Infected BHK-21 Cells Research Articles

Detection of FMDV RNA amplified by the polymerase chain reaction (PCR)

Molecular detection of foot-and-mouth disease virus (FMDV) using the polymerase chain reaction (PCR) is a rapid and accurate method. In this study we present PCR for the detection of FMDV RNA in infected BHK cells. Using PCR and two primers selected from the RNA polymerase gene, a conserved sequence in all types and subtypes of FMDV, we were able to detect FMDV RNA present in RNA extracted from the FMDV-infected cells. RNA from uninfected BHK cells gave negative results. Another set of primers selected from the nucleotide sequence of the variable VP1 gene permitted the demonstration of variations among different FMDV Israeli isolates by PCR. Two 01 type FMDV isolates out of a total of 6 FMDV field isolates (including 01 Geshur) gave a positive PCR while two other 01 isolates and two ASIA isolates were detected with the RNA polymerase gene primers but not with the VP1 primers. Serial dilutions of the RNA used in each reaction showed that a very small amount of RNA may be detected by PCR. The PCR products from the RNA polymerase and the VP1 genes were sequenced and the nucleotide sequences obtained were compared with a known nucleotide sequence of the FMDV 01 genome.

Fate of the 6K membrane protein of semliki forest virus during virus assembly

The Semliki Forest virus directs the synthesis of three virus-specific transmembrane proteins p62, 6K, and E1, which all are made in equimolar amounts from a polyprotein precursor molecule. The p62 and E1 spike proteins form heterodimeric complexes in the endoplasmic reticulum before being transported to the cell surface where virus budding occurs. In this study we show that the 6K protein becomes associated to the p62E1 complex in the endoplasmic reticulum and transported with the complex to the cell surface. During virus budding, E1 and p62 (which has matured into the E2 protein) are incorporated into new virions whereas the 6K is mostly excluded. Virus particles released from infected BHK cells contain only about 3% of 6K in their membrane as compared to the spike protein content. The relevance of these findings for the mechanism of SFV assembly is discussed.

Properties of human parainfluenza virus type 3 RNA polymerase/replicase activity in vitro: consensus with other negative-stranded RNA viruses.

A cell-free system supporting transcription, replication, and nucleocapsid assembly of the genome RNA of human parainfluenza virus type 3 (HPF3) is described. Cytoplasmic extracts from infected CV-1 or BHK cells catalyzed the transcription of the entire HPF3 genome, the replication of genome RNA, and the assembly of this RNA into nucleocapsidlike structures. Newly replicated RNA was resistant to micrococcal nuclease digestion and was stable in CsCl gradients, exhibiting the density of authentic HPF3 nucleocapsids. After fractionation of the extracts, the nucleocapsid-containing pellet fraction synthesized viral mRNAs. Reconstitution with the soluble protein fraction was necessary for genome RNA replication and nucleocapsid assembly.

Proteins of Bovine Ephemeral Fever Virus

The proteins of bovine ephemeral fever virus (BEFV) were examined in purified virions and in infected BHK-21 cells. Five structural proteins were named L (180K), G (81K), N (52K), M1 (43K) and M2 (29K). The 81K G protein incorporated [3H]glucosamine, was removed from virions by treatment with Triton X-100 and bound monoclonal antibodies which were both neutralizing and protective. Treatment of virions with Triton X-100 and 0.2 to 1.0 M-NaCl progressively released L, M1 and M2. The N protein remained associated with nucleocapsids in up to 2.5 M-NaCl. The glycoprotein (G), nucleoprotein (N) and matrix protein (M2) were phosphorylated. In BEFV-infected BHK-21 cells, five virus-induced proteins were detected from 12 h post-infection. The L, N, M1 and M2 proteins corresponded to those detected in virions whereas the G protein existed in two forms. In tunicamycin-treated cells these occurred as 67K and 71K non-glycosylated precursors. In the absence of tunicamycin, 77K and 79K glycosylated forms were further modified to produce the 81K virion G protein and a 90K cell-associated form. Five viral proteins were also detected in cells infected with the closely related Berrimah virus; the Berrimah virus G protein was also present in two forms.

Unique amino acid substitutions in the capsid proteins of foot-and-mouth disease virus from a persistent infection in cell culture

Maintenance of a persistent foot-and-mouth disease virus (FMDV) infection in BHK-21 cells involves a coevolution of cells and virus (J. C. de la Torre, E. Martínez-Salas, J. Díez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). The resident FMDV undergoes a number of phenotypic changes, including a gradual decrease in virion stability. Here we report the nucleotide sequence of the P1 genomic segment of the virus rescued after 100 passages of the carrier cells (R100). Only 5 of 15 mutations in P1 of R100 were silent. Nine amino acid substitutions were fixed on the viral capsid during persistence, and three of the variant amino acids are not represented in the corresponding position of any picornavirus sequenced to date. Cysteine at position 7 of VP3, that provides disulfide bridges at the FMDV fivefold axis, was substituted by valine, as determined by RNA, cDNA, and protein sequencing. The modified virus shows high buoyant density in cesium chloride and depicts the same sensitivity to photoinactivation by intercalating dyes as the parental FMDV C-S8c1. Amino acid substitutions fixed in VP1 resulted in altered antigenicity, as revealed by reactivity with monoclonal antibodies. In addition to defining at the molecular level the alterations the FMDV capsid underwent during persistence, the results show that positions which are highly invariant in an RNA genome may change when viral replication occurs in a modified environment.

SV LM21 , a Sindbis virus mutant resistant to methionine deprivation, encodes an altered methyltransferase

The replication of Sindbis virus (SV STD) in cultured Aedes albopictus mosquito cells is sensitive to methionine deprivation. We have suggested from earlier work that this sensitivity is primarily because of a decreased pool of S-adenosyl methionine (ado met) and the resultant failure to methylate the 5′ cap of the viral mRNAs. SV LM21, a strain of Sindbis virus derived in our laboratory from SV STD by serial passage on mosquito cells maintained after infection in low concentrations of methionine, is resistant to methionine starvation. It was proposed that this adaptation to low methionine, and to the resultant low intracellular levels of ado met, reflected the accumulation of mutations which led to the generation of a viral RNA cap methyltransferase with an increased affinity for ado met. We report here kinetic data which distinguished the enzymes coded for by SV STD and SV LM21. Using guanylylimidodiphosphate (GIDP) as the methyl acceptor, radioactively labeled ado met as the methyl donor, and lysates from infected BHK cells as the enzyme source, we calculated from our results that SV LM21, generated a methyltransferase with a K m for ado met 10-fold lower than that generated by either SV STD or the related alphavirus, Semliki Forest virus. In addition, we found that BHK cells infected with SV LM21, generated higher levels of methyltransferase activity than did cells infected with SVSTD and that the SV STD and SV LM21, enzymes differed with respect to their relative activities at elevated temperatures. We conclude from these results that the SV LM21, phenotype is associated with an altered methyltransferase and suggest that this is the basis of the resistance of SV LM21, to methionine deprivation.

A 165 kd protein of the herpes simplex virion shares a common epitope with the regulatory protein, ICP4

We have investigated the possibility that immediate-early (IE) protein ICP4 could be a part of herpes simplex virus type 1 (HSV-1) virion particle. Immunodetection with a monoclonal antibody against ICP4 reveals that a component of the virion, migrating at 165 kd, shares a common epitope with this immediate-early protein. Immunolocalization studies on purified virions indicate that the antigen can be detected only in virions without membranes, and is located outside the capsid, most probably in the tegument. Ultrastructural localizations on HSV-1 infected BHK cells extracted with a nonionic detergent confirm that the protein immunoreacting with anti-ICP4 is present in virions.

Intracellular transport of the glycoprotein of VSV is inhibited by CCCP at a late stage of post-translational processing

The appearance of newly synthesized glycoprotein (G) of vesicular stomatitis virus at the surface of infected BHK cells is inhibited reversibly by treatment with carbonylcyanide m-chlorophenylhydrazone (CCCP). Under the conditions used, CCCP treatment depleted the cellular ATP levels by 40-60%, consistent with inhibition of transport at energy-requiring stages. The G protein that accumulates in cells treated with CCCP is heterogeneous. Most of it is larger than the newly synthesized G protein, is acylated with palmitic acid, and is resistant to endoglycosidase H (Endo H). Most of the arrested G protein is also sensitive to digestion with neuraminidase, indicating that it has undergone at least partial sialylation. A minority of G protein accumulates under these conditions in a less-mature form, suggesting its inability to reach the mid-Golgi compartment. The oligosaccharides of this G protein are Endo-H-sensitive and seem to be partly trimmed. Whereas sialylated G protein was arrested intracellularly, fucose-labelled G protein was able to complete its transport to the cell surface, indicating that a late CCCP-sensitive step separates sialylation from fucosylation. These post-translational modifications indicate that G protein can be transported as far as the trans-Golgi in the presence of CCCP and is not merely arrested in the endoplasmic reticulum.

Ultrastructural study on Japanese isolates of spotted fever group rickettsiae.

Japanese isolates of spotted fever group rickettsiae were observed under a transmission electron microscope. In Vero cells persistently infected with Japanese isolates, small numbers of intracytoplasmic rickettsiae were seen. On the other hand, moderate numbers of rickettsiae were found in the cytoplasm of productively infected BHK cells. The electron-lucent, halo-like zone was found to surround organisms in the cytoplasm of their host cells, which is a prominent characteristic of spotted fever group rickettsiae. Fine structural features of the cell wall revealed thin outer and thick inner leaflets like those observed in other spotted fever group rickettsiae.

Semliki Forest Virus-specific Non-structural Protein nsP3 Is a Phosphoprotein

Antisera were raised in rabbits against fusion proteins consisting of beta-galactosidase and partial amino acid sequences of Semliki Forest virus (SFV)-specific non-structural proteins nsP1, nsP2, nsP3 and nsP4. The antisera were specific since each of them precipitated only one labelled protein of a size expected for nsP1, nsP2, nsP3 or nsP4 from lysates of [35S]methionine-labelled SFV-infected BHK-21 cells. The specific antisera also precipitated p220 (with sequences of nsP1, nsP2 and nsP3), p155 (nsP1 and nsP2) and p135 (nsP3 and nsP4) which have been previously shown to be cleavage products of the polyprotein precursor of the non-structural proteins. nsP1, nsP4 and most of nsP3, together with the virus-specific RNA polymerase activity, were in the mitochondrial pellet (P15) fraction of infected BHK-21 cells whereas nsP2 was evenly distributed between P15 and the supernatant fraction (S15). Only antisera directed against nsP3 sequences precipitated a labelled protein from cells incubated with [32P]orthophosphate during SFV infection. Treatment of the immunoprecipitate with calf alkaline intestinal phosphatase reduced the amount of labelled nsP3 considerably. Immunoprecipitated 32P-labelled nsP3, isolated by SDS-PAGE, was subjected to acid hydrolysis. Both phosphoserine and phosphothreonine but not phosphotyrosine could be identified in the hydrolysate. Approximately twice as much [32P]serine as [32P]threonine was detected in nsP3. P15 and S15 fractions were prepared from [35S]methionine- and 32P-labelled SFV-infected cells and the 35S/32P ratio of nsP3 was determined after immunoprecipitation and SDS-PAGE. The nsP3 in S15 was less heavily phosphorylated (about 50%) than P15-associated nsP3. Anti-nsP3 serum revealed large cytoplasmic vesicles in SFV-infected cells in indirect immunofluorescence microscopy.

Expression of Theiler's murine encephalomyelitis virus protease 3C and polymerase 3D in Escherichia coli and characterization of monospecific sera

Defined DNA fragments of cloned Theiler's murine encephalomyelitis virus genome were used to construct procaryotic recombinant plasmids expressing viral genes 3C and 3D. In these plasmids (pEX-EMBL vectors), viral sequences are fused in-phase behind the Escherichia coli lac Z′ gene which is under the control of the inducible λ Pr promotor. Partially purified fusion proteins were used to immunize Balb/c mice. Sera monospecific for the viral protease 3C and polymerase 3D were obtained. These sera detected their corresponding antigens in situ in infected BHK cells using immunocytochemical reactions.

Absence of antiviral activity of the broad-spectrum picornavirus inhibitor disoxaril (WIN 51711) against hepatitis A virus : Anders Widell, Laboratory of Clinical Virology, University of Lund, Malmö General Hospital, Malmö, Sweden

The replication of Sindbis virus (SVSTD) in cultured Aedes albopictus mosquito cells is sensitive to methionine deprivation. We have suggested from earlier work that this sensitivity is primarily because of a decreased pool of S-adenosyl methionine (ado met) and the resultant failure to methylate the 5′ cap of the viral mRNAs. SVLM21, a strain of Sindbis virus derived in our laboratory from SVSTD by serial passage on mosquito cells maintained after infection in low concentrations of methionine, is resistant to methionine starvation. It was proposed that this adaptation to low methionine, and to the resultant low intracellular levels of ado met, reflected the accumulation of mutations which led to the generation of a viral RNA cap methyltransferase with an increased affinity for ado met. We report here kinetic data which distinguished the enzymes coded for by SVSTD and SVLM21. Using guanylylimidodiphosphate (GIDP) as the methyl acceptor, radioactively labeled ado met as the methyl donor, and lysates from infected BHK cells as the enzyme source, we calculated from our results that SVLM21, generated a methyltransferase with a Km for ado met 10-fold lower than that generated by either SVSTD or the related alphavirus, Semliki Forest virus. In addition, we found that BHK cells infected with SVLM21, generated higher levels of methyltransferase activity than did cells infected with SVSTD and that the SVSTD and SVLM21, enzymes differed with respect to their relative activities at elevated temperatures. We conclude from these results that the SVLM21, phenotype is associated with an altered methyltransferase and suggest that this is the basis of the resistance of SVLM21, to methionine deprivation.

Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer

Evidence is presented which indicates that the dengue-2 virus nonstructural protein NS1 (soluble complement fixing antigen) exists in infected BHK and mosquito cell cultures as part of a stable oligomer. Identification of the dissociation products of the isolated oligomer and comparison of the number of N-linked glycans in native and denatured NS1 is consistent with the idea that the high-molecular-weight form of NS1 is a homodimer. By analyzing lysates of BHK cells infected with St. Louis encephalitis virus or Powassan virus and proteins from dengue-2 virus-infected mouse brain we have demonstrated that the appearance of the high-molecular-weight form of NS1 is a general feature of flavivirus infection. It is formed between 20 and 40 min after NS1 is synthesized and before the protein passes the Golgi apparatus. Both soluble and pelletable extracellular NS1 are also found as the high-molecular-weight form.

Herpes simplex virus type 1-induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis: isolation and characterization of an ICP6 lacZ insertion mutant.

Herpes simplex virus (HSV) encodes a ribonucleotide reductase consisting of two subunits (140 and 38 kilodaltons) whose genes map to coordinates 0.56 to 0.60 on the viral genome. Host cell lines containing the HpaI F fragment which includes the reductase subunit genes of HSV type 1 strain KOS (coordinates 0.535 to 0.620) were generated. Transfection of these cells with a plasmid containing the immediate-early ICP0 gene resulted in the expression of ICP6; interestingly, ICP4 plasmids failed to induce expression, indicating an unusual pattern of ICP6 regulation. One such cell line (D14) was used to isolate a mutant with the structural gene of lacZ inserted into the ICP6 gene such that the lacZ gene is read in frame with the N-terminal region of ICP6. This mutant generated a protein containing 434 amino acids (38%) of the N terminus of ICP6 fused to beta-galactosidase under control of the endogenous ICP6 promoter. Screening for virus recombinants was greatly facilitated by staining virus plaques with 5-bromo-4-chloro-3-indoyl-beta-D-galactoside (X-gal). Enzyme assays of infected BHK cells indicated that the mutant is incapable of inducing viral ribonucleotide reductase activity. Surprisingly, although plaque size was greatly reduced, mutant virus yield was reduced only four- to fivefold compared with that of the wild type grown in exponentially growing Vero cells. Mutant virus plaque size, yields, and ability to synthesize viral DNA were more severely compromised in serum-starved cells as compared with the wild type grown under the same condition. Although our evidence suggests that the HSV type 1 ribonucleotide reductase is not required for virus growth and DNA replication in dividing cells, it may be required for growth in nondividing cells.

Glucose trimming and mannose trimming affect different phases of the maturation of Sindbis virus in infected BHK cells

The roles of glucose and mannose trimming in the maturation of Sindbis virus in BHK cells have been investigated using inhibitors of glycoprotein oligosaccharide processing. In the presence of the glucosidase inhibitor N-methyl-1-deoxynojirimycin the viral glycoproteins were equipped with oligosaccharides of the composition GIc 3Man 8,9(GIcNAc) 2 and the yield of virus in the extracellular medium was reduced as a result of a block in the proteolytic cleavage of the precursor (pE2) of the E2 viral envelope glycoprotein. The mannosidase I inhibitor 1-deoxymannojirimycin (dMM) also inhibited the appearance of virus in the medium and the oligosaccharides on the viral glycoproteins had the composition Man 9(GIcNAc) 2. However, pE2 was cleaved to E2 under these conditions, and it was found that when the yield of virus from the cells and medium together was considered, there was no difference between untreated and dMM-treated cultures, suggesting the presence of intracellular virus particles in the dMM-treated cultures. When examined by electron microscopy, the dMM-treated cultures were found to contain intracellular virus particles. In addition, nucleocapsids were found lining intracellular membranes. These observations taken together with the plaque test data intimate that Sindbis virus preferentially buds from internal membranes in BHK cells treated with dMM. The results confirm the essential role of glucose trimming in the Sindbis virus-BHK cell system and suggest that the initial stages of mannose removal maybe important too.

Phosphorylation of a ribosomal protein in invertebrate cells infected with ultraviolet-inactivated iridovirus type 6

Summary Chilo iridescent virus (CIV) infection induces a rapid shut-off of macromolecular synthesis in insect host cells. We have previously shown that CIV infection induced the appearance of new phosphoproteins in permissive cells of Choristoneura fumiferana (Cf-124) [15]. Among them, phosphorylation of a 31 Kd ribosomal protein (very similar to the S6 protein) was observed 3 h after infection of Cf-124 cells with UV-inactivated CIV. However, such phosphorylation could not be detected in cells infected native CIV in the same conditions. As previously postulated [15], these results could be interpreted either as an absence of phosphorylation of this ribosomal protein, or as a dephosphorylation step occurring very rapidly during the early stages of the replication cycle, in cells infected with native CIV, as reported for the infection of BHK21 cells by vaccinia virus [1].

Regulation of Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) growth in McCoy cells by amino acid antagonism.

Chlamydiae have amino acid requirements for growth in tissue culture as defined by those amino acids whose individual omission from the growth medium prevents chlamydial multiplication. We have tested the hypothesis that this inhibition of growth arises as a result of antagonism between particular amino acids such that inhibition occurs when the concentration of one amino acid is reduced in the presence of the antagonist amino acid at high concentration. Using the Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), in the presence of cycloheximide, the requirement for valine was abrogated by the simultaneous omission of isoleucine, that for phenylalanine by simultaneous omission of tryptophan and that for leucine by simultaneous omission of isoleucine plus valine. The antagonism shown between leucine and isoleucine plus valine appears to be unique among bacteria. In the absence of cycloheximide, GPIC had an additional need for tryptophan, tyrosine and isoleucine; these amino acid requirements were shown for both infected McCoy, HeLa and BHK cells. The results are consistent with a mechanism for regulation of parasite growth which depends on the balance of amino acid concentrations in the extracellular environment.

Expression at the cell surface of native fusion protein of the Newcastle disease virus (NDV) strain Italien from cloned cDNA.

A cDNA library was constructed with poly(A)+-mRNAs from NDV-Italien infected BHK-21 cells. A clone, that hybridized to the F gene mRNA, was sequenced. A long open reading frame encodes for a protein of 553 amino acids, with a calculated molecular weight of 59,153, consisting of twelve cysteine residues and six potential glycosylation sites. The protein sequence contains a hydrophobic region at the N-terminus of F1 and a presumptive long transmembrane fragment near the C-terminus. Comparison of the F proteins from NDV strains Italien and Australia-Victoria shows that the sequences are very similar, with conservation of most cysteine residues and of the potential glycosylation sites. The F coding sequence was inserted into the genome of vaccinia virus under the control of vaccinia P7.5 transcriptional regulatory sequences. Expression of F protein was demonstrated by indirect immunofluorescence with five anti-F monoclonal antibodies known to react with conformational epitopes.

Protein structure and expression among arenaviruses.

The proteins of arenaviruses were first studies by SMADEL and his colleagues (1939, 1940, 1942), with reference to their antigenicity. These workers described the presence of a virus-specific soluble (S) antigen detectable by complement fixation (CF) in homogenates of spleen and lung from LCMV-infected guinea pigs. Soluble antigen could be separated from infectious virus by ultracentrifugation. Repeatedly washed virions reacted poorly in CF tests while the S antigen lost none of its immunoreactivity after ultracentrifugation. These studies were not extended until nearly 3 decades later when Brown and Kirk (1969), Chastel (1970), Simon (1970), and Bro-Jorgensen (1971) described antigens detectable by C F and immunodiffusion in tissues or cell cultures infected with L C M V. Bro-Jorgensen (1971) found two antigenic species by immunodiffusion using infected BHK-21 cells as the antigen source. One antigen was heat stable and resistant to proteolysis, while the second was degraded by both heating and pronase digestion. Both antigens sedimented at a rate of 3.5 S in rate zonal sucrose gradient centrifugation, and based on this S value the molecular weight of the thermolabile S antigen was estimated to be 48 000.

Cytostatic and antiherpesvirus type 1 and 2 activities of 1-beta-D-arabinofuranosylthymine (ara T) prodrugs.

A series of derivatives of the antibiotic 1-beta-D-arabinofuranosylthymine (ara T) was synthesized by esterification of the hydroxy group in the 5'-position of the arabinose moiety of the nucleoside with straight-chain and branched-chain carboxylic acids: acetyl-ara T, butyryl-ara T, valeroyl-ara T, pivaloyl-ara T and palmitoyl-ara T. These ara T prodrugs were evaluated for their effect on growth of L5178y mouse lymphoma cells and noninfected BHK-21 cells as well as for their antiviral activity in Herpes simplex virus type 1 and 2 infected BHK-21 cells. All compounds exhibited a marked antiherpes virus activity, whereas the cytostatic activity of two of them, the pivaleric ester and the palmitic ester, was extremely weak. The relative antiviral indices of the 5'-pivaloyl-ara T and 5'-palmitoyl-ara T were found to be much better than the index of ara T itself.