AD169-infected Cells Research Articles

Rescue of Pentamer-Null Strains of Human Cytomegalovirus in Epithelial Cells by Use of Histone Deacetylase Inhibitors Reveals an Additional Postentry Function for the Pentamer Complex.

Human cytomegalovirus (HCMV) tropism for epithelial cells is determined by the pentameric glycoprotein complex found on the viral envelope. Laboratory-adapted strains, such as AD169, typically develop loss-of-function mutations for the pentamer, thus losing the ability to efficiently initiate lytic replication in epithelial cells. Using our human salivary gland-derived epithelial (hSGE) cell model, we observed that 3 chemically distinct histone deacetylase (HDAC) inhibitors can rescue infection in hSGE cells using pentamer-null strains of HCMV. Additionally, infection in ARPE-19 epithelial cells was rescued in a similar manner. We isolated nuclei from AD169-infected cells, quantified viral genomes by quantitative PCR (qPCR), and discovered that while HDAC inhibitors increased immediate early (IE) gene expression, they did not increase the amount of viral DNA in the nucleus. Using immunofluorescence microscopy, we observed that pentamer-null strains showed punctate patterning of pp71 in proximity to the nucleus of infected cells, while pp71 was localized to the nucleus after infection with pentamer-containing strains. Upon treatment with HDAC inhibitors, these punctae remained perinuclear, while more cells displayed entry into the lytic cycle, noted by increased IE-positive nuclei. Taken together, our data indicate that HCMV pentamer-null viruses are able to infect epithelial cells (albeit less efficiently than pentamer-positive viruses) and traffic to the nucleus but fail to initiate lytic gene expression once there. These studies reveal a novel postentry function of the pentamer in addition to the recognized role of pentamer in mediating entry. IMPORTANCE Human cytomegalovirus has a wide cellular tropism, which is driven by one of its glycoprotein complexes, the pentamer. Laboratory-adapted strains continuously passaged on fibroblasts readily lose pentamer function and thus lose their ability to infect diverse cell types such as epithelial cells. Pentamer has been attributed an entry function during infection, but mechanistic details as to how this is achieved have not been definitely demonstrated. In this study, we investigate how pharmacological rescue of pentamer-null strains during epithelial infection by histone deacetylase inhibitors implicates a novel role for the pentamer downstream of entry. This work expands on potential functions of the pentamer, will drive future studies to understand mechanistically how it affects tropism, and provides a new target for future therapeutics.

Inhibition of IKKα by BAY61-3606 Reveals IKKα-Dependent Histone H3 Phosphorylation in Human Cytomegalovirus Infected Cells.

Protein kinase inhibitors can be used as tools to identify proteins and pathways required for virus replication. Using virus replication assays and western blotting we found that the widely used protein kinase inhibitor BAY61-3606 inhibits replication of human cytomegalovirus (HCMV) strain AD169 and the accumulation of HCMV immediate-early proteins in AD169 infected cells, but has no effect on replication of HCMV strain Merlin. Using in vitro kinase assays we found that BAY61-3606 is a potent inhibitor of the cellular kinase IKKα. Infection of cells treated with siRNA targeting IKKα indicated IKKα was required for efficient AD169 replication and immediate-early protein production. We hypothesized that IKKα was required for AD169 immediate-early protein production as part of the canonical NF-κB signaling pathway. However, although BAY61-3606 inhibited phosphorylation of the IKKα substrate IκBα, we found no canonical or non-canonical NF-κB signaling in AD169 infected cells. Rather, we observed that treatment of cells with BAY61-3606 or siRNA targeting IKKα decreased phosphorylation of histone H3 at serine 10 (H3S10p) in western blotting assays. Furthermore, we found treatment of cells with BAY61-3606, but not siRNA targeting IKKα, inhibited the accumulation of histone H3 acetylation (H3K9ac, H3K18ac and H3K27ac) and tri-methylation (H3K27me3 and H3K36me3) modifications. Therefore, the requirement for IKKα in HCMV replication was strain-dependent and during replication of an HCMV strain requiring IKKα, IKKα-dependent H3S10 phosphorylation was associated with efficient HCMV replication and immediate-early protein production. Plus, inhibition of HCMV replication by BAY61-3606 is associated with acetylation and tri-methylation modifications of histone H3 that do not involve IKKα.

Maintenance of large numbers of virus genomes in human cytomegalovirus-infected T98G glioblastoma cells.

After infection, human cytomegalovirus (HCMV) persists for life. Primary infections and reactivation of latent virus can both result in congenital infection, a leading cause of central nervous system birth defects. We previously reported long-term HCMV infection in the T98G glioblastoma cell line (1). HCMV infection has been further characterized in T98Gs, emphasizing the presence of HCMV DNA over an extended time frame. T98Gs were infected with either HCMV Towne or AD169-IE2-enhanced green fluorescent protein (eGFP) strains. Towne infections yielded mixed IE1 antigen-positive and -negative (Ag(+)/Ag(-)) populations. AD169-IE2-eGFP infections also yielded mixed populations, which were sorted to obtain an IE2(-) (Ag(-)) population. Viral gene expression over the course of infection was determined by immunofluorescent analysis (IFA) and reverse transcription-PCR (RT-PCR). The presence of HCMV genomes was determined by PCR, nested PCR (n-PCR), and fluorescence in situ hybridization (FISH). Compared to the HCMV latency model, THP-1, Towne-infected T98Gs expressed IE1 and latency-associated transcripts for longer periods, contained many more HCMV genomes during early passages, and carried genomes for a greatly extended period of passaging. Large numbers of HCMV genomes were also found in purified Ag(-) AD169-infected cells for the first several passages. Interestingly, latency transcripts were observed from very early times in the Towne-infected cells, even when IE1 was expressed at low levels. Although AD169-infected Ag(-) cells expressed no detectable levels of either IE1 or latency transcripts, they also maintained large numbers of genomes within the cell nuclei for several passages. These results identify HCMV-infected T98Gs as an attractive new model in the study of the long-term maintenance of virus genomes in the context of neural cell types. Our previous work showed that T98G glioblastoma cells were semipermissive to HCMV infection; virus trafficked to the nucleus, and yet only a proportion of cells stained positive for viral antigens, thus allowing continual subculturing and passaging. The cells eventually transitioned to a state where viral genomes were maintained without viral antigen expression or virion production. Here we report that during long-term T98G infection, large numbers of genomes were maintained within all of the cells' nuclei for the first several passages (through passage 4 [P4]), even in the presence of continual cellular division. Surprisingly, genomes were maintained, albeit at a lower level, through day 41. This is decidedly longer than in any other latency model system that has been described to date. We believe that this system offers a useful model to aid in unraveling the cellular components involved in viral genome maintenance (and presumably replication) in cells carrying long-term latent genomes in a neural context.

Effects of allitridin on transcription of immediate-early,early and late genes of human cytomegalovirus in vitro

The effect of allitridin on the transcription levels of immediate-early (ie), early(e) and late (1) genes of human cytomegalovirus (HCMV) was investigated in order to explore the mechanism of allitridin against HCMV. Established the models of HCMV AD169 strain infected cells and AD169 strain infected cells treated with allitridin (9.6 mg x L(-1)), and they were compared with the appropriate dose(2.3 mg x L(-1)) of ganciclovir (GCV). All groups of cells were infected at 2.5 multiplicity of infection (MOI), using SYBR Green real-time PCR method to detect the dynamic change of ul122, ul123, ul54 and ul83 mRNA expression at 0.5, 2, 4, 6, 12, 24 h post-infection. The mRNA levels of ul122 and ul123 in AD169 infected cells treated with allitridin at all time points were markedly lower than those of AD169 infected controls (P<0.05), but there were no significant difference of ul122 gene in AD169 infected cells treated with GCV and AD169 infected cells at 0.5-6 h post-infection. The inhibitory rates of allitridin to AD169 ul122 and ul123 mRNA reached 75.2% and 70.4% at 24 h post-infection, respectively. The expression of ul54 mRNA in two drug-treatment groups at all time points were lower than those of AD169 infected cells group (P<0.05). The inhibitory rates of alltridin and GCV to AD169 ul54 mRNA were 45.4% and 27.2% at 24 h post-infection,respectively. The expression of HCMV ul83 mRNA in all groups rapidly increased after 6 h of infection,which is most obvious in AD169 infected cells group. The inhibitory rates of alltridin and GCV to AD169 ul83 mRNA were 45.9% and 26.2% at 24 h post-infection, respectively. Allitridin could effectively suppress the transcription of ie genes (ul122 and ul123) of HCMV AD169 strain, led the expression of mRNA significantly lowerd. It was able to supress the transcription of egene (ul54) and l gene (ul83) too, indicating that HCMV ie genes may be the key target of allitridin against HCMV.

The human cytomegalovirus protein UL16 mediates increased resistance to natural killer cell cytotoxicity through resistance to cytolytic proteins.

Several reports have shown that human cytomegalovirus (HCMV)-infected cells are resistant to NK lysis. These studies have focused on receptor-ligand interactions, and different HCMV proteins have been indicated to mediate inhibitory NK signals. Here, we report that the HCMV protein UL16 is of major importance for the ability of HCMV-infected cells to resist NK cell-mediated cytotoxicity. Fibroblasts infected with the UL16 deletion mutant HCMV strain exhibited a 70% increased sensitivity to NK killing at 7 days postinfection compared to AD169-infected cells. Interestingly, HCMV-infected cells did not appear to engage inhibitory molecules on NK cells, since the levels of granzyme B were not reduced in supernatants obtained from NK cell cocultures with infected target cells compared to uninfected target cells. Furthermore, HCMV-infected cells, but not cells infected with the UL16 deletion mutant HCMV strain, exhibited a significantly increased resistance to the action of cytolytic proteins, including perforin, granzyme B, streptolysin O, and porcine NK lysin. In addition, fluorescence-activated cell sorting for UL16-positive transfected cells resulted in protection levels of 90% compared to control cells carrying the green fluorescent protein vector. Thus, the UL16 protein mediates an increased protection against the action of cytolytic proteins released by activated NK cells, possibly by a membrane-stabilizing mechanisms, rather than by delivering negative signals to NK cells.

NK Cell-Mediated Lysis of Autologous HCMV-Infected Skin Fibroblasts Is Highly Variable among NK Cell Clones and Polyclonal NK Cell Lines

Lysis of human cytomegalovirus (HCMV)-infected fibroblasts by autologous natural killer (NK) cells was examined in vitro. For NK cell clones, receptor expression was determined at the level of mRNA and cell-surface protein and compared to the lysis of HCMV AD169 strain-infected fibroblasts in which HLA class I was >70% downregulated. The clones ranged broadly in their ability to lyse AD169-infected fibroblasts, correlating neither with the expression of inhibitory KIR, leukocyte inhibitory receptor-1, or CD94:NKG2A receptors nor with the number of different inhibitory KIR expressed per clone. Some lines of polyclonal NK cells preferentially lysed AD169-infected cells and similarly lysed fibroblasts infected with mutant virus RV798, which lacks the genes for downregulating HLA class I. These results demonstrate that NK cell lysis of HCMV-infected autologous fibroblasts is more complex than a simple missing-self mechanism involving downregulation of HLA class I and failure to engage inhibitory self-specific KIR.

Enhanced endothelial cytopathogenicity induced by a cytomegalovirus strain propagated in endothelial cells

Endothelial involvement has been implicated in cytomegalovirus (CMV) infection, a source of major complications in immunosuppressed individuals (e.g., those with acquired immune deficiency syndrome [AIDS] and organ transplants). Traditionally, CMV has been grown in fibroblasts; however, propagation in these cells may alter characteristics of the virus. In developing an in vitro model system of CMV/endothelial cell interaction, we have addressed this issue by propagating a clinical isolate, CMV VHL 1, in human umbilical vein endothelial (HUVE) cells by serial cocultivation of heavily infected cultures with fresh HUVE monolayers and have compared its infectious properties with those of the fibroblast-raised strain, CMV AD169. In situ hybridization using a biotinylated DNA probe, as well as immunofluorescent staining for CMV-specific antigen, has confirmed infection of HUVE cells inoculated with either strain of the virus. Infection of HUVE by VHL was accompanied by dramatic cytopathology not observed in AD169-infected cells. Plaque assay of culture supernatants revealed greater virus production in VHL-infected HUVE as compared with equivalently inoculated fibroblasts. In contrast, AD169 production in inoculated fibroblasts exceeded that in HUVE. These studies demonstrate the suitability of cultured endothelial cells as a substrate for CMV propagation and suggest that a strain of virus thus propagated may offer an accurate model of CMV/endothelial cell interaction in human disease.

Antiviral activity of 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodocytosine against human cytomegalovirus in human skin fibroblasts.

2'-Deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodocytosine (FIAC) was shown to be a selective anti-human cytomegalovirus agent in vitro with a 50% antiviral effective dose of 0.6 microM (J. M. Colacino and C. Lopez, Antimicrob. Agents Chemother. 26:505-508, 1983) and a 50% cell growth inhibitory dose of 8 microM. Antiviral activity was more readily reversed with 10-fold excess thymidine, whereby the 50% effective dose was increased to 11.3 microM. FIAC-induced cytotoxicity was more readily reversed with 10-fold excess of deoxycytidine, whereby the 50% inhibitory dose was increased to greater than 100 microM. Thymidine was unable to reverse completely the antiviral activity of FIAC. Although, the extent of phosphorylation of thymidine, deoxycytidine, and deoxyuridine was 6-, 4-, and 4-fold greater, respectively, in human cytomegalovirus-infected cell lysates than in uninfected cell lysates, the extent of phosphorylation of FIAC was only 1.3-fold greater in human cytomegalovirus-infected cell lysates than in uninfected cell lysates. By comparison, the extent of FIAC phosphorylation was 500 times greater in herpes simplex virus type 1-infected cells than in uninfected cell lysates. Methotrexate was 400 times more effective against human cytomegalovirus replication than it was against herpes simplex virus type 1 replication, indicating that thymidylate synthetase may be important for human cytomegalovirus replication. However, 10 microM FIAC did not inhibit thymidylate synthetase activity in uninfected or virus-infected cells as determined by their metabolism of [6-3H]deoxyuridine in the presence or absence of drug. FIAC at 1 microM suppresses and FIAC at 10 microM completely inhibits human cytomegalovirus DNA replication as indicated by Southern blot analysis. This inhibition was reversible. FIAC incorporation into the DNA of human cytomegalovirus strain AD169-infected cells was stimulated relative to that in nondividing, uninfected cells.