Viral Ribonucleic Acid Synthesis Research Articles

Role of structural and functional proteins of SARS -COV-2

Severe acute respiratory corona virus-2 (SARS-CoV-2) is a ribonucleic acid (RNA) virus with enveloped no-segmented positive sense belonging to a beta (β) - corona virus family. It has 29,903 nucleotides sized genome with 10 open reading frames (ORF). ORF1 (ab) encodes two polypeptides pp1a and pp1b cleaved into 16 functional proteins, which are mainly intended to form replication transcription complex (RTC). The cleavage process of pp1a and pp1b polypeptides to 16 functional proteins of SARs-CoV-2 is mainly facilitated by main protease and papain-like protease. The replication transcription complex (RTC) formed by the action of 16-functional proteins of SARs-CoV-2 is mainly involved as viral RNA synthesis machinery in the transcriptional and replication process of viral RNAs. ORF (2-10) encodes for structural (for example: spike (S), membrane (M), nucleocapsid (N), and envelop (E)) and accessory proteins of SARs-CoV-2. The main functions of structural proteins are viral assembly, viral coating, viral entry into host cells and assembly of the RNA genome. Accessory proteins are proteins that are not involved in the viral synthesis machinery, as 16 functional proteins, and in the viral assembly, coating, entry into host cells and packaging of Viral RNAs, as structural proteins. Rather, these are proteins that may play central role by enhancing viral assembly process, virulence and pathogenesis of SARs-CoV-2. Our aim in the current review was to elaborate the specific role of these structural and functional proteins on viral genomic replication and transcription, viral assembly, host cell attachment and pathogenesis. Multiple literatures have been reviewed to achieve the objective of this review.

Anesthetic action and virus replication: inhibition of measles virus replication in cells exposed to halothane.

Replication of measles virus in BSC cells was studied in the presence of halothane, a commonly used volatile anesthetic. At clinical concentrations of the anesthetic, appearance of progeny virus was decreased in a dose-related manner. This inhibition was reversible as the removal of halothane allowed virus replication to be resumed. Studies attempting to elucidate the mechanism of action of the anesthetic inhibition of virus replication revealed that halothane did not directly inactivate the virus particle or prevent viral adsorption to the cell. Infectious virus and nucleocapsid production were decreased or stopped, depending on the anesthetic dosage used. Direct immunofluorescent staining for measles virus antigen was negative in cells treated at the higher concentrations of halothane. Recovery of nucleocapsid production started within a few hours after removal of halothane. Furthermore, the combined inhibitory effects on viral ribonucleic acid synthesis of 5-azacytidine and halothane were additive. This evidence suggests that inhibition of measles virus replication occurs at or before ribonucleic acid synthesis.

Chemotherapy of plant virus disease with methyl benzimidazol-2yl-carbamate: effects on plant growth and multiplication of tobacco mosaic virus

Methyl benzimidazol-2yl-carbamate inhibited the accumulation of tobacco mosaic virus in actively growing parts of infected tobacco plants. It also prevented the inhibition of leaf growth by the virus. In expanding leaves, phytotoxic effects of methyl benzimidazol-2ylcarbamate were seen only at levels 50 to 100 times those required to inhibit viral multiplication. No direct inhibition of tobacco mosaic virus ribonucleic acid synthesis was found when the effects of methyl benzimidazol-2yl-carbamate on incorporation of 32P-orthophosphate into nucleic acid were studied. From experiments on time of application, dose level and leaf age, it is suggested that methyl benzimidazol-2yl-carbamate inhibits viral ribonucleic acid synthesis indirectly, by maintaining the host in a state unsuitable for viral multiplication. This may occur through the known cytokinin activity of the compound. In some circumstances, higher concentrations of tobacco mosiac virus were found to result from treatment with methyl benzimidazol-2yl-carbamate. The relationships between leaf growth, symptom formation and viral multiplication are discussed.

Effects of Some Antiviral Isatinisothiosemicarbazones on Cellular and Viral Ribonucleic Acid Synthesis in Mengovirus-Infected FL Cells

Effects of Some Antiviral Isatinisothiosemicarbazones on Cellular and Viral Ribonucleic Acid Synthesis in Mengovirus-Infected FL Cells

Effects of Some Antiviral Isatinisothiosemicarbazones on Cellular and Viral Ribonucleic Acid Synthesis in Mengovirus-Infected FL Cells

Three antiviral isatinisothiosemicarbazones strongly inhibited the incorporation of [(3)H]uridine into the ribonucleic acid (RNA) of FL cells as a consequence of the inhibition of uridine transport. After prelabeling of cells at a low temperature (1 h at 16 C) with uptake of [(3)H]uridine into the acid-soluble nucleotide pool, the later addition of the test compounds revealed only a small or negligible influence on host-directed RNA synthesis. The pulse-labeled soluble nucleotide pool of FL cells was sufficient to give a gradual increase in incorporation into RNA over a period of 7 h. With the same method of prelabeling at the beginning of the experiment, it was also possible to detect virus-induced RNA synthesis in the presence of actinomycin D. In this way the specific inhibitory action of the three isatinisothiosemicarbazones on viral RNA synthesis could be demonstrated.

Inhibition of early viral ribonucleic acid synthesis in interferon-treated cells infected with frog polyhedral cytoplasmic deoxyribovirus

Inhibition of early viral ribonucleic acid synthesis in interferon-treated cells infected with frog polyhedral cytoplasmic deoxyribovirus

Mechanism of action of gliotoxin: elimination of activity by sulfhydryl compounds.

Gliotoxin and two other compounds, with antiviral activity against a number of ribonucleic acid (RNA) viruses and structurally related via the epidithiapiperazinedione moiety, appeared to be equally active in their oxidized and reduced forms. However, the ability of the reduced forms to inhibit viral RNA synthesis was abolished when these compounds were maintained in the reduced state by the simultaneous presence of a large molar excess of dithiothreitol or reduced glutathione. The active form therefore appeared to be that containing a disulfide bridge, and the apparent activity of the dithiol was due to cellular oxidation. Possible mechanisms by which the compounds could interact with viral proteins, e.g., viral RNA-dependent RNA polymerase, are proposed.

Specific Membranous Structures Associated with the Replication of Group A Arboviruses

INTRACYTOPLASMIC MEMBRANOUS STRUCTURES OF A UNIQUE TYPE WERE ASSOCIATED WITH THE REPLICATION OF THREE GROUP A ARBOVIRUSES: Semliki Forest virus (SFV), Sindbis virus, or Western equine encephalomyelitis virus. The structures, referred to as type 1 cytopathic vacuoles (CPV-1), were membrane-limited and characteristically lined by regular membranous spherules measuring 50 nm in diameter. The membranous spherules typically contained a fine central density, but were neither virus cores nor virions. Detection of CPV-1 by electron microscopy at 3 to 6 hr postinfection coincided with the time of rapid virus growth and preceded the accumulation of virus nucleocapsids. A range of 20 to 100 CPV-1 profiles were counted per 100 ultrathin cell sections at 6 to 9 hr postinfection when viruses were grown in chick embryo, baby hamster kidney, or mouse L cells. Maximum counts remained in the same range even when the multiplicity of infection was varied over 100-fold. Inhibition of cellular ribonucleic acid (RNA) and protein synthesis by actinomycin D during SFV infection did not decrease the counts of CPV-1; however, biogenesis of CPV-1 was decreased when viral replication was limited by inhibitors of viral RNA synthesis (guanidine) or of viral protein synthesis (cycloheximide). On the basis of present and earlier findings, we concluded that formation of CPV-1 must result from a virus-specified modification of pre-existing host cell macromolecules.

Synthesis of viral ribonucleic acid during restricted adenovirus infection.

The mechanism by which simian virus 40 converts the abortive adenovirus type 7 infection of monkey cells into an efficient lytic infection has been investigated. Analysis of ribonucleic acid (RNA) synthesis during unenhanced and enhanced infection of monkey cells has shown that adenovirus RNA synthesized in the abortive infection contains both "early" and "late" sequences. In hybridization competition experiments, early adenovirus RNA from human cells prevented the hybridization of only 20% of the adenovirus RNA transcribed in unenhanced infection. Further, the RNA from unenhanced cells was able to completely block the hybridization of RNA synthesized during enhanced infection. Finally, virus-associated RNA, which is a late RNA transcribed in lytic adenovirus infection, is also produced in the unenhanced infection. An accompanying paper describes a marked deficiency in adenoviral capsid protein synthesis in the unenhanced infection. We conclude that RNA sequences, which are sufficient to code for the synthesis and assembly of structural proteins of adenovirus, are transcribed but are not efficiently translated in the unenhanced adenovirus infection of monkey cells.

Viral Ribonucleic Acid Synthesis by Newcastle Disease Virus Mutants Isolated from Persistently Infected L Cells: Effect of Interferon

The synthesis of different viral ribonucleic acid (RNA) species was studied in chick embryo (CE) and mouse L-cell cultures infected with the Herts strain of Newcastle disease virus (NDV(o)) and a mutant isolated from persistently infected L cells (NDV(pi)). In CE cell cultures, both viruses synthesized significant amounts of 54, 36, and 18S RNA. However, in L cells, synthesis of 54S virion RNA was markedly reduced. From these results, it seems likely that the low yield of infective virus in L cells is due to a deficient synthesis of 54S RNA in this host. On this basis, however, it is apparent that the "covert" replication of NDV(o) in L cells is due to factors other than viral RNA synthesis. When low concentrations of interferon were used to pretreat CE cells, a differential effect on the synthesis of various RNA species was observed. The 18S RNA of NDV(o) was more sensitive to interferon action than the 36 and the 54S RNA species. In contrast, the 18S RNA of NDV(pi) was less sensitive than the 36S and the 54S RNA. The inhibition of 54S RNA synthesis correlated with the reduction of viral yield and explained the greater sensitivity of NDV(pi) to interferon.

Inhibition of early vaccinia virus ribonucleic acid synthesis in interferon-treated chicken embryo fibroblasts.

Pretreatment of chicken embryo cells with homologous but not heterologous interferon inhibits the synthesis of vaccinia virus early messenger ribonucleic acid (mRNA). This inhibition is seen in the presence of cycloheximide, i.e., in the absence of protein synthesis, suggesting that the virion-bound transcriptase may be the target of the antiviral activity associated with interferon treatment. The inhibition of viral mRNA synthesis is dependent on the amount of chicken interferon used. The nonviral interferon inducer, polyriboinosine.polyribocytidine, similarly inhibits viral early mRNA synthesis in a dose-specific manner. The helical polynucleotide polydeoxyinosine.polyribocytidine, which is not an effective interferon inducer in chicken embryo cells, has no effect on viral ribonucleic acid synthesis.

Polysomal localization of R17 bacteriophage-specific protein synthesis.

Synthesis of viral ribonucleic acid (RNA) polymerase, maturation protein, and coat protein in Escherichia coli infected with bacteriophage R17 occurs mainly on polysomes containing four or more ribosomes. The 30S ribosomal subunits through trimer-size polysomes, which are associated with all of the R17-specific proteins and are predominant in the infected cell, synthesize only coat protein. These structures may accumulate as products derived from larger polysomes as a result of failure in the release of nascent polypeptides after termination of chain growth. Appreciable amounts of viral coat protein remain attached to ribosomes and polysomes during R17 bacteriophage replication, supporting the hypothesis of the repressor role of this protein. The time course of synthesis of virus-specific proteins obtained from the polysomes of infected cells demonstrated regulated R17 messenger RNA translation consistent with the idea that coat protein is preferentially synthesized whereas the synthesis of noncoat proteins is suppressed.

Preliminary Physiological Characterization of Temperature-Sensitive Mutants of Vesicular Stomatitis Virus

Different temperature-sensitive mutants of vesicular stomatitis virus have been characterized in terms of their ability to induce synthesis of viral ribonucleic acid (RNA) in BHK-21 cells at 39 C (the restrictive temperature for these mutants). Mutants belonging to complementation groups I and IV (and probably II) did not induce actinomycin-resistant RNA synthesis in infected cells incubated at 39 C. All three mutants comprising complementation group III induced viral RNA synthesis at 39 C. The temperature sensitivity of the defective viral functions has also been studied by temperature-shift experiments. The functions associated with the mutants of groups I, II, and IV were required early, whereas the function associated with the group III mutants was not required until a late stage of the viral cycle. The heat sensitivity of extracellular virion was not correlated with complementation group.

Temperature-sensitive mutants of vesicular stomatitis virus: synthesis of virus-specific proteins.

Viral proteins synthesized in L cells infected with temperature-sensitive (ts) mutants of vesicular stomatitis (VS) virus at permissive (31 C) and nonpermissive (39 C) temperatures were compared by polyacrylamide gel electrophoresis. Mutant ts 5, deficient in synthesis of viral ribonucleic acid (RNA(-)), failed to synthesize any of the five identifiable viral proteins at 39 C. Each of three RNA(+) mutants, representing three separate complementation groups, showed distinctive patterns of viral protein synthesis at nonpermissive temperature. Equivalent amounts of (3)H-amino acids were incorporated into the five viral proteins made in cells infected with RNA(+) mutant ts 45 at 31 and 39 C. Complete virions of ts 45 could be identified by electron microscopy of infected cells incubated at the nonpermissive temperature; the defect in ts 45 appeared to be due in part to greater thermolability of virions as compared with the wild-type. RNA(+) mutant ts 23 was deficient in synthesis of viral envelope protein S and failed to make detectable virions at the nonpermissive temperature. Infection of cells at 39 C with the third RNA(+) mutant, ts 52, resulted in synthesis of all five viral proteins, but the peak of radioactivity representing the viral membrane glycoprotein migrated more rapidly on gels than coelectrophoresed authentic virion (14)C-glycoprotein or viral (3)H-glycoprotein extracted from cells infected at 31 C. These data and results of experiments on incorporation of radioactive glucosamine suggest that the primary defect in mutant ts 52 at nonpermissive temperature is failure of glycosylation of the viral glycoprotein. The viral structural proteins made in cells infected with ts 52 at the nonpermissive temperature did not assemble into sedimentable components as they did at permissive temperature; this observation indicates failure of insertion of the nonglycosylated protein (G') into cell membrane. In support of this hypothesis was the finding that antiviral-antiferritin hybrid antibody did not detect VS viral antigen on the plasma membrane of L cells infected at 39 C with ts 52. In contrast, VS viral antigen localized in plasma membrane of L cells infected at 39 C with mutants ts 23 and ts 45 was readily detected by electron microscopy and fluorescence microscopy.

Assay of chick interferons by the inhibition of viral ribonucleic acid synthesis.

A method for assaying chick interferons by their inhibition of viral ribonucleic acid synthesis was devised and evaluated. The technique yielded results faster and had more flexibility than other methods with similar sensitivity and reproducibility.

Effects of elevated temperatures on mengovirus ribonucleic acid synthesis and virus production in Novikoff rat hepatoma cells.

The production of mengovirus in Novikoff rat hepatoma cells is progressively reduced with an increase in incubation temperature of the cells from 34 to 40 C, in spite of the fact that about the same amounts of single-stranded and double-stranded viral ribonucleic acid (RNA) are synthesized at 34, 37, and 40 C; the rate of overall protein synthesis is as high at 40 C as at 37 C. At 40 C, progeny viral RNA accumulates in an undegraded form without being incorporated into virus particles. The results suggest that virus maturation is preferentially inhibited at supraoptimal temperatures. At 42 C, on the other hand, no viral RNA is produced and no viral RNA polymerase activity is detectable in cell lysates. Failure of infected cells to form viral RNA polymerase at 42 C is probably due to an impairment of protein synthesis since most of the polyribosomes are rapidly lost during incubation at 42 C and the rate of amino acid incorporation into protein is 70% lower at 42 C than at 37 C. When infected cells are shifted from 37 to 42 C during the period of active viral RNA synthesis, viral RNA polymerase activity is rapidly lost from the cells, and viral RNA synthesis ceases within 45 min. In contrast, the RNA polymerase is as active in vitro at 42 C as at 37 C, and the activity is relatively stable at 42 C.

Transport as the rate-limiting step in the incorporation of uridine into mengovirus ribonucleic acid in Novikoff rat hepatoma cells.

The incorporation of uridine into the nucleotide pool of actinomycin-treated, mengovirus-infected Novikoff rat hepatoma cells in culture follows simple Michaelis-Menten kinetics, and the apparent V(max) and K(m) values are similar to those for uridine transport by uninfected cells. Incorporation of uridine into mengovirus-specific ribonucleic acid (RNA) also follows Michaelis-Menten kinetics, and the apparent K(m) (about 10 mum) is approximately the same as for uridine transport. Inhibition of uridine transport by the presence of adenosine, persantin, or phenethyl alcohol inhibits simultaneously and to the same extent the incorporation of uridine into the nucleotide pool and into viral RNA, without affecting viral RNA synthesis per se. Phenethyl alcohol, however, also inhibits virus maturation. The inhibition of uridine incorporation into the nucleotide pool and into viral RNA is of the simple competitive type, indicating that transport into the cells is the rate-limiting step in the incorporation of uridine into mengovirus RNA. The results also indicate that treatment with actinomycin D or mengovirus infection does not affect uridine transport.

Assay of Chick Interferons by the Inhibition of Viral Ribonucleic Acid Synthesis

A method for assaying chick interferons by their inhibition of viral ribonucleic acid synthesis was devised and evaluated. The technique yielded results faster and had more flexibility than other methods with similar sensitivity and reproducibility.

Cell Surface Antigen Induced by Venezuelan Equine Encephalomyelitis Virus

By immunofluorescence staining, a specific surface antigen induced by Venezuelan equine encephalomyelitis virus was detected on L-929 cells. Formation of the antigen was independent of viral ribonucleic acid synthesis.

Phospholipid synthesis in Sindbis virus-infected cells.

We investigated the metabolic requirements for the decrease in phospholipid synthesis previously observed by Pfefferkorn and Hunter in primary cultures of chick embryo fibroblasts infected with Sindbis virus. The incorporation of (32)PO(4) into all classes of phospholipids was found to decline at the same rate and to the same extent; thus, incorporation of (14)C-choline into acid-precipitable form provided a convenient measure of phospholipid synthesis that was used in subsequent experiments. Experiments with temperature-sensitive mutants suggested that some viral ribonucleic acid (RNA) synthesis was essential for the inhibition of choline incorporation, but that functional viral structural proteins were not required. The reduction in phospholipid synthesis was probably a secondary effect of infection resulting from viral inhibition of the cellular RNA and protein synthesis. All three inhibitory effects required about the same amount of viral RNA synthesis; the inhibition of host RNA and protein synthesis began sooner than the decline in phospholipid synthesis; and both actinomycin D and cycloheximide inhibited (14)C-choline incorporation in uninfected cells. In contrast, incorporation of (14)C-choline into BHK-21 cells was not decreased by 10 hr of exposure to actinomycin D and declined only slowly after cycloheximide treatment. Growth of Sindbis virus in BHK cells did not cause the marked stimulation of phospholipid synthesis seen in picornavirus infections of other mammalian cells; however, inhibition was seen only late in infection.