Inhibitory effect of hexamethylene bisacetamide on replication of human cytomegalovirus

Summary. The effect of hexamethylane bisacetamide (HMBA), a hybrid polar compound, on gene expression and replication of human cytomegalovirus (HCMV) was studied. When HCMV-infected human thyroid papillary carcinoma (TPC-1) and human embryonic lung (HEL) fibroblast cells were maintained with medium containing 2.5 and 5 mM HMBA for 10 days, there was a greater than 2- to 3-log reduction in virus yield compared to that in untreated cells. Infection of TPC-1 cells with HCMV resulted in an establishment of persistent infection and the cells continuously produced virus with titer of over 10⁵ PFU/ml, whereas HMBA prevented the infected cells from entering into the persistent infection. Moreover, treatment of the persistently infected cultures with HMBA reduced production of infectious HCMV more efficiently than did ganciclovir, and eventually ceased HCMV production. Western blotting analysis revealed that HMBA blocks accumulation of the immediate early 2 (IE2) protein in TPC-1 cells and delays synthesis of this protein in HEL cells, but has little effect on the level of the IE1 protein during the early times after infection. Synthesis of the viral early and late proteins in both cells was also substantially blocked by HMBA. The results indicate that the inhibition or the delay of the critical IE2 protein synthesis in the presence of HMBA would actually be a process that fails to proceed beyond the IE stages in HCMV replication cycle.     

Stimulating effect of heat shock on the early stage of human cytomegalovirus replication cycle

The effect of mild heat shock on the replication of human cytomegalovirus (HCMV) was studied in human embryo fibroblasts. Treatment of cell cultures at 44 degrees C for 10 min just before infection or at 24 h post infection (p.i.) shortened HCMV eclipse period and enhanced viral replication, while heat shock performed at 48 h p.i. had no effect on the HCMV replication cycle. Study of HCMV-induced early and late antigens confirmed that the cellular response to heat shock influences HCMV replication in the early stage of the viral replication cycle.     

Effect of natural human interferon-beta on the replication of human cytomegalovirus

The antiviral effect of natural human interferon-beta (HuIFN-beta) against human cytomegalovirus (CMV) was evaluated in human embryonic lung fibroblasts (HEL). Natural HuIFN-beta, like other HuIFNs, inhibited the replication of CMV. Pretreatment of the cells with natural HuIFN-beta inhibited the appearance of immediate-early antigen (IEA) or pre-early nuclear antigen (PENA) as well as the production of infectious CMV. After a single treatment with natural HuIFN-beta, intracellular 2', 5'-oligoadenylate (2-5A) synthetase activity was induced and maintained at a high level for several days. The anti-CMV effect of natural HuIFN-beta correlated with the intracellular 2-5A synthetase activity.     

Lytic cytomegalovirus replication and the hormones of human pregnancy

The frequency of human cytomegalovirus (CMV) excretion during pregnancy denoting active infection has been demonstrated to increase as gestation advances and at term involves a significant percentage of women. This increase enhances the risk of congenital infection of the fetus. Thus it appears that some factor(s) unique to the condition of pregnancy favors susceptibility to maternal CMV infection. We designed our studies to investigate the possible association of the continuously rising levels of selected hormones and this increased susceptibility. Progesterone, 17 beta-estradiol, and cortisol were added to tissue culture media in final concentrations to match those occurring in term pregnancy serum. Two strains of human foreskin cells, one neonatal and the other fetal, were treated with either single or paired combinations of hormone-containing media. Lytic CMV replication in neonatal foreskin cells was enhanced by a maximum of 5.7-fold when these cells were treated with cortisol. Such enhancement did not occur in the fetal cells. No synergistic effects were seen when cortisol was used in combination with other hormones nor when neonatal foreskin cells were replicated for at least three generations in either single or paired combinations of hormones prior to use. Differential hormonal enhancement of CMV replication in vitro suggests a possible mechanism for the increased incidence of CMV infection observed during human pregnancy.     

Strain-dependent differences in the human cytomegalovirus replication origin

Summary The nucleotide sequence of the human cytomegalovirus replication origin of strain Towne (anAatII-SacI fragment corresponding to nt 90372–94637 of strain AD169) was determined and compared with AD169. Two differences were found in the nucleotide sequence level. One was the alteration of structural organization (a major difference): a 189-bp region of AD169 (nt 93337–93525) was directly repeated three times in Towne. The other was a change in the nucleotide residue level including substitution, insertion, or deletion (a minor difference). The divergent residues were predominantly localized within the nt 92591–92855 region of AD169. A replication assay revealed that replication ability remained after deletion of the 189-bp repeat but disappeared after either a 1.5-kb deletion from theAatII end or a 0.9-kb deletion from theSacI end. The 1.5- and 0.9-kb regions were relatively conserved. These results indicate that at least two regions essential for replication ability lie outside of both the relatively variable region and the 189-bp repeat and suggest that these essential regions support replication even with a spatial separation of either one (AD169) or three repeats (Towne) of the 189-bp region.     

Effect of DNA polymerase inhibitors on the replication of human cytomegalovirus

Summary Aphidicolin, a specific inhibitor of cellular DNA polymerase and of viral DNA polymerase, inhibits production of infectious virus and cellular and viral DNA synthesis of human cytomegalovirus (HCMV)-infected cells. On the other hand, 2, 3-dideoxynucleosides, inhibitors of DNA polymerases and , do not affect HCMV replication. The data show that the DNA polymerases of either viral or cellular origin are required for viral DNA synthesis, and cannot be substituted by the cellular DNA polymerase and .With 2 Figures     

The effect of cyclic AMP on expression of the major immediate-early genes and replication of human cytomegalovirus in human central nervous system cell lines

Summary.  The effect of dibutyryl cyclic AMP (dbcAMP) on interaction of human cytomegalovirus (HCMV) with human central nervous system cell lines was examined. Activation of the major immediate-early (IE) promoter (MIEP) of HCMV by dbcAMP was observed in human neuroblastoma IMR-32 cells, but not in glioma 118MGC and astrocytoma U373-MG cells. The 19 bp repeat element in the enhancer of the MIEP was most likely to be the cis-element that responded to dbcAMP in IMR-32 cells as we expected. In IMR-32 cells activation of the MIEP led to enhanced synthesis of the major IE proteins and infectious HCMV. Received May 7, 1998 Accepted December 2, 1998     

The influence of polymyxin B on virus-induced cytopathogenic effect

Polymyxin B was added to cell cultures for isolation of humanpathogenic viruses. The CPE in cultures infected with entero-, adeno- and vacciniavirus at low multiplicity of infection appeared earlier in the presence of 50 Μg/ml polymyxin B. The final virus titre was as a rule unaffected. With two enterovirus strains the addition of polymyxin B was required to establish the CPE. This antibiotic may therefore prove helpful for isolation and identification of viruses from fecal specimens. With a heavy inoculation of measles virus a direct CPE of non-fused cells was seen. Otherwise the drug-treated cultures did not differ significantly from the control cultures. The CPE induced by two enveloped viruses (mumps and herpes simplex), was adversely influenced, appearing later and being less marked. The titre of herpes simplex virus was in addition reduced by about 2 log 10 steps. Polymyxin B can consequently not be used for primary isolation of virus from sources where these viruses may be found. On a secondary passage, however, the antibiotic may prove helpful in establishing the CPE of a weakly cytopathogenic virus. Polymyxin B showed no deleterious effect on uninfected cells, and bacterial contaminations were not so often observed in cultures with this antibiotic.     

Inhibition by monensin of human cytomegalovirus DNA replication

Summary Monensin, at concentrations which depended on the multiplicity of infection, was found to prevent DNA replication of human cytomegalovirus (HCMV) as well as production of viral progeny in human foreskin fibroblasts. The drug did not affect DNA replication of herpes simplex virus. Inhibition of consecutive HCMV DNA synthesis was also observed following delayed addition of the drug within 12–24 hours postinfection, but was fully reversible upon its removal. Viral replication proceeded, however, without impairment in cultures treated with monensin prior to infection. Induction of viral DNA polymerase activity was not impeded by the inhibitor. Analysis of protein- and glycoprotein synthesis revealed that monensin interfered with the production of a number of HCMV-specific polypeptides. Furthermore, evidence was obtained that the drug may hinder intracellular transport of a 135 kd glycopolypeptide.With 6 Figures     

Dexamethasone enhances human cytomegalovirus replication in human epithelial cell cultures

An epithelial human hepatoma cell line (PLC/PRF/5) and a primary epithelial human baby kidney (HBK) cell culture showed restricted growth of human cytomegalovirus (HCMV). Treatment of these two epithelial cell cultures with dexamethasone greatly enhanced their ability to support HCMV replication. Growth kinetic experiments and infectious center assay revealed that in both the hormone-treated cultures infectious progeny virus appeared earlier by 1 or 2 days and 5- or 10-fold more cells are able to produce infectious virus. There was an approximate 50- or 100-fold increase in virus yield compared to that in the untreated control cultures. Enhanced HCMV replication in the hormone-treated cultures was not due to differences in the cell growth or the virus adsorption and was supported by evidence of increased synthesis of HCMV-specific immediate early antigens and DNA.     

Complete replication of human cytomegalovirus in explants of first trimester human placenta

Tissue integrity and viability of first trimester placenta explants were obtained in culture for 3 weeks. Explants were infected with human cytomegalovirus (HCMV), several cycles of HCMV replication were obtained and the progression of the infection was observed within a tissue that maintains its normal cellular organization. In agreement with recent clinical data, 3 weeks were necessary for the virus to colonize the placenta fully. Complete HCMV replication was observed in trophoblasts, followed by subsequent transmission of the infection to the stromal fibroblasts and fetal endothelial capillary cells. Viral DNA replication was monitored and the production of infectious viral progeny documented.     

Action of human leukocyte and immune interferons on the reproduction and cytopathogenic effect of the encephalomyocarditis virus in mice

A comparative study of the antiviral effect of human immune and leukocyte interferons in homologous diploid cells showed that the resistant condition of the cells produced by interferon depended on the type and concentration of the inhibitor, its presence in the culture medium and time of action on the cells. Reproduction of EMC virus and its cytopathic effect were inhibited to a much greater extent when the cells were treated with leukocyte interferon. The resistance of the cells in both cases was evident as early as one hour after their contact with interferon. The resistance of the cells induced by leukocyte interferon was of longer duration than after induction with immune interferon (greater than 72 h and approximately 48 h, respectively).     

Effect of human cytomegalovirus on replication of SV40 origin and the expression of T antigen

Human cytomegalovirus (HCMV) induces replication of the cloned SV40 origin of DNA replication in both productive and nonproductive infections. HCMV-induced replication of the SV40 DNA origin required the presence of T antigen. Human embryonic lung (HEL) cells were found to be fully permissive for SV40 origin replication only in the presence of HCMV gene expression. In addition, expression of plasmid encoded SV40 T antigen in HEL cells was only induced in the presence of HCMV. Cotransfection of the SV40-cloned origin of replication and seven overlapping cosmids containing the entire HCMV genome is capable of stimulating replication of the SV40 origin. We conclude that induction of the SV40 origin of replication by HCMV is (i) not due to any component of the HCMV virion, (ii) has a requirement for T antigen expression, (iii) occurs in an SV40 nonproductive cell, when T antigen expression is induced by HCMV gene expression.     

Productive and abortive replication of human cytomegalovirus at different environmental pH values

Summary The influence of environmental pH on cytomegalovirus replication cycle was studied. A productive cycle was observed at pH values of 6.5 or higher while at pH values of 5.8, 6.0, 6.2 only virus-induced early events were expressed.With 2 Figures     

Sodium butyrate-inducible replication of human cytomegalovirus in a human epithelial cell line.

Replication of human cytomegalovirus (HCMV) in a human epithelial thyroid papillary carcinoma cell line (TPC-1) was restricted. However, pretreatment of these cells with 5 mM sodium butyrate (NaB) for 24 hr before infection enhanced both HCMV yield and infectious center titer to a similar level of that seen in human embryonic lung fibroblast cells. Immunofluorescence staining, gel electrophoresis, and Northern blot analysis revealed that TPC-1 cells are nonpermissive for expression of HCMV major immediate early (IE1) functions, but many of the cells become permissive after being treated with NaB. The presence of cycloheximide during NaB pretreatment of the cells efficiently diminished the stimulatory effect of NaB on expression of the IE1 gene. Therefore, it appeared that NaB induces the synthesis of a cellular protein(s) which apparently plays an important role in the conversion of nonpermissive cells to a permissive state for expression of this critical viral gene. Transient chloramphenicol acetyltransferase (CAT) assay experiments indicated that in TPC-1 cells the HCMV-CAT construct which contains the complete IE1 promoter regulatory region was expressed poorly, whereas a high level of CAT activity was detectable in the NaB-treated cells. Therefore, these results suggest that the enhancing effect of NaB on HCMV replication is expressed through some host cellular factor(s), and the HCMV IE1 promoter regulatory region is most likely to be the primary target of NaB action.     

Immunosuppressive dose of azathioprine inhibits replication of human cytomegalovirus in vitro

Summary Azathioprine (Aza) was found to have anti-human cytomegalovirus (HCMV) activity in vitro at concentrations used for immunosuppression therapy. The dose of Aza for 50% plaque reduction was 0.592µg/ml for HCMV in human embryonic lung (HEL) cells, but those of Aza for 50% plaque reduction for herpes simplex virus (HSV) and varicella-zoster virus were more than 20µg/ml. The dose of Aza for 50% reduction of the HCMV yield in infected cells was 0.25µg/ml, while that for 50% reduction of the HSV yield in infected cells was more than 50µg/ml. The dose of Aza for 50% growth inhibition of HEL cells was 30µg/ml, and 50.7 and 120 times greater than the doses for 50% reduction of the plaque formation and the yield of HCMV, respectively. Thus Aza was found to have a strong anti-HCMV activity at concentrations used for immunosuppression. When HCMV infected cells were treated with cyclosporine (CsA: 0.2µg/ml) and prednisolone (Pred: 0.3µg/ml) simultaneously with Aza, the doses of Aza for 50% reduction of plaque formation and the yield of HCMV were 0.73 and 0.32µg/ml, respectively. Thus an inhibitory effect of Aza was also observed in HCMV-infected cells treated with CsA and Pred at their concentrations used for immunosuppression. Maintenance of an anti-HCMV dose of Aza in combination with CsA and Pred might establish not only satisfactory immunosuppression but also suppression of HCMV infection in transplant recipients.