Temperature-sensitive mutants of measles virus produced from persistently infected HeLa cells

Summary A persistent infection with the Edmonston strain of measles virus was established in HeLa cells in the absence of measles virus antibody (HeLa_PI cells). By hemadsorption or immunofluorescence virtually 100 per cent of the cells possessed measles virus components. HeLa_PI cells produced no interferon and were not resistant to superinfection with Newcastle disease virus. HeLa_PI cells contained both smooth (15–18 nm) and rough (20–35 nm) nucleocapsids as detected by electron microscopy. The virus produced from the HeLa_PI cells (MV_PI) varied in titer between 1.5 × 10² and 5.5 × 10⁴ PFU/ml, had a smaller plaque size and was more heat resistant than wild-type measles virus. MV_PI was also found to be temperature-sensitive. The temperature-sensitivity of MV_PI was determined by the efficiency of plaquing at 33° and 39° C in Vero cell monolayers. When HeLa_PI cells were incubated at 33° C, there was a 50-fold increase in virus production as well as a slight increase in the percentage of cells forming infectious centers compared to HeLa_PI cells grown at 37° C. MV_PI readily established a persistent infection in HeLa cells which also released temperature-sensitive virus.With 2 Figures     

Surface antigens on HeLa cells persistently infected with HVJ (Sendai Virus)

Summary Surface antigens of HeLa_HVJ cells, a cell line persistently infected with HVJ, were studied by fluorescent antibody staining. After absorption with concentrated HVJ virions and HeLa cells, anti-HeLa_HVJ antiserum was able to demonstrate specific surface fluorescence on HeLa_HVJ cells, while this serum no longer reacted with original HeLa cells nor with HVJ virions. During cytolytic infection of HeLa cells with HVJ, this specific surface antigen appeared at an early stage of infection prior to the appearance of newly synthesized HVJ viral antigens and moreover appeared in spite of the inhibition of viral protein synthesis. This antigen was detected neither on HeLa cells infected with other myxoviruses except HVJ nor on various other kinds of cells infected with HVJ. The specific surface antigen was still found on the HeLa_HVJ cell surface after incubation at 38° C for two days, while HVJ structural antigens on the cell surface no longer could be detected. Mild short-term treatment of HeLa cells with trypsin, neuraminidase fromvibrio cholerae, phospholipase-C and hyaluronidase failed to expose specific antigen. The antigen was distinguishable from the Forssman and human blood type antigens. The mechanism of appearance of a new antigen on the surface of HeLa_HVJ cells remains unclear.With 2 Figures     

Rhinovirus type 14 persistence in HeLa cells studied by use of guanidine.

Guanidine at 2 mM completely prevents replication of rhinovirus type 14 (RV14) in HeLa cells and spares these cells from RV14-induced cytopathic effects. Three serial subcultivations of these cells in the continued presence of guanidine reduced infectious virus titers in cell lysates to undetectable levels. However, the capacity of RV14-infected guanidine-cultured cells to form infectious centers when cultured in the absence of guanidine was not completely abolished until the fifth serial subcultivation (14 days post-inoculation). Production of defective-interfering particles of RV 14 in HeLa cells was not observed. Thermal stability and transmission of uncoated RV 14 to daughter cells in the presence of guanidine can account for the persistence. Cells inoculated with RV14 and cultured in the presence of guanidine synthesized genomic RNA associated with a structure of 1.394 gm/ml density in CsCI, and these structures exhibited low infectivity by standard plaque assay. Nascent RV14 RNA did not appear in virions in cells treated with guanidine. HeLa cell lines (HeLa_V+G), established following six serial subcultures of cells initially infected with RV 14 and continuously cultured in the presence of guanidine, were negative for RV 14 antigens by specific immunofluorescence tests. HeLa_V+G cells were as efficient as normal HeLa cells in production of interferon; replication of RV 14 and mengovirus; and plaque assay of RV 14, rhinovirus type 2, and coxsackievirus B3.     

Nef-mediated MHC class I down-regulation unmasks clonal differences in virus suppression by SIV-specific CD8 T cells independent of IFN-γ and CD107a responses

CD8⁺ T lymphocytes (CTL) play a role in controlling HIV/SIV infection. CTL antiviral activity is dependent on recognition of antigenic peptides associated with MHC class I molecules on infected target cells, and CTL activation can be impaired by Nef-mediated down-regulation of MHC class I molecules. We tested the ability of a series of rhesus macaque CD8⁺ T-cell clones specific for the SIV Gag CM9 peptide to suppress SIV infection of autologous CD4⁺ T cells. We used a set of SIV_mac239 viruses with either wild-type Nef or Nef mutations that impair MHC class I down-regulation. All CTL clones efficiently suppressed virus replication in cells infected with mutant viruses with altered Nef function, phenotypically MHC class I^high or MHC class I^intermediate. However, the ability of the clones to suppress virus replication was variably reduced in the presence of wild-type Nef (MHC class I^low) despite the observations that all CTL clones showed similar IFN-γ responses to titrated amounts of cognate peptide as well as to SIV-infected cells. In addition, the CTL clones showed variable CD107a (CTL degranulation marker) responses that did not correlate with their capacity to suppress virus replication. Thus, the clonal differences are not attributable to TCR avidity or typical effector responses, and point to a potential as yet unknown mechanism for CTL-mediated suppression of viral replication. These data emphasize that current assays for evaluating CTL responses in infected or vaccinated individuals do not fully capture the complex requirements for effective CTL-mediated control of virus replication.     

Defective interfering Tacaribe virus and persistently infected cells

Persistently infected (PI) BHK 21 and Vero cell lines were established by infection with wild-type (wt) Tacaribe virus and passage of surviving cells. These cells release defective interfering (DI) Tacaribe virus particles, which caused complete inhibition of plaque formation by wt Tacaribe virions. This interference activity was reduced when the DI virus was previously uv inactivated or treated with antiviral serum, and no interferon-like activity was detected. Cells pretreated with DI virus were only partially resistant to challenge with heterotypic arenaviruses, and no interference was detected with plaque formation by vesicular stomatitis virus. PI-BHK and PI-Vero cells contained Tacaribe virus-specific polypeptides as demonstrated by immunofluorescence, and by immunoprecipitation and analysis by SDS-polyacrylamide gel electrophoresis; the major virus-specific polypeptide observed was related to the N protein. PI-Vero cells were resistant to homologous superinfection and slightly resistant to superinfection by heterotypic arenaviruses, but supported normal plaque formation by vesicular stomatitis virus. Purified DI virus had a polypeptide profile similar to wt Tacaribe virions, although the DI virus released from PI-BHK cells had a nucleoprotein which migrated slower than the nucleoprotein of wt virions. The L and S segments of standard virions were not detected in DI particles from PI-BHK cells; but five discrete smaller RNA species were observed (MW ～ 1.1?0.2 × 10⁶).     

Characterization of herpes simplex virus persistence in a human T lymphoblastoid cell line.

Persistent, dynamic-state infection with herpes simplex virus (HSV) type 1 has been maintained in human T lymphoblastoid (CEM) cells for many months after initial infection with the wild-type virus (HSV0) (input virus/cell multiplicity of 1.0). Persistently infected cells grew as well as uninfected cells, except during occasional periods of crisis (increased viral replication and cytopathic effect). Cells could survive the crisis when they were maintained for twice the usual time interval (8 to 10 rather than 4 to 5 days) before subculture. Interferon was not detectable in the cultures. HSV0 was compared with HSVp1, a small plaque-forming isolate from persistently infected CEM cells. Primary infection of CEM cells with HSV0 at a low input multiplicity (0.01) led to abortive replication, whereas infection with HSVp1 at the same multiplicity resulted in either rapidly lytic or persistent infection depending upon the time interval of subculture. Approximately 55% of plaque-purified clones of HSVp1, as compared with only 5% of HSV0 clones, displayed temperature-sensitive growth in Vero cells. Defective interfering virus was not detectable in uncloned HSVp1 by interference assay. Persistently infected cultures "cured" by treatment with HSV antiserum or incubation at 39 degrees C were resistant to reinfection with HSV but permissive for vesicular stomatitis virus replication, suggesting that these treatments modulated a shift from the dynamic-state of the static-state, latent infection. These studies provide a model for characterization of HSV persistence and latency in a highly differentiated human cell line.     

Plaque variants of Sindbis virus

Summary Large plaque (G) and small plaque (8) variants were cloned from stock culture of Sindbis virus. Addition of protamine to agar overlay was shown to have little or no effect on the size of plaques produced by G variant, whereas S virus plaque size was significantly increased. This finding suggests that the S variant is more susceptible than the G variant to the inhibitory action of agar factor. The G and S variants differed markedly in their sensitivity to the antiviral action of interferon. Evidence was obtained that the S variant is more susceptible to the action of interferon. The difference of G and S variants in susceptibility to the action of interferon and agar factor may account for the difference of their plaque size. Growth and cytopathic effect of the S variant in a HVJ carrier HeLa cell culture (HeLa_HVJ) was greatly enhanced in contrast to those in normal HeLa cells. Enhancement of growth of wild-type virus in HeLa_HVJ cells was not so significant as in the case of the S variant. These observations were discussed in relations to the properties of the S variant and the cells of HeLa_HVJ.Dedicated to ProfessorJohn F. Enders on the occasion of his 70th birthday.     

Chronic infection of HeLa cells with Japanese encephalitis virus. General characteristics of the system

Chronic infection of HeLa cells was induced by an attenuated variant of Japanese encephalitis (JE) virus (HeLa-K3 cell line). The chronic infection was characterized by alternating phases of degeneration and recovery of the cell monolayer. JE virus was regularly released into the medium of chronically infected cell cultures and virus-specific antigen was regularly demonstrated in the cytoplasm of 15--25% of cells. JE virus persisting in HeLa-K3 cells was sensitive to pancreatic ribonuclease and resistant to treatment with 4 M urea. HeLa-K3 cells did not undergo cytological or karyological transformation; they were susceptible to superinfection with heterologous viruses but resistant to reinfection with homologous virus.     

Persistent infection with human cytomegalovirus in a lymphoblastoid cell line.

Cells from a line of human lymphocytes originating from a leukemic patient were persistently infected with human cytomegalovirus (HCMV). The infected culture has persistently yielded HCMV with titers ranging from 2 × 10⁴ to 3 × 10⁵ PFU/ml over a period of 1 year. Infectious center and fluorescent antigen assays and electron microscopic examination indicated that 1–10% of the cells were infected. It appears that persistent infection is due to a balance between release of virus and the growth of uninfected cells rather than to a defective or temperature-sensitive mutant of HCMV. The treatment of persistently infected cultures with anti-HCMV serum resulted in curing the virus infection. Cured cells in culture grew at the same rate as normal uninfected cells and became resistant to HCMV infection and relatively resistant to HSV infection.     

Host-dependent restriction of influenza virus maturation.

Host-dependent restriction of influenza virus replication in nonpermissive HeLa cells was studied under single cycle conditions using as inoculum bovine kidney (MDBK)-grown virus which was relatively free of defective interfering particles. Biochemical and ultrastructural changes in infected HeLa cells were compared to MDBK cells, which are permissive for influenza virus replication [Choppin and Pons (1970), Virology, 42, 603–610]. Two subclasses of virus-specific nucleoproteins separated in renografin density gradients were present in the nucleus and cytoplasm of both MDBK and HeLa cells; however, the assembly of the 1.26 g/ml virus-specific RNPs occurred at a faster rate in HeLa cells than in MDBK cells. The overall patterns of synthesis of polypeptides in HeLa cells were similar to those seen in MDBK cells, and the polypeptide compositions of virus released from HeLa and MDBK cells were also similar. Electron microscopy showed that elongated virus particles accumulated in tightly packed arrays on the plasma membrane of HeLa cells; however, only a small number of spherical virions were observed budding from the plasma membrane of MDBK cells. Large, intracytoplasmic vesicles filled with budding virus particles were numerous in HeLa cells but were not observed in MDBK cells. Many finely striated inclusions and dense granular inclusions were observed in the cytoplasm of HeLa cells within 24 hr after infection. In contrast, MDBK cells showed much less cytopathic effect at 24 hr after infection, and virus-induced inclusions occurred much less frequently than in HeLa cells. These results suggest that. in HeLa cells the final stages of maturation of virus particles at the plasma membrane may be,blocked, allowing the accumulation of viral products within the cytoplasm and budding virus particles on. the surface of nonpermissive cells. Thus, in the absence of defective interfering particles in the inoculum, the host-dependent restriction in influenza virus replication in HeLa cells does not occur early in the virus growth cycle, but appears to be due to a defect in a late event involving the participation of the plasma membrane. in virus maturation.     

Multiplicity activation of herpes simplex virus in mouse neuroblastoma (C1300) cells

Summary The virus yields and number of infectious centres of HSV infected mouse neuroblastoma C1300 cells (clone 41 A₃) infected at different multiplicities of infection (MOI) were found to vary more than the differences of HSV concentrations of the virus suspensions used for infection of the cells. This suggested that a C1300 cell had to be infected with more than one HSV particle in order to produce progeny virus—multiplicity activation. The greater than expected enhancement of virus production of C1300 cell cultures receiving increasing MOI of HSV was probably not due to improved virus adsorption, nor influenced by non-virus factors in the virus inoculum stimulatory for HSV replication. A hypothesis, that the block in virus replication was promoted by an inhibitor of an HSV specified regulatory protein and could be overcome by the addition of HSV DNA copies in the infected cell, was supported by the results of two types of experiments. Presence of phosphonoformic acid, an inhibitor of the HSV specified DNA polymerase, in the culture medium of HSV infected permissive GMK cells resulted in non-linear relationships between virus yields and MOI. An HSV temperature sensitive mutant (ts B5), defective in a late structural protein, rescued wild type HSV in C1300 cells.With 4 Figures     

An attenuated mutant of Venezuelan encephalitis virus: Biochemical alterations and their genetic association with attenuation

A temperature-sensitive mutant of Venezuelan encephalitis virus derived by chemical mutagenesis from the hamster-virulent 68U201 wild-type (wt) strain was previously found to be attenuated. This mutant, is 126, replicated in infected hamsters and elicited production of protective antibodies. Further phenotypic differences between is 126 and 68U201 wt have been characterized in an attempt to localize the genetic basis of the mutant's attenuated virulence. The mutant was shown to differ from the parent virus with respect to virion surface structure-dependent characteristics: temperature lability, plaque sizes in Vero cells, and binding properties to hydroxylapatite. The surface difference was identified by isoelectric focusing of the virion envelope glycoproteins as an alteration in the E₁ glycoprotein. The common genetic basis of all these phenotypic differences was demonstrated by the isolation of independently arising, stable genetic revertants of ts 126, which exhibited characteristics identical in every respect to 68U201 wt. It appears from these studies that the mutation which gave rise to the is 126 mutant virus occurred in the structural gene coding for the E₁ envelope glycoprotein and that the resultant phenotypic alteration in this glycoprotein is genetically associated with the mutant's lack of virulence.     

Monoclonal antibodies to respiratory syncytial virus: Detection of virus neutralization and other antigen-antibody systems using infected human and murine cells

Monoclonal antibodies to human respiratory syncytial (RS) virus-specific antigens can be obtained without preliminary recourse to large-scale culture and purification of the virion. Lytically infected human and persistently infected murine cultured cells expressing RS virus-specific cell surface and cytoplasmic antigens were substituted as priming immunogens and as substrates in solid-phase antibody radioimmunoassays. Seven hybridoma clones secreting murine IgG of either the γ₁ or the γ_2A subclass bearing κ light chains were isolated. Two of the antibodies were specific for cell surface viral antigens, but only one was able to neutralize RS virus infectivity. The five remaining antibodies did not neutralize virus infectivity and were specific for viral antigens associated with large cytoplasmic inclusions as judged by indirect immunofluorescence (IF) analysis on fixed infected cells. Similar IF analysis using live cells revealed that those antigens, associated with the cytoplasmic inclusions in both the human and murine infected cells, were not expressed on the cell surface of the live infected human cells, but were expressed on the cell surface of the live infected murine cells. Monoclonal antibodies generated via the present system will prove useful in the immunological analysis of viral components which are associated with specific pathogenic functions, such as infectivity, and those which may be abnormally exposed at the surface of persistently infected cells.     

Evaluation of five temperature-sensitive mutants of respiratory syncytial virus in primates: I. Viral shedding,immunologic response,and associated illness

Approximately 10⁴ plaque-forming units (pfu) of five temperature-sensitive (ts) mutants of respiratory syncytial (RS) virus were administered intranasally to seronegative chimpanzees to evaluate their level of attenuation. When the ts-1 mutant was given to two chimpanzees, the pattern of viral growth and illness (rhinorrhea) was similar to that seen during wild-type RS virus infection. Two further defective subclones of ts-1, ts-1 NG-1 and ts-1 NG-16, appeared more attenuated than ts-1 with respect to severity of rhinorrhea. The ts-7 mutant infected the one animal tested; illness was similar to that induced by ts-1 or wild-type virus. 10⁴ pfu of the ts-2 mutant failed to infect two chimpanzees; virus was not isolated, a serum antibody response was not detected, and unlike chimpanzees given the other ts mutants, these animals were not resistant to challenge with wild-type virus. A 40-fold larger inoculum of ts-2, 10^{5, 6}pfu, infected each of four chimpanzees, but the peak quantity of virus shed was approximately 500-fold lower than during wild-type virus infection. Significantly, the ts-2 infected chimpanzees did not become ill, but infection did stimulate the development of RS virus serum neutralizing antibodies. The one chimpanzee given 10^{7, 6} pfu of the ts-2 mutant shed as much virus as the chimpanzees infected with wild-type virus; however, there were no signs of upper respiratory tract illness. Owl monkeys resembled chimpanzees in shedding s large amount of virus and developing rhinorrhea following administration of wild-type virus or the ts-1 mutant. Significantly, 10^{5, 3} pfu of ts-2 infected only one of two owl monkeys tested, but a small quantity of virus (>10 pfu/ml) was shed. These observations suggested that the ts-2 mutant was the most attenuated vaccine cadidate strain tested, and that extent of infection with rs-2 appeared to be related to the quantity of virus administered.     

Persistence of viral genes in a variant of MDBK cell after productive replication of a mutant of influenza virus A/WSN

Summary The MDBK-R cell line is a variant of the MDBK cell line, which was derived by three consecutive high multiplicity superinfections of MDBK cells with AWBY-140 virus, a mutant of influenza virus A/WSN (H 1N 1). MDBK-R cells are permissive for productive replication of AWBY-140, but resist lysis by the virus and grew normally without producing infectious virus after replication of the mutant occurred there. By polymerase chain reaction (PCR), we demonstrated nucleotide sequences specific to all the 8 genes of AWBY-140 in MDBK-R cells which had been infected with the mutant at a high multiplicity and subsequently received 25 passages. This suggests that the genes of influenza virus mutant persisted in the dividing host cells for a long time after productive infection, when none of the cells was producing virus. We were also able to amplify the M gene related sequence of the mutant from both poly(A)⁺ and poly(A)^– fractions of the RNA extracted from the cells at 27th passage level by PCR, which suggests that the persisting genes were replicated and transcribed, but we failed to demonstrate any viral protein in the cells by Western blotting.     

Attenuation of a field Sendai virus isolate through egg-passages is associated with an impediment of viral genome replication in mouse respiratory cells

Summary.  We investigated the mechanisms responsible for attenuation of mouse pathogenicity of Sendai virus (SeV) through passages in eggs. A highly virulent clone, E0, derived from the field SeV Hamamatsu strain, was successively passaged in hen‘s eggs. Analysis of the mouse lethal dose 50% (MLD₅₀) of virus clones obtained from the viruses at egg-passages 1, 15, 30 and 50 demonstrated that attenuation of E0 by egg-passage occurred due to the gradual appearance of and replacement by virus variants possessing higher MLD₅₀. Comparison of viral replication in the mouse lung and mouse pathogenicity with the representative SeV clones, E0, E15cl2, E30cl2 and E50cl9, obtained from the respective egg-passages revealed that the low pathogenicity of the egg-passaged clones was due to poor multi-cycle viral replication in the lung. Furthermore, MLD₅₀s of the SeV clones were found to be negatively correlated with the replication capability in primary mouse pulmonary epithelial (MPE) cells; the egg-passaged clones with more attenuated phenotypes showed lower replication capability in MPE cells. In the MPE cells infected with the SeV clones at m.o.i. 10, however, viral protein and mRNA syntheses of the egg-passaged clones were enhanced or comparable to those of the parental E0 clone at 1 day and 2 days post infection (p.i.) but decreased more rapidly thereafter. In contrast, viral genome synthesis of the egg-passaged clones in the cells at 2 days p.i. was several times lower than that of E0. These results strongly suggest that attenuation of a virulent field SeV strain by egg-passage occurs due to the appearance and selection of virus variants possessing poor propagation capacity in mouse respiratory epithelial cells, which is caused primarily by an impediment of viral genome replication. Received July 21, 2000 Accepted January 10, 2001     

The influence of serum on the cytopathic effect of measles virus

Summary HeLa cell cultures grown in media containing sheep serum form a uniform monolayer of polyhedral cells with active mitosis. These cells, when infected with measles virus, develop large, characteristic syncytia. In contrast, HeLa cells adapted to calf serum do not usually form a complete sheet, the cells are more elongated and mitotic figures are rarely present. After infection with measles, they show only occasional syncytia and the cytopathic effects are delayed and confined to isolated cells. Comparative titration of measles virus in monkey kidney and HeLa cells gave higher TCID₅₀ and yields in the latter species.     

Varicella-zoster virus open reading frame 47 (ORF47) protein is critical for virus replication in dendritic cells and for spread to other cells

Varicella-zoster virus infects human dendritic cells (DCs). We found that VZV infection of DCs resulted in down-regulation of Fas expression on the surface of cells. VZV ORF47 was critical for replication of virus in human immature, but not mature DCs. Immature DCs infected with a mutant virus unable to express ORF47 expressed similar levels of a VZV immediate-early protein as cells infected with parental virus; however, cells infected with the ORF47 mutant expressed lower levels of glycoprotein E. Thus, in the absence of ORF47 protein, there is a block in viral replication between immediate-early and late gene expression. VZV unable to express ORF47 was severely impaired for spread of virus from DCs to melanoma cells. Infection of DCs with parental VZV resulted in a different pattern of phosphoproteins compared with the ORF47 mutant virus. Thus, VZV ORF47 is important for replication in immature DCs and for spread to other cells.     

Construction and properties of a recombinant pseudorabies virus with tetracycline-regulated control of immediate-early gene expression

A study was carried out to determine whether altering the control of expression of the IE180 gene of pseudorabies virus (PRV), by replacing the IE180 promoter with the tetracycline-responsive promoter (Ptet), affects virus replication and virulence. This PRV-BT90 mutant virus was constructed by complementation and recombination in Hela Tet-Off cells. The virus yield produced by infection of Hela Tet-Off cells with PRV-BT90 was similar to that of the parental virus vBecker2. Viral replication of PRV-BT90 was reduced in Vero cells as reflected by a reduction of virus yield and plating efficiency compared to vBecker2. PRV-BT90 plaque formation in Hela Tet-Off cells was inhibited in the presence of doxycycline, whereas vBecker2 plaque formation was not affected. Subcutaneous infection of mice with the two viruses revealed a LD₅₀ higher than 10⁶ TCID₅₀ for the PRV-BT90 mutant virus while the LD₅₀ was 178 TCID₅₀ for the vBecker2 parental virus.