Infection of quiescent fibroblasts with human cytomegalovirus (HCMV) was found to cause a rapid activation of cellular phosphatidylinositol 3-kinase (PI3-K). Maximum PI3-K activation occurred from 15 to 30 min postinfection. This activation was transient, and by 2 h postinfection (hpi), PI3-K activity had declined to preinfection levels. However, at 4 hpi, a second tier of PI3-K activation was detected, and PI3-K activity remained elevated relative to that of mock-infected cells for the remainder of infection. The cellular kinases Akt and p70S6K and the transcription factor NF-B were activated in a PI3-K-dependent manner at similar times following HCMV infection. Analysis using UV-irradiated virus indicated that no viral protein synthesis was necessary for the first phase of PI3-K activation, but viral protein expression was required for the second tier of PI3-K activation. Treatment of infected fibroblasts with LY294002, a potent and specific inhibitor of PI3-K kinase activity, caused a 4-log decrease in viral titers. LY294002 did not inhibit viral entry, but it did decrease viral immediate-early gene expression. In addition, the protein levels of two viral early genes required for DNA replication, UL84 and UL44, were significantly lower in the presence of LY294002. Furthermore, viral DNA replication was strongly inhibited by LY294002 treatment. This inhibition of viral DNA replication could be reversed by adding back the products of PI3-K activity (PI-3,4-P 2 and PI-3,4,5-P 3 ), demonstrating that the effect of LY294002 on the viral life cycle was specifically due to the inhibition of PI3-K activity. These results are the first to suggest that PI3-K mediates HCMV-induced activation of host cell mitogenic pathways. They also provide strong evidence that PI3-K activation is important for initiation of viral DNA replication and completion of the viral lytic life cycle.Human cytomegalovirus (HCMV) is a widespread human pathogen that does not cause significant clinical manifestations in healthy individuals (29,32,50). On the other hand, it causes severe diseases in immunocompromised individuals that, if left untreated, can be fatal. In addition, it is a leading cause of certain types of birth defects (29,32,50). Individuals suffering from diseases caused by HCMV are currently treated with chemical compounds, such as ganciclovir and phosphocarnet, which block the viral lytic life cycle by inhibiting viral DNA replication (48,51,66). However, the substantial toxicity of these drugs and the emergence of drug-resistant strains of HCMV indicate that better antiviral compounds are needed (5, 66, 69). Recently, we have begun to identify and characterize signal transduction pathways that are activated following HCMV infection of human fibroblasts. By studying these pathways, we hope not only to better understand HCMV pathogenesis at the molecular level but also to eventually identify unique, virus-specific targets which can be utilized for the development of potent anti-HCMV compounds (33,34).Like all herpesviruses, the ly...

During human cytomegalovirus (HCMV) infection, a series of regulated events take place following virus binding and entry into the cell, including the upregulation of cellular transcription factors, such as NF-B, which play an essential role in the viral life cycle. We show here that NF-B message is induced during HCMV infection and that the induction is biphasic, suggesting an initial induction at immediate-early (IE) times and a second round of induction at early times. This hypothesis is supported by experiments using cyclohexamide, which showed that the first tier of induction was drug insensitive, while the second tier was drug sensitive. We then show that virus binding alone is sufficient to stimulate NF-B DNA binding activity, supporting its role in the initial induction of NF-B. To begin to elucidate the mechanism(s) for the second tier of NF-B regulation, we examined promoter constructs from the NF-B subunits (p105/p50 and p65) for responsiveness following HCMV infection. HCMV infection transactivated the p105/p50 and p65 promoters. The viral IE proteins (IE1-72, IE2-55, and IE2-86) are expressed during the time we see NF-B induction, so we examined their role in NF-B induction. The IE1-72, IE2-55, and IE2-86 proteins transactivated the p65 promoter, while only the IE2-55 protein transactivated the p105/p50 promoter. The p105/p50 promoter has NF-B sites; therefore, upregulation could also be caused by an autoregulatory mechanism. The p65 promoter, however, has been demonstrated to contain only SP1 sites. To investigate the potential role of SP1, we examined nuclear extracts from HCMV-infected cells. Here, we show that there is a biphasic increase in SP1 activity during viral infection and that there is apparently an absolute requirement for SP1 in the transactivation of the p65 promoter. In conclusion, we suggest a model in which the initial induction of NF-B occurs through viral modulation of cellular factors and the sustained levels of NF-B induction are regulated by a combination of cellular and viral factors.

Antiviral therapy of primary and recurrent infections with human cytomegalovirus is reserved for severe manifestations and faces several limitations. Presently candidates for novel drugs with lower adverse side effects and a minimized frequency of resistance formation are under investigation. Here we demonstrate that artesunate, an antimalaria drug with highly valuable pharmacological properties, possesses antiviral activity. A concentration-dependent inhibition of the replication of human cytomegaloviruses with wild-type phenotype was demonstrated in several cell lines. Inhibition was quantified using recombinant green fluorescent protein expressing virus variants. The IC50 values were in the same range for ganciclovir-sensitive and ganciclovir-resistant human cytomegalovirus, as calculated with 5.8+/-0.4 microM and 6.9+/-0.2 microM, respectively. This indicated a strong antiviral potential and a lack of cross-resistance. The optimal antiviral concentrations of artesunate were separable from those inducing cytotoxicity. In addition, the replication of viruses from three genera was seen to be artesunate-sensitive to varying degrees. This suggests a mechanism linked to cellular activation pathways. Both the protein levels and the DNA binding activity of the two virus-induced cellular transcription factors Sp1 and NF-kappaB were found to be markedly reduced in the presence of artesunate. We also analyzed the cellular signaling kinase phosphoinositide 3-kinase, required for the activation of factors such as Sp1 and NF-kappaB in infected fibroblasts. The phosphorylation of two downstream effectors of phosphoinositide 3-kinase, Akt and p70S6K, was markedly inhibited in the presence of artesunate. Thus, artesunate possesses attractive antiviral characteristics which are suggestively based on the interference with essential steps in the host cell kinase cascades.

Recent evidence indicates activated mitogen-activated protein kinase (MAPK) p38 has a critical function in human cytomegalovirus (HCMV) viral DNA replication in infected human fibroblasts. To elucidate the mechanism of HCMV-mediated p38 activation, we have performed a detailed analysis of p38 activation and the kinases associated with this activation at different times postinfection. We demonstrate that p38 kinase activity is strongly increased following viral infection. Inhibition of this activity significantly inhibited HCMV-induced hyperphosphorylation of pRb and phosphorylation of heat shock protein 27, suggesting that p38 activation is involved in virus-mediated changes in host cell metabolism throughout the course of infection. We then provide evidence that p38 activation is mediated by different mechanisms at early times versus later times of infection. At early times of infection (8 to 14 h postinfection [hpi]), when p38 activation is first observed, no significant activation of the three kinases which can directly phosphorylate p38 (namely, MKK3, MKK6, and MKK4) is detected. Using vectors which express dominant negative proteins, we demonstrate that basal MKK6 kinase activity is necessary for HCMV-mediated p38 activation at these early times of infection (12 hpi). Then, we use ATP depletion to show that at 12 hpi, HCMV inhibits dephosphorylation of activated p38. These two experiments suggest that HCMV activates p38 by inhibition of dephosphorylation of p38. In contrast to early times of infection, at later times of infection (48 to 72 hpi), increased MKK3/6, but not MKK4, activity is observed. These results indicate that at early times of HCMV infection, increased steady-state levels of activated p38 is mediated at least in part by inhibition of dephosphorylation of p38, while at later times of infection p38 activation is due to increased activity of the upstream kinases MKK3 and MKK6. These findings indicate that HCMV has developed multiple mechanisms to ensure activation of the MAPK p38, a kinase critical to viral infection.

We report antiviral activity against human cytomegalovirus for certain dietary flavonoids and their likely biochemical mechanisms of action. Nine out of ten evaluated flavonoids blocked HCMV replication at concentrations that were significantly lower than those producing cytotoxicity against growing or stationary phase host cells. Baicalein was the most potent inhibitor in this series (IC(50)=0.4-1.2 microM), including positive control ganciclovir. Baicalein and genistein were chosen as model compounds to study the antiviral mechanism(s) of action for this series. Both flavonoids significantly reduced the levels of HCMV early and late proteins, as well as viral DNA synthesis. Baicalein reduced the levels of HCMV immediate-early proteins to nearly background levels while genistein did not. The antiviral effects of genistein, but not baicalein, were fully reversible in cell culture. Pre-incubation of concentrated virus stocks with either flavonoid did not inhibit HCMV replication, suggesting that baicalein did not directly inactivate virus particles. Baicalein functionally blocked epidermal growth factor receptor tyrosine kinase activity and HCMV nuclear translocation, while genistein did not. At 24h post infection HCMV-infected cells treated with genistein continued to express immediate-early proteins and efficiently phosphorylate IE1-72. However, HCMV induction of NF-kappaB and increases in the levels of cell cycle regulatory proteins--events that are associated with immediate-early protein functioning--were absent. The data suggested that the primary mechanism of action for baicalein may be to block HCMV infection at entry while the primary mechanism of action for genistein may be to block HCMV immediate-early protein functioning.

The human cytomegalovirus immediate-early gene product 2 (IE2) is able to transactivate homologous and heterologous promoters alone or augmented by immediate-early gene product 1 (IE1). IE2 has also been shown to autoregulate the major immediate-early promoter by directly binding to a cis repression signal located between the TATA box and the cap site. However, IE2 has not been shown to act directly through a specific DNA sequence in transactivating various promoters. To understand whether IE2 can be indirectly involved in DNA sequence-specific transactivation through interactions with other transcriptional factors, we performed a study of the interactions of IE2 with cellular proteins. In order to study these interactions, IE cDNAs were subcloned into a bacterial expression vector, pGEX2T, by polymerase chain reaction amplification to produce fusion proteins which were full-length as well as proteins which contained various functional domains. We were able to demonstrate IE2's ability to interact directly or indirectly with several cellular proteins ranging from > 200 to 14 kDa through glutathione S-transferase-fusion protein precipitation and far-Western analysis. These interactions have been mapped to domains within IE2 which are known to be necessary for either transactivation or both transactivation and autoregulation. All of the IE2-associated proteins are nuclear proteins, and a subset are phosphorylated. In vitro-synthesized 35S-IE2 protein and bacterially expressed IE2 fusion proteins were used to study IE2-IE2 interaction by binding assay and far-Western analysis. IE2-IE2 interactions were mapped to a domain containing a putative helix-turn-helix motif located near the C terminus of IE2, between amino acids 456 and 539. However, IE2 was unable to directly interact with either IE1, an alternatively spliced variant of IE2 (55 kDa), or IE2 deletion mutants that did not contain the multimerization domain.

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Genomic complexity of human CMV is one of the largest among various DNA viruses. With such genome complexity and widespread distribution, even a minimal frequency of mutation will be enough to create a complicated genetic heterogeneity in this virus. The virus genome is relatively stable during subcultivation in tissue culture. Variants with minor modification in restriction enzyme sites do gradually develop. These variants share substantial restriction fragment pattern homology with the parental virus. Virus DNA has a molecular weight of 150 X 10(6) daltons for a complete genome. The DNA contains 2 covalently linked L and S components which are separated by internal inverted repetitious sequences of both terminal-end sequences. L and S components are oriented invertably with 4 isomeric arrangements. No tandem repeated sequences have been found. Based on DNA restriction pattern analysis, we conclude that the majority of recurrent infections represent reactivation of existing latent viruses; however, reinfections by a new virus strain do occur occasionally. By studying virus strains isolated from the consecutive congenitally infected infants and strains from mothers and their congenitally infected offspring, we furthermore conclude that the latent virus in women is relatively stable genetically. Moreover, the virus strains after being transmitted to the offspring are still genetically similar to that of the mothers. As in vitro, spontaneous minor variations occur after the in vivo residence. In a long period of evolution the accumulation of minor variations might create great diversity with major similarity.