René C. L. Olsthoorn

A conformational switch at the 3′ end of a plant virus RNA regulates viral replication

3′ untranslated regions of alfamo‐ and ilar‐virus RNAs fold into a series of stem–loop structures to which the coat protein binds with high affinity. This binding plays a role in initiation of infection (‘genome activation’) and has been thought to substitute for a tRNA‐like structure that is found at the 3′ termini of related plant viruses. We propose the existence of an alternative conformation of the 3′ ends of alfamo‐ and ilar‐virus RNAs, including a pseudoknot. Based on (i) phylogenetic comparisons, (ii) in vivo and in vitro functional analyses of mutants in which the pseudoknot has been disrupted or restored by compensatory mutations, (iii) competition experiments between coat protein and viral replicase, and (iv) investigation of the effect of magnesium, we demonstrate that this pseudoknot is required for replication of alfalfa mosaic virus. This conformation resembles the tRNA‐like structure of the related bromo‐ and cucumo‐viruses. A low but specific interaction with yeast CCA‐adding enzyme was found. The existence of two mutually exclusive conformations for the 3′ termini of alfamo‐ and ilar‐virus RNAs could enable the virus to switch from translation to replication and vice versa. The role of coat protein in this modulation and in genome activation is discussed.

Translation of a nonpolyadenylated viral RNA is enhanced by binding of viral coat protein or polyadenylation of the RNA

On entering a host cell, positive-strand RNA virus genomes have to serve as messenger for the translation of viral proteins. Efficient translation of cellular messengers requires interactions between initiation factors bound to the 5′-cap structure and the poly(A) binding protein bound to the 3′-poly(A) tail. Initiation of infection with the tripartite RNA genomes of alfalfa mosaic virus (AMV) and viruses from the genus Ilarvirus requires binding of a few molecules of coat protein (CP) to the 3′ end of the nonpolyadenylated viral RNAs. Moreover, infection with the genomic RNAs can be initiated by addition of the subgenomic messenger for CP, RNA 4. We report here that extension of the AMV RNAs with a poly(A) tail of 40 to 80 A-residues permitted initiation of infection independently of CP or RNA 4 in the inoculum. Specifically, polyadenylation of RNA 1 relieved an apparent bottleneck in the translation of the viral RNAs. Translation of RNA 4 in plant protoplasts was autocatalytically stimulated by its encoded CP. Mutations that interfered with CP binding to the 3′ end of viral RNAs reduced translation of RNA 4 to undetectable levels. Possibly, CP of AMV and ilarviruses stimulates translation of viral RNAs by acting as a functional analogue of poly(A) binding protein or other cellular proteins.

Kissing Interaction between 3′ Noncoding and Coding Sequences Is Essential for Porcine Arterivirus RNA Replication

ABSTRACT We used an infectious cDNA clone of Porcine reproductive and respiratory syndrome virus (PRRSV) to investigate the presence of essential replication elements in the region of the genome encoding the structural proteins. Deletion analysis showed that a stretch of 34 nucleotides (14653 to 14686) within ORF7, which encodes the nucleocapsid protein, is essential for RNA replication. Strand-specific reverse transcription-PCR analysis of viral RNA isolated from transfected BHK-21 cells revealed that this region is required for negative-strand genomic RNA synthesis. The 34-nucleotide stretch is highly conserved among PRRSV isolates and folds into a putative hairpin. A 7-base sequence within the loop of this structure was suggested to base-pair with a sequence present in the loop of a hairpin located in the 3′ noncoding region, resulting in a kissing interaction. Mutational analyses confirmed that this kissing interaction is required for RNA replication.

A conserved hairpin structure in Alfamovirus and Bromovirus subgenomic promoters is required for efficient RNA synthesis in vitro

The coat protein gene in RNA 3 of alfalfa mosaic virus (AMV; genus Alfamovirus, family Bromoviridae) is translated from the subgenomic RNA 4. Analysis of the subgenomic promoter (sgp) in minus-strand RNA 3 showed that a sequence of 37 nt upstream of the RNA 4 start site (nt +1) was sufficient for full sgp activity in an in vitro assay with the purified viral RNA-dependent RNA-polymerase (RdRp). The sequence of nt -6 to -29 could be folded into a potential hairpin structure with a loop represented by nt -16, -17, and -18, and a bulge involving nt -23. By introducing mutations that disrupted base pairing and compensatory mutations that restored base pairing, it was shown that base pairing in the top half of the putative stem (between the loop and bulge) was essential for sgp activity, whereas base pairing in the bottom half of the stem was less stringently required. Deletion of the bulged residue A-23 or mutation of this residue into a C strongly reduced sgp activity, but mutation of A-23 into U or G had little effect on sgp activity. Mutation of loop residues A-16 and A-17 affected sgp activity, whereas mutation of U-18 did not. Using RNA templates corresponding to the sgp of brome mosaic virus (BMV; genus Bromovirus, family Bromoviridae) and purified BMV RdRp, evidence was obtained indicating that also in BMV RNA a triloop hairpin structure is required for sgp activity.

Leeway and constraints in the forced evolution of a regulatory RNA helix.

The start of the coat protein gene of RNA phage MS2 adopts a well‐defined hairpin structure of 12 bp (including one mismatch) in which the start codon occupies the loop position. An earlier expression study using partial MS2 cDNA clones had indicated that the stability of this hairpin is important for gene expression. For every ‐1.4 kcal/mol increase in stability a 10‐fold reduction in coat protein was obtained. Destabilizations beyond the wild‐type value did not affect expression. These results suggested that the hairpin was tuned in the sense that it has the highest stability still compatible with maximal ribosome loading. Employing an infectious MS2 cDNA clone, we have now tested the prediction that the delta G 0 of the coat protein initiator helix is set at a precise value. We have introduced stabilizing and destabilizing mutations into this hairpin in the intact phage and monitored their evolution to viable species. By compensatory mutations, both types of mutants quickly revert along various pathways to wild‐type stability, but not to wild‐type sequence. As a rule the second‐site mutations do not change the encoded amino acids or the Shine‐Dalgarno sequence. The return of too strong hairpins to wild‐type stability can be understood from the need to produce adequate supplies of coat protein. The return of unstable hairpins to wild‐type stability is not self‐evident and is presently not understood. The revertants provide an evolutionary landscape of slightly suboptimal phages, that were stable at least for the duration of the experiment (approximately 20 infection cycles).(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of ribosomal frameshifting by RNA G-quadruplex structures

Guanine-rich sequences can fold into four-stranded structures of stacked guanine-tetrads, so-called G-quadruplexes (G4). These unique motifs have been extensively studied on the DNA level; however, exploration of the biological roles of G4s at the RNA level is just emerging. Here we show that G4 RNA when introduced within coding regions are capable of stimulating −1 ribosomal frameshifting (−1 FS) in vitro and in cultured cells. Systematic manipulation of the loop length between each G-tract revealed that the −1 FS efficiency positively correlates with G4 stability. Addition of a G4-stabilizing ligand, PhenDC3, resulted in higher −1 FS. Further, we demonstrated that the G4s can stimulate +1 FS and stop codon readthrough as well. These results suggest a potentially novel translational gene regulation mechanism mediated by G4 RNA.

Influenza Virus RNA Structure: Unique and Common Features

The influenza A virus genome consists of eight negative-sense RNA segments. Here we review the currently available data on structure-function relationships in influenza virus RNAs. Various ideas and hypotheses about the roles of influenza virus RNA folding in the virus replication are also discussed in relation to other viruses.

Phage display selects for amylases with improved low pH starch-binding

Directed evolution of secreted industrial enzymes is hampered by the lack of powerful selection techniques. We have explored surface display to select for enzyme variants with improved binding performance on complex polymeric substrates. By a combination of saturation mutagenesis and phage display we selected alpha-amylase variants, which have the ability to bind starch substrate at industrially preferred low pH conditions. First we displayed active alpha-amylase on the surface of phage fd. Secondly we developed a selection system that is based on the ability of alpha-amylase displaying phages to bind to cross-linked starch. This system was used to probe the involvement of specific beta-strands in substrate interaction. Finally, a saturated library of alpha-amylase mutants with one or more amino acid residues changed in their Cbeta4 starch-binding domain was subjected to phage display selection. Mutant molecules with good starch-binding and hydrolytic capacity could be isolated from the phage library by repeated binding and elution of phage particles at lowered pH value. Apart from the wild type alpha-amylase a specific subset of variants, with only changes in three out of the seven possible positions, was selected. All selected variants could hydrolyse starch and heptamaltose at low pH. Interestingly, variants were found with a starch hydrolysis ratio at pH 4.5/7.5 that is improved relative to the wild type alpha-amylase. These data demonstrate that useful alpha-amylase mutants can be selected via surface display on the basis of their binding properties to starch at lowered pH values.

Stem-loop structures can effectively substitute for an RNA pseudoknot in -1 ribosomal frameshifting.

−1 Programmed ribosomal frameshifting (PRF) in synthesizing the gag-pro precursor polyprotein of Simian retrovirus type-1 (SRV-1) is stimulated by a classical H-type pseudoknot which forms an extended triple helix involving base–base and base–sugar interactions between loop and stem nucleotides. Recently, we showed that mutation of bases involved in triple helix formation affected frameshifting, again emphasizing the role of the triple helix in −1 PRF. Here, we investigated the efficiency of hairpins of similar base pair composition as the SRV-1 gag-pro pseudoknot. Although not capable of triple helix formation they proved worthy stimulators of frameshifting. Subsequent investigation of ∼30 different hairpin constructs revealed that next to thermodynamic stability, loop size and composition and stem irregularities can influence frameshifting. Interestingly, hairpins carrying the stable GAAA tetraloop were significantly less shifty than other hairpins, including those with a UUCG motif. The data are discussed in relation to natural shifty hairpins.

Group-specific structural features of the 5'-proximal sequences of coronavirus genomic RNAs.

Abstract Global predictions of the secondary structure of coronavirus (CoV) 5′ untranslated regions and adjacent coding sequences revealed the presence of conserved structural elements. Stem loops (SL) 1, 2, 4, and 5 were predicted in all CoVs, while the core leader transcription-regulating sequence (L-TRS) forms SL3 in only some CoVs. SL5 in group I and II CoVs, with the exception of group IIa CoVs, is characterized by the presence of a large sequence insertion capable of forming hairpins with the conserved 5′-UUYCGU-3′ loop sequence. Structure probing confirmed the existence of these hairpins in the group I Human coronavirus-229E and the group II Severe acute respiratory syndrome coronavirus (SARS-CoV). In general, the pattern of the 5′ cis-acting elements is highly related to the lineage of CoVs, including features of the conserved hairpins in SL5. The function of these conserved hairpins as a putative packaging signal is discussed.