Virus-specific RNA Synthesis Research Articles

Role and Virology of RSV and RSV RNA Polymerase

Respiratory syncytial virus (RSV) is a highly contagious, respiratory virus that commonly infects children under the age of five. RSV infections can present as cold-like symptoms which include a runny nose, coughing, and fever among other common symptoms; however, RSV can cause more severe infections such as bronchiolitis and pneumonia in young children and older, immunosuppressed patients. Despite RSV being a leading cause for respiratory illness in small children, there is limited research on its molecular biology, and especially its RNA polymerase. That being said, there has been slow progress in the development of a vaccine, partly due to the history of failed trials in the past. What is known is that the viral infection of RSV is initiated through the delivery of a virus-specific RNA synthesis machine into the host cell. This machine is responsible for both genome replication and gene transcription, thus including the RNA polymerase as a component. The RSV RNA polymerase consists of two units, a core large (L) protein, and a modulatory phosphoprotein (P). The L protein is 250 kDa with three functional domains: the RNA dependent RNA polymerization (RdRp), the capping (Cap), and the cap methyltransferase (MT) domains. As for the P protein, it has two domains that are understood to be the Oligomerization domain (POD) and the C-terminal domain (PPTD). These two domains are known to interact with the RdRp and Cap domains of the L protein which is essential in stabilizing the structure of the overall protein. The L protein serves as the core of the RSV structure and is responsible for the catalyzation of the RNA polymerization in replication and transcription, the cap addition, and cap methylation of nascent viral mRNAs. When synthesizing RNA, RSV RNA uses the nucleoprotein coated genomic RNA (N:RNA) as the template. In this process, the L protein requires the tetrameric P protein to displace the nucleoproteins. The synthesis of the mRNA caps first begins when a polyribonucleotide transferase forms the mRNA caps. This is a notable distinction from eukaryotes and other viral families which use guanylyltransferase instead. The caps are then methylated by the RNA polymerase at the 2’-O position, followed by the N-7 position, a pattern opposite from the order followed in mammalian mRNA. The refined mechanisms of these processes have yet to be described, but it is speculated that they share similarities with the cap addition and methylation assays of vesicular stomatitis virus (VSV), whose assays have been reported. The Walton High School MAPS team worked in partnership with the MSOE Center for Biomolecular Modeling to design a 3D model of RSV RNA Polymerase using a recently published cryo-electron microscopy structure to further investigate and examine the structure-function relationship of RSV RNA polymerase.

C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage

Rubella virus (RUB) replicons are derivatives of the RUB infectious cDNA clone that retain the nonstructural open reading frame (NS-ORF) that encodes the replicase proteins but not the structural protein ORF (SP-ORF) that encodes the virion proteins. RUB defective interfering (DI) RNAs contain deletions within the SP-ORF and thus resemble replicons. DI RNAs often retain the 5′ end of the capsid protein (C) gene that has been shown to modulate virus-specific RNA synthesis. However, when replicons either with or without the C gene were passaged serially in the presence of wt RUB as a source of the virion proteins, it was found that neither replicon was maintained and DI RNAs were generated. The majority DI RNA species contained in-frame deletions in the SP-ORF leading to a fusion between the 5′ end of the C gene and the 3′ end of the E1 glycoprotein gene. DI infectious cDNA clones were constructed and transcripts from these DI infectious cDNA clones were maintained during serial passage with wt RUB. The C-E1 fusion protein encoded by the DI RNAs was synthesized and was required for maintenance of the DI RNA during serial passage. This is the first report of a functional novel gene product resulting from deletion during DI RNA generation. Thus far, the role of the C-E1 fusion protein in maintenance of DI RNAs during serial passage remained elusive as it was found that the fusion protein diminished rather than enhanced DI RNA synthesis and was not incorporated into virus particles.

RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells.

RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects >270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both a messenger RNA and replication template, making it an attractive target for the study of RNA interference. Double-stranded small interfering RNA (siRNA) molecules designed to target the HCV genome were introduced through electroporation into a human hepatoma cell line (Huh-7) that contained an HCV subgenomic replicon. Two siRNAs dramatically reduced virus-specific protein expression and RNA synthesis to levels that were 90% less than those seen in cells treated with negative control siRNAs. These same siRNAs protected naive Huh-7 cells from challenge with HCV replicon RNA. Treatment of cells with synthetic siRNA was effective >72 h, but the duration of RNA interference could be extended beyond 3 weeks through stable expression of complementary strands of the interfering RNA by using a bicistronic expression vector. These results suggest that a gene-therapeutic approach with siRNA could ultimately be used to treat HCV.

Influenza A virus NEP (NS2 protein) downregulates RNA synthesis of model template RNAs.

The influenza A virus NEP (NS2) protein is an structural component of the viral particle. To investigate whether this protein has an effect on viral RNA synthesis, we examined the expression of an influenza A virus-like chloramphenicol acetyltransferase (CAT) RNA in cells synthesizing the four influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA) and NEP from recombinant plasmids. Influenza A virus NEP inhibited drastically, and in a dose-dependent manner, the level of CAT expression mediated by the recombinant influenza A virus polymerase. This inhibitory effect was not observed in an analogous artificial system in which expression of a synthetic CAT RNA is mediated by the core proteins of an influenza B virus. This result ruled out the possibility that inhibition of reporter gene expression was due to a general toxic effect induced by NEP. Analysis of the virus-specific RNA species that accumulated in cells expressing the type A recombinant core proteins and NEP showed that there was an important reduction in the levels of minireplicon-derived vRNA, cRNA, and mRNA molecules. Taken together, the results obtained suggest a regulatory role for NEP during virus-specific RNA synthesis, and this finding is discussed regarding the biological implications for the virus life cycle.

Comparative study of reproduction of sindbis virus sensitive and resistant to adamantane derivatives

The sensitivity of Sindbis virus gradually decreases during serial passages in the presence of 1-adamantane carbonic acid amide. After 4 passages, viral reproduction was not suppressed even by subtoxic concentrations of the inhibitor. Sensitive Sindbis strain principally differed from the resistant one: the latency of reproduction of resistant virus was 2 h longer than that of the sensitive strain. Cells infected with the sensitive and resistant strains of Sindbis virus contained the major structural proteins (C, E1+E2), their precursors pE2, p130, p90, and p78 protein, which probably represent a nonstructural virus-specific protein nsP3. The synthesis of virus-specific polypeptides and RNA of resistant variant is resistant to the inhibitor during incubation.

Influenza C virus persistence depends on exceptional steps in viral RNA synthesis and transport.

The cell line MDCK-pi, which is persistently infected with a variant of influenza C/AnnArbor/1/50 virus (C/AA-pi), was studied as a long-term persistence model by means of a strand-specific in situ hybridization assay. As a typical feature of the persistence, we identified a continuous synthesis of antigenomic positive-strand RNA encoded by segment 7 (NS) during virus production. In contrast, infection with the parental wild-type virus led to a rapid reduction of antigenomic RNA as observed in the late period of replication particularly for RNA segment 7. Furthermore, the replication cycle of the persistent variant did not show the switch from early to late replication events followed by clearance of intracellular virus, but was regulated in terms of productive and nonproductive phases. Nonproductive phases were reversible and characterized by a low level of virus-specific RNA signals. In the productive phase, a difference in cytoplasmic RNA transport was detected for the two viruses: a marked cytoplasmic accumulation of negative- and positive-strand wild-type virus RNAs stood in contrast to a RNA localization in different cellular compartments for the persistent virus. Also, Vero cells infected with the C/AA-pi variant were restricted to a transient, non-persistent replication cycle and produced a wild-type-like course of virus-specific RNA transport. These data indicate that influenza C virus persistence depends on a distinctly modified and cell type-specific regulation of virus-specific RNA synthesis and transport.

Infectious bovine viral diarrhea virus (strain NADL) RNA from stable cDNA clones: a cellular insert determines NS3 production and viral cytopathogenicity.

Bovine viral diarrhea virus (BVDV), strain NADL, was originally isolated from an animal with fatal mucosal disease. This isolate is cytopathic in cell culture and produces two forms of NS3-containing proteins: uncleaved NS2-3 and mature NS3. For BVDV NADL, the production of NS3, a characteristic of cytopathic BVDV strains, is believed to be a consequence of an in-frame insertion of a 270-nucleotide cellular mRNA sequence (called cIns) in the NS2 coding region. In this study, we constructed a stable full-length cDNA copy of BVDV NADL in a low-copy-number plasmid vector. As assayed by transfection of MDBK cells, uncapped RNAs transcribed from this template were highly infectious (>10(5) PFU/microg). The recovered virus was similar in plaque morphology, growth properties, polyprotein processing, and cytopathogenicity to the BVDV NADL parent. Deletion of cIns abolished processing at the NS2/NS3 site and produced a virus that was no longer cytopathic for MDBK cells. This deletion did not affect the efficiency of infectious virus production or viral protein production, but it reduced the level of virus-specific RNA synthesis and accumulation. Thus, cIns not only modulates NS3 production but also upregulates RNA replication relative to an isogenic noncytopathic derivative lacking the insert. These results raise the possibility of a linkage between enhanced BVDV NADL RNA replication and virus-induced cytopathogenicity.

Multiple factors including subgenomic RNAs and reduced viral protein expression are associated with a persistent infection by porcine rubulavirus (LPMV).

The synthesis of virus specific RNA and the expression of viral proteins in PK-15 cells persistently infected with the porcine rubulavirus LPMV have been studied at two different cell-passages following establishment of persistency (passages 25 and 65). Protein analysis of persistently infected cells and the virus particles released from these failed to demonstrate the presence of the polymerase (L) protein. A decrease in the amount of the phospho- (P) protein was also noted. The genome and mRNAs, both mono- and bicistronic, could readily be identified in the persistently infected cells with the exception of the L mRNA. By analysis of transcription gradients generated using the NIH Image analysis software, as well as analysis of the editing frequency, it was concluded that the changes in viral protein levels in persistently infected cells could be associated with a reduction in the amount of L mRNA and a shift in editing of the P gene. In addition, several large subgenomic RNAs of both the internally deleted and copy-back type were found in the persistently infected cells. The relevance of these findings to the persistent state is discussed.

Temperature-sensitive mouse cell factors for strand-specific initiation of poliovirus RNA synthesis

Two cell lines, TgSVA and TgSVB, were established from the kidneys of transgenic mice carrying the human gene encoding poliovirus receptor. The cells were highly susceptible to poliovirus infection, and a large amount of infectious particles was produced in the infected cells at 37 degrees C. However, the virus yield was greatly reduced at 40 degrees C. This phenomenon was common to all mouse cells tested. To identify the temperature-sensitive step(s) of the virus infection cycle, different steps of the infection cycle were examined for temperature sensitivity. The results strongly suggested that the growth restriction observed at 40 degrees C was due to reduced efficiency of the initiation process of virus-specific RNA synthesis. Furthermore, this restriction appeared to occur only on the synthesis of positive-strand RNA. Virus-specific RNA synthesis in crude replication complexes was not affected by the nonpermissive temperature of 40 degrees C. In vitro uridylylation of VPg seemed to be temperature sensitive only after prolonged incubation at 40 degrees C. These results indicate that a specific host factor(s) is involved in the efficient initiation process of positive-strand RNA synthesis of poliovirus and that the host factor(s) is temperature sensitive in TgSVA and TgSVB cells.

The Receptor-Destroying Enzyme of Influenza C Virus Is Required for Entry into Target Cells

The hemagglutinin-esterase (HE) protein of influenza C viruses possesses an acetylesterase activity, which appears essential for replication, as determined by reduced infectivity after inhibition of the viral enzyme [Vlasak et al., J. Virol. 63, 2056-2062 (1989)]. Analysis revealed the absence of virus-specific RNA and protein synthesis in infected cells after inhibition of the receptor-destroying enzyme. In addition, hemolytic activity was reduced after incubation of influenza C/JJ/50 virus with diisopropyl-fluorophosphate or 3,4-dichloro-isocoumarin. Further analysis revealed that inhibition of hemolysis depends on virus and erythrocyte concentrations. It is suggested that an active receptor-destroying enzyme is required for entry of influenza C virus into target cells at a step prior to fusion of the vital and cellular membrane. Our data indicate that cleavage of receptors bound to the HE protein is a prerequisite for the low pH-triggered conformational change required for fusion.

Rescue of Sindbis virus-specific RNA replication and transcription by using a vaccinia virus recombinant

A heterologous system expressing functional Sindbis virus nonstructural proteins (nsPs) has several possible uses for studying Sindbis virus-specific RNA replication and transcription in vivo and in vitro. Of the many possible approaches, vaccinia virus offers an attractive transient expression system given that Sindbis virus replication can occur in cells which have been previously infected by vaccinia virus. In this report, a vaccinia virus recombinant (called vSINNS), which contains the cDNA encoding the Sindbis virus nsPs under the control of either the vaccinia virus 7.5K promoter or the bacteriophage T7 promoter, has been constructed and characterized. Upon infection of several cell types with vSINNS, Sindbis virus nsP precursors and processed forms, including nsP1, nsP2, and both phosphorylated and nonphosphorylated forms of nsP3, were synthesized. Proteins containing the putative RNA-dependent RNA polymerase domain (nsP4 and nsP34), which are normally produced in small amounts by readthrough of an opal termination codon, were not detected in vSINNS-infected cells. However, all nsP functions necessary for Sindbis virus-specific RNA synthesis must have been expressed, since both replication and subgenomic mRNA transcription of an engineered Sindbis virus defective interfering RNA in cells infected with vSINNS was observed. Furthermore, vSINNS could be used as a helper virus to amplify, to relatively high titers, a replication-defective Sindbis virus mutant containing an in-frame deletion in the conserved N-terminal domain of nsP3. These data, as well as the observation that normal yields of parental Sindbis virus are produced in cells which have been previously infected with vSINNS, indicate that expression of Sindbis virus nsPs, in the absence of Sindbis virus-specific RNA replication, is not sufficient to block the formation of active RNA replication complexes by superinfecting Sindbis virus.

Synthesis and Localization of Japanese Encephalitis Virus RNAs in the Infected Cells

Synthesis and localization of virus-specific RNA in cells infected with Japanese encephalitis virus (JEV) were examined. To prepare specific RNA probes, we constructed four kinds of plasmids which contained DNA fragments corresponding to JEV genomic RNA. Minus probes, JT18V and JT19III, transcribed by T7 RNA polymerase were able to recognize a negative strand of JEV-specific RNA synthesized in cells as early as 6 hr postinfection (p.i.). In the experiments using a plus-strand probe JT19V to hybridize the 3' end of JEV-RNA, not only full-length 42S(+) RNA but also 10S(+) RNA were detected in the infected cells at 24 hr p.i. The positive-strand 42S RNA was found in much greater abundance in the membrane fraction than in the supernatant fraction of the infected cells. In contrast, larger amounts of the negative-strand RNAs existed in the supernatant fraction. It is suggested from the data that the JEV-specific negative- and positive-strand RNAs accumulate at different sites in the infected cells.

Genetic and phenotypic studies on Ross River virus variants of enhanced virulence selected during mouse passage

We have passaged Ross River virus (RRV) in mice to generate variants with increased mouse virulence and attempted to relate changes in virulence to genome sequence changes. RRV NBO (zero passage in mice) is a plaque-purified clone of the mouse-avirulent strain RRV NB5092, and is of low virulence for day-old mice. During RRV NBO replication in infant mice, its virulence for day-old mice increased markedly with time. By 7 days postinfection the LD 50 value of harvested virus (passage level one) was ≌10 4-fold less than that of the parental virus. No further decrease in LD 50 followed 10 serial passages in infant mice. However, 10th passage level virus showed increased clinical effects in week-old mice by comparison with virus from passage levels one and two. The growth kinetics of RRV variants in mice suggested that the rate and extent of RRV replication in the brain tissue determined the enhanced mouse virulence of serially passaged virus. Seven out of eight independently passaged, 10th passage level variants had changes in the E2 gene leading to one or two amino acid substitutions. The changes were at residues 212, 232, 234, 251, 341, 27 and 172, and 72 and 134 in these variants; all changes except two were nonconservative. Residues 212, 234, and 251 form part of a neutralization determinant in RRV. Changes in epitope b2 (which includes amino acids 246, 248, and 251) alter the kinetics of RRV entry into cells (P. Kerr, R. C. Weir, and L. Dalgarno, unpublished data). First and second passage level virus of enhanced virulence was unchanged in E2 or El gene sequences from RRV NBO. However, 1st, 2nd, and 10th passage level virus induced higher levels of virus-specific RNA synthesis than did RRV NBO in cultured BHK cells. We propose a model for the mechanism of virulence enhancement on passaging RRV NBO in mice.

Inhibition of murine coronavirus RNA synthesis by hydroxyguanidine derivatives

A series of hydroxyguanidine derivatives, which are substituted salicylaldehyde Schiff-bases of 1-amino-3- hydroxyguanidine tosylate, were tested for the inhibition of RNA synthesis of mouse hepatitis virus (MHV). It was shown that these compounds could selectively inhibit virus-specific RNA synthesis. Every aspect of viral RNA synthesis, including synthesis of negative-stranded RNA, subgenomic mRNA transcription and genomic RNA replication, was inhibited to roughly the same extent. These compounds are the first known inhibitors of coronaviral RNA synthesis and should prove useful for understanding the mechanism of viral RNA synthesis.

Synthesis of virus-specific RNA in permeabilized murine coronavirus-infected cells

We have developed a permeabilized cell system for assaying mouse hepatitis virus-specific RNA polymerase activity. This activity was characterized as to its requirements for mono- and divalent cations, requirements for an exogenous energy source, and pH optimum. This system faithfully reflects MHV-specific RNA synthesis in the intact cell, with regard to both its time of appearance during the course of infection and the products synthesized. The system is efficient and the RNA products were identical to those observed in intact MHV-infected cells as judged by agarose gel electrophoresis and hybridization. Permeabilized cells appear to be an ideal system for studying coronavirus RNA synthesis since they closely mimic in vivo conditions while allowing much of the experimental flexibility of truly cell-free systems.

Carrier-Mediated Delivery of Antiviral Agents

Publisher Summary Of the seven compounds currently approved by the U.S. Food and Drug Administration (FDA) as antiviral agents for use in humans, six are nucleoside or nucleotide analogs. Amantadine (l-adamantanamine hydrochloride), the first antiviral compound to be licensed for clinical use, is the sole exception. Effective in the prophylaxis and therapy of influenza A infections, amantadine appears to inhibit the initiation of transcription at an early stage between uncoating and virus-specific RNA synthesis. Idoxyuridine (IDU, 5-iodo-2 ‘-deoxyuridine) represents the first antiviral drug to be successfully used for preventing development of severe ocular lesions and blindness because of herpesvirus infections. Its activity is limited to DNA viruses, specifically herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) and varicella zoster virus (VZV). Being poorly soluble in water, aqueous solutions of IDU are ineffective against infections other than those localized in the eye. Trifluorothymidine (TFT, 5-trifluoromethyl-2’-deoxyuridine) is another halogenated pyrimidine nucleoside analog licensed for use in humans.

The immunosorbtion of influenza virus nucleocapsids from cell lysates as a technique for the studies on viral RNA synthesis

Radioimmunosorbtion of influenza virus nucleocapsids from lysates of the infected cells was applied for studies on virus-specific RNA synthesis. The method allowed the isolation of intact-labelled viral RNA segments. The procedure included preadsorbtion of antiviral antibodies on protein A containing sorbents. The protein A containing sorbent with attached antibodies was mixed with lysates of influenza virus-infected [ 3H]uridine-labelled cells. Viral nucleocapsids bound by the antibodies to the sorbent were used for RNA extraction. The isolated RNA was analysed by polyacrylamide gel electrophoresis with subsequent autoradiography. The method allows the isolation of nondegraded labelled virus-specific RNA by means of a relatively simple procedure.

Influenza virus gene expression: control mechanisms at early and late times of infection and nuclear-cytoplasmic transport of virus-specific RNAs.

Single-stranded M13 DNAs specific for various influenza virus genomic segments were used to analyze the synthesis of virus-specific RNAs in infected cells. The results show that influenza virus infection is divided into two distinct phases. During the early phase, the syntheses of specific virion RNAs, viral mRNAs, and viral proteins were coupled. Thus, the NS (nonstructural) virion RNA was preferentially synthesized early, leading to the preferential synthesis of NS1 viral mRNA and NS1 protein; in contrast, M (matrix) RNA synthesis was delayed, leading to the delayed synthesis of M1 viral mRNA and M1 protein. This phase lasted for 2.5 h in BHK-21 cells, the time at which the rate of synthesis of all the viral mRNAs was maximal. During the second phase, the synthesis of all the virion RNAs remained at or near maximum until at least 5.5 h postinfection, whereas the rate of synthesis of all the viral mRNAs declined dramatically. By 4.5 h, the rate of synthesis of all the viral mRNAs was 5% of the maximum rate. Viral mRNA and protein syntheses were also not coupled, as the synthesis of all the viral proteins continued at maximum levels, indicating that protein synthesis during this phase was directed principally by previously synthesized viral mRNAs. Short pulses (3 min) with [3H]uridine and nonaqueous fractionation of cells were used to show that influenza virion RNA synthesis occurred in the nucleus, demonstrating that all virus-specific RNA synthesis was nuclear. Virion RNAs, like viral mRNAs, were efficiently transported to the cytoplasm at both early and late times of infection. In contrast, the full-length transcripts of the virion RNAs, which are the templates for virion RNA synthesis, were sequestered in the nucleus. Thus, the template RNAs, which were synthesized only at early times, remained in the nucleus to direct virion RNA synthesis throughout infection. These results enabled us to present an overall scheme for the control of influenza virus gene expression.

The three influenza virus polymerase (P) proteins not associated with viral nucleocapsids in the infected cell are in the form of a complex.

The three influenza virus polymerase, or P, proteins (PB1, PB2, and PA) that are associated with viral nucleocapsids and are responsible for viral mRNA synthesis are in the form of a complex that moves down the template in association with the growing mRNAs during transcription (J. Braam, I. Ulmanen, and R.M. Krug, Cell 34:609-618, 1983). We determined whether infected cells contained a pool of P proteins not associated with viral nucleocapsids and, if so, whether the P proteins in this pool were in the form of a complex with each other. The cytoplasmic and nuclear extracts from infected cells were depleted of nucleocapsids by centrifugation, and the resulting supernatants were subjected to immunoprecipitation with an antiserum specific for either the PB1 protein or the PB2 protein. Both antisera precipitated all three P proteins, indicating that the P proteins were in a complex that was largely resistant to disruption by the detergents present in the immunoprecipitation buffer. Sucrose density gradient analysis showed that the P protein complexes ranged from about 11S to 22S and that almost all of the PB1 and PB2 protein molecules synthesized during a 1-h period (2.5 to 3.5 h postinfection) were in these complexes. Little or no free PB1 or PB2 protein was detected. The possible role of these nonnucleocapsid P protein complexes in the initiation and reinitiation of virus-specific RNA synthesis is discussed.

The in vitro synthesis of velvet tobacco mottle virus-specific double-stranded rna by a soluble fraction in extracts from infected Nicotiana clevelandii leaves

RNA-dependent RNA polymerase activity was detected in a membrane-free cytoplasmic fraction from the leaves of Nicotiana clevelandii 4 days after inoculation with velvet tobacco mottle virus (VTMoV). The amount of enzyme activity was still increasing 8 days after inoculation when virus-induced local lesions were becoming necrotic. RNA synthesis in extracts from leaves infected for 7 days proceeded linearly for about 10 min and required all four ribonucleoside triphosphates and magnesium ions. Enzyme activity was significantly increased by the addition of EGTA to the assay mixture. The polymerase activity was insensitive to actinomycin D, DNase, rifampicin and a-amanitin showing that it was RNA-dependent. The virus-specific RNA-dependent RNA polymerase-template complex in crude extracts from VTMoV-infected leaves and in partially purified preparations was shown to synthesize ds-RNAs of M, about 3.5 X 106 and 0.72 X 106, specific to VTMoV RNAs 1 and 3, respectively. The in vitro synthesis appears to have involved only the elongation of positive strands of RNAs 1 and 3 on their negative strand templates.