Transient Inhibition of Polyoma Virus Synthesis by Sendai Virus (Parainfluenza I) II. Mechanism of the Interference by Inactivated Virus

The mechanism of the transient inhibition of polyoma virus synthesis by betapropiolactone-inactivated Sendai virus was studied. Polyoma virus early functions did not appear to be affected, although deoxyribonucleic acid (DNA) and structural protein synthesis were inhibited 60 and 35% respectively. The inhibition of macromolecular synthesis was not sufficient to account for the 90% inhibition of infectious progeny formation. Encapsidation of polyoma DNA into mature virions appears to be completely inhibited after superinfection by beta-propiolactone-inactivated Sendai virus. Ultraviolet irradiation of live or beta-propiolactone-inactivated Sendai virus preparations abolishes the interfering capacity, indicating that a functional Sendai virus ribonucleic acid molecule is the interfering component.     

Homologous Interference by Incomplete Sendai Virus Particles: Changes in Virus-Specific Ribonucleic Acid Synthesis

Incomplete Sendai virus particles (I particles) interfered with the replication of several strains of infectious Sendai virions (standard virus) but not with the replication of Newcastle disease virus, mumps virus, or Sindbis virus. I particles did not induce interferon, and ultraviolet irradiation of I particles abolished their ability to interfere. Protein synthesis was not necessary to establish interference. The degree of interference depended on the interval between exposure of cells to the I particles and challenge by standard virus, and this was reflected in the degree of inhibition of virus-specific ribonucleic acid (RNA) synthesis in infected cells. The most dramatic change was decreased accumulation of 50S virus-specific RNA in infected cells. RNA species sedimenting slower than 50S were not as markedly reduced in total amount, but hybridization experiments showed that a substantial portion of these slowly sedimenting RNA species were plus strands, presumably representing replicas of the RNA species in I particles. When I particles in insufficient numbers to interfere were added to cells as late as 8 hr after standard virus, there were no obvious changes in virus-specific RNA species in the cells; however, significant amounts of 19 and 25S RNA species, representing progeny of the I particles, appeared in the culture medium. It was concluded that interference was an intracellular event affecting an early step in virus replication. Competition by I particles for cell sites or substrates needed by standard virus seemed a less likely mechanism of interference than competition for enzymes specified by standard virus.     

Effect of the Inhibition of Protein Synthesis on the Establishment of Transformation by Polyoma Virus

Puromycin was used to study the effect of the inhibition of protein synthesis on transformation of hamster cells (BHK21) by polyoma virus. The drug was used at a concentration (10(-4)m) which caused in these cells a drastic but fully reversible inhibition of protein synthesis. A two- to threefold enhancement of transformation rate was obtained when the cells were exposed to puromycin for a period of 5 hr that started at the end of the virus adsorption period. No further enhancement was produced by prolonging puromycin treatment up to 13 hr after infection. The possibility that the observed effect on transformation rate could be mainly attributed to cell selection by puromycin was excluded. In addition, the relevance of a number of possible secondary effects of puromycin (inhibition of cell division, inhibition of deoxyribonucleic acid synthesis, etc.) was also ruled out. The effect of puromycin on transformation appeared to be dependent on the time (relative to infection) of addition of the drug. In fact, no transformation enhancement was observed when the cells were exposed to puromycin prior to infection or beyond the 10th hr after infection. Since another drug known to affect protein synthesis (p-fluorophenylalanine) was also shown to produce similar effects, it is suggested that transformation enhancement results from the inhibition of protein synthesis during a sensitive period closely following adsorption of the virus.     

Inhibition of Host Cell Ribosomal Ribonucleic Acid Methylation by Foot-and-Mouth Disease Virus

A study of protein and ribonucleic acid (RNA) synthesis in cells infected by foot-and-mouth disease virus has indicated possible mechanisms of viral control over host cell metabolism. Foot-and-mouth disease virus infection of baby hamster kidney cells resulted in 50% inhibition of host cell protein synthesis at 180 min postinfection. A viral-induced interference with host cell RNA methylation was observed to be more rapidly inhibited than protein synthesis. To determine the nature of methylation inhibition, the kinetics of several host cell methylated RNA species were examined subsequent to virus infection. Data from sucrose zonal centrifugation and methylated albumin kieselguhr chromatography showed that methylation of nuclear RNA was inhibited 50% at 60 min postinfection. Inhibition of nuclear ribosomal RNA precursors and formation of nascent ribosomes correlated with inhibition kinetics of nuclear RNA methylation. It is suggested that the viral interference with the host nuclear RNA methylation is directly responsible for the observed loss of nascent ribosome formation. Moreover, early in the infectious cycle, methylation inhibition of host cell RNA could, in part, account for the cessation of host protein synthesis.     

Enhancement of 32P Incorporation into Phospholipids in Cultured Cells by Sendai Virus of Parainfluenza Type 1

Sendai virus infection induced enhancement of ³²P incorporation into phospholipids in chick embryo, monkey kidney and bovine kidney cells, as previously observed in chorioallantoic membranes of chick embryos. These findings indicate that phospholipid synthesis is enhanced upon Sendai virus infection. Ultraviolet irradiation abolished the ability of the virus to induce the enhanced synthesis of phospholipids, a fact suggesting that the phenomenon depends upon infectivity of the virus. Gamma irradiation of host cells little affected the enhanced ³²P labeling of cellular phospholipids, suggesting that the function of host cell DNA may not be directly involved in the phenomenon.     

Effect of Ultraviolet Irradiation and Actinomycin D on Polyoma Virus Replication in Mouse Embryo Cell Cultures

Ultraviolet irradiation and actinomycin D impair the capacity of mouse embryo (ME) cells to support the replication of polyoma virus, but not of encephalomyocarditis (EMC) virus. The loss in capacity for polyoma virus synthesis was an “all-or-none” effect and followed closely upon the loss in cellular capacity for clone formation. Cells treated with either agent produced polyoma “T” antigen, but did not synthesize polyoma structural protein. Infection of untreated ME cells with polyoma virus produced marked stimulation of both deoxyribonucleic acid (DNA) synthesis and ribonucleic acid (RNA) synthesis. ME cell cultures irradiated with ultraviolet for 30 sec at 60 μw/cm² or treated with actinomycin D at 0.1 μg/ml for 6 hr prior to infection were incapable of synthesizing DNA or RNA, even after infection with polyoma virus. Irradiation of cells during infection produced cessation of synthesis of both RNA and DNA. Addition of actinomycin D during infection did not inhibit DNA synthesis but abolished RNA synthesis and reduced the yield of polyoma virus to 10% of that in untreated infected cultures. Both agents lost the ability to prevent replication of a full yield of polyoma virus when administered 30 hr after infection or later. The period after which neither agent inhibited polyoma replication corresponded with the period at which maximal RNA synthesis in untreated infected cultures had subsided. It can be concluded on the basis of the data presented that the functional integrity of the mouse embryo cell genome is required for the replication of polyoma virus, but not for EMC virus. Whereas the requirement for cellular DNA-dependent RNA synthesis for polyoma virus replication has been demonstrated, the exact nature of the host-cell function remains to be elucidated.     

Chilling of somatic cells unnecessary for Sendai virus-induced heterokaryon formation.

Incubation of cell suspensions or monolayers at 4 degrees C for 20 min after addition of beta-propiolactone-inactivated Sendai virus did not enhance heterokaryon formation as compared to parallel cell cultures incubated at 37 degrees C immediately after the addition of Sendai virus. These findings show that the routine chilling of somatic cells in fusion experiments can be omitted.     

Trypsin Action on the Growth of Sendai Virus in Tissue Culture Cells: I. Restoration of the Infectivity for L Cells by Direct Action of Trypsin on L Cell-Borne Sendai Virus

Sendai virus grown in fertile eggs (egg Sendai) infects L cells in which the synthesis of L Sendai (grown in L cells) occurs by the one-step mechanism. L Sendai is not infectious for L cells when tested by the tube titration method although it is infectious for chick embryos. When L cells infected with egg Sendai were dispersed by trypsin and plated on a monolayer culture of L cells, the viral agents spread to the adjacent recipient cells in which the synthesis of L Sendai occurred. The newly infected L cells became infectious for L cells again by trypsin treatment. Kinetic experiments suggested that the target of trypsin is the mature virus, of L Sendai nature, just budding from the L-cell surface. By using an immunofluorescent cell-counting technique, recovery of the infectivity of L Sendai for L cells due to a direct enzymatic action of trypsin was demonstrated. Under the optimal condition, the infectivity increased 1,000-fold for L cells and 10-fold for chick embryos, and both the titers could favorably be compared. No increasing effect of trypsin was observed on the infectivity of egg Sendai. Density centrifugation studies revealed a difference between egg Sendai and L Sendai in the density. Trypsin treatment which induced the maximal enhancement of L Sendai infectivity did not affect both the densities, showing that variations of Sendai virus in the infectivity for L cells and in the density are independent types of host-controlled modification.     

Polyoma-induced stimulation of cellular RNA synthesis is paralleled by changed expression of the viral genome.

We studied synthesis of viral and cellular RNA in the presence and absence of 5-fluorodeoxyuridine (FdU, an inhibitor of DNA synthesis) during lytic infection with polyoma virus in confluent, primary mouse kidney cell cultures. In the presence of FdU, synthesis of early 19S polyoma mRNA and of polyoma tumor (T)-antigen, i.e. expression of the early viral gene, is rapidly followed by a mitogenic reaction of the host cell; it leads to an increase of 30 +/- 5% in cellular, mainly 28S and 18S rRNA, followed by activation of the cellular DNA-synthesizing apparatus. Polyoma-induced cellular RNA synthesis is paralleled by increased production of early 19S mRNA and begin of expression of the late viral genes, leading to synthesis of small amounts of late 19S and 16S mRNAs. Changed expression of the viral genome occurs in the absence of detectable synthesis of polyoma DNA I. Infection in the absence of FdU induces the same sequence of events; it is followed, however, by duplication of the mouse cell chromatin (S-phase) and production of progeny virus.     

DNA synthesis in polyoma virus infection. IV. Mechanism of formation of closed-circular viral DNA deficient in superhelical turns.

A marked reduction in the rate of viral DNA synthesis is accompanied by an alteration to the superhelicity of progeny DNA in polyoma virus-infected cells in which protein synthesis has been inhibited by cycloheximide. Viral DNA molecules formed in the presence of cycloheximide consist predominantly of closed-circular monometric species (referred to as form Ic) characterized by a decreased superhelix density, corresponding to deltasigmao = 0.0195, as compared to form I DNA by propidium diiodide-cesium chloride isopycnic analysis. Form Ic is synthesized on pre-existing form I templates without the intervention of progeny form I as an intermediate. It is concluded that inhibition of protein synthesis results in the alteration of some process in the closure of daughter DNA that leads to a marked reduction of superhelical turns of progeny molecules. About two-thirds of form Ic molecules return to the form I conformation upon reversal of cycloheximide inhibition by a mechanism independent of DNA replication.     

State of the viral DNA in rat cells transformed by polyoma virus. I. Virus rescue and the presence of nonintergrated viral DNA molecules.

The interaction of polyoma virus with a continuous line of rat cells was studied. Infection of these cells with polyoma did not cause virus multiplication but induced transformation. Transformed cells did not produce infectious virus, but in all clones tested virus was rescuable upon fusion with permissive mouse cells. Transformed rat cells contained, in addition to integrated viral genomes, 20 to 50 copies of nonintegrated viral DNA equivalents per cell (average). "Free" viral DNA molecules were also found in cells transformed by the ts-a and ts-8 polyoma mutants and kept at 33 C. This was not due to a virus carrier state, since the number of nonintegrated viral DNA molecules was found to be unchanged when cells were grown in the presence of antipolyoma serum. Recloning of the transformed cell lines produced subclones, which also contained free viral DNA. Most of these molecules were supercoiled and were found in the muclei of the transformed cells. The nonintegrated viral DNA is infectious. Its specifici infectivity is, however, about 100-fold lower than that of polyoma DNA extracted from productively infected cells, suggesting that these molecules contain a large proportion of defectives.     

Inhibition of Herpes Simplex Virus Type 2 Replication by Thymidine

Replication of herpes simplex virus type 2 (HSV-2) was impeded in KB cells which were blocked in their capacity to synthesize DNA by 2 mM thymidine (TdR). The degree of inhibition was dependent upon the concentration of TdR. In marked contrast, HSV-1 is able to replicate under these conditions. The failure of HSV-2 to replicate is probably due to the inhibition of viral DNA synthesis; there was a marked reduction in the rate of DNA synthesis as well as the total amount of HSV-2 DNA made in the presence of 2 mM TdR. We postulated that the effect of TdR on viral replication occurs at the level of ribonucleotide reductase in a manner similar to KB cells. However, unlike KB cells, an altered ribonucleotide reductase activity, highly resistant to thymidine triphosphate inhibition, was found in extracts of HSV-2-infected KB cells. This activity was present in HSV-2-infected cells incubated in the presence or absence of TdR. Ribonucleotide reductase activity in extracts of HSV-1-infected KB cells showed a similar resistance to thymidine triphosphate inhibition. These results suggest that the effect of TdR on HSV-2 replication occurs at a stage of DNA synthesis other than reduction of cytidine nucleotides to deoxycytidine nucleotides.     

Origin of the Thymidine Kinase Induced by Polyoma Virus in Productively Infected Cells

Cells of the 3T3 mouse line efficiently supported the multiplication of polyoma virus, and the infectious process was accompanied by a marked increase in thymidine kinase (TK) activity. Two lines of 5-bromodeoxyuridine-resistant 3T3 cells have been isolated. As expected, these cells incorporated practically no exogenous thymidine into their deoxyribonucleic acid (DNA) and contained negligible TK activity. Like the parental 3T3 cells, TK(-) lines were susceptible to productive infection by polyoma virus, but infection did not lead to an increase in TK activity. Since kinase activity did appear after infection with another virus (vaccinia) known to contain the gene(s) for that enzyme, it is concluded that TK is not one of the gene products of polyoma virus. As induction of cellular DNA synthesis by polyoma virus occurs normally when the TK(-) cells are infected in the stationary phase, TK cannot play a role in the determination of this phenomenon.     

Nature of Transient Inhibition of Deoxyribonucleic Acid Synthesis in HeLa Cells by Parainfluenza Virus 1 (Sendai)

Adsorption of ultraviolet-inactivated Sendai virus, at high or low multiplicity, to HeLa cells caused a transient increased incorporation of (3)H-thymidine into the cellular deoxyribonucleic acid (DNA). In HeLa cells synchronized by a double-thymidine block, this increased incorporation of thymidine during the S phase lasted from about 30 to 90 min after virus adsorption. The observations that the kinetics of accumulation of radioactive thymidine in the nucleotide pool did not differ in control and in the virus-treated cells and that the (32)P incorporation into the DNA of the virus-treated cells was inhibited at the same time indicate that the augmented incorporation of (3)H-thymidine into DNA results from a transient block in the endogenous pathway of thymidine synthesis. Chromatographic analysis of the nucleotide pool of the virus-treated cells labeled with (14)C-formate indicates that methylation of deoxyuridine monophosphate to thymidine monophosphate is inhibited. It is suggested that the inhibition is caused by a block of either the thymidilate synthetase or some step in the tetrahydrofolate cycle.     

Transient Inhibition of Avian Myeloblastosis Virus Reproduction by Amethopterin and Fluorodeoxyuridine

Avian myeloblastosis virus cannot initiate its reproduction in the presence of amethopterin or fluorodeoxyuridine. This inhibition is reversed by thymidine. Addition of either inhibitor after virus production has started does not inhibit further virus synthesis. In presence of either inhibitor, deoxyribonucleic acid synthesis is inhibited by over 90%, but ribonucleic acid synthesis is not affected. Cells resume their normal growth rate 24 hr after removal of either inhibitor.     

RNA干扰抑制麻疹病毒体外复制的实验研究

为了研究短发夹RNA(shRNA)介导的RNA干扰对麻疹病毒体外复制的抑制作用,构建靶向与麻疹病毒复制密切相关的宿主细胞基因Rab9 GTPase基因特异性shRNA表达载体,分别转染Vero-E6和B95a细胞后感染麻疹病毒Edmonston株和野生株。逆转录聚合酶链反应(RT-PCR)和免疫印迹技术(Western-blot)检测转染细胞内Rab9 GTPase基因表达水平;标准蚀斑试验测定麻疹病毒滴度。结果显示转染细胞内Rab9 GTPase mRNA和蛋白质的表达水平同对照组相比明显降低,标准蚀斑试验显示麻疹病毒的复制受到显著抑制,抑制率达到90%以上。结果表明载体介导的shRNAs能通过特异性下调Rab9 GTPase基因表达抑制麻疹病毒体外复制,Rab9 GTPase可能成为治疗麻疹病毒感染的RNA干扰靶。     

Inhibition of Host-Cell Protein and Ribonucleic Acid Synthesis by Newcastle Disease Virus

The mechanisms of Newcastle disease virus-(NDV) induced inhibition of cell protein and ribonucleic acid (RNA) synthesis were investigated. It was observed that the ability of NDV to inhibit cell RNA synthesis is dependent on the virus strain. The inhibitors, azauridine and cycloheximide, were added to cell cultures at different times after infection to study the roles of protein and RNA synthesis in the viral inhibition process. Viral inhibition of cell RNA synthesis and viral inhibition of cell protein synthesis become resistant to cycloheximide at a different time after infection than that in which they become resistant to azauridine. The results indicate that the inhibition of cell RNA synthesis by the Texas strain involves the synthesis of inhibitory proteins which are coded by the viral genome. The Texas and Beaudette strains of NDV appear to employ different mechanisms for the inhibition of host-cell protein synthesis. Viral inhibition of cell protein synthesis does not appear to cause, or be the result of, viral inhibition of cell RNA synthesis.     

Inhibition of Mitosis and Macromolecular Synthesis in Rat Embryo Cells by Kilham Rat Virus

The effects of Kilham rat virus multiplication were studied in cultured rat embryo cells to examine the mechanisms by which virus infection might be related to developmental defects in rats and hamsters. The virus was found to inhibit motosis and deoxyribonucleic acid (DNA) synthesis within 2 to 10 hr after infection. However, total ribonucleic acid synthesis was relatively unaffected until about 20 hr after infection, and total protein synthesis did not decline significantly until loss of viable cells was apparent in the cultures. No effect on chromosomes was detected. The effect of Kilham rat virus on DNA synthesis appears to be due to inhibition of macromolecular synthesis rather than to an inhibition of uptake of precursors into cells. The effect of the virus on mitosis may be an addition to the effect on DNA synthesis, since mitosis is inhibited even in cultures in which cells are able to divide at the time of infection and which have presumably completed DNA synthesis.     

Direct Inhibition of Cellular Fatty Acid Synthase Impairs Replication of Respiratory Syncytial Virus and Other Respiratory Viruses

Fatty acid synthase (FASN) catalyzes the de novo synthesis of palmitate, a fatty acid utilized for synthesis of more complex fatty acids, plasma membrane structure, and post-translational palmitoylation of host and viral proteins. We have developed a potent inhibitor of FASN (TVB-3166) that reduces the production of respiratory syncytial virus (RSV) progeny in vitro from infected human lung epithelial cells (A549) and in vivo from mice challenged intranasally with RSV. Addition of TVB-3166 to the culture medium of RSV-infected A549 cells reduces viral spread without inducing cytopathic effects. The antiviral effect of the FASN inhibitor is a direct consequence of reducing de novo palmitate synthesis; similar doses are required for both antiviral activity and inhibition of palmitate production, and the addition of exogenous palmitate to TVB-3166-treated cells restores RSV production. TVB-3166 has minimal effect on RSV entry but significantly reduces viral RNA replication, protein levels, viral particle formation and infectivity of released viral particles. TVB-3166 substantially impacts viral replication, reducing production of infectious progeny 250-fold. In vivo, oral administration of TVB-3166 to RSV-A (Long)-infected BALB/c mice on normal chow, starting either on the day of infection or one day post-infection, reduces RSV lung titers 21-fold and 9-fold respectively. Further, TVB-3166 also inhibits the production of RSV B, human parainfluenza 3 (PIV3), and human rhinovirus 16 (HRV16) progeny from A549, HEp2 and HeLa cells respectively. Thus, inhibition of FASN and palmitate synthesis by TVB-3166 significantly reduces RSV progeny both in vitro and in vivo and has broad-spectrum activity against other respiratory viruses. FASN inhibition may alter the composition of regions of the host cell membrane where RSV assembly or replication occurs, or change the membrane composition of RSV progeny particles, decreasing their infectivity.