Studies on the interaction between coxsackievirus A9 and HeLa cells. I. Plaque-forming ability of coxsackievirus A9 in HeLa cell cultures.

Most of the coxsackievirus A9 (CA 9 virus) including the prototype strain formed plaques in HeLa cell monolayers under agar overlay, although they showed little or no cytopathogenicity under fluid medium. These viruses were isolated or passaged in primary cynomolgus monkey kidney (MK) cell cultures, and the infectivity of any strain in terms of plaque-forming units was much higher in MK cells than in HeLa cells, even after plaque purification of the virus in HeLa cell cultures. CA 9 virus contained in the original throat swabs as well as some clones obtained by plaque purification in MK cells failed to form plaques in HeLa cells, but virus preparations obtained after several undiluted passages through MK cells included plaque-formers in HeLa cells, suggesting that such plaque (HeLa)-forming viruses may have developed at a certain rate during multiplication of the original non-plaque (HeLa)-forming virus population in MK cells. Out of four lines of HeLa cells examined, two, including a clonal line S3, failed to support plaque formation by CA 9 virus.     

The coxsackievirus A9 RGD motif is not essential for virus viability.

An RGD (arginine-glycine-aspartic acid) motif in coxsackievirus A9 has been implicated in internalization through an interaction with the integrin alpha v beta 3. We have produced a number of virus mutants, lacking the motif, which have a small-plaque phenotype in LLC-Mk2 and A-Vero cells and are phenotypically normal in RD cells. Substitution of flanking amino acids also affected plaque size. The results suggest that interaction between the RGD motif and alpha v beta 3 is not critical for virus viability in the cell lines tested and therefore that alternative regions of the CAV-9 capsid are involved in internalization.     

Persistent infection of cells in culture by measles virus. I. Development and characteristics of HeLa sublines persistently infected with complete virus

Rustigian, Robert (Tufts University School of Medicine, Boston, Mass.). Persistent infection of cells in culture by measles virus. I. Development and characteristics of HeLa sublines persistently infected with complete virus. J. Bacteriol. 92:1792-1804. 1966.-After the development of marked cytopathic effects in HeLa cultures infected with the Edmonston strain of measles virus, renewed cell growth occurred, and HeLa sublines persistently infected with measles virus were obtained. Persistent infection has occurred in a large fraction of the cells of infected clonal lines for more than 300 to 500 cell generations during a period of 6 years. One mechanism by means of which infection was maintained in the clonal lines is transmission of virus or viral subunits from cell to cell at division. Continued subculture of the persistently infected populations resulted in the virtual disappearance of cytopathic effects, a marked decrease in the amount of extracellular virus, and alterations in the cytopathogenicity of virus recovered from persistently infected populations. The intracellular virus-host cell events in late passages of the infected clonal lines appeared to be similar to those in cells of primary infected cultures at early stages of infection, as judged by the pattern of viral immunofluorescence and the very low incidence of cells with intranuclear inclusion bodies. Cultures of the persistently infected clonal lines were highly resistant to super infection by parent Edmonston virus. Cultures of one of these clonal lines were just as susceptible as normal HeLa cultures to vaccinia, herpes simplex, and polio type 2 viruses, and a simian agent, with a possible low degree of resistance to the simian agent.     

Defective interfering influenza viruses and host cells: establishment and maintenance of persistent influenza virus infection in MDBK and HeLa cells.

WSN (H0N1) influenza virus upon undiluted passages in different species of cells, namely, bovine kidney (MDBK), chicken embryo (CEF), and HeLa cells, produced a varying amount of defective interfering (DI) virus which correlated well with the ability of the species of cell to produce infectious virus. However, the nature of the influenza DI viral RNA produced from a single clonal stock was essentially identical in all three cells types, suggesting that these cells do not exert a great selective pressure in the amplification of specific DI viral RNAs either at early or late passages. DI viruses produced from one subtype (H0N1) could interfere with the replication of infectious viruses belonging to other subtypes (H1N1, H3N2). DI viral RNAs could also replicate with the helper function of other subtype viruses. The persistent infection of MDBK and HeLa cells could be initiated by coinfecting cells with both temperature-sensitive mutants (ts-) and DI influenza viruses. Persistently infected cultures cultures at early passages (up to passage 7) showed a cyclical pattern of cell lysis and virus production (crisis), whereas, at later passages (after passage 20), they produced little or no virus and were resistant to infection by homologous virus but not by heterologous virus. The majority of persistently infected cells, however, contained the complete viral genome since they expressed viral antigens and produced infectious centers. Selection of a slow-growing temperature-sensitive variant rather than the presence of DI virus or interferon appears to be critical in maintaining persistent influenza infection in these cells.     

Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade.

BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: protection against CB3 on HeLa, protection against CB3-RD on rhabdomyosarcoma (RD) cells, and protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of [35S]methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1-RD, CB3-RD, and CB5-RD virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and 125I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses.     

Lytic and transforming functions of individual products of the adenovirus E1A gene.

To distinguish the individual roles of the 13S, 12S, and 9S adenovirus E1A gene products, we isolated the corresponding cDNA clones and recombined them into both plasmids and viruses. Only the expected E1A mRNA products were made from the corresponding 12S and 13S viruses. The 9S mRNA was detected when the 9S virus was coinfected with the 13S virus but not when either virus was infected alone. The 13S virus formed plaques equally well in 293 cells, HeLa cells, and A549 cells, a human lung oat cell carcinoma line. Plaque titers of the 12S virus were much reduced in HeLa and A549 cells compared with 293 cells, although the 12S virus is multiplicity-dependent leaky in both HeLa and A549 cells. A549 cells were significantly more permissive than HeLa cells for growth of the 12S virus. In A549 cells even at low multiplicities of infection the final yield of 12S virus eventually approached the maximum yield from 293 cells. Expression from the adenovirus early region 2 and early region 3 promoters in HeLa cells was activated in the presence of a 13S cDNA E1A region but not in the presence of a 12S E1A cDNA region. Although defective for lytic growth in HeLa cells, the 12S virus immortalized BRK cells at very high efficiency, whereas infection of these cells with 13S virus, as with wild-type E1A virus, resulted mainly in cell death. The 13S product does have an immortalization function, however, revealed in the absence of adenovirus lytic functions when a plasmid containing the E1A 13S cDNA region was transfected into BRK cells. The 9S virus failed to immortalize infected BRK cells or to interfere with focus formation when coinfected with the 12S virus.     

A group B coxsackievirus/poliovirus 5' nontranslated region chimera can act as an attenuated vaccine strain in mice

The linear, single-stranded enterovirus RNA genome is flanked at either end with a nontranslated region (NTR). By replacing the entire 5' NTR of coxsackievirus B3 (CVB3) with that from type 1 poliovirus, a progeny virus was obtained following transfection of HeLa cells. The chimeric virus, CPV/49, replicates like the parental CVB3 strain in HeLa cells but is attenuated for replication and yield in primary human coronary artery endothelial cell cultures, in a human pancreas tumor cell line, and in primary murine heart fibroblast cultures. Western blotting analyses of CPV/49 replication in murine heart fibroblast cultures demonstrate that synthesis of CPV/49 proteins is significantly slower than that of the parental CVB3 strain. CPV/49 replicates in murine hearts and pancreata, causing no disease in hearts and a minor pancreatic inflammation in some mice that resolves by 28 days postinoculation. A single inoculation with CPV/49 induces protective anti-CVB3 neutralizing antibody titers that completely protect mice from both heart and pancreatic disease when mice are challenged 28 days p.i. with genetically diverse virulent strains of CVB3. That a chimeric CVB3 strain, created from sequences of two virulent viruses, is sufficiently attenuated to act as an avirulent, protective vaccine strain in mice suggests that chimeric genome technology merits further evaluation for the development of new nonpoliovirus enteroviral vectors.     

Properties and origins of infectious rhinovirus type 14 particles of different buoyant densities.

Isopycnic centrifugation of rhinovirus type 14 (RV14), purified from infected HeLa or KB cell cultures, into CsCl gradients resolved two bands of infectious virus particles with buoyant density values of 1.409 +/- 0.007 (H virus) and 1.386 +/- 0.004 (L virus) g/ml. Only H virus was detected by incorporation of radiolabeled uridine into viral RNA, and H virus accounted for the majority of infectivity in gradients. H and L virus could not be differentiated by plaque morphology, extent of neutralization by RV14-specific antiserum, or particle size. Electron microscope studies showed that most L-virus particles were associated with an amorphous material. Treatment of L virus with proteolytic enzymes or rebanding L virus in CsCl gradients resulted in recovery of the majority of infectivity as H virus. Virus purified from cell-free fluids from infected HeLa or KB cell cultures banded only as H virus. HeLa cell cultures challenged with purified H virus and harvested at 3 h postinoculation for virus purification yielded only infectious H virus. Both H and L viruses were detected in cell cultures that had been challenged with purified H virus and harvested at 12 h postinoculation. The data suggest that H virus represents progeny virus, whereas L virus represents sequestered infectious virus particles which become associated with an amorphous material and do not enter into viral replicative processes.     

干扰素膜蛋白Tet-on系统稳定细胞系的建立及对CA16病毒的抑制作用

通过Tet-on调控系统,构建受多西环素诱导表达干扰素诱导的跨膜蛋白(interferon-induced transmembrane proteins 1/2/3,IFITM1/2/3)基因的HeLa细胞系,并初步探索了IFITM蛋白对柯萨奇病毒A16(CA16)的抑制作用.首先将调控质粒pTet-on转染进入HeLa细胞,通过G418筛选出阳性克隆细胞系,在此细胞系基础上共同转染反应质粒pTRE2-IFITM1/2/3和伴侣质粒pTK-Hyg,通过潮霉素筛选出单克隆细胞系,加入多西环素后利用Western印迹筛选出可诱导表达IFITM1/2/3蛋白的单克隆细胞系.使用实时荧光定量PCR(RT-qPCR)检测发现,多西环素诱导表达的IFITM蛋白对不同感染复数(multiplicity of infection,MOI)的CA16具有明显的抑制作用,其中IFITM 3对CA16的抑制效果最为明显.Tet调控IFITM1/2/3基因表达HeLa细胞系的成功建立,为进一步研究IFITM基因的功能及其抗病毒机理提供了一个理想的细胞模型.     

Limited proteolysis of the coxsackievirus and adenovirus receptor (CAR) on HeLa cells exposed to trypsin

Trypsin treatment of HeLa cells results in a limited proteolysis of the coxsackievirus and adenovirus receptor (CAR) after which the cleaved CAR remains cell-associated and tryptic peptides remain associated through disulfide bonds. Trypsin-treated HeLa cells remain susceptible to infection with coxsackievirus B and produce progeny virus at 8 h post-infection in amounts comparable to cells with intact CAR. HeLa cells remove the proteolysed CAR within 15 h and require over 24 h to restore intact CAR to control levels. As turnover is relatively slow, physiological functions that require intact CAR protein may be compromised for more than 24 h following trypsin treatment. Moreover, since removal of proteolysed CAR proceeds at more than twice the replacement rate, trypsin treatment disrupts the receptor-per-cell steady state for at least 24 h.     

Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa sublines and recovery of a HeLa clonal line persistently infected with incomplete virus

Rustigian, Robert (Tufts University School of Medicine, Boston, Mass.). Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa clonal line persistently infected with incomplete virus. J. Bacteriol. 92:1805-1811. 1966.-The effect of viral antibody on persistent infection of HeLa cells by the Edmonston strain of measles virus was investigated by culturing cells from three persistently infected clones in medium supplemented with human immune globulin. The three infected HeLa clones were isolated from a persistently infected parent line. Two sublines which were grown in the presence of measles antibody developed a nonyielder state, wherein there is no detectable virus infectious for normal HeLa cultures. There is, however, continued synthesis of intracellular viral antigen and formation of viral intracytoplasmic inclusion bodies. The development of a nonyielder state was associated with a marked decrease in the degree of hemadsorption in cultures of both sublines. Further studies of the viral properties of non-yielder HeLa cell populations were made with a clone obtained from one of these sublines by plating under antibody. Persistent infection in this line was characterized by synthesis of incomplete virus even when the cells were cultured thereafter in anti-body-free medium. This was evidenced by (i) failure to recover infectious virus from the clonal population despite continued formation of intracellular viral antigen and viral intracytoplasmic inclusion bodies in a majority of the cells, (ii) the presence of only a few cells with surface viral antigen(s) including hemagglutinin, and (iii) the relatively weak antibody response to viral envelope antigen(s) after injection of cells into guinea pigs.     

麻疹病毒沪191对HeLa肿瘤细胞抑制作用的体内外实验研究

研究麻疹病毒减毒疫苗沪191株(MV沪191)在组织培养中和裸鼠体内对HeLa肿瘤细胞的抑制作用.用空斑实验测定MV沪191感染HeLa细胞后细胞裂解液中病毒量;用MTF试验测定MV沪191感染对细胞活性的影响;用流式细胞仪分析测定MV沪191感染引起的细胞凋亡和对细胞周期的影响;HeLa肿瘤细胞背部皮下接种BALB/C裸鼠引起的肿瘤,评估MV沪191体内抑瘤作用.MV沪191感染HeLa细胞后可引起广泛的CPE,感染的HeLa细胞与对照组相比细胞活性明显降低.MV沪191感染HeLa细胞后随着时间延长,G1/G0细胞率明显增多,S期率明显减少,细胞凋亡率明显增加(P＜0.01).给药第60天时瘤内治疗组、静脉治疗组和对照组肿瘤体积平均分别为15.5、64.6、156.4 mm3.瘤内治疗组与对照组相比有显著差异(P＜0.01);静脉治疗组与对照组相比有明显差异(P＜0.05).MV沪191减毒株在组织培养中和裸鼠体内对HeLa肿瘤有明显的杀伤作用.     

Stabilization of "A" particles of coxsackievirus B3 by a HeLa cell plasma membrane extract.

Previous studies in our laboratory showed that HeLa cell plasma membranes were recovered from sucrose gradients in two major bands and that the heavier band possessed a putative inhibitor of uncoating of coxsackievirus B3. It has now been found that the mechanism of inhibition is the stabilization of "A" particles against inactivation at 37 degrees C. [3H]uridine-labeled virions converted to A particles by band 4, the heavier band, were four times more stable at 37 degrees C than those produced by band 3. Partially purified A particles from both bands were equally unstable. It was found that the stabilizing factor was extractable by saline from band 4 and remained soluble after centrifugation (109,000 X g for 2 h). Addition to A particles of this soluble factor isolated from either band 4 or band 3 stabilized the A particles. The stabilizing factor could not be replaced by an extract from band 3 or by bovine serum albumin. Thus, the finding that the membrane factor inhibits virus uncoating by stabilizing A particles against spontaneous disruption at 37 degrees C focuses attention on an inherent problem associated with defining receptor-mediated virus uncoating.     

Measles Virus-Specified Polypeptide Synthesis in Two Persistently Infected HeLa Cell Lines

Measles virus-directed protein synthesis was examined in two HeLa cell lines (K11 and K11A) that are persistently infected with wild-type measles virus. Four viral proteins (H, hemagglutination protein; P, nucleocapsid-associated protein; NP, the major nucleocapsid protein; and M, the matrix protein) were readily detected in both cell lines by immune precipitation of [(35)S]methionine-labeled cell extracts followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, three (H, NP, and M) of the four viral proteins in both K11 and K11A cells differed from the corresponding viral proteins synthesized in HeLa cells acutely infected with the parental wild-type virus. In addition, the M protein from K11A cells migrated significantly more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the M protein from K11 cells, and there appeared to be slight differences in the H and NP proteins between these two persistently infected cell lines. The altered viral proteins detected in K11 and K11A cells appeared to be the result of viral mutations rather than changes in the host cell, since virus recovered from these cells directed the synthesis of similar aberrant viral proteins in HeLa cells. Virus recovered from K11 cells and virus recovered from K11A cells were both temperature sensitive and grew more slowly than wild-type virus. HeLa cells infected with virus recovered from K11 cells readily became persistently infected, resembling the original persistently infected K11 cells. Thus, viral mutations are associated with persistent measles virus infections in cell cultures.     

Apoptosis: a mechanism of cell killing by influenza A and B viruses.

In previous studies, we observed that the virulent avian influenza A virus A/Turkey/Ontario/7732/66 (Ty/Ont) induced severe lymphoid depletion in vivo and rapidly killed an avian lymphocyte cell line (RP9) in vitro. In examining the mechanism of cell killing by this virus, we found that Ty/Ont induced fragmentation of the RP9 cellular DNA into a 200-bp ladder and caused ultrastructural changes characteristic of apoptotic cell death by 5 h after infection. We next determined that the ability to induce apoptosis was not unique to Ty/Ont. In fact, a variety of influenza A viruses (avian, equine, swine, and human), as well as human influenza B viruses, induced DNA fragmentation in a permissive mammalian cell line, Madin-Darby canine kidney (MDCK), and this correlated with the development of a cytopathic effect during viral infection. Since the proto-oncogene bcl-2 is a known inhibitor of apoptosis, we transfected MDCK cells with the human bcl-2 gene; these stably transfected cells (MDCKbcl-2) did not undergo DNA fragmentation after virus infection. In addition, cytotoxicity assays at 48 to 72 h after virus infection showed a high level of cell viability for MDCKbcl-2 compared with a markedly lower level of viability for MDCK cells. These studies indicate that influenza A and B viruses induce apoptosis in cell cultures; thus, apoptosis may represent a general mechanism of cell death in hosts infected with influenza viruses.     

GRP78, a coreceptor for coxsackievirus A9, interacts with major histocompatibility complex class I molecules which mediate virus internalization.

It is becoming apparent that over the years cell infection by virus seems to have evolved into a multistep process in which many viruses employ distinct cell surface molecules for their attachment and cell entry. In this study the attachment and entry pathway of coxsackievirus A9 (CAV-9), a member of the Picornaviridae family, was investigated. It has been known that, although integrin alpha(v)beta3 is utilized as a receptor, its presence alone is insufficient for CAV-9 infection and that CAV-9 also requires a 70-kDa major histocompatibility complex class I (MHC-I)-associated protein (MAP-70) as a coreceptor molecule. We document by protein isolation and peptide sequencing that the 70-kDa protein is GRP78, a member of the heat shock protein 70 family of stress proteins. Furthermore we show by using fluorescence resonance energy transfer (FRET) that GRP78 is also expressed on the cell surface and associates with MHC-I molecules. In addition CAV-9 infection of permissive cells requires GRP78 and also MHC-I molecules, which are essential for virus internalization. The identification of GRP78 as a coreceptor for CAV-9 and the revelation of GRP78 and MHC-I associations have provided new insights into the life cycle of CAV-9, which utilizes integrin alpha(v)beta3 and GRP78 as receptor molecules whereas MHC-I molecules serve as the internalization pathway of this virus to mammalian cells.     

Plasma membrane budding as an alternative release mechanism of the extracellular enveloped form of vaccinia virus from HeLa cells

In HeLa cells the assembly of modified vaccinia virus Ankara (MVA), an attenuated vaccinia virus (VV) strain, is blocked. No intracellular mature viruses (IMVs) are made and instead, immature viruses accumulate, some of which undergo condensation and are released from the cell. The condensed particles may undergo wrapping by membranes of the trans-Golgi network and fusion with the plasma membrane prior to their release (M. W. Carroll and B. Moss, Virology 238:198-211, 1997). The present study shows by electron microscopy (EM), however, that the dense particles made in HeLa cells are also released by a budding process at the plasma membrane. By labeling the plasma membrane with antibodies to B5R, a membrane protein of the extracellular enveloped virus, we show that budding occurs at sites that concentrate this protein. EM quantitation revealed that the cell surface around a budding profile was as strongly labeled with anti-B5R antibody as were the extracellular particles, whereas the remainder of the plasma membrane was significantly less labeled. To test whether budding was a characteristic of MVA infection, HeLa cells were infected with the replication competent VV strains Western Reserve strain (WR) and International Health Department strain-J (IHD-J) and also prepared for EM. EM analyses, surprisingly, revealed for both virus strains IMVs that evidently budded at the cell surface at sites that were significantly labeled with anti-B5R. EM also indicated that budding of MVA dense particles was more efficient than budding of IMVs from WR- or IHD-J-infected cells. This was confirmed by semipurifying [(35)S]methionine-labeled dense particles or extracellular enveloped virus (EEVs) from the culture supernatant of MVA- or IHD-J-infected HeLa cells, respectively, showing that threefold more labeled dense particles were secreted than EEVs. Finally, although the released MVA dense particles contain some DNA, they are not infectious, as assessed by plaque assays.     

Comparative sensitivity of various cell culture systems for isolation of viruses from wastewater and fecal samples.

In efforts to define the most sensitive cell culture systems for recovery of viruses from wastewaters, 181 samples were inoculated in parallel into tube cultures of various cell types and were plaqued in bottle and petri dish cultures of three types of monkey kidney cells. Polioviruses were recovered most frequently in the RD line of human rhabdomyosarcoma cells, group A coxsackieviruses in RD and human fetal diploid kidney (HFDK) cells, group B coxsackieviruses in the BGM line of African green monkey kidney cells, echoviruses in RD and primary rhesus monkey kidney (RhMK) cells, and reoviruses in RhMK cells. BGM cells were unsatisfactory for recovery of viruses other than polioviruses and group B coxsackieviruses, and a line of fetal rhesus monkey kidney (MFK) was not a satisfactory substitute for primary RhMK. With RhMK cells, comparable numbers of virus isolations were made in tube cultures and in plaque assays conducted in bottle cultures, but with BGM and MFK cells, fewer isolations were made by plaquing than by inoculation of tube cultures. In comparative plaque assays on fecal samples under three different overlays in bottle and plate cultures of RhMK, BGM, and MFK cells, it was found that plaquing in the most sensitive system, RhMK, was less efficient for virus recovery than was inoculation of tube cultures of RhMK or HFDK cells. Overall, plaque assays performed in petri dishes in a CO(2) incubator yielded fewer virus isolates than did parallel plaque assays performed in closed bottle cultures. Other limitations of plaque assays for recovery of human enteric viruses are discussed.     

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.     

Double-stranded RNA-dependent protein kinase and 2-5A system are both activated in interferon-treated, encephalomyocarditis virus-infected HeLa cells.

Activation of the interferon-inducible, double-stranded RNA-dependent protein kinase was monitored in monolayer cultures of control and interferon-treated HeLa cells infected with encephalomyocarditis virus. The extent of phosphorylation in the intact cell of the alpha-subunit of eucaryotic protein synthesis initiation factor eIF2 by the kinase was determined for the first time in this type of system, using a two-dimensional immunoblot technique. Virus protein synthesis and the kinetics of activation of the ppp(A2'p)nA (n greater than or equal to 2) system were analyzed in parallel. Enhanced phosphorylation of eIF2-alpha was obvious at 9 h and increased by 12 h postinfection. ppp(A2'p)nA and ppp(A2'p)nA-mediated rRNA cleavage were observed from 6 h. No viral protein synthesis was detected in cells in which a general inhibition of protein synthesis developed with time. It can be concluded that both the kinase and ppp(A2'p)nA system are active in interferon-treated, encephalomyocarditis virus-infected HeLa cells.