The replicative intermediate molecule of bovine viral diarrhoea virus contains multiple nascent strands

Summary.  The replication of bovine viral diarrhoea virus (BVDV) RNA is considered to involve replicative intermediates (RI) and replicative forms (RF) of virus RNA. These RNA species were radiolabelled metabolically by ³H-uridine in BVDV-infected cells and separated by sucrose gradient centrifugation. RNA in the fractions was then digested with RNase A in high salt (2 × SSC) and the RNase-resistance was determined. Using the Baltimore formula, this led to the calculation that the RI of BVDV contained 6–7 nascent strands on the template. This suggests that the structure of the BVDV RI is similar to that of poliovirus and dengue 2 virus. Accepted September 15, 1997 Received May 26,1997     

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.     

Replicative forms of Sendai virus RNA

Summary The formation of replicative forms of Sendai virus RNA in virus-infected cells was studied. Several RNA species with sedimentation coefficients 7 S to 48S were revealed in the course of infection, and a certain sequence in their appearance was observed. Replicative forms of Sendai virus RNA were found as multistranded replicative intermediate (RI) with sedimentation coefficients ranging from 20S to 30S, and as double-stranded replicative form (RF) with a sedimentation coefficient of about 22–23S. Parental RNA was found in the intranuclear structures and in the cytoplasm. Participation of parental RNA in the formation of double-stranded RF was evidenced.     

Replication strategy of Kunjin virus: evidence for recycling role of replicative form RNA as template in semiconservative and asymmetric replication

Only three forms of Kunjin virus-specified RNA were isolated from cytoplasm early after the latent period (about 15 hr) viz., 44 S genomic-sized single-stranded RNA, 20 S double-stranded "replicative form" (RF), and 20-28 S partially ribonuclease-resistant (about 70%) "replicative intermediate" (RI). The RF and RI were resolved by electrophoresis in aqueous-agarose gel only following LiCl fractionation. The RI did not enter urea-polyacrylamide gels. After denaturation of untreated or RNase-treated RI and RF, only 44 S RNA was present in electropherograms. RNA polymerase activity at 8 hr postinfection was detected by in vitro assays of cytoplasmic extracts and reached a maximum at 24 hr, the only major labeled product being RF; a trace amount of free 44 S RNA was also produced. These results, and the kinetics of incorporation of [3H]uridine into RI, RF, and 44 S RNA in pulse and pulse-chase experiments, formed the basis of a model in which flavivirus RF functions as a recycling template for semiconservative and (mainly) asymmetric replication, on which only one nascent strand is synthesized per cycle.     

Replication of tobacco mosaic virus RNA in tobacco mesophyll protoplasts inoculated in vitro

Synthesis of tobacco mosaic virus (TMV)-specific RNAs was investigated using tobacco mesophyll protoplasts inoculated in vitro. Three species of TMV-specific RNA were separated by polyacrylamide gel electrophoresis, and were identified as single-stranded viral RNA, its replicative form (RF), and replicative intermediate (RI) by examining their chromatographic behavior on cellulose, solubility in 1 M NaCl, and susceptibility to denaturation by dimethylsulfoxide. The molecular weight of RF was shown to be about 3.8 × 10⁶ by coelectrophoresis with rice dwarf virus RNA. Time course of the synthesis of RF and RI as well as the results of pulse-chase experiments were consistent with the possible precursor role of these structures in the synthesis of TMV-RNA. No other forms of TMV-specific RNA were detected in infected protoplasts. Kinetics of the replication of TMV-RNA in synchronously infected protoplasts showed the presence of three successive phases in virus replication. During the initial phase, viral RNA replicated exponentially and was encapsidated 4–5 hr later, so that most of the viral RNA synthesized remained free or only partially coated. The rate of viral RNA replication became linear at the end of the initial phase and remained so throughout the subsequent phases. Active formation of virus particles continued during the intermediate phase to encapsidate the bulk of viral RNA synthesized by this time. In the final phase, the synthesis of viral RNA was closely followed by encapsidation.     

Partial characterisation of citrus leaf blotch virus, a new virus from Nagami kumquat

Summary.  Citrus leaf blotch virus (CLBV) was purified from leaves of Nagami kumquat SRA-153 that showed bud union crease when propagated on Troyer citrange. Virions were filamentous particles (960 × 14 nm) containing a 42 kDa protein and a single-stranded RNA (ssRNA) of about 9,000 nt (Mr 3 × 10⁶). Infected tissue contained three species of double-stranded RNA (dsRNA) of Mr 6, 4.5 and 3.4 ×  10⁶. The nucleotide sequence of several complementary DNA (cDNA) clones showed significant similarities with replication-related proteins from plant filamentous viruses in several genera. A digoxigenin-labelled probe from one of these cDNA clones hybridised in Northern blots with ssRNA from virions and with the three dsRNA species, suggesting that the ssRNA is the genomic RNA of the virus, the largest dsRNA is its replicative form, and the two smaller dsRNAs probably replicative forms of 5′ co-terminal subgenomic RNAs. CLBV was also detected in several citrus cultivars from Spain and Japan including Navelina sweet orange field trees propagated on Troyer citrange showing bud union crease; however, no virus could be detected in other citrus trees with similar symptoms. This indicates that CLBV is not restricted to kumquat SRA-153, but its involvement in causing the bud union disorder remains unclear. Received February 22, 2000 Accepted June 23, 2000     

Replication of tobacco mosaic virus : III. Viral RNA metabolism in separated leaf cells

The metabolism of RNA has been studied in cell suspensions prepared from tobacco mosaic virus (TMV)-infected tobacco leaves. The cell suspensions incorporated [³H]-uridine into encapsidated viral RNA at a constant rate for as long as 44 hr. Gel electrophoretic analyses of RNA extracted from infected and uninfected cells which had been exposed to [³H]uridine, showed that both incorporated label into ribosomal RNA species. However, RNA preparations from infected cells contained five additional species of RNA not observed in preparations from healthy cells; in addition to TMV RNA, label was detected in two species of double-stranded RNA identified as replicative form (RF) and replicative intermediate (RI), a single-stranded RNA component of low molecular weight (LMC) and one with a molecular weight greater than that of TMV RNA. Synthesis of all five species of virus-specific RNA was insensitive to actinomycin D indicating their independence of cellular DNA.     

Infectivity of mengovirus replicative form. IV. Intracellular conversion into replicative intermediate.

The replicative form (RF) of mengovirus was previously shown to be infectious, and the infectivity of this virus-induced, double-stranded RNA, to depend on the host-cell macromolecular synthesis. Analysis of the ³H-labeled RNAs recovered from the cytoplasm of ³H-RF-infected cells revealed that the parental RF molecules sedimented faster in neutral sucrose density gradients as the infectious cycle proceeded. When ³H-RF-infected cells were pulse-labeled with [¹⁴C]uridine at 3 hr after infection most of the [¹⁴C]RNA was found in a rapidly sedimenting structure derived from (or tightly associated with) the parental ³H-RF. This structure had all the characteristics of the replicative intermediate (RI). Treatment of the host cell with interferon abolished the infectivity of RF, but did not prevent the intracellular conversion of RIP into RI. By contrast, exposure of the cultures to actinomycin D suppressed both the infectivity of RF and its transition to RI. The role of a host cell factor in the mechanism of infectivity of picornavirus RF is discussed.     

Synthesis of dengue virus RNA in vitro: initiation and the involvement of proteins NS3 and NS5

Summary An assay for flavivirus RNA-dependent RNA polymerase activity in vitro was established using extracts of Vero cells infected with dengue virus type 2 (DEN-2) or Kunjin virus (KUN). RNA synthesis was initiated on a template of viral replicative form (RF) and RF was converted to the replicative intermediate (RI). The RNA-dependent RNA polymerase complex of DEN-2 utilised either DEN-2 or KUN RF as template, and similarly the KUN polymerase complex utilised either DEN-2 or KUN RF template. In addition, antibodies against the nonstructural proteins NS3 and NS5 inhibited the conversion of RF to RI, indicating that NS3 and NS5 are involved in viral RNA replication.     

Characterization of RNA-dependent RNA polymerases in uninfected and cowpea chlorotic mottle virus-infected cowpea leaves: selective removal of host RNA polymerase from membranes containing CCMV RNA replicase.

Extracts from Cowpea chlorotic mottle virus (CCMV)-infected Cowpea leaves contained membrane-bound (31,000 g pellet) and soluble (31,000 g supernatant) RNA-dependent RNA polymerase activities. The membrane-bound RNA-dependent RNA polymerase (CCMV RNA replicase) increased 12-fold 4 days after inoculation. The viral RNA synthesis in vitro proceeded linearly for 20 min and required the four nucleoside triphosphates and Mg²⁺ ions for activity. Manganese ion was a poor substitute for Mg²⁺. Optimal enzymatic activity in vitro was unaffected by exogenous RNA or KCI. The CCMV RNA replicase product was predominantly heterodisperse single-stranded RNAs, some of which comigrated with CCMV virion RNA. Small amounts of large double-stranded RNAs were also products of the replicase reaction. The soluble RNA-dependent RNA polymerase from CCMV-infected or healthy Cowpea leaves required the four nucleoside triphosphates and Mg²⁺ ions for activity. Its activity in the in vitro assay was stimulated by adding exogenous RNAs but was inhibited by KCI. The product of the soluble RNA-dependent RNA polymerase was predominantly double-stranded RNA of approximately 4 to 6 S. RNA-dependent RNA polymerase activity, similar to that detected in the soluble fraction, was detected in the membrane pellet. This activity, which complicates the analysis of viral replicase assay, was removed without affecting CCMV RNA replicase activity by washing the 31,000 g pellets with buffer containing 0.5 M KCI. The KCI treatment aids in preparation of membrane-bound fractions devoid of host RNA-dependent RNA polymerase activity and high in viral replicase activity.     

Genome structure and complete sequence of genomic RNA of Daphne virus S

Summary. The complete genomic nucleotide sequence and structure of Daphne virus S (DVS), a daphne-infecting member of the genus Carlavirus, were determined. The genome of DVS was 8,739 nucleotides long, excluding the poly (A) tails. The genome of DVS contained six open reading frames coding for proteins of Mr 227 kDa (viral replicase), 25 kDa, 11 kDa and 7 kDa (triple gene block TGB) proteins 1, 2 and 3), 35 kDa (coat protein; CP), and 12 kDa from the 5′ to 3′ ends; respectively. This is the typical genome structure of members of the genus Carlavirus. Overall amino acid sequence similarities for the six ORFs of DVS were from 58.5% to 13.2% to those of the other carlaviruses. The 227 kDa replicase of DVS shared 45.5–39.2% amino acid similarities to that of 8 other known carlaviruses. Results from phylogenetic analyses of viral replicases and CPs demonstrated that DVS is a close relative of Helenium virus S and Chrysanthemum virus B. A total of 13 isolates of DVS shared 100–95.9% identities for the amino acid level and 99.5–81.0% identities for the nucleotide level. This is the first report of the complete genome sequence and structure of DVS and supports the conclusion that DVS is a typical species of the genus Carlavirus.     

Characterization and in vitro translation analysis of pelargonium flower break virus

Summary.  Pelargonium flower break virus (PFBV) is one of the common viruses in the glasshouses of Western Europe and has been assigned to the genus Carmovirus. A Spanish isolate obtained from nursery-grown Pelargonium zonale plants (PFBV-m) has been characterized. The molecular weight of genomic RNA and coat protein of PFBV-m were determined to be 1.36 × 10⁶ (corresponding to approximately 4 kb) and 36,000, respectively. Only genomic-size RNA was encapsidated in PFBV virions; making necessary to purify double-stranded RNA from infected tissue in order to detect putative PFBV subgenomic RNAs. PFBV RNA directed the synthesis of a major polypeptide of 34 kDa and three other relevant polypeptides of estimated sizes 88–90 kDa, 42 kDa and 35–36 kDa. Antisera specific to PFBV immunoprecipitated the in vitro translated 35–36 kDa polypeptide indicating that this polypeptide is the PFBV coat protein. The PFBV in vitro translation pattern was very similar to that of CarMV although the relative levels of translated coat protein differed dramatically between the two viruses, most probably due to the lack of encapsidation of subgenomic PFBV. In vitro translation studies with a different biological clone obtained from the same PFBV-m isolate revealed a prominent additional polypeptide which is postulated to be a truncation of the 5′ proximal ORF. Received November 27, 1998 Accepted March 24, 1999     

Regulation of single-strand RNA synthesis of alfalfa mosaic virus in non-transgenic cowpea protoplasts by the viral coat protein

Summary.  We have compared the RNA synthesis of alfalfa mosaic virus in complete (by RNAs 1, 2 and 3) and incomplete infections (by RNAs 1 and 2) of cowpea protoplasts. Both viral RNA polymerase activity and accumulation of viral RNA were measured. By annealing RNA in solution with ³²P-labelled probes of plus and minus polarity followed by treatment with ribonucleases, we determined viral RNAs quantitatively in both single- and double-stranded RNA fractions. The accumulation of single-stranded RNA of positive polarity differed considerably between the two types of infection (250 ng vs. less than 1 ng per 10⁵ protoplasts), although viral RNA polymerase activities as measured in vitro and the concentrations of minus RNA were similar. Since the method also measured fragmented RNA, this difference is probably not due to lack of protection of viral RNA by coat protein during incomplete infection. Synthesis of single-stranded plus RNA requires either RNA 3 itself or one of its gene products. We postulate that coat protein is the stringent regulator of alfalfa mosaic virus genomic expression. Accepted November 3, 1997 Received August 14, 1997     

HIV DNA integration during cell-to-cell transmission of infection: evidence for partially integrated DNA structures in acutely infected cells

Summary.  A one-step cell-to-cell transmission model of human immunodeficiency virus (HIV) infection was used to study viral DNA integration in the early phase of viral replication. Co-culturing H3B cells as virus donors with CD4+ Hut78 recipient cells in a ratio of 1:4 produced a synchronous, one-step viral infection with de novo synthesis of unintegrated HIV DNA within 4 h p.i., which subsequently integrates in the host genomic DNA to form provirus. To study the kinetics of viral DNA integration, cellular chromosomal DNA was isolated at different times after co-culturing and extensive electrophoresis was used to remove residual unintegrated viral DNA. Removal of contaminating, unintegrated viral DNA in the purified chromosomal DNA fraction was confirmed by various experiments. When purified chromosomal DNA (free of contaminating unintegrated viral DNA) – from the mix of acutely infected cells – was digested with restriction enzymes KpnI, BamHI or PstI and analysed by Southern blot hybridization, integration of viral DNA into chromosomal DNA was first observed at 8 h p.i. and was essentially complete by 72 h p.i. In addition, evidence was found for a relatively stable, partially integrated HIV DNA structure within the chromosomal DNA, that was first detectable at 8 h p.i. and did not become fully integrated until 72 hours post infection. Received December 12, 2000 Accepted March 30, 2001     

The effect of ribonuclease on the replicative forms of Sindbis virus RNA

Summary Three species of double-stranded RNA, designated RF I, RF II, and RF III in order of decreasing size (25), are produced by ribonuclease treatment of extracts of chicken embryo cells infected for 6 hours with Sindbis virus. Only one class of replicative form RNA is present in extracts not treated with ribonuclease; this class contains some molecules which can be enzymatically cleaved to produce the other two replicative forms. At a low level of enzyme (0.001 µg/ml) the major species obtained was RF I, the replicative form of the genome. When the enzyme concentration was increased 10-, 100-, and 1000-fold, there was a progressive increase in the proportions of RF's II and III and a concomitant decrease in the proportion of RF I. The generation of RF's II and III by nuclease resulted in the ratio expected for these two species if they are produced by cleavage of RF I-like molecules. In preparations of isolated double-stranded RNA, only RF I and replicative intermediate RNA were present. Mild nuclease treatment of these preparations converted the replicative intermediates primarily to RF I. Higher enzyme levels generated greater proportions of RF II and RF III, but RF I-like molecules were the major source for these increased proportions. Treatment of the isolated naturally occurring replicative form with 0.01 µg of ribonuclease per ml cleaved some molecules migrating as RF I during gel electrophoresis into molecules which migrated as RF II and RF III.With 5 Figures     

A possible replicative form of brome mosaic virus RNA 4

A double-stranded RNA of a size expected for the replicative form of BMV RNA 4, the smallest of the four RNAs of brome mosaic virus, was isolated from infected leaves by a procedure involving removal of single-stranded RNA by precipitation in 2 M LiCl, followed by cellulose chromatography and polyacrylamide gel electrophoresis. Like the other double-stranded RNAs of BMV, it had characteristics of a replicative form, such as distinctive elution patterns from cellulose and hydroxyapatite columns, and purple staining with toluidine blue O. It coelectrophoresed in 2.5% polyacrylamide gels with double-stranded RNA synthesized in vitro by Qβ replicase with BMV RNA 4 as template. The origin and mode of replication of BMV RNA 4 is discussed.     

Synergism in replication of cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) RNA in orchid protoplasts

Summary.  An in vitro orchid protoplast isolation method to study replication kinetics of CymMV and ORSV was developed. This method allows the isolation of viable and raphid-free petal protoplasts from an orchid hybrid, {\it Dendrobium} Sonia (Dendrobium Caesar × Dendrobium Tomie Drake). The optimum field strength for both viral RNA to achieve good efficiency of electroporation was 750 V/cm and the optimum viral RNA concentration required was 1 μg and 4 μg per 2 × 10⁶ protoplasts for CymMV and ORSV, respectively. Autoradiographs of Northern blots depicting the viral genomic and subgenomic RNA in the extracts, referred to as the “Replication Footprint Profiles” (RFP) of specific CymMV/ORSV virus were prepared at different time intervals. Viral RNA synthesis reached a maximum at 18 h for CymMV and 24 h for ORSV. When CymMV and ORSV viral RNA were electroporated into the protoplasts simultaneously, detection signals of both the positive and negative strand viral RNA increased as compared to the singly infected protoplasts. Thus, synergism in replication of CymMV and ORSV was observed in orchid protoplasts. Received November 20, 1997 Accepted February 25, 1998     

Synthesis of genomic and subgenomic RNAs by a membrane-bound RNA-dependent RNA polymerase isolated from oat plants infected with cereal yellow dwarf virus

Summary. A membrane-bound RNA-dependent RNA polymerase (RdRp) complex was isolated by differential sedimentation from oat plants infected with cereal yellow dwarf virus (CYDV). When incubated with ³²P-labelled UTP, unlabelled ATP, CTP and GTP, and Mg²⁺ ions, the RdRp preparation catalysed the synthesis of double-stranded (ds) RNAs corresponding in size to the virus genomic RNA (5.7 kbp) and two putative subgenomic RNAs (2.8 and 0.7 kbp). Hybridisation using strand-specific hybridization targets showed that the 5.7-kbp dsRNA was labelled mainly in the plus strand, whereas the 2.8- and 0.7-kbp dsRNAs were labelled only in the minus strand. Genomic-length single-stranded, plus-strand RNA of 5.7 kb and single-stranded, plus-strand subgenomic RNAs of 2.8 and 0.7 kbp were detected in RNA isolated from oat plants infected with CYDV. Mapping experiments were consistent with the genomic and subgenomic RNAs having common 3′ ends, but different 5′ ends, whether produced in vitro or in vivo. The RdRp-encoding region of the CYDV genome was cloned and expressed in Escherichia coli, and the purified protein was used to raise antibodies in a rabbit. In immunoblots, the antibodies detected a protein of about 68 kDa in RdRp preparations from CYDV-infected oat plants, but not from equivalent preparations from healthy oats. As far as we are aware, this is the first report of an in vitro RNA synthesis system for a phloem-limited virus.     

Evidence for different replicative strategies in the plant tombusviruses

Analysis of the replicative form (RF) RNAs of three viruses assigned to the Tombusvirus group-tomato bushy stunt virus (TBSV), carnation ringspot (CRSV), and turnip crinkle (TCV) viruses-indicated that all three have different replicative strategies. Viral specific double-stranded (ds)RNAs were isolated from infected plant tissues by phenol extraction and cellulose chromatography and analyzed by polyacrylamide gel electrophoresis. A single RF species with a molecular weight of 3.0 x 10(6) daltons, twofold that of the viral single-stranded RNA, was isolated from TCV-infected plants. Two major RF species of 3.0 and 1.0 x 10(6) daltons and a minor one with 1.2 x 10(6) daltons were isolated from CRSV-infected tissues corresponding to the three genomic RNAs of this multicomponent virus. TBSV-infected tissues yielded two replicative intermediate RNA species in addition to two dsRNAs with molecular weights of 3.2 and 1.5 x 10(6) daltons. Hybridization experiments indicated that both dsRNAs were TBSV-specific. It is proposed that the 1.5 x 10(6) dalton dsRNA represents the replicative form of a subgenomic fragment of the TBSV RNA with a postulated MW of 0.75 x 10(6) daltons. A model for the replication of TBSV is suggested and its possible significance discussed.