Hisashi Ogura

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.

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.

Restricted Synthesis of the Fusion Protein of Measles Virus at Elevated Temperatures

The elevation of culture temperatures from 35 degrees C to 39 degrees C led to the cessation of the synthesis of the fusion (F) protein of measles virus. This effect was caused by inhibition of the translation of the corresponding mRNA rather than by a decrease in the synthesis or stability of the mRNA or by increased degradation of the F protein at elevated temperatures. The haemagglutinin (H) and F mRNAs were distributed differently in gradients on which polysomes were fractionated. The H mRNA was present almost exclusively in the largest polysomes whereas the F mRNA was more evenly distributed over large and small polysomes. The distribution was not affected by a temperature shift. The inhibition of F protein synthesis thus appeared to be related to a cessation of elongation of the nascent polypeptide chain rather than to a defect in initiation of the translation of the F mRNA at 39 degrees C.

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.

Synthesis of M Protein of HVJ (Sendai Virus) in Rat Glial Cells is Selectively Restricted at a Non-permissive Temperature

Production of HVJ (Sendai virus) wild-type in rat glial cells was characteristically restricted at high temperature. The synthesis of the M protein was selectively decreased in infected cells at 39 degrees C. This temperature-sensitive (ts) character of HVJ was not observed in infection of LLCMK2 cells or chick embryo fibroblast cells. Newcastle disease virus, mumps virus and vesicular stomatitis virus did not show ts growth in glial cells.

Effect of Various Sodium Taurocholate Preparations on the Recovery of Clostridium difficile Spores

The effect of four sodium taurocholate preparations, which are easily available in Japan, on recovery of Clostridium difficile spores was examined. All preparations, except for one, enabled the recovery of nearly all spores counted microscopically. Moreover, by using 69 toxigenic and 34 nontoxigenic C. difficile strains, the relationship between the recovery of spores in the medium with sodium taurocholate and toxigenicity of C. difficile was analyzed. It was noted that the number of strains with recovery rate of more than 70% was greater in toxigenic strains than in nontoxigenic strains, suggesting a more abundant recovery of toxigenic C. difficile strains in the presence of sodium taurocholate.

Difference in Susceptibility of Various Cell Cultures to Cytotoxic Culture Filtrates of Clostridium sordellii

Recently we demonstrated that the lethal toxin-producing strains of Clostridium sordellii were cytotoxigenic for baby hamster kidney cell cultures (4), which had never been used to test the cytotoxigenicity of C. sordellii (1-3, 6), and that some of the cytotoxigenic C. sordellii strains produced a positive reaction in the ligated rabbit ileal loop (7). The purpose of this study was to find cultured cell lines, other than baby ham sterkidney cells, susceptible to the cytotoxic culture filtrates of C. sordellii and those specifically susceptible to the culture filtrates of ligated ileal loop response-positive strains of C. sordellii. Two cytotoxigenic and ileal loop response-positive strains and 13 cytotoxigenic but ileal loop response-negative strains (4, 7) were used. Of these, one cytotoxigenic and ileal loop response-positive strain, 3703, and one cytotoxigenic but ileal loop response-negative strain, KZ 1047, were mainly used. Cytotoxic culture filtrates were prepared from cultures of C. sordellii strains incubated in m-BHI medium according to the method previously described (4, 7). The cell cultures tested were eight established cell lines and one primary human embryonic lung cell culture, HEL, which was prepared from a 6-month embryonic lung in our laboratory. Baby hamster kidney (BHK-21/WI-2) and human amnion (FL) cells were cultured in Eagles minimal essential medium (Eagles MEM) (Nissui, Tokyo) containing 5% calf serum; human embryonic intestine (Intestine 407), human embryonic lung

Antigenic variation of HVJ (Sendai virus) HN glycoprotein detectable by monoclonal antibodies during persistent infection.

Three newly established monoclonal hybridoma antibodies to the haemagglutinin molecule of HVJ, designated A7, B3 and F11, recognize operationally non-overlapping antigenic determinants and have neutralizing activity. Using these antibodies, the frequencies of occurrence of neutralization-resistant antigenic variants were analysed in virus populations released from four cell lines persistently infected with HVJ, namely GM2-HVJ, LLCMK2-HVJ, Vero-HVJ and GEsl-HVJ at various passage stages. Antigenic variants were selected from culture fluids of these HVJ carrier cells at a total frequency of 10(-3.3), 10(-3.8) and 10(-3.6) by monoclonal antibodies A7, B3 and F11, respectively. These values were considerably higher than those of 10(-4.7) to 10(-5.2) detected in a stock preparation of wild-type virus with these antibodies. All the variant viruses isolated as above were negative in neutralization, haemagglutination inhibition and immunofluorescent staining tests with each monoclonal antibody used for their isolation, but were positive with the other antibodies.

Relation of HVJ (Sendai virus) production to cell growth phase in persistently infected mouse 3T3 cells

SummaryUsing 3T3 mouse fibroblasts persistently infected with temperature-sensitive HVJ (3T3-HVJpi), the relationship between production of HVJ and cell growth was investigated. In these cells the highest virus release into the culture media occurred from 24 to 48 hours after seeding. The release gradually decreased as the cultures approached confluency, at which time it was reduced below 1–2 per cent of that on day 2 in parallel with the decrease of both cellular DNA and RNA synthesis rates. Comparative examination in growing and resting HVJ carrier 3T3 cells showed that amounts of nucleocapsids and rate of viral structural protein synthesis in the latter phase was reduced to 4–5 per cent of those in the former phase. In addition, viral replication and transcription rates in the resting phase were found to be suppressed to 8–9 per cent of levels detected in the growing phase. These results suggest that the reduced virus production in the resting HVJ carrier cells may be mainly due to the suppression of viral RNA synthesis.