Studies on the Persistent Infection with Measles Virus in HeLa Cells

HeLa/MV cells which have previously been found to contain measles antigen in more than 90% of cells were very similar to normal HeLa cells in their morphology and growth. Although almost all of HeLa/MV cells may be infected, only 10% of them released infectious virus particles. The hemadsorption test, however, showed that most of the infected cells produced by hemagglutinin. Two kinds of clones were obtained by cloning HeLa/MV cells in the presence of anti-measles serum. One was the virus-releasing clone and the other the uninfected clone which did not contain any measles antigen. The proportions of virus-releasing clones to all clones varied between 20 to 54%, and did not show any increase even after recloning of the virus-releasing clones. The susceptibility of the uninfected clones to the standard measles virus was not different from that of normal HeLa cells.     

Persistent Infection of Cells in Culture by Measles Virus III. Comparison of Virus-Specific RNA Synthesized in Primary and Persistent Infection in HeLa Cells

The pattern of actinomycin D-resistant RNA synthesis was examined during primary infection of HeLa cells by virulent Edmonston measles virus and in two HeLa clones persistently infected by the same strain of virus. One of these clones, K11, produces infectious virus of low virulence for HeLa cells, and the other, K11A-HG-1, has thus far failed to yield infectious virus. The patterns of virus-specific RNA synthesized in these three types of infection are qualitatively similar to each other and to the patterns of virus-specific RNA synthesis in other paramyxovirus infections. There were, however, quantitative differences. In addition, virions of the virulent Edmonston strain of measles virus were found to contain high-molecular-weight RNA with a sedimentation constant identical to that of Newcastle disease virus.     

Activation of a Latent Measles Virus Infection in Hamster Cells

The characteristics of infectious measles virus released from latently infected hamster embryo fibroblast cells are described. Low levels of virus were released spontaneously when the cultures were incubated at 37 C; this phenomenon was observed 19 passages after the cells had been exposed to the virus and has continued through cell passage 45. The virus yield could be significantly increased by cocultivation of the hamster cells with BSC-1 cells or incubation of the latently infected cells at 33.5 C rather than at 37 C. Measles virus released after cocultivation demonstrated increased cytopathology in cell culture and reduced temperature sensitivity when compared to the virus released at 33.5 C. After cell passage 45, there was an increase in spontaneous release of virus. However, the viruses recovered by cocultivation or temperature release after cell passage 45 were nearly identical. These observations suggest a possible mechanism for measles virus activation in cells latently infected with this virus.     

Factors Influencing the Establishment of Persistent Infection of HVJ (Sendai Virus) in L Cells

Infection of a subline of L cells adapted to grow in suspension (Ls) with Fushimi strain of HVJ (HVJ-F) resulted in a virus carrier state. Ls cells, when cultured in monolayer, showed morphological changes following infection of HVJ-F and were detached from the glass wall. However, when the detached cells were transferred to a new environment of suspension culture within 5 days after infection, the carrier state was again established. HVJ-F caused only lethal infection in L cells maintained exclusively in monolayer (Lm). On the other hand, both Ls and Lm, irrespective of their culture conditions, were lethally infected by Nagoya 1–60 strain of HVJ. The overall results showed that culture condition as well as the kind of host cells or virus strains is an important factor regulating the establishment and maintenance of the virus carrier state.     

Measles Virus Infection Induces Terminal Differentiation of Human Thymic Epithelial Cells

Measles virus infection induces a profound immunosuppression that may lead to serious secondary infections and mortality. In this report, we show that the human cortical thymic epithelial cell line is highly susceptible to measles virus infection in vitro, resulting in infectious viral particle production and syncytium formation. Measles virus inhibits thymic epithelial cell growth and induces an arrest in the G₀/G₁ phases of the cell cycle. Moreover, we show that measles virus induces a progressive thymic epithelial cell differentiation process: attached measles virus-infected epithelial cells correspond to an intermediate state of differentiation while floating cells, recovered from cell culture supernatants, are fully differentiated. Measles virus-induced thymic epithelial cell differentiation is characterized by morphological and phenotypic changes. Measles virus-infected attached cells present fusiform and stellate shapes followed by a loss of cell-cell contacts and a shift from low- to high-molecular-weight keratin expression. Measles virus infection induces thymic epithelial cell apoptosis in terminally differentiated cells, revealed by the condensation and degradation of DNA in measles virus-infected floating thymic epithelial cells. Because thymic epithelial cells are required for the generation of immunocompetent T lymphocytes, our results suggest that measles virus-induced terminal differentiation of thymic epithelial cells may contribute to immunosuppression, particularly in children, in whom the thymic microenvironment is of critical importance for the development and maturation of a functional immune system.     

Persistent Infection of Human Fetal Endothelial Cells with Rubella Virus

Cardiovascular abnormalities are the leading cause of neonatal death among patients with congenital rubella syndrome (CRS). Although persistence of rubella virus (RV) in fetal endothelium has been repeatedly suggested as a possible cause of cardiovascular birth defects, evidence of the permissiveness of fetal endothelial cells to RV is lacking. In this study we evaluated the ability of RV to infect and persist in primary fetal endothelial cells derived from human umbilical vein (HUVEC). We found that wild type (wt) low passage clinical RV productively infected HUVEC cultures without producing cytopathology or ultrastructural changes. RV did not inhibit host cell protein synthesis, cell proliferation, or interfere with the cell cycle. Persistently infected cultures were easily established at low and high multiplicities of infection (MOI) with both laboratory and wt clinical RV strains. However, synchronous infections of entire HUVEC monolayers were only observed with clinical RV strains. The release of infectious virions into media remained at consistently high levels for several subcultures of infected HUVEC. The results indicate that macrovascular fetal endothelial cells are highly permissive to RV and allow slow persistent RV replication. The findings provide more evidence for the suggestion that vascular pathologies in CRS are triggered by persistent rubella virus infection of the endothelium.     

Bunyamwera Virus Replication in Cultured Aedes albopictus (Mosquito) Cells: Establishment of a Persistent Viral Infection

Bunyamwera virus replication was examined in Aedes albopictus (mosquito) cell cultures in which a persistent infection is established and in cytopathically infected BHK cells. During primary infection of A. albopictus cells, Bunyamwera virus reached relatively high titers (10⁷ PFU/ml), and autointerference was not observed. Three virus-specific RNAs (L, M, and S) and two virion proteins (N and G1) were detected in infected cells. Maximum rates of viral RNA synthesis and viral protein synthesis were extremely low, corresponding to <2% of the synthetic capacities of uninfected control cells. Viral protein synthesis was maximal at 12 h postinfection and was shut down to barely detectable levels at 24 h postinfection. Virus-specific RNA and nucleocapsid syntheses showed similar patterns of change, but later in infection. The proportions of cells able to release a single PFU at 3, 6, and 54 days postinfection were 100, 50, and 1.5%, respectively. Titers fell to 10³ to 10⁵ PFU/ml in carrier cultures. Persistently infected cultures were resistant to superinfection with homologous virus but not with heterologous virus. No changes in host cell protein synthesis or other cytopathic effects were observed at any stage of infection. Small-plaque variants of Bunyamwera virus appeared at approximately 7 days postinfection and increased gradually until they were 75 to 95% of the total infectious virus at 66 days postinfection. Temperature-sensitive mutants appeared between 23 and 49 days postinfection. No antiviral activity similar to that reported in A. albopictus cell cultures persistently infected with Sindbis virus (R. Riedel and D. T. Brown, J. Virol. 29: 51-60, 1979) was detected in culture fluids by 3 months after infection. Bunyamwera virus replicated more rapidly in BHK cells than in mosquito cells but reached lower titers. Autointerference occurred at multiplicities of infection of 10. Virus-specific RNA and protein syntheses were at least 20% of the levels in uninfected control cells. Host cell protein synthesis was completely shut down, and nucleocapsid protein accumulated until it was 4% of the total cell protein. We discuss these results in relation to possible mechanisms involved in determining the outcome of arbovirus infection of vertebrate and mosquito cells.     

Persistent Infection of L Cells with Vesicular Stomatitis Virus: Evolution of Virus Populations

A previous report (Youngner et al., J. Virol. 19:90-101, 1976) documented that noncytocidal persistent infection can be established with wild-type vesicular stomatitis virus (VSV) in mouse L cells at 37°C and that a rapid selection of RNA⁻, group I temperature-sensitive (ts) mutants consistently occurs in this system. To assess the selective advantage of the RNA⁻ts phenotype, evolution of the virus population was studied in persistent infections initiated in L cells by use of VSV ts 0 23 and ts 0 45, RNA⁺ mutants belonging to complementation groups III and V. In L cells persistently infected with ts 0 23, the ts RNA⁺ virus population was replaced gradually by viruses which had a ts RNA⁻ phenotype. VSV ts 0 45 (V) has another marker in addition to reduced virus yield at 39.5°C: a defective protein (G) which renders virion infectivity heat labile at 50°C. Persistent infections initiated with this virus (ts, heat labile, RNA⁺) evolved into a virus population which was ts, heat resistant, and RNA⁻. These findings suggest that the ts phenotype itself is not sufficient to stabilize the VSV population in persistently infected L cells and also indicate that the ts RNA⁻ phenotype may have a unique selective advantage in this system. In addition to the selection of ts RNA⁻ mutants, other mechanisms which also might operate in the maintenance of persistent VSV infections of L cells were explored. Whereas defective-interfering particles did not seem to mediate the carrier state, evidence was obtained that interferon may play a role in the regulation of persistent infections of L cells with VSV.     

Effect of Persistent Fibroma Virus Infection on Susceptibility of Cells to Other Viruses

Shope fibroma virus establishes a persistent cytoplasmic infection in primary (RK) and serially cultivated (DRK(3)) rabbit kidney cells which is accompanied by a morphological alteration of the cells. The response of such cells to superinfection by other viruses was compared with that of control cells by determining plaque production and virus yield of superinfecting viruses. It was found that the growth of other poxviruses, myxoma and vaccinia, was greatly inhibited in the fibroma virus-infected cells, but that of pseudorabies and herpes simplex viruses, which are unrelated deoxyribonucleic acid viruses, was virtually unaffected. The ribonucleic acid (RNA) viruses, poliovirus 1 and coxsackievirus B1, did not produce plaques on either RK or fibroma virus-infected (F-RK) monolayers. However, the growth of several other RNA viruses, vesicular stomatitis virus, encephalomyocarditis virus, Sindbis virus, and Newcastle disease virus, was enhanced in F-RK cells. None of these latter RNA viruses produced any infectious progeny in DRK(3) cells, but they all plaqued on and produced good yields in DRK(3) cells persistently infected with fibroma virus. This phenomenon is termed facilitation. Facilitation results from the infection of DRK(3) cells by fibroma virus. Neither interference nor facilitation were due to changes in the adsorption or eclipse of the superinfecting virus.     

Saffold Virus Type 3 (SAFV-3) Persists in HeLa Cells

Saffold virus (SAFV) was identified as a human cardiovirus in 2007. Although several epidemiological studies have been reported, they have failed to provide a clear picture of the relationship between SAFV and human diseases. SAFV genotype 3 has been isolated from the cerebrospinal fluid specimen of patient with aseptic meningitis. This finding is of interest since Theiler’s murine encephalomyelitis virus (TMEV), which is the closely related virus, is known to cause a multiple sclerosis-like syndrome in mice. TMEV persistently infects in mouse macrophage cells in vivo and in vitro, and the viral persistence is essential in TMEV-induced demyelinating disease. The precise mechanism(s) of SAFV infection still remain unclear. In order to clarify the SAFV pathogenicity, in the present study, we studied the possibilities of the in vitro persistent infection of SAFV. The two distinct phenotypes of HeLa cells, HeLa-N and HeLa-R, were identified. In these cells, the type of SAFV-3 infection was clearly different. HeLa-N cells were lyticly infected with SAFV-3 and the host suitable for the efficient growth. On the other hand, HeLa-R cells were persistently infected with SAFV-3. In addition, the SAFV persistence in HeLa-R cells is independent of type I IFN response of host cells although the TMEV persistence in mouse macrophage cells depends on the response. Furthermore, it was suggested that SAFV persistence may be influenced by the expression of receptor(s) for SAFV infection on the host cells. The present findings on SAFV persistence will provide the important information to encourage the research of SAFV pathogenicity.     

Measles Virus Infection and Replication in Undifferentiated and Differentiated Human Neuronal Cells in Culture

Measles virus (MV) infection of the human central nervous system (CNS) typically involves widespread infection of neurons. However, little is known about how they become infected, how defective virus arises and accumulates, or how virus spreads among the cells of the CNS. In vitro studies of viral interactions with human neuronal cells may contribute to the resolution of such issues. In mixed cultures containing differentiated human neuronal (hNT2) cells and neuroepithelial cells, immunofluorescence studies show that the neurons, unlike both their NT2 progenitors and the neuroepithelial cells, are not initially susceptible to MV infection. This is possibly due to their lack of expression of CD46, a known cell surface receptor for MV. Later in the course of infection, however, both MV proteins and genomic RNA become detectable in their processes, where they contact infected, fully permissive neuroepithelial cells. Such a mechanism of virus transfer may be involved in the initiation and spread of persistent MV infection in diseases such as subacute sclerosing panencephalitis. Furthermore, mutated defective virus may readily accumulate and spread without the need, at any stage, for viral maturation and budding.     

Enhanced Gene Silencing in Cells Cured of Persistent Virus Infection by RNA Interference

We compared HEp-2-derived cells cured of persistent poliovirus infection by RNA interference (RNAi) with parental cells, to investigate possible changes in the efficiency of RNAi. Lower levels of poliovirus replication were observed in cured cells, possibly facilitating virus silencing by antiviral small interfering RNAs (siRNAs). However, green fluorescent protein (GFP) produced from a measles virus vector and also GFP and luciferase produced from plasmids that do not replicate in human cells were more effectively silenced by specific siRNAs in cured than in control cells. Thus, cells displaying enhanced silencing were selected during curing by RNAi. Our results strongly suggest that the RNAi machinery of cured cells is more efficient than that of parental cells.Small interfering RNAs (siRNAs) mediate RNA interference (RNAi), a natural biological phenomenon regulating a wide range of cellular pathways (8, 20). RNAi-based therapies with siRNAs or small hairpin RNAs (shRNAs) have been developed against several viral infections, and a reduction of the viral yield by several orders of magnitude has frequently been obtained (4, 9). However, virus clearance from cells and the complete cure of persistent virus infections have only rarely been reported (24, 25). We have developed several models of persistent virus infection by using poliovirus (PV), a positive-strand RNA virus of the Picornaviridae family (5, 7, 16, 21). We previously studied the effects of antiviral siRNAs applied months after the infection of HEp-2 cells with a persistent PV mutant (7, 25). We used a mixture (“the Mix”) of two synthetic siRNAs targeting the viral RNA genome in the 5′ noncoding (NC) region and the 3D polymerase (3D^pol) (siRNA-5′NC and siRNA-3D^pol, respectively; synthesized by Sigma-Proligo). When repeated transfections with the Mix were performed in persistently PV-infected cultures, most cultures stopped producing virus (25). Here, we investigate the important issue of changes in RNAi efficacy following siRNA treatment, 2 to 5 months after the cure. The efficiency of gene silencing in cells was stable during this period.We used the HEp-Q4 and -Q5 cell lines, which were cured of persistent PV infection after transfections with the Mix (25). The cured cells and their parental cell line, HEp-2, had similar growth rates (data not shown). To compare PV silencing efficiencies in the three cell lines, they were transfected either with the Mix or with an irrelevant siRNA (siRNA-IRR) in the presence of Lipofectamine 2000 (Invitrogen) in 24-well plates as previously described (25). Treated and mock-treated cells were infected 16 h posttransfection with PV strain Sabin 3, at a multiplicity of infection (MOI) of 1 50% infectious dose (ID₅₀) per cell. The viral progeny was titrated 24 h postinfection, as previously described (16). HEp-Q4 and HEp-Q5 were permissive to PV infection, although viral yields were about 1 log lower in these cells than in HEp-2 cells (Fig. ​(Fig.1A).1A). Virus silencing was observed in all three cell lines treated with the Mix; however, silencing was significantly more efficient in HEp-Q4 (≈2.2 times more efficient; P = 0.013, Student''s t test) and HEp-Q5 (≈5.6 times more efficient; P = 0.015) than in HEp-2 cells (Fig. 1A and B). Similar results were obtained with an shRNA (Thermo Scientific) targeting the same region as the siRNA-5′NC (data not shown).Open in a separate windowFIG. 1.Efficiency of enterovirus silencing in HEp-2, HEp-Q4, and HEp-Q5 cells after transfection with specific siRNAs. (A) Yield of progeny virus produced by cells infected at an MOI of 1 ID₅₀, 16 h posttransfection with the antiviral Mix containing two anti-PV siRNAs (20 pmol), the irrelevant siRNA-IRR (20 pmol), or no siRNA. Samples were harvested 24 h postinfection. Each bar represents the mean value ± SEM of six infected cultures from three independent experiments. (B to E) For each cell line, silencing efficiency is expressed as the ratio of infectious virus yield (titer in ID₅₀/ml) in the presence of the irrelevant siRNA-IRR to infectious virus yield (titer in ID₅₀/ml) in the presence of the antiviral siRNAs in cured cells, normalized with respect to the silencing efficiency in HEp-2 cells. S2, PV strain Sabin 2. (F) GFP silencing efficiency for each cell line is expressed as a ratio [1 − (mean GFP levels in the presence of siRNA-eGFP)/(mean GFP levels in the presence of siRNA-IRR)] in cured cells, normalized with respect to the efficiency of silencing in HEp-2 cells. Each bar represents the mean value ± SEM of at least four cultures from two independent experiments. *, P < 0.05 based on Student''s t test comparing HEp-Q4 and HEp-Q5 with HEp-2 cells.We investigated whether the differences in silencing efficacies between the three cell lines were due to differences in siRNA transfection efficiency by transfecting HEp-2, HEp-Q4, and HEp-Q5 cells with fluorescein isothiocyanate-conjugated siRNA (siRNA-FITC; 20 pmol/well; Cell Signaling) and testing them between 4 and 48 h posttransfection. The fluorescence of transfected cells was measured with a FACScan flow cytometer (Becton Dickinson), and data were analyzed with CellQuest software (Becton Dickinson). The percentages of siRNA-FITC-positive cells were similar for all cell types (Fig. ​(Fig.2A).2A). The mean fluorescence per positive cell and the percentage of cells displaying fluorescence peaked 16 and 24 h posttransfection, respectively, and decreased thereafter (Fig. ​(Fig.2).2). These findings suggest both that the presence of siRNAs in cells was similarly transient in the three cell types, as previously reported (27), and that the high silencing efficiencies in cured cells were not a consequence of higher transfection efficiencies. All subsequent experiments were performed between 16 and 40 h posttransfection.Open in a separate windowFIG. 2.Transfection efficiencies of fluorescein-conjugated siRNAs in HEp-2, HEp-Q4, and HEp-Q5 cells. A fluorescent siRNA-FITC (20 pmol) was used to transfect each of the three cell lines in the presence of Lipofectamine 2000. Fluorescent cells were analyzed 4 to 48 h posttransfection by using a FACScan flow cytometer (Becton Dickinson). The percentage of fluorescent cells (A) and the mean fluorescence per positive cell, in arbitrary units (B), are shown. Each bar represents the mean value ± SEM. (C) Representative FACS plots (cell granularity versus cell size), showing the similarities between the three cell populations.Fluorescence-activated cell sorting (FACS) plots for granularity versus cell size were very similar for the three cell lines (Fig. ​(Fig.2C),2C), as were those for cell numbers versus fluorescence (not shown), suggesting highly related cell populations. Although highly probable, it remains to be confirmed that the cured cells originated from a subpopulation of HEp-2 cells.Virus silencing was also investigated in cured cells infected with Sabin 2 or coxsackievirus A17 (CAV17) strain 67591 (22) or in cells transfected with Sabin 2 RNA. The experimental conditions used for Sabin 2 and CAV17 were identical to those for Sabin 3, except that only the 3D polymerase was targeted by siRNAs. Sabin 2 RNA (1 μg) was prepared as previously described (12) and used with siRNA-3D^pol (20 pmol/well) for the cotransfection of cells in the presence of Lipofectamine 2000. Virus yields were determined 7.5 h after transfection. In all cases, virus silencing was more effective in HEp-Q4 and -Q5 cells than in HEp-2 cells (Fig. 1C to E). Additional experiments were performed with a PV replicon encoding the green fluorescent protein (GFP), PV-eGFP (28) (2 μg/well), which was used with siRNA-eGFP (20 pmol/well; Ambion) for cotransfection. GFP fluorescence was measured by flow cytometry, 16 h after transfection. As for PV, a higher silencing efficiency was observed in cured cells than in HEp-2 cells (Fig. ​(Fig.1F1F).We then investigated whether the lower level of viral multiplication in HEp-Q4 and -Q5 cells in the absence of siRNAs involved an entry or postentry step. We quantified the expression of the PV receptor (CD155) at the surface of cells. We used flow cytometry after indirect immunofluorescence labeling with anti-CD155 antibodies, as previously described (16). More than 98.4% ± 2% (mean ± standard error of the mean [SEM]) of cured cells, like HEp-2 cells, tested positive for CD155 (data not shown). In the absence of siRNAs, a decrease in viral replication was also observed in HEp-Q4 and -Q5 cells infected with the Sabin 2 PV strain in cells, in which the early stages of the viral cycle were bypassed by transfection with Sabin 2 RNA, and in cells infected with the CAV17 virus, which uses a cell receptor other than CD155 (12) (data not shown). Together, these results suggest that PV multiplication is reduced at a postentry step, probably at replication, in cured cells.We investigated whether PV silencing was also enhanced in other HEp-derived cells in which Sabin 3 PV multiplication was reduced by using HEp-S31 (cl18) cells that had been cured of persistent PV infection by growth at a supraoptimal temperature rather than by RNAi (2). PV yield was ≈1.6 logs lower in HEp-S31 (cl18) cells than in HEp-2 cells (data not shown). Sabin 3 PV silencing in HEp-S31 (cl18) cells was 1.7 ± 0.9 times more effective (mean of six experiments) than that in HEp-2 cells (relative efficacy of 1) (data not shown), but this difference was not significant. However, these results do not exclude the possibility that reduced PV replication facilitates PV silencing by the Mix in cured cells. We therefore pursued our work with a different virus.We investigated whether the high silencing efficiency in HEp-Q4 and -Q5 cells was specific to enteroviruses by using a measles virus expressing GFP, MV-eGFP (26), and siRNA-eGFP to silence GFP expression. Cells were transfected with either siRNA-eGFP or siRNA-IRR, infected with MV-eGFP (1 ID₅₀ per cell, 16 h posttransfection), and the GFP silencing efficiency was determined 40 h posttransfection by flow cytometry. For each cell line, silencing efficiency was expressed as a percentage {[1 − (percentage of siRNA-eGFP-transfected cells expressing GFP)/(percentage of siRNA-IRR-transfected cells expressing GFP)] × 100}. GFP silencing was significantly stronger in HEp-Q4 cells (≈14%; P = 0.048) and HEp-Q5 cells (≈17%; P = 0.010) than in HEp-2 cells (Fig. ​(Fig.3A).3A). There was no significant difference in the silencing efficiency of GFP between HEp-Q4 and -Q5 cells (Fig. ​(Fig.3A).3A). The anti-PV Mix did not silence GFP expression (data not shown), indicating that the silencing of GFP was not due to anti-PV siRNAs persisting in cured cells months after the initial treatment.Open in a separate windowFIG. 3.Efficiency of GFP and luciferase silencing in HEp-2, HEp-Q4, and HEp-Q5 cells after transfection with specific siRNAs. (A and B) GFP silencing, expressed as a percentage calculated for each cell line as follows: {[1 − (GFP expression in the presence of siRNA-eGFP)/(GFP expression in the presence of the irrelevant siRNA-IRR)] × 100}. (A) Cells were infected 16 h posttransfection with a measles virus encoding eGFP (MV-eGFP [26]) at an MOI of 1 ID₅₀/cell, and fluorescent cells were analyzed 24 h after infection (40 h posttransfection). Each bar represents the mean value ± SEM of three independent experiments. (B) Cells were cotransfected with pEGFP-C1 and siRNA-eGFP or siRNA-IRR and analyzed 40 h later. Each bar represents the mean value ± SEM of four independent experiments. (C) Luciferase silencing efficiency for each cell line, expressed as the ratio of luciferase activity in the presence of the irrelevant siRNA-IRR to luciferase activity in the presence of the specific siRNAs in cured cells, normalized with respect to silencing efficiency in HEp-2 cells. Relative efficiencies are shown as in Fig. ​Fig.11 for luciferase, because the enzymatic reaction amplified the signal. Each bar represents the mean value ± SEM of triplicates from three independent experiments. *, P < 0.05 based on Student''s t test comparing HEp-Q4 and HEp-Q5 with HEp-2 cells.To test whether the high silencing efficiency in HEp-Q4 and -Q5 cells was dependent on viral infection, plasmid vectors pEGFP-C1 (Clontech Laboratories) and pRL-CMV (Promega) were used to generate GFP (6) and Renilla luciferase (18), respectively. These plasmids do not replicate in human cells. Cells (10⁶) were cotransfected with pEGFP-C1 (1 μg) and siRNAs (20 pmol) in the presence of Lipofectamine 2000, as recommended by the manufacturer. GFP fluorescence was analyzed by flow cytometry 40 h posttransfection. Silencing efficiencies were expressed as a percentage {[1 − (mean GFP levels in the presence of siRNA-eGFP)/(mean GFP levels in the presence of siRNA-IRR)] × 100)}. Mean silencing efficiency was significantly higher in HEp-Q4 (≈15%; P = 0.003) and HEp-Q5 (≈15%; P = 0.002) cells than in HEp-2 cells (Fig. ​(Fig.3B).3B). The efficiency with which the GFP encoded by pEGFP-C1 was silenced was similar in HEp-Q4 and -Q5 cells.The efficacy of siRNAs was then assessed with pRL-CMV, which encodes the Renilla luciferase and Silencer Renilla luciferase (AM4630; Ambion). Cells (10⁶) were cotransfected with the plasmid (100 ng) and either specific or irrelevant siRNA (7 pmol) in the presence of Lipofectamine 2000. Luciferase assays were performed with a Dual-Glo luciferase assay system (Promega), as recommended by the manufacturer at 40 h posttransfection, and luminescence was measured with a luminometer (Centro LB960; Berthold). The results of the sensitive luciferase assays confirmed that the relative efficiency of silencing was significantly higher in cured than in parental cells (Fig. ​(Fig.3C).3C). By contrast, results obtained in HEp-S31 (cl18) cells, cured without siRNAs, were not significantly different from those obtained in control HEp-2 cells (data not shown), strongly suggesting that the treatment of HEp-Q4 and -Q5 cells with specific siRNAs selected cells in which siRNAs mediated silencing more efficiently than in parental cells.The difference in silencing efficiency between cured and HEp-2 cells may be due to differences in the abundance and/or efficacy of cellular factors involved in gene silencing. Some major actors of the RNAi pathway, particularly those associated with the RNA-induced silencing complex (RISC), have been identified (3, 10, 13, 19). The active endonucleolytic core of the RISC includes the guide strand of the siRNA and a slicer protein called Argonaute 2 (Ago2) (17). We used Western blotting to study Ago-2 and other factors contributing to the function of RISC (3, 10, 11, 14, 19, 23): the endonuclease Dicer, the transactivation response RNA binding protein (TRBP), the protein activator of double-stranded RNA-dependent protein kinase (PACT), and the RNA helicase A (RHA) (Fig. ​(Fig.4).4). Exportin 5, which plays a role upstream from the dicing process in the export of small RNA precursors (29), was included as a control.Open in a separate windowFIG. 4.Comparative analysis of proteins involved in RNAi in HEp-2, HEp-Q4, and HEp-Q5 cell lines. Whole-cell lysates were tested for Exportin 5 (A), Dicer (B), Ago-2 (C), the helicase RHA (D), TRBP (E to H) and PACT (I) by Western blotting with the corresponding specific antibodies. Blots were subsequently stripped and reprobed with antiactin antibodies to confirm equal protein loading. (E and F) TRBP levels in HEp-Q4 and HEp-Q5 cells were determined by densitometry and are plotted in arbitrary units, as ratios relative to the level of actin and to the level of TRBP in HEp-2 cells. In panel F the symbols correspond to TRBP levels determined in nine different experiments. (G) TRBP levels in HEp-2 cells transfected with pcDNA-TRBP (14) and in cells cotransfected with pcDNA-TRBP and siRNA-TRBP. (H) TRBP levels were compared in human IMR5 cells, HEpS31 (cl18) cells previously cured of persistent PV infection by growth at a supraoptimal temperature, and the control HEp-2 cell line. TRBP/actin densitometry and PACT/actin densitometry results are indicated in arbitrary units in the histograms below the corresponding Western blot results shown in panels H and I.Proteins (30 to 50 μg) from each cell line were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10 to 20% Tricine gels; Invitrogen) and transferred to nitrocellulose membranes (Amersham Biosciences) as previously described (1). The membranes were incubated with one of the following primary antibodies (1): anti-Ago2 monoclonal antibody (MAb; Abcam), anti-RHA MAb (Abcam), and anti-TRBP2 MAb (Santa Cruz Biotechnology); rabbit antibodies against Dicer (Santa Cruz Biotechnology); anti-PACT MAb (Santa Cruz Biotechnology), and anti-Exportin 5 MAb (Abcam). The antiactin MAb (AC-40; Sigma-Aldrich) was used to check for equal protein loading. Membranes were then washed and treated with appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) for 2 h at room temperature. Protein bands were detected with an enhanced chemiluminescence detection kit (ECL⁺; Amersham Biosciences) and a G:box (Syngene).Exportin 5, Dicer, Ago-2, and RHA were similarly abundant in all three cell lines (Fig. 4A to D), suggesting that quantitative differences in protein levels were unlikely to be responsible for the enhanced silencing in HEp-Q4 and -Q5 cells. There was significantly more TRBP in HEp-Q4 (≈21%; P = 0.026) and HEp-Q5 (≈28%; P = 0.016) cells than in HEp-2 cells, as indicated by the results of nine experiments (Fig. 4E and F). The specificity of the anti-TRBP antibody was checked on extracts of HEp-2 cells transfected with a plasmid encoding TRBP, pcDNA-TRBP (14), with and without silencing by siRNA-TRBP (Fig. ​(Fig.4G).4G). GFP silencing was not enhanced in HEp-2 cells overproducing TRBP, and it was not decreased by downregulating TRBP gene expression with siRNA-TRBP (data not shown). These results suggest that the high levels of TRBP in the cured cell lines are not the cause of the enhanced silencing in these cells.There was less TRBP protein in HEp-S31 (cl18) cells (2) than in HEp-2 and other control cells (IMR5) (Fig. ​(Fig.4H),4H), indicating that high levels of TRBP are not necessarily selected in cells persistently infected with PV. PACT was slightly downregulated in the cured cells (Fig. ​(Fig.4I).4I). Moreover, PACT is unlikely to be involved in the enhanced silencing in cured cells, because we used synthetic siRNAs and PACT functions principally during siRNA production by Dicer (14). We did not investigate the activities or subcellular distributions of the various factors involved in RNAi in the three cell lines, and they may differ. It is also possible that other factors, not tested here, contribute to the efficacy of siRNAs in cured cells. The molecular details of the mechanism involved remain to be determined.Overall, our results suggest that both a decrease in viral replication and the enhancement of gene silencing contributed to the mechanism by which cells persistently infected with poliovirus were cured by RNAi. Our results also indicate that cells displaying enhanced silencing may be selected during treatment with siRNAs. This may result in profound changes to cell phenotype, because RNAi plays an essential role in the regulation of cellular gene expression (15).     

Latency of Human Measles Virus in Hamster Cells

A latent system employing measles virus (Schwarz strain) was developed in hamster embryo fibroblasts (HEF). Measles virus-specific antigen was detected by immunofluorescence in 30 to 50% of HEF cells, and these cells released infectious virus when co-cultivated with a susceptible monkey cell line, BSC-1 cells. No infectious virus could be detected in the cells when measures were taken to exclude passage of viable latent cells onto the indicator BSC-1 cells. Infectious center assays demonstrated that about 1 in 10 of the latently infected cells in the population could release infectious virus. Infectious virus appeared within 6 hr after co-cultivation of the HEF cells with BSC-1 cells, as compared to 24 hr required for normal replication of measles virus in the BSC-1 cells. Furthermore, labeling of progeny virus ribonucleic acid (RNA) by using tritiated uridine, and inhibition of RNA or protein synthesis by 5-azacytidine or cycloheximide suggested that neither additional RNA nor protein synthesis is required after co-cultivation of the cells to effect early virus release. It can therefore be postulated that there is a block at a late step in virus replication in the latently infected hamster cells. The most obvious site would concern maturation of infectious virions at the cell membrane.     

油桐尺蠖核型多角体病毒感染Bs484细胞的研究

本文报道利用油桐尺蠖核型多角体病毒感染处于不同培养基及不同培养时间的Ｂｓ４８４细胞的部分感染效果和特征。结果表明,细胞培养基的类型和细胞传代后的时间对病毒感染率和多角体产量以及其它感染特征都有比较明显的影响;经过比较,处于Ｇｒａｃｅ培养基组的传你后三天的Ｂｓ４８４细胞是一个理想ＢｓＮＰＶ感染受试系统。同时发现,健康的油桐尺蠖血淋巴具有提高细胞活性及病毒感染效果的作用。     

Purification of Host DNA Synthesis-Suppressing Factor (DSF) Produced by Infection with Measles Virus

Host DNA synthesis-suppressing factor (DSF) produced into culture fluid of cloned HeLa cells (HeLa C-9) infected with a small plaque variant of Toyoshima strain of measles virus was purified by precipitation with ammonium sulfate, chromatography on CM-cellulose and DEAE-cellulose, and gel-filtration on Sephadex G-100 and G-200. The specific activity of the finally purified DSF was 302 units/mg of protein representing approximately 300-fold purification. The molecular weight of DSF was estimated to be about 55 000. By isoelectric focusing, two kinds of DSF having isoelectric points of 4.24 and 5.24 were detectable. The purified DSF was able to suppress host DNA synthesis of HeLa cells, continuous human lymphoid cells (NC-37), mouse L cells and Meth-A cells derived from an ascitic tumor of the mouse. The activity of the purified DSF was inactivated by heating at 56 C for 30 min or by treatment with trypsin.     

Selection of Temperature-Sensitive Mutants During Persistent Infection: Role in Maintenance of Persistent Newcastle Disease Virus Infections of L Cells

Virus mutants (NDV(pi)) recovered from L cells persistently infected with Newcastle disease virus (NDV, Herts strain) are temperature-sensitive (ts) at 43 C, although the wild-type virus (NDV(o)) which initiated the persistent infection replicates normally at that temperature. To study the relationship between the ts marker of NDV(pi) and the other properties which distinguish this virus from NDV(o), NDV(pi) ts(+) revertants were selected at the nonpermissive temperature and NDV(o) ts mutants were generated by treating NDV(o) with nitrous acid. Spontaneously-occurring ts mutants in the Herts NDV population were also isolated. The different virus populations were characterized with regard to plaque size, virulence for eggs, and thermal stability of infectivity, hemagglutinin, and neuraminidase. The NDV(pi) ts(+) revertants, although no longer temperature-sensitive, retained NDV(pi) properties, whereas both spontaneously-occurring and mutagen-induced ts mutants remained wild-type in their other properties. These findings showed that the properties which characterized NDV(pi) were independent of the ts marker. However, the ts marker and the other markers of NDV(pi) were coselected during the persistent infection, and the combination of those markers appeared to be important in the outcome of NDV infection of L cells. NDV(pi) replicated productively in L cells, whereas NDV(o), the NDV(pi) ts(+) revertants, and the spontaneously-occurring ts mutants all yielded covert infections in L cells. The role of the selection of ts mutants in persistent infection was confirmed as follows: L cells were persistently infected with NDV(pi) ts(+) revertants and NDV(o) ts mutants. Virus recovered from the persistently infected cultures after eight cell passages was always temperature-sensitive and of smaller plaque size than the parental virus in chicken embryo cell cultures. Similar results were obtained with virus recovered from L-cell cultures persistently infected with two other velogenic strains of NDV, the Texas-GB and Kansas-Man strains. These results strongly suggest that selection of ts mutants during the persistent infection was not random and played a role in establishment or maintenance of the persistent infection, or both.     

Proteins Specified by Sindbis Virus in HeLa Cells

HeLa cells infected with Sindbis virus were found to contain five species of nonvirion proteins besides the structural proteins of the virus. Some of the nonvirion proteins were found to serve as precursors to the viral structural proteins in a pulse-chase experiment.