The cis-acting replication elements define human enterovirus and rhinovirus species

Replication of picornaviruses is dependent on VPg uridylylation, which is linked to the presence of the internal cis-acting replication element (cre). Cre are located within the sequence encoding polyprotein, yet at distinct positions as demonstrated for poliovirus and coxsackievirus-B3, cardiovirus, and human rhinovirus (HRV-A and HRV-B), overlapping proteins 2C, VP2, 2A, and VP1, respectively. Here we report a novel distinct cre element located in the VP2 region of the recently reported HRV-A2 species and provide evolutionary evidence of its functionality. We also experimentally interrogated functionality of recently identified HRV-B cre in the 2C region that is orthologous to the human enterovirus (HEV) cre and show that it is dispensable for replication and appears to be a nonfunctional evolutionary relic. In addition, our mutational analysis highlights two amino acids in the 2C protein that are crucial for replication. Remarkably, we conclude that each genetic clade of HRV and HEV is characterized by a unique functional cre element, where evolutionary success of a new genetic lineage seems to be associated with an invention of a novel cre motif and decay of the ancestral one. Therefore, we propose that cre element could be considered as an additional criterion for human rhinovirus and enterovirus classification.     

tRNA-like structures of plant viral RNAs: conformational requirements for adenylation and aminoacylation

Bromo- and cucumovirus RNAs contain a tRNA-like structure as an integral part of their genome. This structure is located at the 3' end of the viral RNA and is an acceptor of tyrosine. The 3' regions of representative viral RNAs have been sequenced and quite unorthodox secondary foldings have been proposed for these 3' ends. The question therefore remained as to how these structures could be recognized by tRNA-specific enzymes. We have established the minimum number of nucleotides from the 3' end of the brome mosaic virus and broad bean mottle virus RNAs required for the formation of structures recognized by the tyrosyl-tRNA synthetase and/or the tRNA nucleotidyltransferase. The results obtained delineate the length of the tRNA-like region, and indicate that the 5' region of the tRNA-like structure participates in the formation of the amino acid stem. This has led us to propose an 'L'-shaped secondary structure for these tRNA-like regions.     

A program for the identification of tRNA-like structures in DNA sequence data.

A computer algorithm has been developed which identifies tRNA genes and tRNA-like structures in DNA sequences. The program searches the sequence string for specific base positions that correspond to the invariant and semi-invariant bases found in tRNAs. The tRNA nature of the sequence is confirmed by the presence of complementary base pairing at the tRNA's calculated 5' and 3' ends (which in situ constitutes the amino-acyl stem region). The program achieves greater than 96% accuracy when run against known tRNA sequences in the Genbank database. The program is modular and is readily modified to allow searching either a file or database. The program is written in "C" and operates on a D.E.C. Vax 750. The utility of the algorithm is demonstrated by the identification of a distinctive tRNA structure in an intron of a published bovine hemoglobin gene.     

Trans- and cis-acting elements for the replication of P1 miniplasmids

Replication-deficient mutants of the unit-copy miniplasmid lambda-P1:5R were isolated after hydroxylamine mutagenesis. Complementation tests showed that the majority of these mutants are defective in the production of the repA protein product. Two of these mutants have suppressible nonsense (amber) mutations. The DNA sequence of one of these, repA103, has been determined. The lesion lies within the repA open reading frame, showing that the repA product is essential for plasmid replication. Complementation of deletion mutants of lambda-P1:5R by repA protein showed that the origin of replication lies to the left of repA and that this 300-base-pair origin region is the only portion of the DNA essential for plasmid replication if repA protein is supplied in trans. Six of the 21 hydroxylamine-induced mutants were not complemented by repA. Replication of three of these could be restored by introduction into the plasmid of a wild-type origin region, suggesting that they were origin-defective. The DNA sequence of two mutants was determined. Mutant rep-11 has a 43-base-pair deletion within the incC sequence (incC is a series of five direct repeats of a 19-base-pair sequence known to be involved in the regulation of plasmid replication). The deletion appears to have been generated by homologous recombination between two repeats. Mutant rep-30 has a single base substitution in a region just to the left of incC that destroys one of five G-A-T-C (dam methylation) sites in this region. As lambda-P1:5R is unable to establish itself as a plasmid in a methylase-defective (dam-) strain, it seems probable that methylation of the G-A-T-C sequences is important for origin function. The incC region and the sequences to its left appear to constitute an essential part of the origin of replication.     

Potato virus X-related single- and double-stranded RNAs: Characterization and identification of terminal structures

Six species of 3′-coterminal poly(A) -containing RNAs of subgenomic (sg) size have been found in plants infected with potato virus X (PVX): two major (0.9 kb — the coat protein mRNA, and 2.1 kb) and four minor (1.4, 1.8, 3.0 and 3.6 kb). The 5′-end of the shortest sgRNA is located 26 nucleotides upstream of the initiating codon of the coat protein gene (812 nucleotides from the 3′-terminal poly(A) tract of the PVX genomic RNA). Double-stranded analogues have been found for most sgRNAs. The genomic-size double-stranded RNA (the replicative form) is shown to carry a poly(A)-poly(U) hybrid of a predominant length of 150–250 bp on one end, and an unpaired G residue on the other (the 3′-end of the negative chain). In contrast to this(—) the chains of double-stranded 0.9 and 2.1 kbp sgRNAs lack the unpaired G and both end in C.     

Functional equivalency and diversity of cis-acting elements among yeast replication origins.

The DNA replication origins of the yeast Saccharomyces cerevisiae require several short functional elements, most of which are not conserved in sequence. To better characterize ARS305, a replicator from a chromosomal origin, we swapped functional DNA elements of ARS305 with defined elements of ARS1. ARS305 contains elements that are functionally exchangeable with ARS1 A and B1 elements, which are known to bind the origin recognition complex; however, the ARS1 A element differs in that it does not require a 3' box adjacent to the essential autonomously replicating sequence consensus. At the position corresponding to ARS1 B3, ARS305 has a novel element, B4, that can functionally substitute for every type of short element (B1, B2, and B3) in the B domain. Unexpectedly, the replacement of element B4 by ARS1 B3, which binds ABF1p and is known as a replication enhancer, inhibited ARS305 function. ARS305 has no short functional element at or near positions corresponding to the B2 elements in ARS1 and ARS307 but contains an easily unwound region whose functional importance was supported by a broad G+C-rich substitution mutation. Surprisingly, the easily unwound region can functionally substitute for the ARS1 B2 element, even though ARS1 B2 was found to possess a distinct DNA sequence requirement. The functionally conserved B2 element in ARS307 contains a known sequence requirement, and helical stability analysis of linker and minilinker mutations suggested that B2 also contains a DNA unwinding element (DUE). Our findings suggest that yeast replication origins employ a B2 element or a DUE to mediate a common function, DNA unwinding during initiation, although not necessarily through a common mechanism.     

Identification of a novel cis-acting RNA element involved in nuclear export of hY RNAs

Ro RNPs are small cytoplasmic RNA-protein complexes of unknown function that have been found in all metazoan cells studied so far. In human cells, Ro RNPs consist of one of four small RNA molecules, termed hY RNAs and at least two well-characterized proteins, Ro60 and La. In previous Xenopus laevis oocyte microinjection studies, we showed that an intact Ro60 binding site (Stem-loop 1) is a prerequisite for efficient nuclear export of hY1 RNA, whereas an intact La-binding site promotes nuclear retention (Simons et al. RNA, 1996, 2:264-273). Here we present evidence that the distal half (Stem 2) of the conserved base-paired stem structure found in all hY RNAs also plays a critical role in the export process. A minimal RNA molecule containing this region, L1S2 RNA, competes effectively for the export of full-length hY1 RNAs and is itself exported very rapidly in a Ro60-independent and RanGTP-dependent manner. Mutational analyses of this RNA shows that a 5'/3' terminal double-stranded stem structure (>10 bp) of no specific nucleotide sequence constitutes a novel nuclear export element (NEE). Cross-competition studies indicate that this type of NEE may also be involved in export of other classes of RNAs. Like full-length hY1 RNA, L1S2 RNA also competes for export of ET-202 RNA, an RNA that was selected for its efficient nuclear export in the presence of the nuclear transport inhibitor, VSV Matrix protein (Grimm et al. Proc Natl Acad Sci USA, 1997, 94:10122-10127). However, export of L1S2 RNA is strongly inhibited by VSV-M protein, showing that these RNAs use partially overlapping, but not identical export pathways. We propose that export of Y RNAs is mediated by two contiguous cis-acting elements in the 5'/3' double-stranded stem region that is conserved between different Y RNAs.     

Identification of cis-acting DNA elements involved in the regulation of angiotensinogen gene expression.

Angiotensinogen is the precursor molecule of one of the most potent vasoactive substances, angiotensin-II. Angiotensinogen is normally synthesized in the liver and secreted into the plasma where it is converted into angiotensin-II by the combined proteolytic action of renin and angiotensin converting enzyme. Angiotensinogen levels in the plasma are modulated by a number of pathological and physiological factors. In order to understand the regulation of angiotensinogen gene expression, we have constructed an expression vector in which 688 bp of the 5'-flanking region of the rat angiotensinogen gene were attached to the chloramphenicol acetyl transferase (CAT) coding sequence. We have also obtained 5'-sequential deletion mutants from the rat angiotensinogen promoter attached to the CAT gene, and have identified multiple cis-acting DNA sequences involved in the regulation of angiotensinogen gene expression by transient transfection of these recombinant DNA molecules in human hepatoma cell lines, Hep3B, and HepG2.     

Structural variation and functional importance of a D-loop-T-loop interaction in valine-accepting tRNA-like structures of plant viral RNAs

Valine-accepting tRNA-like structures (TLSs) are found at the 3′ ends of the genomic RNAs of most plant viruses belonging to the genera Tymovirus, Furovirus, Pomovirus and Pecluvirus, and of one Tobamovirus species. Sequence alignment of these TLSs suggests the existence of a tertiary D-loop–T-loop interaction consisting of 2 bp, analogous to those in the elbow region of canonical tRNAs. The conserved G₁₈·Ψ₅₅ pair of regular tRNAs is found to covary in these TLSs between G·U (possibly also modified to G·Ψ) and A·G. We have mutated the relevant bases in turnip yellow mosaic virus (TYMV) and examined the mutants for symptom development on Chinese cabbage plants and for accumulation of genetic reversions. Development of symptoms is shown to rely on the presence of either A·G or G·U in the original mutants or in revertants. This finding supports the existence and functional importance of this tertiary interaction. The fact that only G·U and A·G are accepted at this position appears to result from steric and energetic limitations related to the highly compact nature of the elbow region. We discuss the implications of these findings for the various possible functions of the valine-accepting TLS.     

cis-acting lesions targeted to the hydrophobic domain of a poliovirus membrane protein involved in RNA replication.

The structural requirements of the hydrophobic domain contained in poliovirus polypeptide 3AB were studied by using a molecular genetic approach in combination with an in vitro biochemical analysis. We report here the generation and analysis of deletion, insertion, and amino acid replacement mutations aimed at decreasing the hydrophobic character of the domain. Our results indicated that the hydrophobicity of this region of 3AB is necessary to maintain normal viral RNA synthesis. However, in vitro membrane association assays of the mutated proteins did not establish a direct correlation between 3AB membrane association and viral RNA synthesis. Some of the lethal mutations we engineered produced polyproteins with abnormal P2- and P3-processing capabilities due to an alteration in the normal cleavage order of the polyprotein. A detailed analysis of these mutants suggests that P2 is not the major precursor for polypeptides 2A and 2BC and that P2 protein products are derived from P2-P3-containing precursors (most likely P2-P3 or P2-3AB). Such precursors are likely to result from primary polyprotein cleavage events that initiate a proteolytic cascade not previously documented. Our results also indicated that the function provided by the hydrophobic domain of 3AB cannot be provided in trans. We discuss the implications of these results on the formation of limited-diffusion replication complexes as a means of sequestering P2- and P3-region polypeptides required for RNA synthesis and protein processing.     

cis-acting genomic elements and trans-acting proteins involved in the assembly of RNA viruses

There is now considerable evidence that a specific site (or sites) in the genome of an RNA virus interacts with a viral protein to initiate the assembly of the virus ribonucleoprotein or nucleocapsid. We describe the progress that has been made in defining these elements for a number of different viruses: the togavirus, Sindbis virus; the coronavirus, mouse hepatitis virus; influenza A virus; several retroviruses; and the hepadnavirus, hepatitis B virus. The importance of cis-acting elements in packaging has been established for all of these viruses. For Sindbis virus, specificity in the binding of the RNA element to a region of the viral capsid protein in vitro has also been demonstrated.     

Picornaviral capsid assembly: similarity of rhinovirus and enterovirus precursor subunits.

Cytoplasmic extracts of rhinovirus 1A-infected HeLa cells, pulsed 15 min with [3H]leucine, contained a 13S subunit which was rich in the capsid precursor, peptide 92. After a 30-min chase, most of the capsid-related protein sedimented in a 14S peak that contained equimolar amounts of the capsid peptides epsilon, alpha, and gamma, and some residual chain 92. The 14S subunit could be dissociated at pH 4.8 into 6S subunits containing only epsilon, alpha, and gamma chains in equal proportions, indicating that the 14S subunit is an oligomer of (epsilon gamma alpha) protomers. These subunits resemble subunits previously identified in the assembly of enteroviruses. These observations support the idea that rhinovirus assembly is basically similar to that of enteroviruses. Comparative studies on the peptide stoichiometry of the virion and the capsid precursor subunits indicate that rhinovirus 1A can contain as many as 11 immature protomers per virion.     

Interaction between the antisense and target RNAs involved in the regulation of IncB plasmid replication.

Physical analysis of RNA I, the small antisense RNA which regulates the replication of IncB miniplasmid pMU720, showed that it is a highly structured molecule containing an imperfectly paired stem closed by a 6-base hairpin loop. Mutational studies revealed that a 3-base sequence in the hairpin loop is critical to the interaction between RNA I and its complementary target in the RepA mRNA (RNA II). Furthermore, a 2-base interior loop in the upper stem was found to play an important role in facilitating effective binding between RNA I and RNA II. From these analyses, a model describing the molecular mechanism of binding between RNA I and RNA II is proposed.