On the nature of spontaneous RNA synthesis by Q beta replicase.

Numerous RNA species of different length and nucleotide sequence grow spontaneously in vitro in Q beta replicase reactions where no RNA templates are added deliberately. Here, we show that this spontaneous RNA synthesis by Q beta replicase is template directed. The immediate source of template RNA can be the laboratory air, but there are ways to eliminate, or at least substantially reduce, the harmful effects of spontaneous synthesis. Solitary RNA molecules were detected in a thin layer of agarose gel containing Q beta replicase, where they grew to form colonies that became visible upon staining with ethidium bromide. This result provides a powerful tool for RNA cloning and selection in vitro. We also show that replicating RNAs similar to those growing spontaneously are incorporated into Q beta phage particles and can propagate in vivo for a number of phage generations. These RNAs are the smallest known molecular parasites, and in many aspects they resemble both the defective interfering genomes of animal and plant viruses and plant virus satellite RNAs.     

Reversion of Q beta RNA phage mutants by homologous RNA recombination.

Q beta phage RNAs with inactivating insertion (8-base) or deletion (17-base) mutations within their replicase genes were prepared from modified Q beta cDNAs and transfected into Escherichia coli spheroplasts containing Q beta replicase provided in trans by a resident plasmid. Replicase-defective (Rep-) Q beta phage produced by these spheroplasts were detected as normal-sized plaques on lawns of cells containing plasmid-derived Q beta replicase, but were unable to form plaques on cells lacking this plasmid. When individual Rep- phage were isolated and grown to high titer in cells containing plasmid-derived Q beta replicase, revertant (Rep+) Q beta phage were obtained at a frequency of ca. 10(-8). To investigate the mechanism of this reversion, a point mutation was placed into the plasmid-derived Q beta replicase gene by site-directed mutagenesis. Q beta mutants amplified on cells containing the resultant plasmid also yielded Rep+ revertants. Genomic RNA was isolated from several of the latter phage revertants and sequenced. Results showed that the original mutation (insertion or deletion) was no longer present in the phage revertants but that the marker mutation placed into the plasmid was now present in the genomic RNAs, indicating that recombination was one mechanism involved in the reversion of the Q beta mutants. Further experiments demonstrated that the 3' noncoding region of the plasmid-derived replicase gene was necessary for the reversion-recombination of the deletion mutant, whereas this region was not required for reversion or recombination of the insertion mutant. Results are discussed in terms of a template-switching model of RNA recombination involving Q beta replicase, the mutant phage genome, and plasmid-derived replicase mRNA.     

Efficient templates for Q beta replicase are formed by recombination from heterologous sequences.

A very efficient replicase template has been isolated from the products of spontaneous RNA synthesis in an in vitro Q beta replicase reaction that was incubated in the absence of added RNA. This template was named RQ135 RNA because it is 135 nucleotides in length. Its sequence consists entirely of segments that are homologous to ribosomal 23 S RNA and the phage lambda origin of replication. The sequence segments are unrelated to the sequence of Q beta bacteriophage genomic RNA. Nonetheless, this natural recombinant is replicated in vitro at a rate equal to the most efficient of the known Q beta RNA variants. Apparently, the structural properties that ensure recognition of an RNA template by Q beta replicase are not confined to viral RNA, but can appear as a result of recombination among other RNAs that usually occur in cells.     

Differential stimulation by polyamines of phage RNA-directed synthesis of proteins

The effect of polyamines on Q beta and MS2 phage RNA-directed synthesis of three kinds of protein in an Escherichia coli cell-free system has been studied. With both phage RNAs, the degree of stimulation of protein synthesis by spermidine was in the order RNA replicase greater than A protein, while the synthesis of coat protein was not stimulated significantly by spermidine. The synthesis of RNA replicase was stimulated by 1 mM spermidine approx. 8-fold. From the results of Q beta RNA direct alanyl-tRNA and seryl-tRNA binding to ribosomes and initiation dipeptide synthesis, it is suggested that the preferential stimulation of the synthesis of RNA replicase by spermidine is due at least partially to the stimulation of the initiation of RNA replicase synthesis.     

An algorithm for searching RNA motifs in genomic sequences

RNA molecules, which are found in all living cells, fold into characteristic structures that account for their diverse functional activities. Many of these RNA structures consist of a collection of fundamental RNA motifs. The various combinations of RNA basic components form different RNA classes and define their unique structural and functional properties. The availability of many genome sequences makes it possible to search computationally for functional RNAs. Biological experiments indicate that functional RNAs have characteristic RNA structural motifs represented by specific combinations of base pairings and conserved nucleotides in the loop regions. The searching for those well-ordered RNA structures and their homologues in genomic sequences is very helpful for the understanding of RNA-based gene regulation. In this paper, we consider the following problem: given an RNA sequence with a known secondary structure, efficiently determine candidate segments in genomic sequences that can potentially form RNA secondary structures similar to the given RNA secondary structure. Our new bottom-up approach searches all potential stem-loops similar to ones of the given RNA secondary structure first, and then based on located stem-loops, detects potential homologous structural RNAs in genomic sequences.     

Stalkless mutants of Caulobacter crescentus.

A stalk, a single falgellum, several pili, and deoxyribonucleic acid (DNA) phage receptors are polar surface structures expressed at a defined time in the Caulobacter crescentus cell cycle. When mutants were isolated as DNA phage phiCbK-resistant or ribonucleic acid (RNA) phage phiCp2-resistant, as well as nonmotile, strains, 5 out of 30 such mutant isolates were found not to possess stalks, but did possess inactive flagella. These stalkless mutants were resistant simultaneously to both DNA and RNA phages and did not possess pili and DNA pendent stalkless mutants. All motile revertants simultaneously regained the capacity to form stalks and susceptibility to DNA and RNA phages. It is suggested that a single mutation pleiotropically affects stalk formation, flagella motility, and coordinate polar morphogenesis of pili and DNA phage receptors. The stalkless mutants grew at a generation time similar to that of the wild-type strain at 30 degrees C. Cell size and morphology of a stalkless mutant, C. crescentus CB13 pdr-819, were also similar to those of the wild-type strain, except for the absence of a stalk. In addition, the CB13 pdr-819 predivisional cells were partitioned into smaller and larger portions, indicating asymmetrical cell division, as in the wild-type strain. From these results, it is suggested that swarmer cells undergo transition to cells of a stalked-cell nature without stalk formation and that the cell cycle of the stalkless mutant proceeds in an ordered sequence similar to that defining the wild-type cell cycle.     

HIV controls the selective packaging of genomic, spliced viral and cellular RNAs into virions through different mechanisms

In addition to genomic RNA, HIV-1 particles package cellular and spliced viral RNAs. In order to determine the encapsidation mechanisms of these RNAs, we determined the packaging efficiencies and specificities of genomic RNA, singly and fully spliced HIV mRNAs and different host RNAs species: 7SL RNA, U6 snRNA and GAPDH mRNA using RT-QPCR. Except GAPDH mRNA, all RNAs are selectively encapsidated. Singly spliced RNAs, harboring the Rev-responsible element, and fully spliced viral RNAs, which do not contain this motif, are enriched in virions to similar levels, even though they are exported from the nucleus by different routes. Deletions of key motifs (SL1 and/or SL3) of the packaging signal of genomic RNA indicate that HIV and host RNAs are encapsidated through independent mechanisms, while genomic and spliced viral RNA compete for the same trans-acting factor due to the presence of the 5′ common exon containing the TAR, poly(A) and U5-PBS hairpins. Surprisingly, the RNA dimerization initiation site (DIS/SL1) appears to be the main packaging determinant of genomic RNA, but is not involved in packaging of spliced viral RNAs, suggesting a functional interaction with intronic sequences. Active and selective packaging of host and spliced viral RNAs provide new potential functions to these RNAs in the early stages of the virus life cycle.     

Sequence homologies between ribosomal and phage RNAs: A proposed molecular basis for RNA phage parasitism

An extensive nucleotide sequence homology between the 3′-end of the 16 S ribosomal RNA and segments of bacteriophage MS2 or Qβ RNA is described. In addition, a notable sequence homology of coliphage RNAs with several other segments of ribosomal RNA is shown. The role of bacterial proteins in the recognition of phage RNA, and the resemblance of phage and host RNAs as the molecular basis of RNA phage parasitism is discussed.The problem of quantifying the degree of homology is discussed in the Appendix with a preliminary attempt towards a solution. A relative measure of homology is described, and used to analyze statistically the data obtained.     

Viral Genomic Single-Stranded RNA Directs the Pathway Toward a T = 3 Capsid

The molecular mechanisms controlling genome packaging by single-stranded RNA viruses are still largely unknown. It is necessary in most cases for the protein to adopt different conformations at different positions on the capsid lattice in order to form a viral capsid from multiple copies of a single protein. We showed previously that such quasi-equivalent conformers of RNA bacteriophage MS2 coat protein dimers (CP₂) can be switched by sequence-specific interaction with a short RNA stem-loop (TR) that occurs only once in the wild-type phage genome. In principle, multiple switching events are required to generate the phage T = 3 capsid. We have therefore investigated the sequence dependency of this event using two RNA aptamer sequences selected to bind the phage coat protein and an analogous packaging signal from phage Qβ known to be discriminated against by MS2 coat protein both in vivo and in vitro. All three non-cognate stem-loops support T = 3 shell formation, but none shows the kinetic-trapping effect seen when TR is mixed with equimolar CP₂. We show that this reflects the fact that they are poor ligands compared with TR, failing to saturate the coat protein under the assay conditions, ensuring that sufficient amounts of both types of dimer required for efficient assembly are present in these reactions. Increasing the non-cognate RNA concentration restores the kinetic trap, confirming this interpretation. We have also assessed the effects of extending the TR stem-loop at the 5′ or 3′ end with short genomic sequences. These longer RNAs all show evidence of the kinetic trap, reflecting the fact that they all contain the TR sequence and are more efficient at promoting capsid formation than TR. Mass spectrometry has shown that at least two pathways toward the T = 3 shell occur in TR-induced assembly reactions: one via formation of a 3-fold axis and another that creates an extended 5-fold complex. The longer genomic RNAs suppress the 5-fold pathway, presumably as a consequence of steric clashes between multiply bound RNAs. Reversing the orientation of the extension sequences with respect to the TR stem-loop produces RNAs that are poor assembly initiators. The data support the idea that RNA-induced protein conformer switching occurs throughout assembly of the T = 3 shell and show that both positional and sequence-specific effects outside the TR stem-loop can have significant impacts on the precise assembly pathway followed.     

The 5' untranslated region of alfalfa mosaic virus RNA 1 is involved in negative-strand RNA synthesis

The three genomic RNAs of alfalfa mosaic virus each contain a unique 5' untranslated region (5' UTR). Replacement of the 5' UTR of RNA 1 by that of RNA 2 or 3 yielded infectious replicons. The sequence of a putative 5' stem-loop structure in RNA 1 was found to be required for negative-strand RNA synthesis. A similar putative 5' stem-loop structure is present in RNA 2 but not in RNA 3.     

Autocatalytic replication of a recombinant RNA

We demonstrate that a heterologous RNA sequence can be copied in vitro by Q beta replicase when it is inserted into a naturally occurring Q beta replicase template. A recombinant RNA was constructed by inserting decaadenylic acid between nucleotides 63 and 64 of MDV-1 (+) RNA, using phage T4 RNA ligase. The insert was located away from regions of the template known to be required for the binding of the replicase and for the initiation of product strand synthesis. To minimize the disruption of template structure, we inserted the heterologous sequence into a hairpin loop on the exterior of the molecule. Q beta replicase copied this recombinant RNA in vitro, and the complementary product strands served as templates for the synthesis of additional copies of the original recombinant RNA. The reaction was therefore autocatalytic and the amount of recombinant RNA increased exponentially. A 300-fold amplification of the recombinant RNA occurred within nine minutes. Insertion of biologically significant RNAs into the MDV-1 RNA sequence should allow them to be replicated autocatalytically.     

Q beta RNA bacteriophage: mapping cis-acting elements within an RNA genome

We have identified, for the first time, regions of cis-acting RNA elements within the bacteriophage Q beta replicase cistron by analyzing the infectivities of 76 replicase gene mutant phages in the presence of a helper replicase. Two separate classes of mutant Q beta phage genomes (35 different insertion mutants, each containing an insertion of 3 to 15 nucleotides within the replicase gene, and 41 deletion genomes, each having from 15 to 935 nucleotides deleted from different regions of the gene) were constructed, and their corresponding RNAs were tested for the ability to direct the formation of progeny virus particles. Each mutant phage was tested for plaque formation in an Escherichia coli (F+) host strain that supplied helper Q beta replicase in trans from a plasmid DNA. Of the 76 mutant genomes, 34% were able to direct virus production at or close to wild-type levels (with plaque yield ratios of greater than 0.5), another 36% also produced virus particles, but at much lower levels than those of wild-type virus (with plaque yield ratios of less than 0.05), and the remaining 30% produced no virus at all. From these data, we have been able to define regions within the Q beta replicase gene that contain functional cis-acting RNA elements and further correlate them with regions of RNA that are solely required to code for functional RNA polymerase.     

Viral escape from antisense RNA

RNA coliphage SP was propagated for several generations on a host expressing an inhibitory antisense RNA complementary to bases 31–270 of the positive-stranded genome. Phages evolved that escaped inhibition. Typically, these escape mutants contained 3–4 base substitutions, but different sequences were observed among different isolates. The mutations were located within three different types of structural features within the predicted secondary structure of SP genomic RNA: (i) hairpin loops; (ii) hairpin stems; and (iii) the 5' region of the phage genome complementary to the antisense molecule. Computer modelling of the mutant genomic RNAs showed that all of the substitutions within hairpin stems improved the Watson–Crick pairing of the stem. No major structural rearrangements were predicted for any of the mutant genomes, and most substitutions in coding regions did not alter the amino acid sequence. Although the evolved phage populations were polymorphic for substitutions, many substitutions appeared independently in two selected lines. The creation of a new, perfect, antisense RNA against an escape mutant resulted in the inhibition of that mutant but not of other escape mutants nor of the ancestral, unevolved phage. Thus, at least in this system, a population of viruses that evolved to escape from a single antisense RNA would require a cocktail of several antisense RNAs for inhibition.     

The coat protein leads the way: an update on basic and applied studies with the Brome mosaic virus coat protein

The Brome mosaic virus (BMV) coat protein (CP) accompanies the three BMV genomic RNAs and the subgenomic RNA into and out of cells in an infection cycle. In addition to serving as a protective shell for all of the BMV RNAs, CP plays regulatory roles during the infection process that are mediated through specific binding of RNA elements in the BMV genome. One regulatory RNA element is the B box present in the 5' untranslated region (UTR) of BMV RNA1 and RNA2 that play important roles in the formation of the BMV replication factory, as well as the regulation of translation. A second element is within the tRNA-like 3' UTR of all BMV RNAs that is required for efficient RNA replication. The BMV CP can also encapsidate ligand-coated metal nanoparticles to form virus-like particles (VLPs). This update summarizes the interaction between the BMV CP and RNAs that can regulate RNA synthesis, translation and RNA encapsidation, as well as the formation of VLPs.     

Nucleotide sequence heterogeneity of an RNA phage population

The nucleotide sequence of 32P-RNA from Q beta phage clones was sampled by two-dimensional polyacrylamide gel electrophoresis of the RNAase T1-resistant oligonucleotides (T1 fingerprinting). About 15% of the clones derived from a multiply passaged Q beta population showed fingerprint patterns which deviated from that of the RNA from the total population. All deviations examined could be attributed to one and, less frequently, to two or more nucleotide transitions. Since the fingerprinting technique allows the analysis of only about 10% of the RNA sequence, we estimate that each viable phage genome in a multiply passaged population differs in one to two positions from the "average" sequence of the parental population. Several deviant clones were tested by growth competition against a "wildtype" population, after 10-20 generations, the resulting phage showed the "wild-type" T1 fingerprint pattern. We propose that a Q beta phage population is in a dynamic equilibrium, with viable mutants arising at a high rate (Batschelet, Domingo and Weissmann, 1976; Domingo, Flavell and Weissmann, 1976) on the one hand, and being strongly selected against on the other. The genome of Q beta phage cannot be described as a defined unique structure, but rather as a weighted average of a large number of different individual sequences.     

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.     

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus.

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus (BSMV) has been determined. The sequence is 3289 nucleotides in length and contains four open reading frames (ORFs) which code for proteins of Mr 22,147 (ORF1), Mr 58,098 (ORF2), Mr 17,378 (ORF3), and Mr 14,119 (ORF4). The predicted N-terminal amino acid sequence of the polypeptide encoded by the ORF nearest the 5'-end of the RNA (ORF1) is identical (after the initiator methionine) to the published N-terminal amino acid sequence of BSMV coat protein for 29 of the first 30 amino acids. ORF2 occupies the central portion of the coding region of RNA beta and ORF3 is located at the 3'-end. The ORF4 sequence overlaps the 3'-region of ORF2 and the 5'-region of ORF3 and differs in codon usage from the other three RNA beta ORFs. The coding region of RNA beta is followed by a poly(A) tract and a 238 nucleotide tRNA-like structure which are common to all three BSMV genomic RNAs.