Human cytomegalovirus IE86 protein binds to cellular Mcm3 protein but does not inhibit its binding to the Epstein-Barr virus oriP in U373MG-p220.2 cells.

UNLABELLED: Human cytomegalovirus (HCMV) or its immediate-early IE86 protein alone induces cell cycle in quiescent primary human foreskin fibroblasts (HFFs), but blocks its progression at the G1/S interphase and inhibits cellular DNA synthesis by a mechanism that is not clearly understood. It is assumed that, in this phenomenon, the binding of minichromosome maintenance (Mcm) proteins to replication origins is blocked. In this work, we analyzed the initiation of DNA replication in HCMV-permissive U373MG cells and used oriP of Epstein-Barr virus (EBV) as a simplified model of a cellular replication origin. Using U373MG cells we found that HCMV IE86 protein was bound to Mcm3, but did not inhibit the cellular DNA synthesis. Using U373MG-p220.2 cells carrying EBV oriP and expressing Epstein-Barr nuclear antigen 1 (EBNA1), we found that EBNA1 as well as Mcm3 were bound to oriP and that neither HCMV nor IE86 protein inhibited the binding of Mcm3 to oriP. Differences between the effects of HCMV on the cell cycle progression in HFFs and U373MG cells are discussed. KEYWORDS: cell cycle; Human cytomegalovirus; DNA replication.

Functional roles of the human cytomegalovirus essential IE86 protein.

The IE86 protein of human cytomegalovirus (HCMV) is unique among viral and cellular proteins because it negatively autoregulates its own expression, activates the viral early and late promoters, and both activates and inhibits cellular promoters. It promotes cell cycle progression from Go/G1 to G1/S and arrests cell cycle progression at the G1/S interface or at G2/M. The IE86 protein is essential because it creates a cellular environment favorable for viral replication. The multiple functions of the IE86 protein during the replication of HCMV are reviewed.

Human cytomegalovirus-induced upregulation of intercellular cell adhesion molecule-1 on villous syncytiotrophoblasts.

Human cytomegalovirus (HCMV) is secreted apically from villous trophoblasts, thus congenital infection is not likely to occur by basal release across the basement membrane. As an alternative route, we hypothesize that an HCMV-infected villous syncytiotrophoblast (ST) upregulates intercellular adhesion molecule (ICAM)-1, causing blood monocytes to bind to the ST and induce apoptosis. Purified (>99.99%) populations of human villous trophoblasts were differentiated into an ST-like culture, infected with HCMV strain AD169, and assessed for ICAM-1 expression by immunofluorescence. Infection strongly upregulated ICAM-1 24 h after challenge. ICAM-1 was also stimulated by transfection with viral genes IE2-55, IE1-72, and IE2-86, but not by UV-inactivated virus. Infection with a green fluorescent protein recombinant virus allowed infection and ICAM-1 expression to be topographically located. We found that ICAM-1 was expressed on both infected and noninfected cells. Furthermore, antibody to tumor necrosis factor (TNF)alpha and, to a lesser extent, interleukin (IL)1 beta inhibited ICAM-1 upregulation on noninfected cells but not on infected cells. We conclude that HCMV IE proteins stimulate ICAM-1 expression on villous trophoblasts by paracrine release of TNF alpha and IL1 beta, as well as by a direct effect on infected cells.

Effect of the R1 element on expression of the US3 and US6 immune evasion genes of human cytomegalovirus.

Human cytomegalovirus (HCMV) has several gene products that are important for escape from immune surveillance. These viral gene products downregulate the expression of HLA molecules on the cell surface. The viral US3 and US6 gene products are expressed at immediate-early and early times after infection, respectively. There are two regulatory regions between the US3 and the US6 transcription units. The first region is an NF-kappaB responsive enhancer that promotes the immediate-early expression of the US3 gene and is designated the R2 enhancer. Upstream of the R2 enhancer is a region designated the R1 element that in transient transfection assays behaves as a silencer by repressing the effect of the enhancer on downstream gene expression (A. R. Thrower et al., J. Virol. 1996, 70, 91; Y.-J. Chan et al., J. Virol. 1996, 70, 5312). We constructed recombinant viruses with wild-type or mutated R1 elements. The expression of the US3 gene at 6 h after infection and the US6 gene at 24 h was higher when the R1 element was present. The R1 element in the context of the viral genome is not a silencer of US3 or US6 gene expression. The R1 element has multiple effects on the US3 and US6 RNAs. It enhances the level of US3 and US6 mRNA; it determines the 3'-end cleavage and polyadenylation of US6 RNA, and it stabilizes read-through viral RNAs. The potential mechanisms of R1 enhancement of US3 and US6 gene expression are discussed.

Activation of transcription of the human cytomegalovirus early UL4 promoter by the Ets transcription factor binding element.

The human cytomegalovirus (HCMV) early UL4 promoter has served as a useful model for studying the activation of early viral gene expression. Previous transient-transfection experiments detected cis-acting elements (the NF-Y site and site 2) upstream of the transcriptional start site (L. Huang and M. F. Stinski, J. Virol. 69:7612-7621, 1995). The roles of two of these sites, the NF-Y site and site 2, in the context of the viral genome were investigated further by comparing mRNA levels from the early UL4 promoter in human foreskin fibroblasts infected by recombinant viruses with either wild-type or mutant cis-acting elements. Steady-state mRNA levels from the UL4 promoter with a mutation in the NF-Y site were comparable to that of wild type. A mutation in an Elk-1 site plus putative IE86 protein binding sites decreased the steady-state mRNA levels compared to the wild type at early times after infection. Electrophoretic mobility shift assays and antibody supershifts detected the binding of cellular transcription factor Elk-1 to site 2 DNA with infected nuclear extracts but not with mock-infected nuclear extracts. The role of cellular transcription factors activated by the mitogen activated protein kinase/extracellular signal-regulated kinase pathway in activating transcription from early viral promoters is discussed.

The human cytomegalovirus IE86 protein can block cell cycle progression after inducing transition into the S phase of permissive cells.

Human cytomegalovirus (HCMV) infection of permissive cells has been reported to induce a cell cycle halt. One or more viral proteins may be involved in halting progression at different stages of the cell cycle. We investigated how HCMV infection, and specifically IE86 protein expression, affects the cell cycles of permissive and nonpermissive cells. We used a recombinant virus that expresses the green fluorescent protein (GFP) to determine the effects of HCMV on the cell cycle of permissive cells. Fluorescence by GFP allowed us to select for only productively infected cells. Replication-defective adenovirus vectors expressing the IE72 or IE86 protein were also used to efficiently transduce 95% or more of the cells. The adenovirus-expressed IE86 protein was determined to be functional by demonstrating negative autoregulation of the major immediate-early promoter and activation of an early viral promoter in the context of the viral genome. To eliminate adenovirus protein effects, plasmids expressing GFP for fluorescent selection of only transfected cells and wild-type IE86 protein or a mutant IE86 protein were tested in permissive and nonpermissive cells. HCMV infection induced the entry of U373 cells into the S phase. All permissive cells infected with HCMV were blocked in cell cycle progression and could not divide. After either transduction or transfection and IE86 protein expression, the number of all permissive or nonpermissive cell types in the S phase increased significantly, but the cells could no longer divide. The IE72 protein did not have a significant effect on the S phase. Since IE86 protein inhibits cell cycle progression, the IE2 gene in a human fibroblast IE86 protein-expressing cell line was sequenced. The IE86 protein in these retrovirus-transduced cells has mutations in a critical region of the viral protein. The locations of the mutations and the function of the IE86 protein in controlling cell cycle progression are discussed.

A strong negative transcriptional regulatory region between the human cytomegalovirus UL127 gene and the major immediate-early enhancer.

The region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region. DNase I protection analysis with human cell nuclear extracts demonstrated multiple protein binding sites in this region of the viral genome (P. Ghazal, H. Lubon, C. Reynolds-Kohler, L. Hennighausen, and J. A. Nelson, Virology 174:18-25, 1990). However, the function of this region in the context of the viral genome is not known. In wild-type human CMV-infected human fibroblasts, cells permissive for viral replication, there is little to no transcription from UL127. We determined that the unique region prevented transcription from the UL127 promoter but had no effect on the divergent MIE promoter. In transient-transfection assays, the basal level of expression from the UL127 promoter increased significantly when the wild-type unique sequences were mutated. In recombinant viruses with similar mutations in the unique region, expression from the UL127 promoter occurred only after de novo viral protein synthesis, typical of an early viral promoter. A 111-bp deletion-substitution of the unique sequence caused approximately a 20-fold increase in the steady-state level of RNA from the UL127 promoter and a 245-fold increase in the expression of a downstream indicator gene. This viral negative regulatory region was also mutated at approximately 50-bp regions proximal and distal to the UL127 promoter. Although some repressive effects were detected in the distal region, mutations of the region proximal to the UL127 promoter had the most significant effects on transcription. Within the proximal and distal regions, there are potential cis sites for known eucaryotic transcriptional repressor proteins. This region may also bind unknown viral proteins. We propose that the unique region upstream of the UL127 promoter and the MIE enhancer negatively regulates the expression from the UL127 promoter in permissive human fibroblast cells. This region may be a regulatory boundary preventing the effects of the very strong MIE enhancer on this promoter.

A cis repression sequence adjacent to the transcription start site of the human cytomegalovirus US3 gene is required to down regulate gene expression at early and late times after infection.

Human cytomegalovirus has two enhancer-containing immediate-early (IE) promoters with a cis repression sequence (CRS) positioned immediately upstream of the transcription start site, designated the major IE (MIE) promoter and the US3 promoter. The role of the CRS upstream of the US3 transcription start site in the context of the viral genome was determined by comparing the levels of transcription from these two enhancer-containing promoters in recombinant viruses with a wild-type or mutant CRS. Upstream of the CRS of the US3 promoter was either the endogenous enhancer (R2) or silencer (R1). The downstream US3 gene was replaced with the indicator gene chloramphenicol acetyltransferase (CAT). Infected permissive human fibroblast cells or nonpermissive, undifferentiated monocytic THP-1 cells were analyzed for expression from the US3 promoter containing either the wild-type or mutant CRS. With the wild-type CRS, the maximum level of transcription in permissive cells was detected within 4 to 6 h after infection and then declined. With the mutant CRS and the R2 enhancer upstream, expression from the US3 promoter continued to increase throughout the viral replication cycle to levels 20- to 40-fold higher than for the wild type. In nonpermissive or permissive monocytic THP-1 cells, expression from the US3 promoter was also significantly higher when the CRS was mutated. Less expression was obtained when only the R1 element was present, but expression was higher when the CRS was mutated. Thus, the CRS in the enhancer-containing US3 promoter appears to allow for a short burst of US3 gene expression followed by repression at early and late times after infection. Overexpression of US3 may be detrimental to viral replication, and its level of expression must be stringently controlled. The role of the CRS and the viral IE86 protein in controlling enhancer-containing promoters is discussed.

The gene product of human cytomegalovirus open reading frame UL56 binds the pac motif and has specific nuclease activity.

Using the cis-acting human cytomegalovirus (HCMV) packaging elements (pac 1 and pac 2) as DNA probes, specific DNA-protein complexes were detected by electrophoretic mobility shift assay (EMSA) in both HCMV-infected cell nuclear extracts and recombinant baculovirus-infected cell extracts containing the HCMV p130 (pUL56) protein. DNA-binding proteins, which were common in uninfected and infected cell extracts, were also detected. Mutational analysis showed that only the AT-rich core sequences in these cis-acting motifs, 5'-TAAAAA-3' (pac 1) and 5'-TTTTAT-3' (pac 2), were required for specific DNA-protein complex formation. The specificity of the DNA-protein complexes was confirmed by EMSA competition. Furthermore, a specific endonuclease activity was found to be associated with lysates of baculovirus-infected cells expressing recombinant p130 (rp130). This nuclease activity was time dependent, related to the amount of rp130 in the assay, and ATP independent. Nuclease activity remained associated with rp130 after partial purification by sucrose gradient centrifugation, suggesting that this activity is a property of HCMV p130. We propose a possible involvement of p130 in HCMV DNA packaging.

Effect of a modulator deletion on transcription of the human cytomegalovirus major immediate-early genes in infected undifferentiated and differentiated cells.

Differentiation-dependent expression of the human cytomegalovirus (HCMV) major immediate-early (MIE) genes, encoding IE1 and IE2, may partly govern virus replication in monocytic THP-1 and embryonal carcinoma (Tera-2) cells. The modulator of the MIE promoter was shown previously in transient transfection assays to repress transcription from promoter segments in undifferentiated THP-1 and Tera-2 cells but not in differentiated cells. To determine the biological importance of these findings, we constructed a recombinant HCMV (r delta MSVgpt) without a modulator. In comparison to wild-type (WT) virus, r delta MSVgpt exhibits a slight delay in growth in human fibroblasts, but there is no appreciable change in IE1 and IE2 transcription. Moreover, there is no appreciable change in the early/late kinetics of transcription of RNAs colinear with the predicted UL128 coding region, which is adjacent to the modulator, although the size distribution and abundance of these RNAs are altered. In infected undifferentiated THP-1 and Tera-2 cells, WT and r alpha MSVgpt viruses produce minimal but comparable amounts of IE1 RNAs. The genomes of both viruses are detectable in similar amounts within these undifferentiated cells. Induction of cellular differentiation before infection overcomes the block in MIE gene transcription. WT and r alpha MSVgpt infections of differentiated THP-1 cells produce similar levels of IE1 and IE2 RNAs. Thus, differentiation-dependent control of MIE gene transcription involves regulatory mechanisms other than the modulator. Possible alternative functions of the modulator are discussed.

Cellular or viral protein binding to a cytomegalovirus promoter transcription initiation site: effects on transcription.

We have previously shown that the IE2 protein of human cytomegalovirus (CMV) represses its own synthesis by binding to the major immediate-early promoter (M. P. Macias and M. F. Stinski, Proc. Natl. Acad. Sci. USA 90:707-711, 1993). The binding of a viral protein (IE2) and a cellular protein in the region of the transcription start site was investigated by site-specific mutational analysis and electrophoretic mobility shift assay. The viral protein and the cellular protein require different but adjacent core DNA sequence elements for binding. In situ chemical footprinting analysis of DNA-protein interactions with purified CMV IE2 protein or HeLa cell nuclear extracts demonstrated binding sites that overlap the transcription start site. The IE2 protein footprint was between bp -15 and +2, relative to the transcription start site, and the cellular protein was between bp -16 and +7. The ability of the unknown human cellular protein of approximately 150 kDa to bind the CMV major immediate-early promoter correlates with an increase in the level of transcription efficiency. Mutations in the core DNA sequence element for cellular protein binding significantly reduced the level of in vitro transcription efficiency. Mutations upstream and downstream of the core sequence moderately reduced the transcription efficiency level. Negative autoregulation of the CMV promoter by the viral IE2 protein may involve both binding to the DNA template and interference with the function of a cellular protein that binds to the transcription start site and enhances transcription efficiency.

Regulation of a human cytomegalovirus immediate-early gene (US3) by a silencer-enhancer combination.

The US3 open reading frame of human cytomegalovirus (HCMV) is transcribed at immediate-early (IE) times after infection. Upstream of the US3 promoter, between -84 and -259 bp relative to the transcription start site, there are five copies of an 18-bp repeat, referred to as 5R2. Between -340 and -560 bp there are seven copies of a 10-bp dyad repeat, referred to as 7R1. We investigated the roles of these repeats in transcription from the US3 promoter in human foreskin fibroblast or HeLa cells. In transient transfection assays, the region containing 5R2 up-regulated transcription and was responsive to the p65 subunit of NF-kappa B. The DNA region containing 7R1 down-regulated transcription from either the US3 promoter or a heterologous promoter in a position- and orientation-independent manner. Mutational analysis and transient transfections indicated that DNA containing the 10-bp dyad or one-half of the dyad was sufficient to cause repression of downstream gene expression. DNA probes containing one or more copies of the pentanucleotide sequence TGTCG specifically bound cellular proteins, as demonstrated by electrophoretic mobility shift assays and cold-competition electrophoretic mobility shift assays. Two different DNA-protein complexes were detected with DNA probes containing one or two copies of the pentanucleotide. In HCMV-infected cell nuclear extracts, one of the DNA-protein complexes was present in amounts inversely proportional to the amount of US3 transcription. Its formation was affected by dephosphorylation of the DNA-binding protein(s). Transient dephosphorylation of the cellular repressor protein may occur during HCMV infection. Repression of US3 transcription may relate to the number of pentanucleotides and the cellular proteins that bind to it. Twenty-one copies of a TRTCG motif (R = purine) were found clustered upstream of the US3 gene and also in the modulator upstream of the HCMV IE1 and IE2 genes.

Regulation of human cytomegalovirus immediate-early gene expression.

The positive and negative cis-acting elements that affect transcription from the human cytomegalovirus major immediate-early (MIE) promoter and the viral and cellular proteins that bind to these elements are discussed. The data obtained using in vitro transcription and transient transfection assays are reviewed and compared to recent data using recombinant viruses with cis-acting elements deleted. The effects of cell type and cellular differentiation on activation of transcription from the MIE promoter are compared with the effects of mitogens and virion-associated tegument proteins that directly or indirectly activate protein kinase pathways. The repressor and enhancer regions upstream of the MIE promoter in the large unique component of the viral genome are compared to the elements upstream of the more simplified IEUS3 promoter in the short unique component of the viral genome.

Binding of cellular repressor protein or the IE2 protein to a cis-acting negative regulatory element upstream of a human cytomegalovirus early promoter.

We have previously shown that the human cytomegalovirus early UL4 promoter has upstream negative and positive cis-acting regulatory elements. In the absence of the upstream negative regulatory region, the positive element confers strong transcriptional activity. The positive element contains a CCAAT box dyad symmetry and binds the cellular transcription factor NF-Y. The effect of the negative regulatory element is negated by the viral IE2 protein (L. Huang, C.L. Malone, and M.F. Stinski, J. Virol. 68:2108, 1994). We investigated the binding of cellular or viral IE2 protein to the negative regulatory region. The major cis-acting negative regulatory element was located between -168 and -134 bp relative to the transcription start site. This element could be transferred to a heterologous promoter, and it functioned in either orientation. Mutational analysis demonstrated that a core DNA sequence in the cis-acting negative regulatory element, 5'-GTTTGGAATCGTT-3', was required for the binding of either a cellular repressor protein(s) or the viral IE2 protein. The cellular DNA binding activity was present in both nonpermissive HeLa and permissive human fibroblast cells but more abundant in HeLa cells. Binding of the cellular repressor protein to the upstream cis-acting negative regulatory element correlates with repression of transcription from the early UL4 promoter. Binding of the viral IE2 protein correlates with negation of the repressive effect.

Adenovirus E1A 13S gene product up-regulates the cytomegalovirus major immediate early promoter.

Latent cytomegalovirus (CMV) infection is often reactivated in the lung. We postulated that this reactivation could occur by stimulation of the CMV major immediate early (IE) promoter by other viruses that infect the lung. The specific aim of this study was to investigate whether adenovirus early proteins could stimulate the CMV IE promoter in inflammatory cells. We transfected the monocyte/macrophage THP-1 cell line and the T-lymphocyte Jurkat cell line with plasmids coding for adenovirus E1A 12S or 13S proteins, along with a plasmid containing the CMV IE promoter region linked to the chloramphenicol acetyltransferase (CAT) reporter gene. In unstimulated THP-1 cells, the E1A 13S gene product increased CMV IE CAT activity by 18-fold compared with cells containing the control E1A plasmid. This effect was not seen in cells transfected with the E1A 12S plasmid. There was a similar effect of the E1A 13S gene product in LPS-stimulated THP-1 cells. In unstimulated Jurkat cells, the E1A 13S gene product stimulated CMV IE CAT activity by 19-fold compared with cells containing the E1A control plasmid; the E1A 12S gene product had no effect. There was a similar effect of the 13S E1A gene product in phorbol myristate acetate-stimulated Jurkat cells. These findings demonstrate that the CMV IE promoter can be stimulated by early viral proteins of adenovirus in inflammatory cells. These observations could be important for understanding the reactivation of latent CMV infection.

A human cytomegalovirus early promoter with upstream negative and positive cis-acting elements: IE2 negates the effect of the negative element, and NF-Y binds to the positive element.

The human cytomegalovirus early promoter for the UL4 gene, which codes for an early viral envelope glycoprotein designated gpUL4, requires immediate-early viral protein two (IE2) synthesis to be activated (C.-P. Chang, C. L. Malone, and M. F. Stinski, J. Virol. 63:281, 1989). We investigated the cis-acting and trans-acting factors that regulate transcription from this UL4 promoter. In transient transfection assays, the viral IE2 protein negated the effect of an upstream cis-acting negative element and enhanced downstream gene expression. A cis-acting positive element contributed to the activity of the viral promoter when an upstream cis-acting negative element was deleted or when the viral IE2 protein was present. The cellular protein(s) that binds to the cis-acting negative element requires further investigation. The cellular protein that binds to the cis-acting positive element was characterized. Two DNA sequence-specific protein complexes were detected with DNA probes spanning the region containing the cis-acting positive element and human cytomegalovirus-infected human fibroblast cell nuclear extracts. The more slowly migrating complex was labeled complex A, and the faster was labeled complex B. Only complex B was detected with mock-infected cell nuclear extracts. Competition experiments confirmed the specificity of the A and B complexes. The protein bound to the DNA in both the complexes contacts a CCAAT box imperfect dyad symmetry (5'CCAATCACTGG3'). Either CCAAT box within the dyad symmetry could compete for binding the nuclear factor. Mutation of the CCAAT box dyad symmetry resulted in a decrease of the transcriptional activity from the UL4 promoter. A cellular transcription factor, antigenically related to nuclear factor-Y (NF-Y), was found in both complexes A and B. Events associated with viral infection caused phosphorylation of protein complex A. Dephosphorylation of the DNA-binding protein converts complex A to complex B. The effect of phosphorylation of NF-Y is not known.

Regulation of expression of the IL-1 receptor antagonist (IL-1ra) gene by products of the human cytomegalovirus immediate early genes.

Human cytomegalovirus (HCMV), which is a major cause of morbidity and mortality in immunosuppressed patients, can itself alter immune function. It has previously been shown that HCMV immediate early (IE) gene products regulate the IL-1 beta promoter. The purpose of these studies was to determine whether HCMV IE gene products regulate expression of the IL-1 receptor antagonist (IL-1ra) gene. THP-1 cells, a myelomonocytic cell line, were transfected with a plasmid containing one or more of the HCMV IE genes downstream of the HCMV major immediate early promoter, or with a control plasmid. IL-1 beta and IL-1ra protein secretion was evaluated by ELISA, and expression of the mRNA for the cytokines was examined by means of Northern blot analysis. The HCMV IE1+2 gene products were found to increase expression of the mRNA for both IL-1 beta and IL-1ra; however, only the IL-1ra protein was released in increased amounts. The individual HCMV IE gene products had different effects on expression of the IL-1ra gene; the HCMV IE1 gene product down-regulated expression of the IL-1ra gene, whereas the IE2 gene product up-regulated expression of the IL-1ra gene. Thus, HCMV IE gene products can either up-regulate or down-regulate expression of the IL-1ra gene, depending on which IE genes are expressed in monocytes-macrophages. This study adds to the understanding of how HCMV can alter immune function during both active and latent infection.

The immediate early genes of human cytomegalovirus upregulate tumor necrosis factor-alpha gene expression.

Cytomegalovirus (CMV) is an important cause of disease in the immunocompromised patient and CMV infection is associated with predominantly mononuclear inflammatory response. Since products of the CMV immediate early (IE) gene region are potent trans-activators, we used the monocyte cell line THP-1 and a transient transfection assay to determine if these viral proteins upregulate expression of the TNF gene. The IE genes of CMV upregulated TNF gene activity as judged by increases in promoter activity, steady state mRNA, and protein production. The presence or absence of the 3' untranslated region of the TNF gene did not affect gene expression induced by the IE gene products. These studies suggest that activation of TNF gene expression by the CMV IE gene products may, in part, account for the inflammatory response associated with CMV infections.

Avian sarcoma virus RNA synthesis, RNA splicing and virus production in human foreskin fibroblasts: effect of co-infection with human cytomegalovirus.

The level of RNA transcripts in human foreskin fibroblast (HFF) cells initiated from the avian sarcoma virus (ASV) long terminal repeat (LTR) promoter was stimulated more than 10-fold when the cells were also infected with human cytomegalovirus (HCMV). HCMV was able to stimulate transcription from the ASV LTR promoter even when all the LTR sequence upstream of the TATA box was deleted, suggesting that only the basal LTR promoter is required for the effect. There were no significant changes in the ASV RNA splicing pattern in stimulated and unstimulated HFF cells. The mRNAs showing an increase during HCMV stimulation included aberrantly spliced ASV RNA species as well as unspliced gag-pol, single-spliced env and single-spliced src mRNAs. This pattern was quite different from ASV splicing in chicken embryo fibroblasts (CEF) but typical of that seen in other mammalian cells. A dramatic increase in infectious ASV production from the normally non-permissive HFF was correlated with the increase in amount of ASV RNA in response to HCMV. Thus, there is not an absolute block to ASV production in human cells. However, infectious ASV production was inefficient in HCMV-stimulated HFF compared to that in CEF cells.

Mutations of the human cytomegalovirus immediate-early 2 protein defines regions and amino acid motifs important in transactivation of transcription from the HIV-1 LTR promoter.

The human cytomegalovirus (HCMV) immediate-early two (IE2) protein of 579 amino acids significantly activates expression from the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter. Using a proviral HIV-1 genome with a mutated tat gene we demonstrate that the IE2 protein effects an increase in the steady-state level of viral RNA similar to a level as from a wild-type proviral genome. The regions of the HCMV IE2 protein required for transactivation of the HIV-1 LTR promoter were analyzed by mutagenizing the IE2 gene and determining the activity of the mutant protein in human fibroblast cells. The region between amino acids 169 and 194 is required to transactivate the HIV-1 LTR promoter, although we have previously shown that this region is not required to activate a representative HCMV early promoter (C. L. Malone, et al., J. Virol. 64, 1498, (1990)). A region downstream of amino acid 290, which is required to activate a representative HCMV early promoter, is also required to activate the HIV-1 LTR promoter. Three types of mutations within this region were shown to greatly decrease IE2 activity: (1) amino acid substitutions of the cysteine or histidine residues in a putative zinc finger motif between amino acids 428 and 452; (2) substitution of the acidic charged residues between amino acids 558 and 561; (3) substitution of the two prolines at residues 556 and 557 immediately upstream of these acidic residues. Substitution of the other acidic residues near the carboxyl terminus also diminished transactivation by IE2. These data indicate that acidic amino acids and the secondary structure in the carboxyl end of the IE2 protein have an important role in transactivation of the HIV-1 LTR promoter. The other regions of the IE2 protein required for transactivation of the HIV-1 LTR are discussed.