Replicative Form RNA Research Articles

A Host Small GTP-binding Protein ARL8 Plays Crucial Roles in Tobamovirus RNA Replication

Tomato mosaic virus (ToMV), like other eukaryotic positive-strand RNA viruses, replicates its genomic RNA in replication complexes formed on intracellular membranes. Previous studies showed that a host seven-pass transmembrane protein TOM1 is necessary for efficient ToMV multiplication. Here, we show that a small GTP-binding protein ARL8, along with TOM1, is co-purified with a FLAG epitope-tagged ToMV 180K replication protein from solubilized membranes of ToMV-infected tobacco (Nicotiana tabacum) cells. When solubilized membranes of ToMV-infected tobacco cells that expressed FLAG-tagged ARL8 were subjected to immunopurification with anti-FLAG antibody, ToMV 130K and 180K replication proteins and TOM1 were co-purified and the purified fraction showed RNA-dependent RNA polymerase activity that transcribed ToMV RNA. From uninfected cells, TOM1 co-purified with FLAG-tagged ARL8 less efficiently, suggesting that a complex containing ToMV replication proteins, TOM1, and ARL8 are formed on membranes in infected cells. In Arabidopsis thaliana, ARL8 consists of four family members. Simultaneous mutations in two specific ARL8 genes completely inhibited tobamovirus multiplication. In an in vitro ToMV RNA translation-replication system, the lack of either TOM1 or ARL8 proteins inhibited the production of replicative-form RNA, indicating that TOM1 and ARL8 are required for efficient negative-strand RNA synthesis. When ToMV 130K protein was co-expressed with TOM1 and ARL8 in yeast, RNA 5′-capping activity was detected in the membrane fraction. This activity was undetectable or very weak when the 130K protein was expressed alone or with either TOM1 or ARL8. Taken together, these results suggest that TOM1 and ARL8 are components of ToMV RNA replication complexes and play crucial roles in a process toward activation of the replication proteins' RNA synthesizing and capping functions.

Survival and Replication of Dengue-2 Virus in Diapausing Eggs of Aedes albopictus (Diptera: Culicidae)

Survival and replication of dengue-2 virus (family Flaviviridae, genus Flavivirus, DENV-2) was determined in diapausing eggs of Aedes albopictus (Skuse) (Diptera: Culicidae) to evaluate the importance of this mechanism for viral maintenance during adverse climatic conditions. Infected and uninfected eggs of Ae. albopictus were induced to diapause under low temperature and short photoperiod. Virus was detected by reverse transcription-polymerase chain reaction and isolated in C6/36 cells from both diapausing eggs and nondiapausing eggs. Nucleic acid hybridization was used to monitor viral replication in diapausing eggs. DENV-2 might survive in infected diapausing eggs in a relatively quiescent status based on the absence of a replicative intermediate RNA. In contrast, the viral replicative intermediate RNA along with the replicative form RNA was detected in nondiapausing eggs. It seemed that the virus replicated more actively in nondiapausing eggs than in diapausing eggs. Infected diapausing eggs may play an important role in the maintenance of DENV during adverse climatic conditions in nature.

Survival and Replication of Dengue-2 Virus in Diapausing Eggs of <I>Aedes albopictus</I> (Diptera: Culicidae)

Survival and replication of dengue-2 virus (family Flaviviridae, genus Flavivirus, DENV-2) was determined in diapausing eggs of Aedes albopictus (Skuse) (Diptera: Culicidae) to evaluate the importance of this mechanism for viral maintenance during adverse climatic conditions. Infected and uninfected eggs of Ae. albopictus were induced to diapause under low temperature and short photoperiod. Virus was detected by reverse transcription-polymerase chain reaction and isolated in C6/36 cells from both diapausing eggs and nondiapausing eggs. Nucleic acid hybridization was used to monitor viral replication in diapausing eggs. DENV-2 might survive in infected diapausing eggs in a relatively quiescent status based on the absence of areplicative intermediate RNA. In contrast, the viral replicative intermediate RNA along with the replicative form RNA was detected in nondiapausing eggs. It seemed that the virus replicated more actively in nondiapausing eggs than in diapausing eggs. Infected diapausing eggs may play an important role in the maintenance of DENV during adverse climatic conditions in nature.

An in vitro Flaviviridae replicase system capable of authentic RNA replication

We have established an in vitro replication system for bovine viral diarrhea virus (BVDV), a surrogate for the closely-related hepatitis C virus. In an in vitro reaction, BVDV replication complexes synthesize vRNA and replicative form (RF) and replicative intermediate (RI) RNAs. Kinetic and heparin trapping experiments demonstrate the recycling of RF and RI products and the initiation of vRNA synthesis in this system. Consistent with this, quantitative hybridization reveals the asymmetric synthesis of positive and negative strand RNA products. These findings support the notion that RF serves as a template and RI as a precursor in the synthesis of vRNA. Furthermore, the antiviral activity of an NS5B inhibitor was similar in BVDV replicase and infectivity assays. Together, these results indicate that the in vitro activity of BVDV replicase complexes recapitulates RNA replication that occurs in infected cells, providing a system in which to study both mechanisms and inhibitors of Flaviviridae replication.

Architecture of the Flaviviral Replication Complex: PROTEASE, NUCLEASE, AND DETERGENTS REVEAL ENCASEMENT WITHIN DOUBLE-LAYERED MEMBRANE COMPARTMENTS

Flavivirus infection causes extensive proliferation and reorganization of host cell membranes to form specialized structures called convoluted membranes/paracrystalline arrays and vesicle packets (VP), the latter of which is believed to harbor flaviviral replication complexes. Using detergents and trypsin and micrococcal nuclease, we provide for the first time biochemical evidence for a double membrane compartment that encloses the replicative form (RF) RNA of the three pathogenic flaviviruses West Nile, Japanese encephalitis, and dengue viruses. The bounding membrane enclosing the VP was readily solubilized with nonionic detergents, rendering the catalytic amounts of enzymatically active protein component(s) of the replicase machinery partially sensitive to trypsin but allowing limited access for nucleases only to the vRNA and single-stranded tails of the replicative intermediate RNA. The RF co-sedimented at high speed from nonionic detergent extracts of virus-induced heavy membrane fractions along with the released individual inner membrane vesicles whose size of 75-100 nm as well as association with viral NS3 was revealed by immunoelectron microscopy. Viral RF remained nuclease-resistant even after ionic detergents solubilized the more refractory inner VP membrane. All of the viral RNA species became nuclease-sensitive following membrane disruption only upon prior trypsin treatment, suggesting that proteins coat the viral genomic RNA as well as RF within these membranous sites of flaviviral replication. These results collectively demonstrated that the newly formed viral genomic RNA associated with the VP are oriented outwards, while the RF is located inside the nonionic detergent-resistant vesicles.

The RNA structures engaged in replication and transcription of the A59 strain of mouse hepatitis virus.

In addition to the RI (replicative intermediate RNA) and native RF (replicative form RNA), mouse hepatitis virus-infected cells contained six species of RNA intermediates active in transcribing subgenomic mRNA. We have named these transcriptive intermediates (TIs) and native transcriptive forms (TFs) because they are not replicating genome-sized RNA. Based on solubility in high salt solutions, approximately 70% of the replicating and transcribing structures that accumulated in infected cells by 5-6 h post-infection were multi-stranded intermediates, the RI/TIs. The other 30% were in double-stranded structures, the native RF/TFs. These replicating and transcribing structures were separated by velocity sedimentation on sucrose gradients or by gel filtration chromatography on Sepharose 2B and Sephacryl S-1000, and migrated on agarose gels during electrophoresis, according to their size. Digestion with RNase T1 at 1-10 units/microgram RNA resolved RI/TIs into RF/TF cores and left native RF/TFs intact, whereas RNase A at concentrations of 0.02 microgram/microgram RNA or higher degraded both native RF/TFs and RI/TIs. Viral RI/TIs and native RF/TFs bound to magnetic beads containing oligo(dT)(25), suggesting that the poly(A) sequence on the 3' end of the positive strands was longer than any poly(U) on the negative strands. Kinetics of incorporation of [(3)H]uridine showed that both the RI and TIs were transcriptionally active and the labelling of RI/TIs was not the dead-end product of aberrant negative-strand synthesis. Failure originally to find TIs and TF cores was probably due to overdigestion with RNase A.

Chromatography of mouse hepatitis virus replicative intermediate and replicative form RNA.

One of our goals is to obtain large amounts of selected size classes of the Coronavirus replicative intermediates (RI) and native replicative form (RF) RNA that contain the templates for viral genome and messenger RNA syntheses. The two replicative molecules differ in their relative amounts of single-stranded and double-stranded character, with the RF RNA considered essentially or completely double-stranded. It is known that there are seven species of RI RNA, each of which is proportional in size and abundance to one of the seven species of viral messenger RNA produced in infected cells (Sawicki and Sawicki 1990; 1998; Schaad and Baric 1994; Baric and Yount 2000). The presence in coronavirus-infected cells of seven viral minus strand templates was first reported by Sethna and Brian (1989). To assist in this effort, we undertook an analysis of how mouse hepatitis virus (MHV-A59) RI and RF RNA fractionate on three gel filtration matrices, Sepharose 2B, Superose 6 and Sephacryl S-1000. A second goal was to attempt to explain the failure by others to find subgenomic RI and their RF cores after ribonuclease treatment and gel filtration chromatography.

MHV subgenomic negative strand function.

Mouse hepatitis virus (MHV), a member of Nidovirales, contains a -32 KB linear, single-stranded, positive polarity RNA genome. Upon entry into the cell, the viral genome is transcribed into 7-8 subgenomic mRNAs ranging in size from ∼1.0-32.0 KB. The positive strand mRNAs are arranged in a 3′ co-terminal nested set, and each contains a 5’ end ∼72 nucleotide (nt) leader RNA sequence which is derived from the 5′ end of the genome. Leader RNA sequences are joined to body sequences of each subgenomic length mRNA at highly conserved transcriptional start (TSE) sites located just upstream from the coding sequences of each viral gene (Baric et al., 1983; Sethna et al, 1989). In addition to the viral mRNAs, both full-length as well as subgenomic length negative strand RNAs and replicative form (RF) RNAs have been detected in porcine transmissible gastro-enteritis virus (TGEV), bovine Coronavirus (BCV) and MHV-infected cells (Baric et al., 1983, Sethna et al., 1989; 1991; Sawicki and Sawicki, 1990). The subgenomic length negative strand RNA contains antileader RNA sequences (Sethna et al., 1991). It has been suggested that the subgenomic length negative strands function as dead end products of transcription (Yokomori et al., 1992; Jeong and Makino, 1992). In this manuscript, we provide direct evidence that the subgenomic negative strands function as the principle templates for the synthesis of each corresponding mRNA.

Nascent Synthesis of Leader Sequence-Containing Subgenomic mRNAs in Coronavirus Genome-Length Replicative Intermediate RNA

Infection with coronavirus results in the accumulation of genomic-sized mRNA and six to eight subgenomic mRNAs that make up a 3′ coterminal nested-set structure. Genome-length negative-strand RNA and subgenomic-length negative-strand RNAs, each of which corresponds to each of the subgenomic mRNAs, also accumulate in infected cells. The present study examined whether the genome-length negative-strand RNA serves as a template for subgenomic mRNA synthesis. Genome-length replicative intermediate (RI) RNA was purified by two-dimensional gel electrophoresis of intracellular RNAs from cells infected with mouse hepatitis virus. RNase A treatment of the purified genome-length RI resulted in the production of the genome-length replicative form RNA, indicating that the genome-length RI included genome-length template RNA. RNase protection assays using the purified genome-length RI and two probes, which corresponded to the 5′ 300-nt region of mRNA 6 and to the same region of mRNA 7, showed the presence of nascent leader sequence-containing subgenomic mRNAs in the genome-length RI. These data demonstrated that the genome-length negative-strand RNA serves as a template for subgenomic mRNA synthesis.

Subgenomic negative-strand RNA function during mouse hepatitis virus infection.

Mouse hepatitis virus (MHV)-infected cells contain full-length and subgenomic-length positive- and negative-strand RNAs. The origin and function of the subgenomic negative-strand RNAs is controversial. In this report we demonstrate that the synthesis and molar ratios of subgenomic negative strands are similar in alternative host cells, suggesting that these RNAs function as important mediators of positive-strand synthesis. Using kinetic labeling experiments, we show that the full-length and subgenomic-length replicative form RNAs rapidly accumulate and then saturate with label, suggesting that the subgenomic-length negative strands are the principal mediators of positive-strand synthesis. Using cycloheximide, which preferentially inhibits negative-strand and to a lesser extent positive-strand synthesis, we demonstrate that cycloheximide treatment equally inhibits full-length and subgenomic-length negative-strand synthesis. Importantly, following treatment, previously transcribed negative strands remain in transcriptionally active complexes even in the absence of new negative-strand synthesis. These findings indicate that the subgenomic-length negative strands are the principal templates of positive-strand synthesis during MHV infection.

Effect of La Crosse virus infection on overwintering of Aedes triseriatus.

The effect of La Crosse (LAC) virus infection on Aedes triseriatus overwintering success was determined. Eggs from LAC virus transovarially infected (LAC TOT+) and uninfected (LAC TOT-) Ae. triseriatus colonies were induced into diapause, held in natural conditions, and returned to the laboratory at predetermined times for assay of diapause, mortality, and filial infection rates, and to examine viral transcription and replication during diapause. Embryos from the LAC TOT+ colony exhibited greater cumulative mortality (16.7%) than the LAC TOT- eggs (7.3%) throughout the overwintering periods. The increased mortality rate in LAC TOT+ eggs corresponded with a decrease in filial infection rates. Eggs from the LAC TOT+ colony terminated diapause more readily than the LAC TOT- colony. An RNA strand-specific reverse transcriptase-polymerase chain reaction technique was used to monitor viral transcription and replication in mosquito eggs during overwintering, and to compare viral replication in diapausing and nondiapausing embryos. Viral messenger and replicative form RNA were present in eggs in all sample periods, suggesting that some virus replication occurred during diapause.

Conversion of dengue virus replicative form RNA (RF) to replicative intermediate (RI) by nonstructural proteins NS-5 and NS-3.

Dengue viruses infect more than 100 million people each year and cause serious clinical manifestations. It is important to understand the replication of these viruses so that therapeutic and/or prophylactic agents may be designed. Dengue virus type 2 nonstructural proteins NS-5 and NS-3 were produced by in vitro transcription and translation of cloned genes. Both proteins possessed RNA-dependent RNA polymerase activity as measured by their ability to convert purified replicative form (RF) RNA to replicative intermediate (RI). The recombinant proteins, however, required one or more cellular protein(s) for their activity. Examination of NS-3 protein sequence revealed heretofore unnoticed sequence similarities with other polymerases.

Molecular characterization of virus-specific RNA produced in the brains of flavivirus-susceptible and -resistant mice after challenge with Murray Valley encephalitis virus.

Natural resistance to flaviviruses in mice is controlled by a single genetic locus, FIv, on chromosome 5. Although the mechanism of this resistance is not fully understood, it is believed to operate at the level of virus replication rather than the immune response. It has been hypothesized that enhanced production of viral defective interfering (DI) particles is responsible for a substantial reduction in the titres of infectious virus in resistant mice. However, this has never been established at the molecular level since such particles have not been isolated and characterized. We have studied the products of virus replication in the brains of flavivirus-susceptible C3H/HeJ (Flv(s)) and -resistant congenic C3H/RV (Flv(r)) mice after an intracerebral challenge (i.c.) with Murray Valley encephalitis (MVE) virus and have found no evidence for the accumulation of truncated viral RNA in the brains of resistant mice. All three major viral RNA species, the replicative intermediate (RI), replicative form (RF) and virion RNA (vRNA) together with a subgenomic RNA species of 0.6 kb, which has not been previously described, were present in the brains of both mouse strains. However, the viral RF and RI RNA forms preferentially accumulated in the brains of resistant mice. Thus, we confirm that the resistance allele Flv(r) interferes with discrete steps in flavivirus replication, although the precise mechanism remains to be determined.

Equine arteritis virus subgenomic mRNA synthesis: analysis of leader-body junctions and replicative-form RNAs.

In addition to the genomic RNA, a 3' coterminal nested set of six subgenomic mRNAs is produced in equine arteritis virus (EAV)-infected cells. The seven viral RNAs are also 5' coterminal, since they all contain a 206-nucleotide common leader sequence which is identical to the 5' end of the genome. A conserved penta-nucleotide sequence motif, 5' UCAAC 3', was shown to be present at the junctions between the leader and body sequences in each of the mRNAs. In addition, two alternative junction sites were detected for mRNA 3. Seven replicative-form (RF) RNAs (RFs I to VII), corresponding to the genomic RNA and each of the subgenomic EAV mRNAs, could be prepared from lysates of infected cells. The minus-strand RNA contents of these RF RNAs were analyzed by using an RNase protection assay with an RNA probe containing the mRNA 2 leader-body junction. It was established that RF II contained a negative-stranded copy of mRNA 2, including a complementary leader sequence. The presence of subgenomic minus-strand RNA in RFs is indicative of a function as a transcription template during the production of EAV subgenomic mRNAs.

Genetics of mouse hepatitis virus transcription: evidence that subgenomic negative strands are functional templates

Mouse hepatitis virus (MHV) A59 temperature-sensitive (ts) mutants belonging to complementation group C were characterized and mapped by standard genetic recombination techniques. Temperature shift experiments early in infection suggested that the group C allele can be divided into two phenotypically distinct subgroups, designated C1 and C2. Since previous data indicated that the group C1 mutants probably contained an early defect which affects negative-strand synthesis, RNA synthesis was further examined by analyzing replicative-form (RF) RNA. Full-length as well as subgenomic-length RF RNAs were radiolabeled from 3 to 12 h postinfection (p.i.) and labeled late in infection after shift to the nonpermissive temperature (39.5 degrees C). The relative percent molar ratios of each mRNA and corresponding RF RNA were roughly equivalent throughout infection. Temperature shift experiments at 5.5 or 6.0 h p.i. resulted in an 83 to 92% reduction in the amount of total RF RNA at 39.5 degrees C. Radiolabeling time course experiments after temperature shift to 39.5 degrees C also demonstrated incorporation (6 to 9 h p.i.) into both subgenomic-length and full-length RF RNAs, suggesting that previously transcribed negative strands were functional templates throughout infection. To determine if the reduction in RF RNA was due to a decrease in positive- or negative-strand RNA synthesis, rates of mRNA synthesis were calculated from both full-length and subgenomic-length templates. The rate of mRNA synthesis after the shift was increased at 39.5 degrees C compared with that at 32 degrees C regardless of the template used; however, transcription rates calculated from subgenomic-length templates were similar to those of other viral and eukaryotic polymerases. These findings support the notion that the group C1 allele regulates negative-strand RNA synthesis and strongly suggest that the subgenomic negative-strand RNAs are probably the predominant functional templates for the synthesis of positive-strand RNAs late in infection.

Use of a flavivirus RNA-dependent RNA polymerase assay to investigate the antiviral activity of selected compounds.

We have developed an assay using flavivirus RNA-dependent RNA polymerase to test the inhibitory activity of potential antiviral agents. The effects of antiviral agents on RNA synthesis were examined in this assay using extracts of Vero cells infected with dengue virus type 2 or Kunjin virus. Several different classes of known polymerase inhibitors were tested. The synthesis of double-stranded replicative form RNA was inhibited in a dose-dependent fashion in the presence of the polyoxometalate HPA-23 [(NH4)18(NaW21Sb9O86)17].14 H2O and several structurally related compounds.

Detection of Sugarcane Mosaic Virus and Fiji Disease Virus in Diseased Sugarcane using the Polymerase Chain Reaction

The reverse transcription-polymerase chain reaction (RT-PCR) was adapted for detection of sugarcane mosaic potyvirus (SCMV) and Fiji disease fijivirus (FDV) in total nucleic acid extracts from diseased sugarcane. The denaturation method developed for RT-PCR of FDV-specific double-stranded RNA (dsRNA) was suitable for the amplification of SCMV RNA and also amplified SCMV-specific replicative form (double-stranded) RNA. RT-PCR amplification of total nucleic acid extracts from SCMV-infected sugarcane yielded a 359-bp product and detected the presence of the virus in a 1:10 4 dilution of a solution containing the total nucleic acids extracted from 250 mg of SCMV-infected tissue [...]

Evidence for New Transcriptional Units Encoded at the 3′ End of the Mouse Hepatitis Virus Genome

Prior studies have demonstrated that the mouse hepatitis virus (MHV) genome is divided into at least seven coding regions each transcribed into a distinct mRNA. The majority of these mRNAs are synthesized from a highly conserved intergenic start site (UCU/CAAAC), contain a 65-72 nt leader RNA at their 5′ end and form a 3′ co-terminal nested set. In this study, we have used radiolabeling experiments to demonstrate the presence of a small ∼900 nt mRNA and its corresponding RF RNA in MHV-infected cells. Surprisingly, PCR amplification and sequence analysis revealed the presence of not one, but two small leader-containing RNAs that initiate from highly conserved intergenic start sites (UCCAAAC and UCUAAAU) which are located within the 3′-most nucleocapsid gene sequence. These studies provide evidence suggesting that one or two additional small mRNAs are encoded from the 3′ end of the MHV genome.

A simple and rapid method of preparing large fragments of dengue virus cDNA from replicative-form RNA using reverse transcriptase and PCR

A method is described for cloning large fragments (1.5 kb to 2 kb) of dengue virus cDNA from replicative-form viral RNA. Aedes albopictus cells (C6/36 clone) infected with dengue virus contain double-stranded, replicative-form RNA molecules which were used as a template for an initial reverse transcription using a primer containing sequence homologous to regions of the genome at or near the 3' end of the gene being studied. The product was then used as a template for polymerase chain reaction (PCR) amplification using the same 3' primer and a second which hybridized to a region at the 5' end of the sequence to be cloned. Both primers were engineered to contain specific restriction enzyme cutting sites which enabled the PCR product to be cut and cloned directly into plasmids for sequencing and expression studies. We have used this method to construct clones of the envelope glycoprotein gene (E) and the non-structural genes 1 and 2a (NS1/2a) and 3 (NS3) of dengue type 2, Tonga 1974 strain, and E and NS1/2a from dengue type 3, H-87 strain, either as discrete genes or as constructs with long and short leader sequences, with or without anchor sequences. The method could be applied to the cloning of any gene from any flavivirus, directly from infected cell extracts, without the necessity for tedious virus purification steps.

Molecular and ultrastructural analysis of heavy membrane fractions associated with the replication of Kunjin virus RNA.

In subcellular extracts of Kunjin virus-infected cells prepared by lysis and differential centrifugation, the viral RNA polymerase, RNA and proteins were associated mainly with cytoplasm. When the cytoplasmic extract (500 g supernate) of infected cells labelled for 3 h from 24 h post-infection was further fractionated by rapid centrifugation through a sucrose density gradient, all viral products were located only in dense or "heavy membrane" fractions, which contained three types of virus-induced morphologically distinct membrane structures. These dense fractions were treated with 0.5% NP40 and the soluble material was again centrifuged through a sucrose gradient for analyses as before. Viral RNA polymerase activity was retained and was associated with replicative intermediate RNA and some replicative form RNA in the peak enzyme fractions sedimenting at 20S to 40S. Enrichment of NS3 and of the small nonstructural proteins NS2A and NS2B/NS4A was apparent in these fractions which were well separated from the slow sedimenting structural proteins. No detergent-resistant structures in the "heavy membrane" fractions other than ribosome-like particles were visible. The data show that the RNA polymerase complex cosedimented with virus-induced membrane structures and remained associated with specific nonstructural proteins and replicative intermediate RNA after detergent treatment.