HCMV Replication Research Articles

Antiviral activity of berberine and related compounds against human cytomegalovirus

Berberine chloride ( 1) and the structurally related compounds were assessed for the anti-human cytomegalovirus (HCMV) activity using the plaque assay. The anti-HCMV activity (IC 50 0.68 μM) of 1 was equivalent to that (IC 50 0.91 μM) of ganciclovir (GCV). The mechanism of action by which 1 inhibits the replication of HCMV is presumed to be different from that of GCV; 1 would interfere with intracellular events after virus penetration into the host cells and before viral DNA synthesis.

HCMV induced IL-6 release in trophoblast and trophoblast like cells

Background Human cytomegalovirus (hCMV) infection of the trophoblasts is a crucial event in virus transmission from mother to child, being one responsible factor for intrauterine infection of the unborne. Differences of virus replication in trophoblasts depending on time point of pregnancy and degree of differentiation of trophoblasts might influence this transmission. Furthermore, immunological reactions of the trophoblasts to hCMV infection might be important defence mechanisms too. Objectives hCMV replication and interleukin-6 release in trophoblasts and trophoblast like cells (choriocarcinoma cells) was investigated. Study design Trophoblasts from term and 1st trimester placentas were isolated and infected with hCMV. hCMV production and release to the supernatant as well as interleukin-6 release and interleukin-6 mRNA production by these infected cells was measured. Choriocarcinoma cell lines (JEG-3, JAR) were treated the same. Non-infected trophoblasts were used as controls. Results In 1st trimester trophoblast, term trophoblasts and JEG-3 permissive hCMV replication was observed, although with different kinetics and efficiency. In JAR no complete virus replication was seen. High levels of interleukin-6 were measured in the supernatants of all hCMV infected cells immediately after infection. IL-6 mRNA upregulation was seen 48 h after infection in those cell types replication of hCMV occurred (1st trimester trophoblasts, term trophoblasts, JEG-3). At that time-point hCMV immediate early proteins appeared. In JARs no virus production and no IL-6 mRNA upregulation was seen, and IL-6 levels in the supernatant of these hCMV infected cells declined significantely until day 6 after infection compared to mock infected cells. Conclusion These observations show that hCMV replication is influenced by the degree of trophoblast differentiation. Interleukin-6 is upregulated by hCMV infection, but is independent of complete virus replication.

Experimental study on the action of allitridin against human cytomegalovirus in vitro: Inhibitory effects on immediate-early genes

Garlic ( Allium sativum) extraction has been reported having anti-HCMV efficacy. This study was aimed to investigate the effect of allitridin (diallyl trisulfide, a compound from A. sativum extraction) on the replication of HCMV and the expression of viral immediate-early genes. In HCMV plaque-reduction assay, allitridin appeared a dose-dependent inhibitory ability with EC 50 value of 4.2 μg/ml (selective index, SI = 16.7). Time-of-addition and time-of-removal studies showed that allitridin inhibited HCMV replication in earlier period of viral cycle before viral DNA synthesis. Both immediate early gene (ie1) transcription and IEA (IE 172 and IE 286) expression was suppressed by allitridin, but not by GCV in a single HCMV cycle format. In addition, allitridin appeared stronger inhibition on IE 286 than on IE 172. Decrease of viral DNA load in infected cells was also detected under allitridin treatment, probably due to an indirect consequence of the reduction in ie gene transcription. In summary, this study indicated that allitridin has anti-HCMV activity and the mechanism is associated with suppression of ie gene transcription.

Human cytomegalovirus-inhibitory flavonoids: Studies on antiviral activity and mechanism of action

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 μM), 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 24 h post infection HCMV-infected cells treated with genistein continued to express immediate-early proteins and efficiently phosphorylate IE1-72. However, HCMV induction of NF-κB 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.

Oral Activity of a Methylenecyclopropane Analog, Cyclopropavir, in Animal Models for Cytomegalovirus Infections

We reported previously that purine 2-(hydroxymethyl)methylenecyclopropane analogs have good activity against cytomegalovirus infection. A second-generation analog, (Z)-9-[[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl]guanine (ZSM-I-62, cyclopropavir [CPV]), has particularly good activity against murine and human cytomegaloviruses (MCMV and HCMV) in vitro. To determine the oral activity of this compound in vivo, BALB/c or severe combined immunodeficient (SCID) mice infected with MCMV and two models using SCID mice implanted with human fetal tissue and subsequently infected with HCMV were used. In MCMV-infected normal mice, CPV at 10 mg/kg of body weight was highly effective in preventing mortality when administered at 24, 48, or 72 h post-viral inoculation and reduced titers of virus in tissues of SCID mice by 2 to 5 log10. In one HCMV model, human fetal retinal tissue was implanted into the anterior chamber of the mouse eye and inoculated with the Toledo strain of HCMV, and in the second, human fetal thymus and liver tissues were implanted under the kidney capsule of mice and then inoculated with HCMV. In general, replication of HCMV in both types of implant tissue increased from 7 through 21 to 28 days and then gradually decreased to undetectable levels by 8 weeks postinfection. Oral treatment with 45 or 15 mg of CPV/kg initiated 24 h after infection was highly effective in reducing replication to undetectable levels in both models and was generally more effective than ganciclovir. These data indicate that the methylenecyclopropane analog, CPV, was highly efficacious in these four animal models and should be evaluated for use in HCMV infections in humans.

Novel Chemical Class of pUL97 Protein Kinase-Specific Inhibitors with Strong Anticytomegaloviral Activity

Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns. The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication. Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication. We present evidence that three related quinazoline compounds are potent inhibitors of the pUL97 kinase activity and block in vitro substrate phosphorylation, with 50% inhibitory concentrations (IC(50)s) between 30 and 170 nM. Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency. The IC(50)s of these three quinazoline compounds (2.4 +/- 0.4, 3.4 +/- 0.6, and 3.9 +/- 1.1 microM, respectively) were in the range of the IC(50) of ganciclovir (1.2 +/- 0.2 microM), as determined by the HCMV green fluorescent protein-based antiviral assay. Importantly, the quinazolines were demonstrated to have strong inhibitory effects against clinical HCMV isolates, including ganciclovir- and cidofovir-resistant virus variants. Moreover, in contrast to ganciclovir, the formation of resistance to the quinazolines was not observed. The mechanisms of action of these compounds were confirmed by kinetic analyses with infected cells. Quinazolines specifically inhibited viral early-late protein synthesis but had no effects at other stages of the replication cycle, such as viral entry, consistent with a blockage of the pUL97 function. In contrast to epithelial growth factor receptor inhibitors, quinazolines affected HCMV replication even when they were added hours after virus adsorption. Thus, our findings indicate that quinazolines are highly efficient inhibitors of HCMV replication in vitro by targeting pUL97 protein kinase activity.

SUMOylation of the human cytomegalovirus 72-kilodalton IE1 protein facilitates expression of the 86-kilodalton IE2 protein and promotes viral replication.

The 72-kDa immediate-early 1 protein (IE1-72kDa) of human cytomegalovirus has been previously shown to be posttranslationally modified by covalent conjugation to the ubiquitin-related protein SUMO-1. Using an infectious bacterial artificial chromosome clone of human cytomegalovirus, we constructed a mutant virus (BADpmIE1-K450R) that is deficient for SUMOylation of IE1-72 kDa due to a single amino acid exchange in the SUMO-1 attachment site. Compared to wild-type virus, this mutant grew more slowly and generated a reduced yield in infected human fibroblasts, indicating that SUMO modification is required for the full activity of IE1-72 kDa. The lack of SUMOylation did not affect the intranuclear localization of IE1-72 kDa, including its ability to target to and disrupt PML bodies and to bind to mitotic chromatin. Likewise, SUMOylation-deficient IE1-72 kDa activated several viral promoters as efficiently as the wild-type protein. However, the failure to modify IE1-72 kDa resulted in substantially reduced levels of the IE2 transcript and its 86-kDa protein (IE2-86 kDa). These observations suggest that SUMO modification of IE1-72 kDa contributes to efficient HCMV replication by promoting the accumulation of IE2-86 kDa.

Synthesis and anti-HCMV activity of 1-acyl-β-lactams and 1-acylazetidines derived from phenylalanine

Different Phe-derived 1-acyl-β-lactams, analogous to a series of 2-azetidinones acting as HCMV serine protease inhibitors, were synthesized. Some of these compounds were modest inhibitors of the HCMV replication. Interestingly, removal of the carbonyl group of the β-lactam ring, most likely acting as the serine trap, resulted in an azetidine derivative with anti-HCMV activity comparable to that of the reference compound ganciclovir.

Human cytomegalovirus stimulates cellular IKK2 activity and requires the enzyme for productive replication.

Human cytomegalovirus (HCMV) exploits the host transcription factor NF-kappaB to enhance its own replication, dissemination, and reactivation from latency. Here we report that HCMV infection activates the upstream IkappaB kinase (IKK) complex and that its catalytic IKK2 subunit is required for HCMV-induced NF-kappaB activation, as well as the replication of different HCMV strains. These results indicate that IKK2 is essential for HCMV replication and emphasize the feasibility of blocking NF-kappaB activation as a way of inhibiting infection.

Thrombin stimulates IL-6 and IL-8 expression in cytomegalovirus-infected human retinal pigment epithelial cells

Recently, we reported that thrombin specifically stimulates protease-activated receptor-1 (PAR-1) signaling in RPE entailing inhibition of Sp1 dependent HCMV replication. We now studied whether thrombin modulates the expression of the proinflammatory cytokine/chemokines IL-6 and IL-8 in mock- and cytomegalovirus-infected human retinal pigment epithelial cells (RPE). Our data show that thrombin/PAR-1 stimulates IL-6 and IL-8 gene transcription and protein secretion in both mock- and HCMV-infected RPE. Thrombin/PAR-1-mediated signaling stimulated PKC and NF-kappaB-dependent IL-6 and IL-8 gene expression via phosphoinositide 3-kinase and further downstream via p42/44 and p38 MAPKs. Thus, thrombin/PAR-1-mediated IL-6/IL-8 gene expression is uncoupled from Sp1 inhibition and may support proinflammatory pathomechanisms probably involved in hemorrhage/HCMV retinitis progression.

“SENSELESS” ANTIVIRAL POLYRIBONUCLEOTIDES: POLY (1-PROPARGYLINOSINIC ACID)

ABSTRACT Previous work has shown that novel amphipathic oligo and polyribo-nucleotides exhibiting secondary structure in solution are potent inhibitors of HIV and HCMV replication and cytopathicity in tissue culture. It was hypothesized that the mechanism(s) of action for these compounds might be inhibition of retroviral reverse transcriptase (RT) and/or viral uptake by cells. Pursuit of the essential pharmacophore has led to the discovery of poly (1-propargylinosinic acid) (10), an HIV and HCMV-active polyribonucleotide lacking the secondary structure previously thought to be essential for the observed antiviral activity.

Degradation of p21cip1 in cells productively infected with human cytomegalovirus.

Human cytomegalovirus (HCMV) stimulates arrested cells to enter the cell cycle by activating cyclin-dependent kinases (Cdks), notably Cdk2. Several mechanisms are involved in the activation of Cdk2. HCMV causes a substantial increase in the abundance of cyclin E and stimulates translocation of Cdk2 from the cytoplasm to the nucleus. Further, the abundance of the Cdk inhibitors (CKIs) p21cip1/waf1 (p21cip1) and p27kip1 is substantially reduced. The activity of cyclin E/Cdk2 increases as levels of CKIs, particularly p21cip1, fall. We have previously shown that these phenomena contribute to priming the cell for efficient replication of HCMV. In this study, the mechanisms responsible for the decrease in p21cip1 levels after HCMV infection were investigated by measuring p21cip1 RNA and protein levels in permissive human lung (LU) fibroblasts after HCMV infection. Northern blot analysis revealed that p21cip1 RNA levels increased briefly at 3 h after HCMV infection and then decreased to their nadir at 24 h; thereafter, RNA levels increased to about 60% of the preinfection level. Western blot analysis demonstrated that the relative abundance of p21cip1 protein roughly paralleled the observed changes in initial RNA levels; however, the final levels of protein were much lower than preinfection levels. After a transient increase at 3 h postinfection, p21cip1 abundance declined sharply over the next 24 h and remained at a very low level through 96 h postinfection. The disparity between p21cip1 RNA and protein levels suggested that the degradation of p21cip1 might be affected in HCMV-infected cells. Treatment of HCMV-infected cells with MG132, an inhibitor of proteasome-mediated proteolysis, provided substantial protection of p21cip1 in mock-infected cells, but MG132 was much less effective in protecting p21cip1 in HCMV-infected cells. The addition of E64d or Z-Leu-Leu-H, each an inhibitor of calpain activity, to HCMV-infected cells substantially increased the abundance of p21cip1 in a concentration-dependent manner. To verify that p21cip1 was a substrate for calpain, purified recombinant p21cip1 was incubated with either m-calpain or mu-calpain, which resulted in rapid proteolysis of p21cip1. E64d inhibited the proteolysis of p21cip1 catalyzed by either m-calpain or mu-calpain. Direct measurement of calpain activity in HCMV-infected LU cells indicated that HCMV infection induced a substantial and sustained increase in calpain activity, although there was no change in the abundance of either m- or mu-calpain or the endogenous calpain inhibitor calpastatin. The observed increase of calpain activity was consistent with the increases in intracellular free Ca2+ and phospholipid degradation in HCMV-infected LU cells reported previously from our laboratory. Considered together, these results suggest that the increase in calpain activity observed following HCMV infection contributes significantly to the reduction of p21cip1 levels and the resultant cell cycle progression.

Functional heterogeneity and high frequencies of cytomegalovirus-specific CD8(+) T lymphocytes in healthy seropositive donors.

Human cytomegalovirus (HCMV) infection is largely asymptomatic in the immunocompetent host, but remains a major cause of morbidity in immunosuppressed individuals. Using the recently described technique of staining antigen-specific CD8(+) T cells with peptide-HLA tetrameric complexes, we have demonstrated high levels of antigen-specific cells specific for HCMV peptides and show that this may exceed 4% of CD8(+) T cells in immunocompetent donors. Moreover, by staining with tetramers in combination with antibodies to cell surface markers and intracellular cytokines, we demonstrate functional heterogeneity of HCMV-specific populations. A substantial proportion of these are effector cytotoxic T lymphocytes, as demonstrated by their ability to lyse peptide-pulsed targets in "fresh" killing assays. These data suggest that the immune response to HCMV is periodically boosted by a low level of HCMV replication and that sustained immunological surveillance contributes to the maintenance of host-pathogen homeostasis. These observations should improve our understanding of the immunobiology of persistent viral infection.

Indolocarbazoles: potent, selective inhibitors of human cytomegalovirus replication

In our search for new, safer anti-HCMV agents, we discovered that the natural product Arcyriaflavin A ( 1a) was a potent inhibitor of HCMV replication in cell culture. A series of analogues (symmetrical indolocarbazoles) was synthesised to investigate structure–activity relationships in this series against a range of herpes viruses (HCMV, VZV, HSV1, and 2). This identified a number of novel, selective and potent inhibitors of HCMV, 12,13-dihydro-2,10-difluoro-5 H-indolo[2,3- a]pyrrolo[3,4- c]carbazole-5,7-(6 H)-dione ( 1d) being the best example (IC 50=40 nM, therapeutic index >1450). Compounds described in this series were generally poor inhibitors of protein kinase C βII, and no correlation was found between the ability to inhibit HCMV and the enzyme PKC.

Inhibition of HCMV replication by the tetrahydroindolizine CMV423

L’émergence de souches de cytomégalovirus (CMV) résistantes aux antiviraux et conduisant à l’échec thérapeutique, constitue un problème majeur survenant au cours de traitements prolongés chez les patients immunodéprimés. Trois inhibiteurs de l’ADN polymérase virale sont actuellement utilisés pour traiter les infections à CMV, le ganciclovir (Cymevan®), le cidofovir (®), et le foscarnet (Foscavir®). Aucune de ces molécules n’est efficace sur le virus latent. Le ganciclovir (GCV) est la molécule la plus utilisée, en traitement curatif, en traitement anticipé et en prophylaxie. La résistance au ganciclovir est donc la plus fréquente et la mieux étudiée. Les mutations de la phosphotransférase virale UL97, sont les premières à apparaître. Cette enzyme est responsable de la primophosphorylation du ganciclovir et de l’aciclovir dans les cellules infectées, indispensable à leur activation. Des mutations de l’ADN polymérase, codée par le gène UL54, surviennent plus tardivement et conduisent à une résistance croisée au cidofovir ou, plus rarement, au foscarnet. La recherche des souches résistantes par l’étude phénotypique est longue et difficile. Elle nécessite l’isolement du virus en culture. L’approche moléculaire, basée sur la détection directe des mutations à partir du prélèvement ou de l’isolat permet une mise en évidence précoce de la résistance. Des techniques rapides ont été développées. Cependant, ces techniques séduisantes sont actuellement limitées à la seule recherche des mutations les plus fréquentes de UL97. L’étude de la séquence complète des gènes UL97 et UL54, seule approche permettant de détecter toutes les mutations, reste peu pratiquée du fait de l’absence de standardisation des techniques. Elle est effectuée par des laboratoires spécialisés. La recherche et l’étude systématique des souches résistantes est toutefois nécessaire au développement de nouvelles molécules antivirales, et pourra guider, lorsqu’un éventail plus large sera disponible, un véritable choix thérapeutique.The emergence of cytomegalovirus (CMV) resistant strains responsible for therapeutic failure is of major concern during prolonged antiviral treatments in immunocompromised hosts. Three polymerase inhibitors are used in clinical practice: ganciclovir (Cymevan®), cidofovir (Vistide®) and foscarnet (Foscavir®). These molecules are not active on latent virus. Ganciclovir (GCV) is the most frequently administered in curative or pre-emptive therapy, and in prophylaxis. Antiviral resistance therefore concerns essentially GCV. UL97 phosfotransferase mutations appear first during treatment. This kinase is responsible for the activation of GCV and acyclovir (ACV) in infected cells. Mutations within the UL54 viral polymerase can occur later. They are responsible for double resistance to GCV and cidofovir (CDV) and/or foscarnet. Resistant isolates can be identified by phenotypic assays. These methods are time-consuming and rely on the multiplication of the isolate in cell-culture. Molecular methods based on the detection of UL97 or UL54 resistance-related mutations may demonstrate resistance early because of its sensitivity. Rapid techniques have been developed. Though, they are limited to the most frequent UL97 mutations, and lack sensitivity in clinical samples. The complete sequence of UL97 and UL54 genes allows the detection of all the mutations, but is restricted to few laboratories, due to its lack of standardisation. However, the research of new resistance-related mutations is necessary to the development of antiviral molecules. It will be useful to enlarge the therapeutic choices in clinical practice when new molecules will be available.

Constrained analogues of 2′-nor cyclic nucleoside monophosphates

The synthesis of constrained analogues of 2′-nor-cyclic nucleosides monophosphates containing a thiomethylene tether was readily accomplished from the oxathiolane intermediate 4. The uracil, cytosine, and 5-bromocytosine spirophosphate analogues 22, 16, and 25 were inhibitory to HCMV replication in Flow 2002 cells.

Inhibition of Cellular Cdk2 Activity Blocks Human Cytomegalovirus Replication

Human cytomegalovirus is a herpesvirus that induces numerous cellular processes upon infection. Among these are activation of cyclin-dependent kinase 2, which regulates cell cycle progression in G1 and S phase. We report here that inhibition of cellular Cdk2 activity blocks HCMV replication. Inhibition of Cdk2 activity by roscovitine inhibits HCMV DNA synthesis, production of infectious progeny, and late antigen expression in infected cells in a dose-dependent manner. HCMV replication is also inhibited by the expression of a Cdk2 dominant negative mutant, whereas expression of wild-type Cdk2 has no effect on viral replication. These data indicate that activation of cellular Cdk2 is necessary for HCMV replication.

Antiviral effects of plasma and milk proteins: Lactoferrin shows potent antiviral activity on both HIV and HCMV replication in vitro in the same concentration range

Antiviral effects of plasma and milk proteins: Lactoferrin shows potent antiviral activity on both HIV and HCMV replication in vitro in the same concentration range

Synthesis and antiviral activity of pyranosylphosphonic acid nucleotide analogues.

Pyranosyl nucleotide analogues have been designed so that the intramolecular base to phosphorus distance closely approximates that of natural nucleotides. This was achieved by attaching the phosphorus directly at the anomeric position and the base at the 4-position of the carbohydrate. A series of compounds incorporating natural bases and having this novel structure were made via a short synthesis starting from commercially available glycals. Addition of triisopropyl phosphite to the glycals furnished alpha- and beta-2-enopyranosylphosphonates which were then substituted with the heterocycle using Mitsunobu chemistry. Deprotection afforded the 2',3'-unsaturated isonucleotide analogue. In some cases the deprotection sequence induced double-bond migration leading to the 1',2'-unsaturated derivative. NMR spectroscopic structural analysis established an axial preference for the base and an equatorial preference for the beta-phosphorus which results in intramolecular base to phosphorus distances within 1 A of that of natural nucleotides. All of the deprotected compounds were screened for inhibition of HCMV, HSV-2, and HIV replication. Several compounds inhibited HCMV and HSV-2, the most potent of which was the unsaturated cytosine analogue 18 (HCMV IC50 = 10 microM, HSV-2 IC50 = 85 microM). None of the compounds were cytotoxic at the highest dose (1 mM) tested. None of the compounds were inhibitory to HIV.

Retinoid activation of retinoic acid receptors but not of retinoid X receptors promotes cellular differentiation and replication of human cytomegalovirus in embryonal cells

The susceptibility of human embryonal cell line NT-2/D1 to replicate human cytomegalovirus (hCMV) is dependent on retinoic acid (RA) stimulation. Physiological responses to retinoic acid involve two distinct subfamilies of nuclear receptors, the RA receptors (RARs) and retinoid X receptors (RXRs), which function by activating transcription as heterodimeric or RXR homodimeric complexes from cis-acting DNA response elements. At present, it is not clear whether the association between these two classes of receptors can lead to multiple distinct induction pathways by signalling one or both receptor partners. Here we have determined, by selectively activating endogenous receptors with novel synthetic ligands specific for either RARs or RXRs, what ligand interaction is physiological in the retinoid receptor pathways necessary for inducing replication of hCMV in differentiated embryonal cells. We show that ligand binding to RAR alone is sufficient and that exclusive ligand activation of RXR is insufficient for inducing replication of hCMV. We also find that differentiation and inhibition of NT-2/D1 cell growth are promoted by compounds that signal the RAR pathway. These results provide direct evidence that RAR ligand-mediated physiological responses are separable and distinct from RXR ligand activation functions. Moreover, our results provide insight into a hormone response pathway for cellular differentiation that might be coopted by hCMV in the host.