Infected Baby Hamster Kidney Cells Research Articles

Sequential modification of translation initiation factor eIF4GI by two different foot-and-mouth disease virus proteases within infected baby hamster kidney cells: identification of the 3Cpro cleavage site

Microbiology Society journals contain high-quality research papers and topical review articles. We are a not-for-profit publisher and we support and invest in the microbiology community, to the benefit of everyone. This supports our principal goal to develop, expand and strengthen the networks available to our members so that they can generate new knowledge about microbes and ensure that it is shared with other communities.

Sequential modification of translation initiation factor eIF4GI by two different foot-and-mouth disease virus proteases within infected baby hamster kidney cells: identification of the 3Cpro cleavage site.

Infection of cells by foot-and-mouth disease virus (FMDV) causes the rapid inhibition of cellular cap-dependent protein synthesis that results from cleavage of the translation initiation factor eIF4G, a component of the cap-binding complex eIF4F. Two FMDV proteins, the leader (L) and 3C proteases, have been shown individually to induce cleavage of eIF4GI at distinct sites within baby hamster kidney (BHK) cells. Here, sequential cleavage of eIF4GI by the L and 3C proteases was demonstrated in FMDV-infected BHK cells. The FMDV 3C cleavage site within hamster eIF4GI was localized to a small region (about 40 aa) of the protein, between the sites cleaved by the poliovirus 2A protease and the human immunodeficiency virus type 2 protease. Human eIF4GI was found to be resistant to the action of the FMDV 3C protease. On the basis of amino acid sequence alignments, it was predicted and then verified that substitution of a single amino acid residue within this region of human eIF4GI conferred sensitivity to cleavage by the FMDV 3C protease within cells. Full-length eIF4GI and both forms of the C-terminal cleavage product must be capable of supporting the activity of the FMDV internal ribosome entry site in directing translation initiation.

Efficacy of an Inactivated Vaccine for Equine Herpesvirus Type 1 in a Novel Hamster Model

A vaccine designated F.EHV1(S^–)BHK, which was prepared by formaldehyde treatment of equine herpesvirus type 1 (EHV1)-infected baby hamster kidney cells, stimulated neutralising antibodies in hamsters and rabbits and protected new-born hamsters against a lethal challenge with EHV1 by vaccination of their pregnant mothers. The preparative method was then modified to eliminate virus particles and intraparticulate DNA, producing a vaccine designated Bg.F.EHV1(S^–)BHK. this modification stimulated neutralising antibodies with evidence of in vivo inactivation of the virus above non-specific levels in a mouse model. There were no adverse effects from these vaccines in rodent or rabbit species.

A different mode of entry by dengue-2 neutralisation escape mutant virus.

Entry processes were compared between dengue-2 (DEN-2) virus and a DEN-2 neutralisation escape mutant virus in baby hamster kidney (BHK) cells. The mutant virus (mu6B2) was resistant to neutralisation by a monoclonal antibody, MAb 6B2. Infection of BHK cells by the wild-type DEN-2 virus resulted in direct penetration of the virions into the cytoplasm whereas the mutant virus entered cells by endocytosis. The continual presence of the monoclonal antibody might have asserted some pressure for the mutant to adopt a different mode of entry.

Altered expression of adenovirus 12 DNA-binding protein but not DNA polymerase during abortive infection of hamster cells

Replication of human adenovirus type 12 DNA is blocked in abortively infected baby hamster kidney cells. The activity and accumulation of adenovirus 12 DNA polymerase is equivalent in infected hamster and human cell extracts. However, the accumulation of adenovirus type 12 DNA-binding protein is approximately 120-fold lower in extracts from infected hamster cells when compared to infected permissive human cells. This difference in accumulation is not due to replication of viral DNA during productive infection, since this difference is observed in the presence of hydroxyurea. The DNA-binding protein from infected hamster cells retains the ability to bind denatured DNA-cellulose. An adenovirus 5 early region 1 transformed hamster cell line competent to complement the adenovirus 12 DNA replication defect also stimulates accumulation of the DNA-binding protein even when the cells are treated with hydroxyurea. Thus, the reduced expression of the viral DNA-binding protein may play a role in the mechanism of abortive infection of hamster cells by adenovirus 12.

Regional distribution of Sindbis virus glycoproteins on the plasma membrane of infected baby hamster kidney cells

Sindbis virus-infected baby hamster kidney (BHK) cells were analysed in surface replicas or conventional thin sections after specific immunolabelling with antiviral glycoprotein antibodies in conjunction with colloidal gold-conjugated protein A. Newly synthesized viral glycoproteins were detected, beginning 1 1 2 h after infection, while the virus maturation started 3 h after infection. The glycoproteins appeared to be inserted on the plasma membrane in large spots located mainly in the central area of the cells: no clustering of the labelling was detected. Later, the glycoproteins appeared to arrange linearly in regions in the medial portion of the cells. No labelling was found in the peripheral area or on the cell edges. A drastic change in the surface labelling was detected following the commencement of virus maturation: gold particles were organized mostly in small clusters, each labelling a budding virus. Very few glycoproteins appeared not to be involved in budding figures. The maturation of the virus was clearly regionalized, but during this time it also involved the peripheral area and the cell edges; preferential budding in narrow cellular processes was often observed. It appeared thus that either isolated glycoproteins soon after infection, or clustered glycoproteins at later times, are strictly regionalized on the plasma membrane: however, the early post-infection distribution is clearly different from that seen later during virus maturation. Our experiments support the concept of discrete plasma membrane domains even in cells that do not display distinct specialization of their surface.

The phosphorylation of ribosomal protein S6 by protein kinases from cells infected with pseudorabies virus.

We examined the ability of protein kinase activities from BHK (baby-hamster kidney) cells infected with pseudorabies virus to catalyse the phosphorylation of ribosomal protein S6 in vitro. When the cytosol from infected cells was fractionated on DEAE-cellulose, 40S ribosomal protein kinase activity was found associated with the two isoforms of the cyclic AMP-dependent protein kinase, protein kinase C and a protein kinase (ViPK, virus-induced protein kinase) only detected in infected cells. The phosphorylation of ribosomal protein by ViPK was of particular interest because the appearance of the protein kinase and the increase in the phosphorylation of protein S6 in infected cells shared a similar time course. At moderate concentrations of KCl the major ribosomal substrate for ViPK was ribosomal protein S7, a protein not found to be phosphorylated in vivo. However, at 600 mM-KCl, or in the presence of 5-10 mM-spermine at 60-150 mM-KCl, the phosphorylation of ribosomal protein S7 was suppressed and ribosomal protein S6 became the major substrate. The maximum stoichiometry of phosphorylation obtained under the latter conditions was 1-2 mol of phosphate/mol of S6, and only mono- and di-phosphorylated forms of S6 were detected on two-dimensional gel electrophoresis. As the infection of BHK cells by pseudorabies virus results in the appearance of phosphorylated species of S6 containing up to 5 mol of phosphate/mol of S6 protein, it appears unlikely that ViPK alone can be responsible for the multiple phosphorylation seen in vivo. Nevertheless, tryptic phosphopeptide analysis did indicate that in vitro ViPK catalysed the phosphorylation of at least one of the sites on ribosomal protein S6 phosphorylated in vivo, so that a contributory role for the enzyme in the phosphorylation in vivo cannot be excluded.

Immunocytochemical study of the partition and distribution of Sindbis virus glycoproteins in freeze-fractured membranes of infected baby hamster kidney cells.

Sindbis virus-infected baby hamster kidney cells were analyzed by thin section fracture-label. Specific immunolabel with antiviral glycoprotein antibodies or with conventional lectin label (wheat germ agglutinin) were used in conjunction with colloidal gold-conjugated protein A or ovomucoid, respectively. In addition, intact infected cells were analyzed with both labeling procedures. Experiments with Sindbis infected-chick embryo fibroblast cells were carried out as controls. Viral transmembrane glycoproteins appeared present in freeze-fractured inner and outer nuclear membrane, endoplasmic reticulum, Golgi stacks and vesicles, and plasma membranes; a clear preferential partition with the exoplasmic faces of all intracellular membranes was observed. By contrast, at the plasma membrane level, Sindbis glycoproteins were found to partition preferentially with the protoplasmic face. It seems likely that this protoplasmic partition is related to the binding with the nucleocapsid that takes place during the budding of the virus. At the cell surface, viral glycoproteins always appeared clustered and were predominantly associated with budding figures: moreover, large portions of the plasma membrane were devoid of both glycoproteins and budding viruses.

The interaction of a topoisomerase-like enzyme from herpes simplex virus type 1-infected cells with non-viral circular DNA.

An enzyme activity from herpes simplex virus type 1 (HSV-1)-infected baby hamster kidney cells has been identified which generates large networks of pBR322 DNA from a monomeric DNA substrate. Extracts derived from cells infected at the non-permissive temperature, with the early regulatory mutants of HSV-1, tsK and tsB2, did not contain activity, suggesting that the enzyme is virus-induced and may be virus-specific. The enzyme is similar to the DNA topoisomerases in that network formation was dependent upon the presence of Mg2+ and a DNA condensing agent, and ATP was not required. Following digestion with EcoRI, the networks could be resolved to a single, linear, monomeric species of pBR322 DNA.

On the role of oligosaccharide trimming in the maturation of Sindbis and influenza virus.

The alpha-glucosidase inhibitor bromoconduritol inhibits the formation of the N-linked, complex-type oligosaccharides of the glycoproteins from influenza viruses (fowl plague virus, influenza virus PR-8) and from sindbis virus. Viral glycoproteins produced in bromoconduritol-treated chicken-embryo and baby-hamster kidney cells are fully glycosylated, but accumulate N-linked, high-mannose oligosaccharides of the composition Glc1Manx (GlcNAc)2 (x = 7, 8, and 9). Other alpha-glucosidase inhibitors (nojirimycin, deoxynojirimycin, acarbose) were not specific inhibitors of oligosaccharide processing under the conditions used in the present investigation. In bromoconduritol-treated, sindbis virus-infected chicken-embryo and baby-hamster kidney cells, the sindbis glycoproteins are metabolically stable. Specific proteolytic cleavage of the polyprotein precursors to form E2 and E1 occurs in bromoconduritol-treated chicken-embryo cells, but cleavage of PE2 to E2 is prevented in the infected baby-hamster kidney cells. Yet, release of infectious sindbis virus particles is inhibited in both cell types indicating that the formation of complex oligosaccharides is required for a late step in virus formation. The release of virus particles from influenza virus PR-8-infected bromoconduritol-treated chicken-embryo cells is not inhibited, and virus with only high-mannose oligosaccharides is formed. In contrast, when chicken-embryo cells were infected with the influenza virus fowl plague virus, release of infectious particles was inhibited. The fowl plague virus hemagglutinin is cleaved in chicken-embryo cells, in contrast to the hemagglutinin of the PR-8 virus. However, the cleavage products HA1 and HA2 do not reach the cell surface. In addition, or as a consequence, HA1 and HA2 are proteolytically broken down, whereas uncleaved hemagglutinin of PR-8 appeared metabolically stable. These results may explain the decrease in formation of fowl plague virus particles and the lack of effect on PR-8 virus in bromoconduritol-treated cells. This work thus shows different biological roles for oligosaccharide processing.

Golgi processed foot-and-mouth disease virus proteins

The Golgi apparatus was isolated from infected baby hamster kidney cells by centrifugation through discontinuous sucrose gradients. Tritium-labeled protein samples were analyzed by polyacrylamide gel electrophoretic autoradiograms. Pulse-chase studies showed that the viral-induced RNA polymerase passed through the Golgi as infection progressed. Some viral coat proteins were also associated with the Golgi as were other as yet unidentified viral proteins (Fig. 1). Immune labeling of isolated and in situ Golgi confirmed the presence of viral RNA polymerase. The isolated (Fig. 2) and in situ Golgi were labeled with guinea pig antipolymerase antibody and ferritin-labeled goat anti-guinea pig sera to show the presence of viral RNA polymerase.Earlier work in this laboratory established that the RNA polymerase was bound to membranes of newly formed smooth vacuoles during infection with FMDV. The smaller protein on the gel in Fig. 1 (arrow) corresponds in size to VPg (a nonstructural protein bound to the 5' end of viral RNA) has also been shown to be membrane bound.

Cell-free translation of frog virus 3 messenger RNAs. Initiation factors from infected cells discriminate between early and late viral mRNAs.

Cell-free protein-synthesizing extracts prepared from rabbit reticulocytes, wheat germ, or cultured baby hamster kidney cells efficiently translated frog virus 3 early mRNAs; in contrast, late mRNAs were translated poorly under similar conditions. However, the translational efficiency of the late viral mRNAs was markedly enhanced in cell-free extracts prepared from frog virus 3 (FV 3)-infected baby hamster kidney cells and in nuclease-treated rabbit reticulocyte extracts by the addition of a 0.5 M KCl wash from FV 3-infected cell ribosomes; the 0.5 M KCl wash (initiation factors) from uninfected cells had no such effect. Total cytoplasmic RNA from infected cells was fractionated according to size on sucrose gradients and fractions containing different concentrations, and relative proportions of early and late mRNAs were translated in either native or initiation factor-supplemented extracts. Under these conditions, the translation efficiency of early mRNAs was unchanged, while the translation of late mRNAs increased 2-7-fold. Thus, the in vitro discriminatory activity of the 0.5 M wash was not dependent on the complexity of the mRNAs present in the translation mixture. We show also that in native extracts, under conditions of blocked polypeptide chain elongation, early mRNAs are initiated preferentially. However, late as well as early mRNAs are initiated equally well in reticulocyte extracts under similar experimental conditions when supplemented with crude initiation factors from infected cells. These data support the conclusion that the translational enhancement of FV 3 mRNAs in vitro is mediated by a virus-specified or virus-modified initiation factor(s) and likely represents a regulatory mechanism of protein synthesis operative in vivo (Willis, D. B., Goorha, R., Miles, M., and Granoff, A. (1977) J. Virol. 24, 326-342).

The effect of polyamines on herpes simplex virus type 1 DNA polymerase purified from infected baby hamster kidney cells (BHK-21/C13).

The DNA polymerase whose synthesis is directed by the herpes simplex virus type 1 (HSV-1) DNA was purified 545-fold from BHK-21/C13 cells 16 h after infection with the virus. Spermidine and spermine stimulated the activity of the polymerase over the concentration range 0.5 mM to 2.5 mM. This effect was enhanced with increased concentrations of polyamine, maximum stimulation being threefold and fourfold for spermidine and spermine respectively. The diamine, putrescine, had little effect on the enzyme at the concentrations used (0.25 to 1.25 mM).

Isolation of foot-and-mouth disease virus messenger RNA from membrane-bound polyribosomes and characterization of its 5′ and 3′ termini

A membrane-bound fraction prepared from foot-and-mouth disease virus (FMDV)-infected baby hamster kidney cells included [ 3H]uridine-labeled 37 S viral messenger RNA (mRNA) and a 20 S RNA species that is partly ribonuclease resistant. The mRNA appeared to contain both a polyadenylic acid (poly (A)) and a polycytidylic acid (poly (C)) tract as demonstrated by its binding to oligo(dT)- and oligo(dG)-cellulose, respectively. Paper chromatography of an RNase T 2 digest of the [ 3H]adenosine-labeled poly(A) fragment from mRNA showed that the poly(A) is at the 3′ end of the mRNA and is approximately 45 nucleotides long. Digestion of 32P-labeled FMDV mRNA with RNase A and T 2 yielded pUp as the 5′ terminus; RNases A and T 2 digested FMD virion RNA lacked this structure.

Nucleic Acid of Rubella Virus and Its Replication in Hamster Kidney Cells

Ribonucleic acid (RNA) has been isolated from partially purified rubella virus preparations and fractionated by rate zonal centrifugation in sucrose density gradients. The bulk of the RNA sedimented as a sharp band with a sedimentation coefficient of 38S. Rubella virus RNA appears to be single-stranded on the basis of its sensitivity to the degrading action of ribonuclease. Fractionation by precipitation with 1 m NaCl, followed by chromatography on cellulose columns, and by rate zonal centrifugation in sucrose density gradients of labeled RNA isolated from actinomycin D-treated and infected baby hamster kidney cells revealed the presence of the following virus-specific types of RNA: (i) single-stranded RNA with a heterogeneous sedimentation pattern, the 38S viral RNA becoming the predominant species only after long periods of labeling late after infection; (ii) double-stranded RNA with a sedimentation coefficient of 20S; (iii) RNA apparently composed of 20S double-stranded RNA and single-stranded branches. On the basis of their properties, the last two species were tentatively identified as the replicative form and the replicative intermediate of rubella virus RNA. Rubella virus RNA was infectious.

The isolation of two enzyme-ribonucleic acid complexes involved in the synthesis of foot-and-mouth disease virus ribonucleic acid.

The foot-and-mouth disease virus-RNA polymerase complex was released from membrane particulates present in the cytoplasm of infected baby hamster kidney cells. The soluble polymerase complex was fractionated by zonal centrifugation in sucrose gradients. Two polymerase complexes (RNA and protein complex) active in the cell-free system were isolated and had S-rate ranges of 20-70S and 100-300S, respectively. The light polymerase complex contained 20S double-stranded RNA; and the heavy polymerase complex contained a polydisperse, partially RNase-resistant RNA. The cell-free product of these two polymerase complexes was analyzed by zonal centrifugation in sucrose gradients. The light polymerase complex synthesized only 20S double-stranded RNA. The product of the heavy polymerase complex contained no detectable 20S double-stranded RNA and only a peak of single-stranded RNA with S-rate corresponding to 37S viral RNA. A third polymerase complex was isolated with S-rate greater than 300S, and it contained a polydisperse, partially RNase-resistant RNA. This third polymerase complex synthesized both 37S viral RNA and 20S double-stranded RNA in the cell-free system, and it is probably the native polymerase complex still bound to cellular particulates.

HERPES ANTIGENS FROM HAMSTER KIDNEY CELL-CULTURES IN THE DIAGNOSIS OF PRIMARY AND RECURRENT INFECTIONS.

Large yields of potent herpes CF antigen were prepared from infected baby hamster kidney cells (BHK21-C13) grown in 80 oz. cylindrical winchester bottles rotating about their long axes at approximately 0.3 r.p.m. Crude antigen extracted from the cells with distilled water as diluent showed no significant drop in titre when retested after 3 and 6 months storage at +4° C or −70° C; antigen stored at −40° C showed a significant drop in titre together with a decrease in sensitivity.