL122), resulting in a 1.4-kb mRNA. The predicted open reading frame codes for a 164-amino-acid protein with a calculated molecular mass of 18 kDa (IE18). Mung bean nuclease analysis and PCR were used to characterize expression of IE18 mRNA in HCMV-infected cells. While the 1.4-kb mRNA was detected in infected human fibroblasts in the presence of a protein synthesis inhibitor, it was not detectable during a normal infection. However, the 1.4-kb mRNA was readily detected in infected human monocyte-derived macrophages at IE times. These results suggest that the novel IE18 mRNA exhibits cell type-specific expression indicating differential regulation of the major IE gene region in different permissive cell types.     

The human cytomegalovirus major immediate-early distal enhancer region is required for efficient viral replication and immediate-early gene expression

The human cytomegalovirus (HCMV) major immediate-early (MIE) genes, encoding IE1 p72 and IE2 p86, are activated by a complex enhancer region (base positions -65 to -550) that operates in a cell type- and differentiation-dependent manner. The expression of MIE genes is required for HCMV replication. Previous studies analyzing functions of MIE promoter-enhancer segments suggest that the distal enhancer region variably modifies MIE promoter activity, depending on cell type, stimuli, or state of differentiation. To further understand the mechanism by which the MIE promoter is regulated, we constructed and analyzed several different recombinant HCMVs that lack the distal enhancer region (-300 to -582, -640, or -1108). In human fibroblasts, the HCMVs without the distal enhancer replicate normally at high multiplicity of infection (MOI) but replicate poorly at low MOI in comparison to wild-type virus (WT) or HCMVs that lack the neighboring upstream unique region and modulator (-582 or -640 to -1108). The growth aberrancy was normalized after restoring the distal enhancer in a virus lacking this region. For HCMVs without a distal enhancer, the impairment in replication at low MOI corresponds to a deficiency in production of MIE RNAs compared to WT or virus lacking the unique region and modulator. An underproduction of viral US3 RNA was also evident at low MOI. Whether lower production of IE1 p72 and IE2 p86 causes a reduction in expression of the immediate-early (IE) class US3 gene remains to be determined. We conclude that the MIE distal enhancer region possesses a mechanism for augmenting viral IE gene expression and genome replication at low MOI, but this regulatory function is unnecessary at high MOI.     

Requirements for enhanced transgene expression by untranslated sequences from the human cytomegalovirus immediate-early gene.

BACKGROUND: The cytomegalovirus immediate early (CMV IE) promoter has been widely used for heterologous expression. Further enhancements of gene expression from this potent promoter may allow for the development of improved gene transfer strategies. We aimed to determine whether inclusion of the first exon (5' untranslated) and first intron of the CMV IE gene would increase heterologous transgene expression in primary target cells and to determine the sequences required for any observed increases. MATERIALS AND METHODS: Comparisons of reporter gene expression were made following transient transfection of vascular smooth muscle cells (VSMCs) with plasmids containing the first exon and intron from the CMV IE gene or deletional mutations. Comparisons were also made using a heterologous promoter (RSV). RESULTS: Gene expression from the CMV IE promoter was increased 5.7-fold in VSMC with the inclusion of the first exon and intron. Similar increases were seen with other target cells and from the heterologous RSV promoter. This increase was associated with an increase in steady-state mRNA. Deletion analyses demonstrated that the enhancement was dependent on the presence of the 5' portion of the first exon while deletion of large segments within the intron was associated with similar levels of expression compared with the parental plasmid. CONCLUSIONS: Inclusion of the first exon and intron from the CMV IE gene increases expression from the CMV IE promoter. This enhancement is seen with the heterologous RSV promoter and is associated with an increase in steady-state mRNA. Deletion analyses suggest that this enhancement is associated with inclusion of sequences within the 5' portion of the first exon and inclusion of an intron.     

Human cytomegalovirus immediate-early two protein region involved in negative regulation of the major immediate-early promoter.

The immediate-early two (IE2) gene products of human cytomegalovirus negatively regulate gene expression from the major immediate-early promoter in permissive human fibroblasts. A mutational analysis of the IE2 proteins indicated that the carboxyl-terminal region is required for negative regulation. The IE2 proteins that lack amino acid residues 365 to 519, or the carboxyl-terminal amino acids failed to negatively regulate. Most of the amino-terminal portion of the IE2 protein was not required for negative regulation. A possible explanation of the negative effect on downstream expression by the IE2 proteins is discussed.     

Identification of the promoter region and gene expression for human acid alpha glucosidase.

Genetic deficiency of acid alpha glucosidase (GAA) results in glycogen storage disease type II. A cDNA containing the complete coding region was constructed and cloned into the expression vector pSV2 and was transiently transfected into an SV40 immortalized GAA deficient human fibroblast cell line which has undetectable levels of GAA enzyme activity and does not express GAA mRNA. Transfected cells had 4.9% of normal human fibroblast enzyme activity. Additionally a 5' 1.8 kb genomic fragment was ligated to the 5' end of the GAA cDNA construct and cloned into pUC19. Transient and stable transfection also resulted in expressed GAA enzyme activity in deficient fibroblast cells, indicating that the genomic fragment has GAA promoter function.     

The IE2 gene products of human cytomegalovirus specifically down-regulate expression from the major immediate-early promoter through a target sequence located near the cap site.

The 82-kDa IE2 protein of human cytomegalovirus (HCMV) acts as both a powerful nonspecific trans activator of heterologous promoters and a negative autoregulator of HCMV immediate-early gene expression in transient assays. We show here that the highly specific down-regulation effect occurs in permissive diploid human fibroblast cells as well as in nonpermissive Vero cells and that the target sequences are conserved within the major immediate-early promoters of both HCMV and simian cytomegalovirus. The response sequences were localized between -67 and +30 in the simian cytomegalovirus IE94 promoter and upstream of position +9 in the HCMV IE68 promoter. Deletion of sequences downstream of -14 in a target IE68-CAT gene abolished the negative phenotype and resulted in a reporter gene that was stimulated instead of inhibited by cotransfection with IE2 effector DNA. Insertion of an oligonucleotide containing sequences from between -17 and +9 into the IE68-CAT deletion construction restored autoregulation in either orientation. Furthermore, this same oligonucleotide transferred the full down-regulation phenotype when inserted at +10 into the nonresponsive IE175 promoter from herpes simplex virus. Therefore, a specific response signal that acts at the DNA level must lie within these boundaries. Additional analysis with inserted oligonucleotides containing deletions or point mutations revealed that essential components of the signal lie between positions -12 and +5. Therefore, negative autoregulation by HCMV IE2 in DNA cotransfection systems resembles that for simian virus 40 large T antigen and herpes simplex virus IE175 by acting through a signal located near the cap site, but the target sequence itself bears no resemblance to those utilized in these other viral systems.     

Identification and characterization of an essential gene in Listeria monocytogenes using an inducible gene expression system

The Listeria monocytogenes gene lmo1594 is a homolog of the Bacillus subtilis cell division gene ezrA. EzrA is a negative regulator of FtsZ ring formation, which is required for efficient cell division as it regulates the frequency and position of Z-rings in the cell and prevents aberrant polar cell division. Previously identified as a putative high pressure (HP) resistance mechanism; conferring enhanced barotolerance when heterologously expressed against an Escherichia coli background; the aim of the current study was to investigate whether lmo1594 plays a role in listerial barotolerance. When the creation of a deletion mutant proved unsuccessful, the role of lmo1594 was addressed by creating a conditional knockout mutant which demonstrated that the gene is in fact essential for cell survival and growth in L. monocytogenes. In order to investigate the effect of lmo1594 on barotolerance, the gene was over-expressed. The over-expression of lmo1594 increased survival levels in L. monocytogenes treated at 300 MPa, but survival levels similar to those of the wild-type strain were observed when treated at a higher pressure (≥400 MPa). In conclusion, this study reveals for the first time that lmo1594 is absolutely essential for listerial cell growth and survival, and also plays an important role in listerial barotolerance.