Motoichi Hatano

Establishment and biological characterization of an in vitro human cytomegalovirus latency model

In an attempt to develop an in vitro human cytomegalovirus (HCMV) latency model system, the growth characteristics of HCMV in a human thyroid papillary carcinoma cell line (TPC-1) were examined. When TPC-1 cultures preheated at 40.5 degrees for 48 hr were infected with HCMV and incubated at a supraoptimal temperature (40.5 degrees), the cultures could be maintained for at least 65 days without detection of infectious virus. In contrast, when the infected cultures were incubated at 37 degrees, HCMV persistently infected cultures were established. HCMV was reactivated from the latently infected cultures by decreasing the incubation temperature from 40.5 to 37 degrees, and the cultures subsequently entered into virus persistent infection. Although HCMV-specific polypeptides which comigrate with the immediate early virus polypeptides and nuclear antigens were continuously detectable in the majority (more than 95%) of the cells during the latent period, a detectable level of virus-specified DNA polymerase (one of the early virus proteins) was not induced, suggesting that the blockage of HCMV replication in the latently infected cultures occurs at the early stages of the HCMV replication cycle. Infectious center assay revealed that 0.002 to 0.2% of the cells contain an HCMV genome that can be activated during the latent period. The latently infected cells were susceptible to superinfection with homologous and heterologous strains of HCMV. In persistently infected cultures approximately 38% of the cells were lysed by reaction with HCMV immune serum and complement, whereas complement-mediated immune cytolysis could not be detected in the latently infected cultures. The data presented suggest that a temperature-sensitive cellular function(s) that controls the expression of the HCMV early functions plays an important role in maintenance of the HCMV genome in the latent state and reactivation of HCMV by decreasing the incubation temperature.

Enhanced replication of human cytomegalovirus in human fibroblasts treated with dexamethasone

The effect of glucocorticoid hormones on the replication of human cytomegalovirus (HCMV) was studied in human embryonic lung (HEL) cells. Treatment of cells with pharmacological concentrations of adrenal glucocorticoids such as dexamethasone enhanced HCMV replication; treatment with oestrogenic or androgenic hormones did not do so. In dexamethasone-treated HEL cells there was an approximately tenfold increase in virus yield, with the virus eclipse period shortened by 1 day compared to control cultures. Treatment of cells with the hormone also enhanced plaquing efficiency of the virus by approximately tenfold. As the synthesis of virus-specific immediate early proteins and antigens was notably enhanced together with an increase of HCMV DNA synthesis, it appeared that the early stages of the HCMV replication cycle might be under hormonal control. Moreover, the data presented suggest that the hormonal enhancement of HCMV replication involves specific receptor proteins and requires the synthesis of a specific cellular mRNA(s).

Human Cytomegalovirus Persistent Infection in a Human Central Nervous System Cell Line: Production of a Variant Virus with Different Growth Characteristics

The susceptibility of human central nervous system cell lines to human cytomegalovirus (HCMV) and the fate of infected cultures were studied. Significant amounts of infectious progeny virus were produced in 118MGC glioma and IMR-32 neuroblastoma, but not in KGC oligodendroglioma cells when the cultures were infected with wild-type virus (HCMVwt) at an m.o.i. of 10 p.f.u. per cell. Further passage of infected 118MGC cells resulted in the establishment of a long-term persistent infection. This infection, designated 118MGC/Towne, continuously produced infectious virus (HCMVpi) with titres ranging from 10(2) to 10(5) p.f.u./10(6) cells up to 360 days post-infection (corresponding to 50 subcultures). Since no temperature-sensitive mutants, defective interfering particles or interferon-like activity were found in the 118MGC/Towne cultures, maintenance of the persistent infection seemed to be due to a balance between the release of infectious virus and the growth of uninfected cells. The HCMVpi produced in long-term persistently infected cultures was shown to be different from the HCMVwt originally used to infect by the following characteristics: HCMVpi replicated slowly and yielded lower amounts of progeny virus than HCMVwt; HCMVpi induced a 73,000 mol. wt. immediate early protein that was not synthesized in HCMVwt-infected cells; HCMVpi had a different DNA structure from that of HCMVwt. These results suggest that HCMVpi is a slower growing variant of HCMVwt and probably plays an important role in the maintenance of the persistent infection.

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.

Epstein-Barr Virus Nuclear Antigen-Positive Nasopharyngeal Hybrid Cells

An epithelial-like hybrid cell line was established by cell fusion of 8-azahypoxanthine-resistant epithelial cells (Ad-AH) with lymphoblastoid cells (A2L), derived from the human nasopharynx. The nasopharyngeal hybrid cells, designated as A2L/AH, were Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA)-positive by the anticomplement immunofluorescence method. Furthermore, the treatment of the hybrid cells with 5-Iodo-2′-deoxyuridine (IUDR) induced early antigen (EA) and viral capsid antigens (VCA), while the treatment of nonproducer lymphoblastoid cells, A2L, induced EA but not VCA. The appearance of IUDR-induced VCA in the hybrid cells suggests that some factor produced by the Ad-AH cells might neutralize a repressed state of VCA and thus activate these antigens with the treatment of IUDR. These established nasopharyngeal hybrid cells might be useful for studies of in vitro nasopharyngeal carcinoma (NPC) since no EBV-carrying NPC cell lines have been established.

Effect of dimethyl sulfoxide on interaction of human cytomegalovirus with host cell: Conversion of a nonproductive state of cell to a productive state for virus replication

The effect of dimethyl sulfoxide (DMSO) on the interaction of human cytomegalovirus (HCMV) with host cell was studied. Confluent state of a human rhabdomyosarcoma cell line (A204) showed a much lower susceptibility to HCMV infection when compared to that in subconfluent actively growing cell cultures. Treatment of confluent cultures with DMSO, however, converted many nonproductive cells in these cultures to a productive state for virus replication. Infectious center assay revealed that approximately 100-fold more cells in the compound-treated cultures are able to produce infectious virus. The amount of infectious virus produced in DMSO-treated confluent cultures was enhanced by approximately 10,000-fold over production in untreated cultures and recovered to the level of that produced in subconfluent cultures productive state for virus replication. This cell physiology-dependent inhibition of HCMV replication and enhancement of virus growth by DMSO did not occur with herpes simplex virus type 2. Immunofluorescence staining, gel electrophoresis, and DNA analyses indicate that block of HCMV replication in confluent cultures probably occurs at the level of early transcription or translation of the viral genome. In contrast, in DMSO-treated confluent cultures appreciable amounts of HCMV DNA polymerase (an early virus function), viral DNA, and late antigens were synthesized. Pretreatment of confluent cultures with DMSO enabled the cells to support HCMV replication. In addition, the most effective enhancement by DMSO was found in cultures that had been treated with the compound up to 5 hr after infection. These results suggest that the enhancing effect by DMSO is primarily expressed through some host cellular function(s) and the early stages in virus growth cycle are most likely under control by DMSO action.

Inhibitors of prostaglandin synthesis inhibit growth of human cytomegalovirus and reactivation of latent virus in a productively and latently infected human cell line

Indomethacin and tetracaine, inhibitors of prostaglandin synthesis, inhibited production of infectious human cytomegalovirus (HCMV) in a human thyroid papillary carcinoma cell line (TPC-1) by 99.9% when added to cultures at the concentration of 2 x 10(-4) M during the first 24 hr after infection. Although immediate early virus proteins were synthesized at similar molar ratios in mock- and compound-treated cultures, induction of HCMV-specific DNA polymerase (one of the early virus proteins) was inhibited by treatment with these compounds, suggesting that the early stages of the virus growth cycle are most likely to be under the control of indomethacin or tetracaine action. We have previously developed an in vitro HCMV latency model system in TPC-1 cultures. This system was used to study the effect of these compounds on reactivation of the latent virus. When TPC-1 cultures preheated for 48 hr at 40.5 degrees were infected with HCMV and incubated at 40.5 degrees, the cultures could be maintained for 30 days without detection of infectious virus. The latent HCMV was reactivated within 10 days by reducing the incubation temperature from 40.5 to 37 degrees. However, when the latently infected cultures were treated with indomethacin or tetracaine immediately after being shifted to 37 degrees, reactivation of the latent virus was not observed.

Rabbit kidney cells abortively infected with human cytomegalovirus are arrested in mitotic phase

SummaryIn rabbit kidney epithelial cells (RK13) abortively infected with human cytomegalovirus (HCMV), DNA synthesis at 1 or 2 days post-infection was enhanced 4 to 5 fold, compared to mock-infected cells. DNA analysis by isopycnic centrifugation revealed that the DNA newly synthesized in the virus infected RK13 cells was of cellular origin. HCMV infection also caused a marked increase in the mitotic activity of RK13 cells. When semi-confluent RK13 cells were infected more than 20 per cent of cells demonstrated mitosis at 72 hours post-infection although the rate of cell growth was considerably reduced compared to that of uninfected cells. The most frequent chromosomal change observed was fragmentation although other aberrations, gap, break, deletion etc. occurred also.Two immediate-early viral polypeptides with apparent molecular weights 72,000 (72K) and 76,000 (76K) daltons were produced in both RK13 cells and human embryonic lung cells (HEL) by 3 hours post-infection. Synthesis of the 76K polypeptide was greater than that of the 72K polypeptide in non-permissive RK13 cells whereas the reverse occurred in permissive HEL cells. Furthermore, of three early polypeptides which were expressed in productively infected HEL cells two, 88K and 80K, were not detected in abortively infected RK13 cells.These results suggest that the arrest in mitosis of the abortively infected RK13 cells and the subsequent chromosomal changes are associated with the altered expression of immediate-early or early virus functions in these cells.Following HCMV infection permissive HEL cells undergo a characteristic sequence of morphological changes (3) of which cell rounding and “contraction” are observed in the early stages and could be related to either the action of cellular contractile proteins or changes in the plasma membrane permeability and a concomitant Ca++ influx (2). ‘Relaxation’ and cell enlargement are other changes considered to be initiated by early virus functions although they occur after the commencement of virus DNA synthesis. Thus, the expression of immediate-early and early virus functions has an important role in controlling the sequential morphological changes in infected cells and may also be important in determining whether infection with HCMV will be productive or abortive.

Evidence for Early Membrane Antigens in Cytomegalovirus-infected Cells

Human cytomegalovirus (HCMV) induces membrane antigens (MA) which are detectable by immunofluorescent techniques as early as 24 h after infection. These antigens appear on the surface of infected cells in non-permissive human epithelioid and animal cells in addition to permissive human cells. The synthesis of the MA occurs in the presence of a DNA synthesis inhibitor, whereas RNA and protein synthesis inhibitors prevent its formation completely. These results demonstrate that the MA is a newly synthesized protein coded by an early function of the viral genome.

Nasopharyngeal Carcinoma in Japan —Clinical Evaluation of EBV Serology and EBV-Associated Nuclear Antigens Test—

This study was aimed at the possible clinical application of two Epstein-Barr virus (EBV) immunological tests to Japanese nasopharyngeal carcinoma (NPC). One of them was serological test to measure antibody titers to EBV-associated antigens (viral capsid antigens (VCA) and virus-induced early antigens (EA)) of 42 NPC sera. The other was for the detection of EBV-associated nuclear antigens (EBNA) in 22 tumour cells of NPC. The results showed that significant elevation of antibody titers to VCA and EA which resided particularly in IgA class as well as a high incidence in the presence of EBNA were found in undifferentiated NPC. Apart from its importance in etiology, EBV serology and the EBNA test may help clinicians to establish a diagnosis of undifferentiated NPC in Japan and there is little doubt that these tests should eventually become a routine biologic test in ENT practice.