Inhibition of host protein synthesis by vaccinia virus: fate of cell mRNA and synthesis of small poly (A)-rich polyribonucleotides in the presence of actinomycin D.

Purified vaccinia virus rapidly inhibited HeLa cell protein synthesis in the presence of actinomycin D. Under these conditions host polyribosomes were extensively degraded but the mRNA was stable as indicated by a greater than 90% recovery of prelabeled polyadenylylated RNA. Although actinomycin D prevented the synthesis of host mRNA and poly(A) in uninfected cells, incorporation of adenosine into poly(A) was inhibited by less than 50% in infected cells. Further analysis indicated that there was little or no normal size viral mRNA but that a unique class of small poly(A)-rich RNA was made in the presence of actinomycin D. From measurements of the RNase resistance and base composition of the RNA, approximately 40% of the nucleotide sequence was estimated to be poly(A). The poly(A)-rich RNA was found associated with small polyribosomes and monoribosomes that were inactive in protein synthesis. It was suggested that the poly(A) segment of the RNA is formed by the poly(A) polymerase previously found in vaccinia virus cores and that the inactive RNA, by competing with host mRNA, may contribute to the virus-mediated inhibition of host protein synthesis observed in the presence of actinomycin D.     

Nucleic acids of respiratory syncytial virus.

Analysis of purified respiratory syncytial virus revealed that the virion RNA was composed of 50S, 28S, 18S, and 4S species. The 18S and 28S species were presumed to represent host rRNA since virus grown in actinomycin D-treated cells contained only 50S and 4S RNAs. Actinomycin D treatment stimulated production of infectious respiratory syncytial virus 5- to 10-fold. The 50S virion RNA was shown to hybridize with polyadenylated mRNA's isolated from infected cells, indicating that respiratory syncytial virus RNA is of negative-strand sense. Six mRNA's were identified by polyacrylamide gel electrophoresis.     

腮腺炎病毒的多肽及其在感染细胞中的合成

以差异离心和蔗糖密度梯度离心祛提纯了在鸡胚尿囊腔中繁殖的腮腺炎病毒粒子。并用SDS—PAGE分析病毒粒子的结构多肽,发现其结构多肽为11种,分子量在35K到72K之间。同时还检测到HN蛋白的多聚体和F蛋白的大亚基F1。将腮腺炎病毒分别感染Hela,Vero和CE细胞,比较这三种细胞对ME株腮腺炎病毒的敏感性,发现CE细胞是ME株的敏感宿主。用[31S]蛋氨酸标记病毒感染的CE细胞,以SDS-PAGE及放射自显影法检测到腮腺炎病毒在宿主细胞中合成了至少8种多肽,分子量在26．5K到94K之间。对这些多肽在细胞中不同时期合成情况进行了研究。还用脉冲追踪(pulsechase)技术在感染细胞中发现了FO到F这一转译后加工(Postttanslational procession)现象。此外也研究了放线菌素D和高沈度氯化钠对细胞蛋白质合成的抑制作用。     

Inhibition of Arbovirus Protein Synthesis by Interferon

Infection of cells treated with guanidine and actinomycin D and then washed to remove the guanidine inhibition of virus growth had no effect on antiviral activity already established by interferon. Protein synthesis in interferon-treated cells infected under these conditions was decreased as compared to control cells similarly treated but not exposed to interferon. In these control cells, analysis by polyacrylamide gel electrophoresis indicated that six proteins were produced during the first hour after guanidine reversal. Five of these proteins have been previously identified as probably being viral in origin. In interferon-treated cells, only a single major protein was produced. Ribonucleic acid (RNA) synthesis by Semliki Forest virus during the first hour after guanidine reversal was markedly depressed by incubation at 42 C, but no inhibition of total virus protein synthesis was seen; this finding suggested that much of the virus protein produced in the first hour after guanidine reversal was carried out by input virus RNA. Interferon was fully active in cells incubated at 42 C. The results suggested that interferon inhibits the production of Semliki Forest virus proteins ordinarily produced under the direction of the virus genome.     

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.     

Contrasting effects of matrix protein on apoptosis in HeLa and BHK cells infected with vesicular stomatitis virus are due to inhibition of host gene expression

Vesicular stomatitis virus (VSV) is a potent inducer of apoptosis in host cells. Recently, it has been shown that two VSV products are involved in the induction of apoptosis, the matrix (M) protein, and another viral product that has yet to be identified (S. A. Kopecky et. al., J. Virol. 75:12169-12181, 2001). Comparison of recombinant viruses containing wild-type (wt) or mutant M proteins showed that wt M protein accelerates VSV-induced apoptosis in HeLa cells, while wt M protein delays apoptosis in VSV-infected BHK cells. Our hypothesis to explain these results is that both effects of M protein are due to the ability of M protein to inhibit host gene expression. This hypothesis was tested by infecting cells with an M protein mutant virus defective in the inhibition of host gene expression (rM51R-M virus) in the presence or absence of actinomycin D, another inhibitor of host gene expression. Actinomycin D accelerated induction of apoptosis of HeLa cells infected with rM51R-M virus and delayed apoptosis in BHK cells infected with rM51R-M virus, similar to the effects of wt M protein. The idea that the induction of apoptosis by M protein in HeLa cells is due to its ability to inhibit host gene expression was further tested by comparing the activation of upstream caspase pathways by M protein versus that by actinomycin D or 5,6-dichlorobenzimidazole riboside (DRB). Expression of M protein activated both caspase-8 and caspase-9-like enzymes, as did treatment with actinomycin D or DRB. Induction of apoptosis by M protein, actinomycin D, and DRB was inhibited in stably transfected HeLa cell lines that overexpress Bcl-2, an antiapoptotic protein that inhibits the caspase-9 pathway. A synthetic inhibitor of caspase-8, Z-IETD-FMK, did not inhibit induction of apoptosis by M protein, actinomycin D, or DRB. Taken together, our data support the hypothesis that the induction of apoptosis by M protein is caused by the inhibition of host gene expression and that the caspase-9 pathway is more important than the caspase-8 pathway for the induction of apoptosis by M protein and other inhibitors of host gene expression.     

Antiviral activities and the mechanism of 9-beta-D-ribofuranosyl-6-alkylthiopurines on several RNA viruses from animals

A series of 9-beta-D-ribofuranosyl-6-alkylthiopurines (6-alkyl TI) were found to inhibit in vitro replication of infectious hematopoietic necrosis virus (IHNV), human influenza virus (IFV) and respiratory syncytial virus (RSV) with IC50 values of about 0.06 microgram/ml, 0.7-1.5 micrograms/ml and 1-3 micrograms/ml, respectively. Viral RNA synthesis in infected cells in the presence of actinomycin D was inhibited by treatment with the compounds dose-dependently. It was also found that the decrease of rNTP pool size in infected cells was remarkably dose-dependent. From these findings, the mode of antiviral action of these compounds may be explained by rNTP imbalance in the treated group.     

Persistent Infection of Cells in Culture by Measles Virus III. Comparison of Virus-Specific RNA Synthesized in Primary and Persistent Infection in HeLa Cells

The pattern of actinomycin D-resistant RNA synthesis was examined during primary infection of HeLa cells by virulent Edmonston measles virus and in two HeLa clones persistently infected by the same strain of virus. One of these clones, K11, produces infectious virus of low virulence for HeLa cells, and the other, K11A-HG-1, has thus far failed to yield infectious virus. The patterns of virus-specific RNA synthesized in these three types of infection are qualitatively similar to each other and to the patterns of virus-specific RNA synthesis in other paramyxovirus infections. There were, however, quantitative differences. In addition, virions of the virulent Edmonston strain of measles virus were found to contain high-molecular-weight RNA with a sedimentation constant identical to that of Newcastle disease virus.     

Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase Activity in Cells Infected with Influenza Virus

Deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase activity was assayed on nuclear preparations of chick embryo fibroblast cells at various times after infection with an influenza A virus (fowl plague virus) and was compared with the activity of uninfected cells. Polymerase activity was increased by about 60% by 2 hr after infection, and this increase coincided with an increase in RNA synthesis in infected cells, as determined by pulse-labeling with uridine. No difference could be detected between the polymerases of infected and uninfected cells as to their requirements for DNA primer, divalent cations, and nucleoside triphosphates, and they were equally sensitive to addition of actinomycin D to the reaction mixture. It is possible that host cell DNA-dependent RNA polymerase is involved in the replication of influenza virus RNA.     

Virological, Immunochemical, and Cytochemical Studies of Four HeLa Cell Lines Infected with Two Strains of Influenza Virus

The production of infectious virus, hemagglutinin, and viral (V) antigens and the changes in ribonucleoprotein (RNP) and lipoprotein metabolism have been studied in four sublines of HeLa cells infected with the PR8 and a PR8 recombinant strain of influenza virus. Much greater amounts of infectious virus and much less hemagglutinin were produced by the PR8 recombinant than by PR8 virus in all four cell lines. Different amounts of infectious virus per infected cell were produced by the recombinant in the four cell lines, whereas very little infectious virus was produced by the PR8 strain in any of the HeLa cells. In all cell lines infected with both strains of virus, "soluble" (S) antigen appeared early in the nucleolus. In cells infected with PR8 recombinant, S antigen subsequently filled the nucleus and later appeared in the cytoplasm. In most cells infected with PR8 virus, nuclear S antigen did not fuse to fill the nucleus, and S antigen was not detected in the cytoplasm. V antigen was observed in the cytoplasm of cells when diffuse nuclear S antigen had formed. The earliest and most frequent change in the RNP of the infected cells was a decrease in stainable RNP spherules (nucleolini) in the nucleolus. This was followed, in a smaller proportion of cells, by the appearance of nuclear and cytoplasmic inclusions containing RNP. There was a characteristic difference in the morphology of the cytoplasmic inclusions produced by the two strains of virus, but the same types of inclusions were observed in all four HeLa lines. A significant increase in lipoprotein was observed only in association with the cytoplasmic inclusions produced by PR8 recombinant virus. There was a striking difference in the proportion of cells with cytochemical changes in RNP in the four cell lines. A significant cytopathic effect (CPE) was observed only in three virus-cell systems in which a high proportion of cells exhibited changes in nucleolinar RNP. It is suggested that disappearance of RNP in the nucleolini may be an indication of shutdown of host ribonucleic acid synthesis and that this in turn results in a CPE. Virus infection resulted in a C-mitotic block that was followed by karyorrhexis. Infection of the cell did not always result in the production of infectious virus, in changes in the RNP of the nucleolini, in the development of nuclear or cytoplasmic RNP inclusions, or in CPE. The results suggest that production of infectious virus, shutdown of cellular RNP synthesis with accompanying CPE, and the formation of inclusions appear to be independent events.     

Newcastle Disease Virus Infection of L Cells

Newcastle disease virus (NDV) California strain reportedly grows poorly in L cells but replicates very well in chicken embryo cells. NDV-infected L cell cultures show a characteristic virus growth curve with respect to uridine incorporation, but plaque assays of the virus produced 24 h postinfection (PI) show no infectious particles when assayed on L cell monolayers and only a very low titer on chick cell monolayers. Plasma membranes isolated and purified from infected L cells 8 h PI contain all of the major virion proteins. In addition, NDV-infected L cells show a 50% loss of H-2 antigenic activity, a phenomenon previously observed in cells productively infected with vesicular stomatitis virus. These results suggest that at least part of the normal process of NDV maturation occurs in NDV-infected L cells. Sodium dodecyl sulfate-polyacrylamide gel patterns of supernatant virus purified from cells radiolabeled with amino acids from 3 to 24 h PI in the presence of actinomycin D show that all the major NDV structural proteins are present. Electron micrographs of NDV-infected L cells show extensive virus maturation at cell membranes. It can be concluded that infection of L cells with NDV results in a normal production of virus-specific RNA, synthesis of all the major structural proteins, association of the viral envelope proteins with the L cell plasma membrane, and the loss of cell surface H-2 antigenic activity. However, most of the virus particles produced are noninfectious.     

Requirement of cell nucleus for Sindbis virus replication in cultured Aedes albopictus cells.

The ability of Sindbis virus to grow in enucleated BHK-21 (vertebrate) and Aedes albopictus (invertebrate) cells was tested to determine the dependence of this virus upon nuclear function in these two phylogenetically unrelated hosts. Although both cell types could be demonstrated to produce viable cytoplasts (enucleated cells) which produced virus-specific antigen subsequent to infection. BHK cytoplasts produced a significant number of progeny virions, whereas mosquito cytoplasts did not. The production of vesicular stomatitis virus in mosquito cells was not significantly reduced by enucleation. That such a host function was not essential for vesicular stomatitis virus growth in insect cells is supported by the observation that the production of this virus by mosquito cells is not actinomycin D sensitive. This result agrees with a previously published report in which it was shown that Sindbis virus maturation in invertebrate cells is inhibited by actinomycin D, indicating a possible requirement for host cell nuclear function (Scheefers-Borchel et al., Virology, 110:292-301, 1981).     

Glucose uptake and lactic acid production of adenovirus type 5-infected HEp-2 cells cultured under exponential growth and stationary phase conditions

Exponentially growing HEp-2 cells have a higher rate of glucose uptake and lactic acid production than stationary phase cells. Infection of cells with adenovirus type 5 stimulates glycolysis irrespective of the original rate of the host cells. Therefore, infected cells cultured under exponential growth conditions have a higher rate of glycolysis than infected stationary phase cells. The rate of host cell glycolysis does not influence the time required for virus replication, the yield of infectious virus particles produced, or the time of appearance and progression of virus-induced cytopathology.     

Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells

The severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 protein has unique biological functions that have not been described in the viral proteins of any RNA viruses; expressed SARS-CoV nsp1 protein has been found to suppress host gene expression by promoting host mRNA degradation and inhibiting translation. We generated an nsp1 mutant (nsp1-mt) that neither promoted host mRNA degradation nor suppressed host protein synthesis in expressing cells. Both a SARS-CoV mutant virus, encoding the nsp1-mt protein (SARS-CoV-mt), and a wild-type virus (SARS-CoV-WT) replicated efficiently and exhibited similar one-step growth kinetics in susceptible cells. Both viruses accumulated similar amounts of virus-specific mRNAs and nsp1 protein in infected cells, whereas the amounts of endogenous host mRNAs were clearly higher in SARS-CoV-mt-infected cells than in SARS-CoV-WT-infected cells, in both the presence and absence of actinomycin D. Further, SARS-CoV-WT replication strongly inhibited host protein synthesis, whereas host protein synthesis inhibition in SARS-CoV-mt-infected cells was not as efficient as in SARS-CoV-WT-infected cells. These data revealed that nsp1 indeed promoted host mRNA degradation and contributed to host protein translation inhibition in infected cells. Notably, SARS-CoV-mt infection, but not SARS-CoV-WT infection, induced high levels of beta interferon (IFN) mRNA accumulation and high titers of type I IFN production. These data demonstrated that SARS-CoV nsp1 suppressed host innate immune functions, including type I IFN expression, in infected cells and suggested that SARS-CoV nsp1 most probably plays a critical role in SARS-CoV virulence.     

Structural and Nonstructural Proteins of an Arbovirus

Purified Semliki Forest virus (SFV) contains three structural proteins while its core (nucleocapsid) contains two of these proteins. To identify all of the proteins synthesized under virus direction, cells were infected with SFV in the presence of actinomycin D and guanidine. Cell protein synthesis was markedly and irreversibly inhibited under these conditions; virus growth was reversibly inhibited by guanidine and began when the cells were washed to remove the guanidine. When cells were treated with guanidine for 4 hr after virus infection and then were washed, five major proteins were produced early in infection. Three of these proteins corresponded to virus structural proteins. None of these five proteins was a major protein of uninfected cells or of virus-infected cells which had been incubated with partially purified interferon before infection. Late in infection, three major proteins, the virus structural proteins, were produced.     

The stimulatory effect of actinomycin D on avian reovirus replication in L cells suggests that translational competition dictates the fate of the infection.

Indirect immunostaining of avian reovirus S1133-infected L-cell monolayers showed that most of the cells can support viral replication. However, the number of cells in which the virus was actually replicating depended on the multiplicity of virus infection. The presence of actinomycin D during infection increased viral protein synthesis, viral growth, and the number of actively infected cells at late infection times. The antibiotic elicited these effects by triggering viral replication in cells that already contained unproductive cytoplasmic virus but that would not get productively infected in the absence of the drug. From these results, we propose a model for the interaction between L cells and avian reovirus S1133 in which viral versus host mRNA competition for the translational machinery determines the fate of the virus infection.     

Physiological State of Human Embryonic Lung Cells Affects Their Response to Human Cytomegalovirus

Cultures of human embryonic lung (HEL) cells in different physiological states were studied for their susceptibility to infection with human cytomegalovirus (CMV) with respect to production of infectious virus, synthesis of viral antigens, and virus-induced stimulation of cellular DNA synthesis. In general, subconfluent, actively growing cells yielded higher amounts of infectious virus than did confluent contact-inhibited cells. The higher yield of infectious virus was correlated with a greater percentage of cells producing viral antigens within the first 48 h after infection. In confluent cultures, 25 to 50% of the cells produced viral antigens within the first 48 h postinfection. This proportion did not change over a 10-fold range of multiplicity of infection, indicating that many of the cells in confluent cultures did not support productive infection. However, virtually all the cells in subconfluent cultures were susceptible. Also, in contrast to herpes simplex virus and pseudorabies virus, infectious CMV is not produced by cells treated with 5-fluorouracil and thymidine. Virus-induced stimulation of cellular DNA synthesis in cells infected at high multiplicities of infection could be detected only in confluent cultures, in which cellular DNA synthesis had been previously suppressed, but could not be detected in similarly treated cultures of subconfluent cells. The lack of detectable stimulation of cellular DNA synthesis in the latter was related to the fact that practically all the cells in the culture synthesized viral antigens within the first 48 h after infection, productive infection and detectable synthesis of cellular DNA being mutually exclusive.