Human cytomegalovirus up-regulates the phosphatidylinositol 3-kinase (PI3-K) pathway: inhibition of PI3-K activity inhibits viral replication and virus-induced signaling.

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-kappaB 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 CMV-IGIV (CytoGam) neutralizes human cytomegalovirus (HCMV) infectivity and prevents intracellular signal transduction after HCMV exposure.

Pretreatment of human cytomegalovirus (HCMV) with human hyperimmune globulin (CytoGam) in human embryonic lung (HEL) fibroblast culture showed successful inhibition of infectivity, and decreased extracellular viral titers and extracellular viral DNA. CytoGam prevented HCMV from inducing intracellular activation of NF-kappaB, Sp-1, and P13-K signaling pathways and the production of immediate-early (IE), early (E), and late (L) viral proteins. CytoGam neutralization of HCMV in this cell culture model prevented the earliest known signal transduction events (NF-kappaB, Sp-1, P13-K activation) after viral specific glycoproteins bind to their cognate cell membrane receptors, suggesting that this agent contains highly effective neutralizing antibodies against HCMV.

Activation of the mitogen-activated protein kinase p38 by human cytomegalovirus infection through two distinct pathways: a novel mechanism for activation of p38.

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.

Inhibitory effect of 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H - imidazole on HCMV DNA replication and permissive infection.

We found that Human Cytomegalovirus (HCMV) infection of human fibroblasts resulted in a dramatic increase in p38 mitogen-activated protein kinase (MAPK) phosphorylation. Recently, drug mediated inhibition of p38 has been demonstrated to exhibit anti-viral activity against HIV (Shapiro, L., Heidenreich, K.A., Meintzer, M.D. and Dinarello, C.A., 1998. Role of p38 mitogen-activated protein kinase in HIV type 1 production in vitro. Proc. Natl. Acad. Sci. USA. 95, 7422-7426). Therefore, we examined the effect of a specific p38 kinase inhibitor on HCMV infection. Inhibiting p38 activity in HCMV infected cells by treating cells with 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole; (FHPI), a p38 inhibitor drug, prevented permissive HCMV infection as measured by plaque assay. In the presence of FHPI, HCMV immediate early gene expression was slightly lower at early times of infection, but there was no inhibition of expression of the early gene UL-84, an HCMV protein essential for viral replication. However, FHPI inhibited HCMV DNA replication and late gene expression. The inhibitory effect of FHPI was reversible, as demonstrated by the induction of HCMV replication upon withdrawal of FHPI. Our data describes FHPI as a novel anti-HCMV compound that inhibits synthesis/activation of cellular and/or viral factors required for initiation of HCMV DNA replication.

Identification of human cytomegalovirus target sequences in the human immunodeficiency virus long terminal repeat. Potential role of IE2-86 binding to sequences between -120 and -20 in promoter transactivation.

OBJECTIVE: Because of the important medical consequences of human cytomegalovirus (HCMV) infection in human immunodeficiency virus (HIV)-infected individuals, we wanted to understand the molecular interactions that occur during co-infection. Specifically, in this study, we wanted to identify the transactivating target sequences on the HIV long terminal repeat (LTR) that responded to HCMV infection. STUDY DESIGN/METHODS: In this study, we transfected the HIV-LTR into human fibroblasts and then mapped the regulation of this promoter following HCMV infection and co-transfection with the HCMV immediate-early (IE) gene product IE2-86. In addition, we examined IE2-86 binding to specific sequences in the HIV-LTR by electrophoretic mobility shift assay. RESULTS: Our results documented that HCMV and IE2-86 could transactivate the HIV-LTR. In mapping the regions of the HIV-LTR that IE2-86 transactivates, we identified discrete target sequences between -120 and -20 that are the major transactivating regions for the IE2-86-mediated effects and determined that IE2-86 could specifically bind to several discrete sequences within this region of the HIV-LTR. CONCLUSIONS: Our discovery of the binding of IE2-86 to the HIV-LTR, coupled with its ability to transactivate the HIV-LTR and induce cellular transcription factors, points to potential molecular mechanisms used by HCMV to upregulate the HIV life cycle and, consequently, exacerbate the conditions observed in individuals co-infected with HCMV and HIV.

Human cytomegalovirus upregulates NF-kappa B activity by transactivating the NF-kappa B p105/p50 and p65 promoters.

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-kappa B, which play an essential role in the viral life cycle. We show here that NF-kappa 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-kappa DNA binding activity, supporting its role in the initial induction of NF-kappa B. To begin to elucidate the mechanism(s) for the second tier of NF-kappa B regulation, we examined promoter constructs from the NF-kappa 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-kappa B induction, so we examined their role in NF-kappa 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-kappa 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-kappa B occurs through viral modulation of cellular factors and the sustained levels of NF-kappa B induction are regulated by a combination of cellular and viral factors.

Human cytomegalovirus immediate-early gene 2 protein interacts with itself and with several novel cellular proteins.

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.

Irreversible inhibition of human cytomegalovirus replication by topoisomerase II inhibitor, etoposide: a new strategy for the treatment of human cytomegalovirus infection.

We demonstrated previously that human cytomegalovirus (CMV) infections could enhance the expression of cellular topoisomerase II and this enzyme activity is essential for CMV to replicate in vitro (Benson and Huang, 1988; Benson and Huang, 1990). In this study, we further show that in addition to m-AMSA and VM26 which we had previously reported, a widely used and clinically available drug, etoposide (VP-16 or VePesid) can irreversibly inhibit CMV replication at the drug concentration (2.5 micrograms/ml) greatly below toxic levels to stationary phase cells. Growing cells were more sensitive to etoposide than stationary phase cells and slight growth inhibition occurred at 2.5 micrograms/ml level. This inhibitor does not prevent the expression of CMV immediate-early and early genes, but can inhibit viral DNA and late viral-proteins synthesis. Because of their irreversible inhibitory effects and approval usage in clinical oncology, it is suggested that this group of compounds, particularly etoposide (VP-16), can be used to control life-threatening CMV infections, such as CMV pneumonitis and CMV retinitis, in cancer and immunocompromised patients or patients with AIDS.

Cytomegalovirus: genetic variation of viral genomes.

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.

Preferential inhibition of herpes-group viruses by phosphonoacetic acid: effect on virus DNA synthesis and virus-induced DNA polymerase activity.

In tissue culture phosphonoacetic acid (PAA) specifically inhibited DNA synthesis of human cytomegalovirus (CMV), murine CMV, simian CMV, Epstein-Barr virus, and Herpesvirus saimiri. Fifty to one hundred micrograms per milliliter PAA completely inhibited viral DNA synthesis with no significant damage to host cell DNA synthesis. In vitro DNA polymerization assays showed that 10 µg/ml of PAA specifically inhibited partially purified human CMV-induced DNA polymerase, while little inhibition of host-cell DNA polymerase activity was found. The specific inhibition of herpes-group virus DNA synthesis with little toxicity to host cells suggests that PAA has great potential as an antiherpesvirus therapeutic agent.