Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis.

The temporal sequence of coronavirus plus-strand and minus-strand RNA synthesis was determined in 17CL1 cells infected with the A59 strain of mouse hepatitis virus (MHV). MHV-induced fusion was prevented by keeping the pH of the medium below pH 6.8. This had no effect on the MHV replication cycle, but gave 5- to 10-fold-greater titers of infectious virus and delayed the detachment of cells from the monolayer which permitted viral RNA synthesis to be studied conveniently until at least 10 h postinfection. Seven species of poly(A)-containing viral RNAs were synthesized at early and late times after infection, in nonequal but constant ratios. MHV minus-strand RNA synthesis was first detected at about 3 h after infection and was found exclusively in the viral replicative intermediates and was not detected in 60S single-stranded form in infected cells. Early in the replication cycle, from 45 to 65% of the [3H]uridine pulse-labeled RF core of purified MHV replicative intermediates was in minus-strand RNA. The rate of minus-strand synthesis peaked at 5 to 6 h postinfection and then declined to about 20% of the maximum rate. The addition of cycloheximide before 3 h postinfection prevented viral RNA synthesis, whereas the addition of cycloheximide after viral RNA synthesis had begun resulted in the inhibition of viral RNA synthesis. The synthesis of both genome and subgenomic mRNAs and of viral minus strands required continued protein synthesis, and minus-strand RNA synthesis was three- to fourfold more sensitive to inhibition by cycloheximide than was plus-strand synthesis.     

Short-lived minus-strand polymerase for Semliki Forest virus

Semliki Forest virus (SFV)-infected BHK-21, Vero, and HeLa cells incorporated [3H]uridine into 42S and 26S plus-strand RNA and into viral minus-strand RNA (complementary to the 42S virion RNA) early in the infectious cycle. Between 3 and 4 h postinfection, the synthesis of minus-strand RNA ceased in these cultures, although the synthesis of plus-strand RNA continued at a maximal rate. At the time of cessation of minus-strand RNA synthesis, two changes in the pattern of viral protein synthesis were detected: a decrease in the translation of nonstructural proteins and an increase in the translation of the viral structural proteins. Addition of cycloheximide and puromycin to cultures of SFV-infected BHK cells actively synthesizing both viral plus- and minus-strand RNA resulted within 15 to 30 min in the selective shutoff of minus-strand RNA synthesis. Removal of the cycloheximide-containing medium led to the resumption of minus-strand synthesis and to an increased rate of viral RNA synthesis. We conclude that the minus-strand polymerase regulates the rate of SFV plus-strand RNA synthesis by determining the number of minus-strand templates and that the synthesis of the minus-strand templates is regulated at the level of translation by a mechanism which utilizes one or more short-lived polymerase proteins.     

The 3'-terminal sequence of Bamboo mosaic virus minus-strand RNA interacts with RNA-dependent RNA polymerase and initiates plus-strand RNA synthesis

A 3'-terminal, 77-nucleotide sequence of Bamboo mosaic virus (BaMV) minus-strand RNA (Ba-77), comprising a 5' stem-loop, a spacer and a 3'-CUUUU sequence, can be used to initiate plus-strand RNA synthesis in vitro . To understand the mechanism of plus-strand RNA synthesis, mutations were introduced in the 5' untranslated region of BaMV RNA, resulting in changes at the 3' end of minus-strand RNA. The results showed that at least three uridylate residues in 3'-CUUUU are required and the changes at the penultimate U are deleterious to viral accumulation in Nicotiana benthamiana protoplasts. Results from UV-crosslinking and in vitro RNA-dependent RNA polymerase competition assays suggested that the replicase preferentially interacts with the stem structure of Ba-77. Finally, CMV/83 + UUUUC, a heterologus RNA, which possesses about 80 nucleotides containing the 3'-CUUUU pentamer terminus, and which folds into a secondary structure similar to that of Ba-77, could be used as template for RNA production by the BaMV replicase complex in vitro .     

Specific Sindbis virus-coded function for minus-strand RNA synthesis.

The synthesis of minus-strand RNA was studied in cell cultures infected with the heat-resistant strain of Sindbis virus and with temperature-sensitive (ts) belonging to complementation groups A, B, F, and G, all of which exhibited an RNA-negative (RNA-) phenotype when infection was initiated and maintained at 39 degrees C, the nonpermissive temperature. When infected cultures were shifted from 28 degrees C (the permissive temperature) to 39 degrees C at 3 h postinfection, the synthesis of viral minus-strand RNA ceased in cultures infected with ts mutants of complementation groups B and F, but continued in cultures infected with the parental virus and mutans of complementation groups A and G. In cultures infected with ts11 of complementation group B, the synthesis of viral minus-strand RNA ceased, whereas the synthesis of 42S and 26S plus-strand RNAs continued for at least 5 h after the shift to 39 degrees C. However, when ts11-infected cultures were returned to 28 degrees C 1 h after the shift to 39 degrees C, the synthesis of viral minus-strand RNA resumed, and the rate of viral RNA synthesis increased. The recovery of minus-strand synthesis translation of new proteins. We conclude that at least one viral function is required for alphavirus minus-strand synthesis that is not required for plus-strand synthesis. In cultures infected with ts6 of complementation group F, the syntheses of both viral plus-strand and minus-strand RNAs were drastically reduced after the shift to 39 degrees C. Since ts6 failed to synthesize both plus-strand and minus-strand RNAs after the shift to 39 degrees C, at least one common viral component appears to be required for the synthesis of both minus-strand and plus-strand RNAs.     

Size and location of poly (A) in encephalomyocarditis virus RNA.

Encephalomyocarditis (EMC) virus RNA contains a covalently bound sequence of polyriboadenylic acid (poly(A). This was determined by two-dimensional gel electrophoresis of complete T1 and pancreatic RNase digests of formamidesucrose gradient-purified RNA and subsequent analysis of the product by alkaline hydrolysis. The size of the EMC virus genomic poly(A) sequence was estimated by formamide-polyacrylamide gel electrophoresis of the RNase-resistant product, or by [3H-]poly(U) hybridization to freshly purified virion RNA, to be, on average, 40 nucleotides in length. The evidence obtained from [3H-]isoniazid labelling and other experiments would indicate that the poly(A) sequence is located at the 3'-terminus of EMC virus RNA.     

Polyadenylic acid on poliovirus RNA. II. poly(A) on intracellular RNAs.

The content, size, and mechanism of synthesis of 3'-terminal poly(A) on the various intracellular species of poliovirus RNA have been examined. All viral RNA species bound to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. At 3 h after infection, the poly(A) on virion RNA, relicative intermediate RNA, polyribosomal RNA, and total cytoplasmic 35S RNA was heterogeneous in size with an average length of 75 nucleotides. By 6 h after infection many of the intracellular RNA's had poly(A) of over 150 nucleotides in length, but the poly(A) in virion RNA did not increase in size suggesting that the amount of poly(A) which can be encapsidated is limited. At all times, the double-stranded poliovirus RNA molecules had poly(A) of 150 to 200 nucleotides. Investigation of the kinetics of poly(A) appearance in the replicative intermediate and in finished 35S molecules indicated that poly(A) is the last portion of the 35S RNA to be synthesized; no nascent poly(A) could be detected in the replicative intermediate. Although this result indicates that poliovirus RNA is synthesized 5' leads to 3' like other RNA's, it also suggests that much of the poly(A) found in the replicative intermediate is an artifact possibly arising from the binding of finished 35S RNA molecules to the replicative intermediate during extraction. The addition of poly(A) to 35S RNA molecules was not sensitive to guanidene.     

Cell-free replication of the hepatitis C virus subgenomic replicon

The hepatitis C virus (HCV) contains a plus-strand RNA genome. The 5' noncoding region (NCR) of the viral genome functions as an internal ribosome entry site, and its unique 3' NCR is required for the assembly of the replication complex during initiation of HCV RNA replication. Lohmann et al. (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilman, and R. Batenschlager, Science 285:110-113, 1999) developed a subgenomic HCV replicon system, which represents an important tool in studying HCV replication in cultured cells. In this study, we describe a cell-free replication system that utilizes cytoplasmic lysates prepared from Huh-7 cells harboring the HCV subgenomic replicons. These lysates, which contain ribonucleoprotein complexes associated with cellular membranes, were capable of incorporating [alpha(32)P]CTP into newly synthesized RNA from subgenomic replicons in vitro. Replicative forms (RFs) and replicative intermediates (RIs) were synthesized from the endogenous HCV RNA templates. Consistent with previous observations, RFs were found to be resistant to RNase A digestion, whereas RIs were sensitive to RNase treatment. The radiolabeled HCV RF-RI complexes contained both minus and plus strands and were specific to the lysates derived from replicon-expressing cells. The availability of a cell-free replication system offers opportunities to probe the mechanism(s) of HCV replication. It also provides a novel assay for potential therapeutic agents.     

Initiation of Genomic Plus-Strand RNA Synthesis from DNA and RNA Templates by a Viral RNA-Dependent RNA Polymerase

In contrast to the synthesis of minus-strand genomic and plus-strand subgenomic RNAs, the requirements for brome mosaic virus (BMV) genomic plus-strand RNA synthesis in vitro have not been previously reported. Therefore, little is known about the biochemical requirements for directing genomic plus-strand synthesis. Using DNA templates to characterize the requirements for RNA-dependent RNA polymerase template recognition, we found that initiation from the 3' end of a template requires one nucleotide 3' of the initiation nucleotide. The addition of a nontemplated nucleotide at the 3' end of minus-strand BMV RNAs led to initiation of genomic plus-strand RNA in vitro. Genomic plus-strand initiation was specific since cucumber mosaic virus minus-strand RNA templates were unable to direct efficient synthesis under the same conditions. In addition, mutational analysis of the minus-strand template revealed that the -1 nontemplated nucleotide, along with the +1 cytidylate and +2 adenylate, is important for RNA-dependent RNA polymerase interaction. Furthermore, genomic plus-strand RNA synthesis is affected by sequences 5' of the initiation site.     

Requirements at the 3' end of the sindbis virus genome for efficient synthesis of minus-strand RNA

The 3'-untranslated region of the Sindbis virus genome is 0.3 kb in length with a 19-nucleotide conserved sequence element (3' CSE) immediately preceding the 3'-poly(A) tail. The 3' CSE and poly(A) tail have been assumed to constitute the core promoter for minus-strand RNA synthesis during genome replication; however, their involvement in this process has not been formally demonstrated. Utilizing both in vitro and in vivo analyses, we have examined the role of these elements in the initiation of minus-strand RNA synthesis. The major findings of this study with regard to efficient minus-strand RNA synthesis are the following: (i) the wild-type 3' CSE and the poly(A) tail are required, (ii) the poly(A) tail must be a minimum of 11 to 12 residues in length and immediately follow the 3' CSE, (iii) deletion or substitution of the 3' 13 nucleotides of the 3' CSE severely inhibits minus-strand RNA synthesis, (iv) templates possessing non-wild-type 3' sequences previously demonstrated to support virus replication do not program efficient RNA synthesis, and (v) insertion of uridylate residues between the poly(A) tail and a non-wild-type 3' sequence can restore promoter function to a limited extent. This study shows that the optimal structure of the 3' component of the minus-strand promoter is the wild-type 3' CSE followed a poly(A) tail of at least 11 residues. Our findings also show that insertion of nontemplated bases can restore function to an inactive promoter.     

RNA Signals in Entero- and Rhinovirus Genome Replication

The VPg-linked, plus-stranded RNA genomes of entero- and rhinoviruses contain very different 5′ and 3′ terminal regions which harbor signals for RNA replication. The terminal cloverleaf-like structure of the 5′-nontranslated region (5′NTR) is known to be required for plus-strand RNA synthesis. Genetic evidence suggest that two stem-loop structures and the poly(A) tail of the 3′NTR have a function in minus-strand synthesis. All of the nonstructural viral proteins, and possibly also some cellular polypeptides, are believed to be involved in RNA replication. RNA synthesis is initiated on a poly(A) template and involves uridylylation of VPg to yield VPgpU(pU). This precursor is likely to serve as primer for the RNA polymerase 3D^polduring both minus- and plus-strand RNA synthesis.     

Synthesis of plus- and minus-strand RNA in rotavirus-infected cells.

The genomes of the rotaviruses consist of 11 segments of double-stranded RNA. During RNA replication, the viral plus-strand RNA serves as the template for minus-strand RNA synthesis. To characterize the kinetics of RNA replication, the synthesis and steady-state levels of viral plus- and minus-strand RNA and double-stranded RNA in simian rotavirus SA11-infected MA104 cells were analyzed by electrophoresis on 1.75% agarose gels containing 6 M urea (pH 3.0). Synthesis of viral plus-strand and minus-strand RNAs was detected initially at 3 h postinfection. The steady-state levels of plus- and minus-strand RNAs increased from this time until 9 to 12 h postinfection, at which time the levels were maximal. Pulse-labeling of infected cells with [3H]uridine showed that the ratio of plus- to minus-strand RNA synthesis changed during infection and that the maximal level of minus-strand RNA synthesis occurred several hours prior to the peak of plus-strand RNA synthesis. No direct correlation was found between the levels of plus-strand and minus-strand RNA synthesis in the infected cell. Pulse-labelling studies indicated that both newly synthesized and preexisting plus-strand RNA can act as templates for minus-strand RNA synthesis throughout infection. Studies also showed that less than 1 h was required between the synthesis of minus-strand RNA in vivo and its release from the cell within virions.     

Modification of the 5' terminus of Sindbis virus genomic RNA allows nsP4 RNA polymerases with nonaromatic amino acids at the N terminus to function in RNA replication

We have previously shown that Sindbis virus RNA polymerase requires an N-terminal aromatic amino acid or histidine for wild-type or pseudo-wild-type function; mutant viruses with a nonaromatic amino acid at the N terminus of the polymerase, but which are otherwise wild type, are unable to produce progeny viruses and will not form a plaque at any temperature tested. We now show that such mutant polymerases can function to produce progeny virus sufficient to form plaques at both 30 and 40 degrees C upon addition of AU, AUA, or AUU to the 5' terminus of the genomic RNA or upon substitution of A for U as the third nucleotide of the genome. These results are consistent with the hypothesis that (i) 3'-UA-5' is required at the 3' terminus of the minus-strand RNA for initiation of plus-strand genomic RNA synthesis; (ii) in the wild-type virus this sequence is present in a secondary structure that can be opened by the wild-type polymerase but not by the mutant polymerase; (iii) the addition of AU, AUA, or AUU to the 5' end of the genomic RNA provides unpaired 3'-UA-5' at the 3' end of the minus strand that can be utilized by the mutant polymerase, and similarly, the effect of the U3A mutation is to destabilize the secondary structure, freeing 3'-terminal UA; and (iv) the N terminus of nsP4 may directly interact with the 3' terminus of the minus-strand RNA for the initiation of the plus-strand genomic RNA synthesis. This hypothesis is discussed in light of our present results as well as of previous studies of alphavirus RNAs, including defective interfering RNAs.     

Defining the roles of cis-acting RNA elements in tombusvirus replicase assembly in vitro

In addition to its central role as a template for replication and translation, the viral plus-strand RNA genome also has nontemplate functions, such as recruitment to the site of replication and assembly of the viral replicase, activities that are mediated by cis-acting RNA elements within viral genomes. Two noncontiguous RNA elements, RII(+)-SL (located internally in the tombusvirus genome) and RIV (located at the 3'-terminus), are involved in template recruitment into replication and replicase assembly; however, the importance of each of these RNA elements for these two distinct functions is not fully elucidated. We used an in vitro replicase assembly assay based on yeast cell extract and purified recombinant tombusvirus replication proteins to show that RII(+)-SL, in addition to its known requirement for recruitment of the plus-strand RNA into replication, is also necessary for assembly of an active viral replicase complex. Additional studies using a novel two-component RNA system revealed that the recruitment function of RII(+)-SL can be provided in trans by a separate RNA and that the replication silencer element, located within RIV, defines the template that is used for initiation of minus-strand synthesis. Collectively, this work has revealed new functions for tombusvirus cis-acting RNA elements and provided insights into the pioneering round of minus-strand synthesis.     

An element of the tertiary structure of Peach latent mosaic viroid RNA revealed by UV irradiation

Following UV irradiation, denaturing polyacrylamide gel electrophoresis and Northern blot hybridization revealed a cross-link in Peach latent mosaic viroid (PLMVd) plus-strand RNA. Primer extension and partial alkaline hydrolysis of the UV-irradiated PLMVd plus-strand RNA resulting from the hammerhead-mediated self-cleavage mapped the cross-link at U81 and at the 3'-terminal C289 (or at a very proximal nucleotide). Supporting this notion, in vitro-synthesized PLMVd plus-strand RNAs with short insertions/deletions at their 3' termini failed to cross-link. Because U81 and C289 are conserved in PLMVd variants and because the initiation site of PLMVd minus-strand RNA maps at a short double-stranded motif containing C289, the UV-photo-cross-linkable element of tertiary structure may be functionally significant. A second cross-linked species similar in size and sequence to the monomeric circular PLMVd form, observed in some PLMVd variants, probably derives from UV-induced ligation of the two termini resulting from self-cleavage.