Japanese encephalitis virus glycoproteins

Mature Japanese encephalitis (JE) virus, or N-form virus, contained three structural proteins: V-1, V-2, and V-3. The large membrane protein V-3 was glycosylated, whereas both V-1 (the small membrane protein) and V-2 (the nucleocapsid protein) were not. Intracellular (I-form), immature virions from infected chick embryo cells did not contain V-1 but a larger protein NV-2, which was glycosylated. T-form virions, released by LLC-MK₂ cells incubated with tris(hydroxymethyl)amino-methane (Tris), also contained the glycoprotein NV-2 instead of the nonglycosylated and smaller V-1. We therefore concluded that JE contained two structural membrane glycoproteins, at least one of which is modified during morphogenesis. The NV-2 polypeptide was heterogeneous, and slight differences in electrophoretic mobility were detected among the NV-2 polypeptide peaks from glucosamine-labeled I-form and T-form virions, glucosamine-labeled cell extracts, and amino acid-labeled cell extracts. The significance of these differences is not clear, but they may indicate that NV-2 is composed of several proteins of similar molecular weight. By analyzing extracts of infected cells labeled with glucosamine or amino acids, we tentatively classified the intracellular polypeptide NV-3 as a virus-specified nonstructural glycoprotein; this polypeptide may be a proteolytic fragment of V-3. The virus-specified polypeptides NV-5, NV-4, and NV-1 were classified as nonglycosylated, nonstructural proteins.     

The proteins of Japanese encephalitis virus

Polyacrylamide gel electrophoresis of Japanese encephalitis virus (JEV) grown in both LLC-MK₂ and chick embryo cell culture revealed three principal polypeptides with molecular weights of 8,700, 13,500, and 53,000 (V-1, V-2, and V-3, respectively). Infected chick cells that were treated with actinomycin D and cycloheximide contained seven polypeptides not present in uninfected cells. In addition to V-2 and V-3, polypeptides with molecular weights of 10,500, 19,000, 45,000, 71,000, and 93,000 (NV-1 through NV-5) were found; V-1 was not regularly detected. A similar pattern of polypeptides was obtained by radioimmune precipitation of soluble antigens from cytoplasmic extracts of infected, actinomycin-D treated, chick cells. When virions were treated with NP-40, a dense, RNA-rich structure was detected which contained V-2. An extracellular, slowly sedimenting, RNA-poor, hemagglutinating particle with a density comparable to the virion was present in virus preparations from cell culture and contained V-1, V-3, and NV-2.     

Membrane-bound proteins of Japanese encephalitis virus-infected chick embryo cells

The seven Japanese encephalitis virus specific polypeptides found in infected chick embryo cells were all bound to membranes. None were completely released from the membranes by treatment with neutral salt, alkaline salt, or dilute detergent, but two of them were partially released by both the neutral and alkaline salts. The polypeptides were released or attacked by trypsin at unequal rates and in the sequence: NV-5≥ NV-4 > V-3. NV-5 was released as a relatively undegraded soluble polypeptide, NV-4 was extensively degraded, and V-3 was degraded but part of its trypsinderived fragment (TF-2) remained membrane bound. We suggest that the three largest viral polypeptides are bound in such a manner that the larger the polypeptide, the more exposed and superficial it is. Treatment of virions with trypsin produced low molecular weight material and three discrete polypeptide fragments, probably all derived from the large virion envelope protein V-3; two (TF-1 and TF-3) had electrophoretic mobilities similar to the two naturally occurring nonvirion virus-specified polypeptides, NV-1 and NV-3.     

Glycoprotein synthesis in cells infected with vaccinia virus. I. Non-virion glycoproteins

When vaccinia-virus infected HeLa cells were incubated in medium containing radioactively labeled glucosamine, the specific activity of the UDP-N-acetylhexosamine pool increased linearly at a rate similar to that of uninfected cells. Nevertheless, starting at 2 hr after infection, hexosamines were incorporated into glycoproteins at a progressively lower rate. Furthermore, from electrophoretic analysis it appeared as though synthesis of the major glycoproteins of uninfected cells was arrested and new glycoproteins were labeled. After equilibrium centrifugation the major vaccinia-induced glycoproteins did not sediment with infectious virus but a large portion was recovered from fractions rich in cell membranes and containing particulate material. Glycoproteins of similar electrophoretic mobilities were made in vaccinia-infected HeLa and chick cells. Both actinomycin D and cycloheximide inhibited the labeling of all virus-induced glycoproteins whereas rifamycin derivatives had a selective effect.     

Inhibition of protein synthesis by vaccinia virus. II. Studies on the role of virus-induced RNA synthesis

Cytoplasmic RNA synthesis can be detected in vaccinia virus-infected HeLa cells in the presence of 2 micrograms/ml but not 20 micrograms/ml of actinomycin D. When RNA synthesis is observed protein synthesis is inhibited in infected, treated cells. We had previously noted that such a correlation may also be observed in infected, cycloheximide-treated cells. If actinomycin D (20 micrograms/ml) is added to these cells at various times after infection and treatment, the inhibition of protein synthesis seen upon removal of cycloheximide does not continue beyond the point to which it had developed before the actinomycin D was added. These results indicate that the inhibition of protein synthesis can be correlated with the amount of cytoplasmic RNA synthesized in infected cells and that this RNA synthesis and the subsequent inhibition of protein synthesis can be prevented by sufficiently high concentrations of actinomycin D. The cytoplasmic RNA which is synthesized does not appear to consist of double-stranded RNA nor of extensive self complementary regions. The cytoplasmic RNA synthesized in infected, cycloheximide treated cells appears to consist of early virus mRNA which can function as mRNA in vitro in a cell-free system derived from normal cells. An examination of the phosphorylation of ribosomal proteins shows six additional phosphoproteins in infected cells, two of which may be observed in infected cycloheximide-treated cells, suggesting that phosphorylation of ribosomal proteins cannot be directly correlated with the inhibition of overall protein synthesis seen in infected cycloheximide-treated cells.     

Equine herpesvirus type 1 infected cell polypeptides: evidence for immediate early/early/late regulation of viral gene expression

EHV-1 polypeptide synthesis was examined in productively infected rabbit kidney and hamster embryo cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of extracts from [35S]methionine- and 3H-amino acid-labeled-infected and mock-infected cultures revealed the presence of 30 infected cell-specific polypeptides (ICPs) which ranged in apparent molecular weights from 16.5K to 213K. Twenty-two of these ICPs comigrated with virion structural proteins. Four ICPs (203K, 176K, 151K, 129K) were detected in extracts of infected cultures labeled in the presence or absence of actinomycin D (Act D) immediately after release from a 4-hr treatment with cycloheximide (CH). These polypeptides, which were designated as EHV-1 immediate early (alpha) ICPs, were not detected in unblocked (non-CH-treated) infected cells. The most abundant ICP was a 31.5K nonstructural protein which, in addition to a 74K protein, was detected in unblocked infected cells at 2-3 hr postinfection. These proteins appeared to be regulated as early (beta) ICPs, since neither protein was observed in Act D-treated cultures released from CH block. Twelve ICPs were classified as late (gamma) polypeptides on the basis of their reduced synthesis in cultures in which viral DNA replication was inhibited by phosphonoacetic acid. All but one (40K) of these late ICPs corresponded to virion structural proteins.     

Inhibition of synthesis of influenza virus proteins: evidence of two host-cell-dependent events during multiplication.

Camptothecin or a critical dose of actinomycin D added to BHK cells before infection selectively inhibits the synthesis of influenza virus haemagglutinin, neuraminidase and membrane proteins but permits the synthesis of other viral proteins. Specifically, camptothecin allows the synthesis of P₁, P₂, nucleoprotein and the large and small nonstructural proteins. However, α-amanitin added to cells on infection does not selectively inhibit the synthesis of any particular group of viral proteins in this way at any of the concentrations tested. If sufficiently high concentrations of α-amanitin or actinomycin D are used, all viral protein synthesis is inhibited completely.We interpret these data as showing that there are two events during the synthesis of influenza virus proteins which are sensitive to the inhibitors used. One event is inhibited by α-amanitin or by a high concentration of actinomycin D and the other by camptothecin or by a critical concentration of actinomycin D.     

Synthesis of the precursor to avian RNA tumor virus internal structural proteins early after infection.

The synthesis of the 76,000-dalton precursor (Pr 76) of the avian RNA tumor virus internal structural proteins was studied as a function of time after infection of chick embryo fibroblasts (CEF) with avian myeloblastosis virus (AMV). During the course of infection, cells were pulse-labeled with [³⁵S]methionine, lysed, and the labeled viral polypeptide precursor was precipitated with antibody against detergent-lysed AMV. Pr 76 was detected by SDS gel electrophoresis of immune precipitates.The earliest time at which Pr 76 synthesis could be detected was 3 hr after infection. Pr 76 synthesis remained low (about 1% of the level of synthesis several days after infection) and constant from 3 until 7 hr after infection. Between 7 and 9 hr after infection, Pr 76 synthesis increased by fivefold.Cells treated with cycloheximide during the first 8 or 12 hr after infection showed an 85–90% inhibition of Pr 76 synthesis and virus production measured late during infection. This finding does not necessarily imply that early viral protein synthesis is required for a productive infection, because cycloheximide also inhibited chick embryo fibroblast DNA synthesis.If cells were treated prior to and during infection with actinomycin D or cytosine arabinoside, precursor synthesis was still observed early (3 to 7 hr after infection). The amount of precursor synthesized early in the presence of inhibitors was similar to that synthesized in the absence of inhibitors, suggesting that the incoming RNA served as a messenger RNA for Pr 76.     

Electron microscopic study of swine embryonic kidney cells infected with the tick-borne encephalitis virus

Electron microscopic studies of morphological lesions in pig embryo kidney cells (PEK) infected with tick-borne encephalitis (TBE) virus as well as morphology and features of TBE morphogenesis after treatment with actinomycin D, cycloheximide and hypertonic NaC1 concentrations in the medium were carried out. Most marked morphological lesions were observed in the cells after combined effect of high NaC1 concentrations in the medium and inhibitors of protein synthesis. After all kinds of treatment, smooth-contour membrane structures were observed in TBE-infected cells. Their number increased considerably with increasing ionic strength of the medium and subsequent return to normal accompanied by treatment with actinomycin D and cycloheximide. No "budding" particles were found in any case and after any treatment. Features of TBE virus morphogenesis are discussed.     

Intracellular distribution of virus-specific RNA in chick embryo cells infected with Japanese encephalitis virus

Japanese encephalitis virus (JEV) infected chick embryo (CE) cells were treated with 4 mug actinomycin D/ml and 5 mM-D-glucosamine at 2 or 3 h before harvesting. Production of JEV was not affected by the short-time treatment of these drugs. The radioactivity in virus-specific RNA in the glucosamine-treated cells was a-parently higher than in non-treated cells. Nuclear and cytoplasmic extracts were prepared from the JEV-infected cells pulse-labelled with 3H-uridine at 15 h after infection. Analysis of virus RNA in nuclear extracts on sucrose density gradients showed that most of the radioactivity was in 23S RNA, 26S RNA and 8 to 12S RNA. The radioactivity of virus RNA in cytoplasmic extracts was found in 42S RNA and RNA fragments sedimenting at less than 8S.     

Association of influenza virus proteins with cytoplasmic fractions

Cytoplasmic extracts of chick embryo fibroblasts infected with fowl plague virus were separated into fractions containing smooth membranes, rough membranes, free ribosomes and polysomes, and a soluble fraction. Viral proteins were analyzed in these fractions by polyacrylamide gel electrophoresis. The hemagglutinin glycoproteins were found to be associated with rough and smooth membranes, and pulse-chase experiments revealed that the large glycoprotein HA migrates from rough to smooth membranes where it is cleaved into glycoproteins HA₁ and HA. The nonglycosylated envelope polypeptide M was located predominantly in smooth membranes, the nucleocapsid protein NP in a fraction of intermediate density, and the nonstructural polypeptide NS in the fractions containing ribosomes. A large amount of protein P was found in the soluble fraction.If glycoprotein synthesis was inhibited by glucosamine or deoxyglucose the predominant virus-specific component located on smooth membranes was protein HA₀ thought to be the unglycosylated or incompletely glycosylated polypeptide of glycoprotein HA. Like HA, HA₀ migrated from rough to smooth membranes where it was also cleaved into two fragments. These data show that polypeptide HA₀ has a high affinity for membranes and they further underline the close relationship between proteins HA and HA₀.     

The inhibition of influenza virus RNA synthesis by actinomycin D and cycloheximide

Chick embryo fibroblast monolayers infected with influenza virus (WSN strain) were treated with either actinomycin D or cycloheximide at various times after infection. Analysis of cell homogenates indicated that in the presence of actinomycin D, synthesis of RNA complementary to virion type RNA was preferentially inhibited. Consequently, no labeled polysomes are found in cells so treated, although virion-type RNA, isolated as the RNP, was found. Cycloheximide, on the other hand, while suppressing the synthesis of complementary type RNA, completely inhibited the synthesis of virion-type RNA.These results confirm and extend the work of Scholtissek and Rott (1970) that showed the same differential effect of these two drugs on RNA synthesis and are added evidence that in influenza virus-infected cells the messenger RNA is that RNA which is complementary to the RNA found in virions.     

Influence of an arbovirus infection (Sindbis virus) on the protein and ribonucleic acid synthesis of cultivated chick embryo cells

Summary Cultured chick embryo cells infected with a group A arbovirus (Sindbis) showed a decreased synthesis of protein and RNA. Protein synthesis was more markedly depressed than RNA synthesis. Inhibition of protein synthesis started 1 to 1 1/2 hours after infection, and cup to 50 per cent inhibition occurred 6 hours postinfection. Results obtained by using p-fluorophenylalanine, actinomycin D and an interferon preparation suggest that the inhibition of cellular protein synthesis depends on the production of protein(s) coded by the viral genome.Dedicated to Prof. Dr. Dr. C.Hallauer on the occasion of his 70th birthday.     

Reversible inhibition of poxvirus replication by cycloheximide during the early phase of infection

Infection of primary chick embryo fibroblasts with Vaccinia WR, IHD-W, and cowpox virus even at high m.o.i. does not cause drastic early inhibition of host cell protein synthesis. This contrasts with the infection by these viruses of many eucaryotic cells. Cellular protein synthesis of mouse L cells is also only partially inhibited after infection with cowpox virus up to a m.o.i. of 2500 e.b. As predicted by Moss and Filler (1970, J. Virol. 5, 99-108) no irreversible inhibition of poxvirus replication is observed in these cells following the addition of cycloheximide early after infection. The viral cores which accumulate in chick embryo fibroblasts in the presence of cycloheximide are further uncoated after removal of the protein synthesis inhibitor. These poxvirus host cell systems can be used to identify in vivo immediate and putative delayed early viral gene products. Formation of progeny virus, viral DNA synthesis, the sequential formation of viral proteins, and sensitivity to interferon has been demonstrated in chick embryo fibroblasts after reversal of the cycloheximide block. These studies indicate a synchronized replication cycle of poxvirus after reversal of the cycloheximide block.     

Sendai virus RNA synthesis and nucleocapsid formation in the presence of cycloheximide

The effect of cycloheximide on viral specific RNA synthesis and viral nucleocapsid formation was studied in chick embryo cells infected with Sendai virus. When cells were infected at a high multiplicity in the presence of cycloheximide, the 18 S and 35 S viral specific RNAs but not the 57 S (virion) RNA were synthesized during a 90-min period indicating that new protein synthesis is not necessary for the initiation of synthesis of the two smaller RNAs. When cells were treated with cycloheximide 18 hr after infection at the time of maximum viral specific RNA synthesis, the rate of synthesis of the 57 S RNA decreased much more rapidly than synthesis of the 35 S and 18 S RNAs. At the same time after infection, newly synthesized 57 S RNA continued to be converted to viral nucleocapsid in the presence of cycloheximide and the rate of incorporation of uridine-5′-³H into nucleocapsid decreased in parallel with the decrease in 57 S RNA synthesis. This indicates that nucleocapsid assembly continues in the absence of active protein synthesis.     

Proteins specified by group B togaviruses in mammalian cells during productive infections

Seven nonstructural and two of the three virion proteins specified by each of Kunjin, dengue type 2, St. Louis encephalitis, and Japanese encephalitis virus were labeled during replication in Vero and in PS cells which had been treated only with actinomycin. After host protein components were eliminated from electrophoretic profiles of infected cytoplasm using a double-label and subtraction method, all virusspecified proteins were readily identified. All profiles are similar but distinguishable from one another. The proteins are synthesized in the same but not in equimolar proportions for long periods. Molecular weight of the major core protein is 13,500 daltons, but the envelope protein ranges from 51,300 daltons (Kunjin) to 59,000 daltons (dengue). The major core protein of the Kunjin virion appeared to be either unstable in cytoplasm or was rapidly lost by incorporation into virions. No evidence was obtained for posttranslational cleavage; the information encoded in the RNA genome of 4.2 × 10⁶ daltons is sufficient to account for the total of about 370,000 daltons of virus-specified proteins.     

Protein synthesis in pig kidney cells infected with herpes simplex virus type 1

Two polypeptides of apparent mol. mass 87,000 and 35,000 were identified in pig kidney cells infected with herpes simplex virus type 1 (HSV-1) after reversing the cycloheximide block. The synthesis of the polypeptide 87,000 declined from 22 hr post infection (p.i.). Its production was prevented by actinomycin D added to the infected cells after removal of cycloheximide. Evidence is presented that the polypeptide 35,000 may be of the cellular origin.