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.

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We coupled the Wells-Riley equation and the susceptible-exposed-infected-recovery (SEIR) model to quantify the impact of the combination of indoor air-based control measures of enhanced ventilation and respiratory masking in containing pandemic influenza within an elementary school. We integrated indoor environmental factors of a real elementary school and aetiological characteristics of influenza to estimate the age-specific risk of infection (P) and basic reproduction number (R(0)). We combined the enhanced ventilation rates of 0.5, 1, 1.5, and 2/h and respiratory masking with 60%, 70%, 80%, and 95% efficacies, respectively, to predict the reducing level of R0. We also took into account the critical vaccination coverage rate among schoolchildren. Age-specific P and R(0) were estimated respectively to be 0.29 and 16.90; 0.56 and 16.11; 0.59 and 12.88; 0.64 and 16.09; and 0.07 and 2.80 for five age groups 4-6, 7-8, 9-10, 11-12, and 25-45 years, indicating pre-schoolchildren have the highest transmission potential. We conclude that our integrated approach, employing the mechanism of transmission of indoor respiratory infection, population-dynamic transmission model, and the impact of infectious control programmes, is a powerful tool for risk profiling prediction of pandemic influenza among schoolchildren.

A 190-nucleotide (nt) packaging signal (PS) located in the 3 end of open reading frame 1b in the mouse hepatitis virus, a group IIa coronavirus, was previously postulated to direct genome RNA packaging. Based on phylogenetic data and structure probing, we have identified a 95-nt hairpin within the 190-nt PS domain which is conserved in all group IIa coronaviruses but not in the severe acute respiratory syndrome coronavirus (group IIb), group I coronaviruses, or group III coronaviruses. The hairpin is composed of six copies of a repeating structural subunit that consists of 2-nt bulges and 5-bp stems. We propose that repeating AA bulges are characteristic features of group IIa PSs.For many plus-stranded RNA viruses, a specific structural element within the genome RNA that serves as a starting point in the assembly of the viral nucleocapsid has been identified. For the Coronaviridae family of RNA viruses (8), which includes important pathogens, such as the severe acute respiratory syndrome coronavirus (SARS CoV), the process of genome encapsidation, which precedes the assembly of the enveloped virus particle, is poorly understood. Previous studies of the mouse hepatitis virus (MHV), a group IIa CoV (4), have led to the identification of a so-called packaging signal (PS) in the 3Ј end of replicase open reading frame 1b (ORF1b) (7,12). A region of 190 nucleotides (nt) was sufficient to drive the encapsidation of the defective interfering RNAs of MHV. Subsequently, a 69-nt fragment of this PS was found to be able, albeit less efficiently, to direct the packaging of heterologous transcripts (3). A secondary structure for MHV PS was proposed (3), but its validity was never experimentally confirmed, nor was it supported by an analysis of the closely related bovine CoVs (2).We have now used BLAST (1) to search for MHV PS homologs in currently available group IIa CoV sequences: human CoV (HCoV) OC43 (GenBank accession number NC_005147), porcine hemagglutinating encephalomyelitis virus (HEV; NC_007732), bovine CoV (NC_003045), HCoV HKU1 genotype A (NC_006577), and HCoV HKU1 genotype B (AY884001). The only common sequence appeared to be an 18-nt stretch that was previously predicted to form the top of the putative bovine CoV PS (2). Subsequently, we folded a 150-nt region surrounding this motif in all group IIa CoVs using Mfold (13) and adjusted the structure manually based on a phylogenetic analysis. The final model, shown in Fig. 1, is supported by at least nine naturally occurring covariations. It partly resembles the Cologna and Hogue structure (2) but contains several previously unnoticed features, such as the repeating AA and GA bulges on its 3Ј side. This feature follows from conserved AGC/GUAAU motifs ( Fig. 1), which are repeated four times at 10-bp intervals. Another, though less-well-conserved, element is the 2-nt bulge at the 5Ј side of the stem-loop, which is also repeated every 10 bp. An internal loop seems to divide the PS into symmetric upper and lower parts in terms of the orientation and the number of the...

In many single-stranded (ss) RNA viruses, the cis-acting packaging signal that confers selectivity genome packaging usually encompasses short structured RNA repeats. These structural units, termed repetitive structural motifs (RSMs), potentially mediate capsid assembly by specific RNA–protein interactions. However, general knowledge of the conservation and/or the diversity of RSMs in the positive-sense ssRNA coronaviruses (CoVs) is limited. By performing structural phylogenetic analysis, we identified a variety of RSMs in nearly all CoV genomic RNAs, which are exclusively located in the 5′-untranslated regions (UTRs) and/or in the inter-domain regions of poly-protein 1ab coding sequences in a lineage-specific manner. In all alpha- and beta-CoVs, except for Embecovirus spp, two to four copies of 5′-gUUYCGUc-3′ RSMs displaying conserved hexa-loop sequences were generally identified in Stem-loop 5 (SL5) located in the 5′-UTRs of genomic RNAs. In Embecovirus spp., however, two to eight copies of 5′-agc-3′/guAAu RSMs were found in the coding regions of non-structural protein (NSP) 3 and/or NSP15 in open reading frame (ORF) 1ab. In gamma- and delta-CoVs, other types of RSMs were found in several clustered structural elements in 5′-UTRs and/or ORF1ab. The identification of RSM-encompassing structural elements in all CoVs suggests that these RNA elements play fundamental roles in the life cycle of CoVs. In the recently emerged SARS-CoV-2, beta-CoV-specific RSMs are also found in its SL5, displaying two copies of 5′-gUUUCGUc-3′ motifs. However, multiple sequence alignment reveals that the majority of SARS-CoV-2 possesses a variant RSM harboring SL5b C241U, and intriguingly, several variations in the coding sequences of viral proteins, such as Nsp12 P323L, S protein D614G, and N protein R203K-G204R, are concurrently found with such variant RSM. In conclusion, the comprehensive exploration for RSMs reveals phylogenetic insights into the RNA structural elements in CoVs as a whole and provides a new perspective on variations currently found in SARS-CoV-2.

The 3 termini of Alfalfa mosaic virus (AMV) RNAs adopt two mutually exclusive conformations, a coat protein binding (CPB) and a tRNA-like (TL) conformer, which consist of a linear array of stem-loop structures and a pseudoknot structure, respectively. Previously, switching between CPB and TL conformers has been proposed as a mechanism to regulate the competing processes of translation and replication of the viral RNA (R. C. L. Olsthoorn et al., EMBO J. 18:4856-4864, 1999). In the present study, the switch between CPB and TL conformers was further investigated. First, we showed that recognition of the AMV 3 untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding enzyme) in vitro is more efficient when the distribution is shifted toward the TL conformation. Second, the recognition of the 3 UTR by the viral replicase was similarly dependent on the ratio of CBP and TL conformers. Furthermore, the addition of CP, which is expected to shift the distribution toward the CPB conformer, inhibited recognition by the CCA-adding enzyme and the replicase. Finally, we monitored how the binding affinity to CP is affected by this conformational switch in the yeast three-hybrid system. Here, disruption of the pseudoknot enhanced the binding affinity to CP by shifting the balance in favor of the CPB conformer, whereas stabilizing the pseudoknot did the reverse. Together, the in vitro and in vivo data clearly demonstrate the existence of the conformational switch in the 3 UTR of AMV RNAs.Alfalfa mosaic virus (AMV) is a plant virus that belongs to one of the five genera in the family Bromoviridae, whose genomes consist of three genomic RNAs (RNAs 1, 2, and 3) and one subgenomic RNA (RNA4) that are capped at the 5Ј end and lack polyadenylation at the 3Ј terminus (3). RNAs 1 and 2 encode the viral subunits P1 and P2 of the replicase, respectively. RNA3 encodes the movement protein and serves as a template for the synthesis of RNA4, which encodes the coat protein (CP).The role of AMV CP has been the subject of extensive research in the past four decades. Initially, it was found that, in contrast to RNAs of the Bromo-, Cucumo-, and Oleavirus genera, the genomic RNAs of AMV and the closely related genus Ilarvirus were not infectious as such but required the presence of CP in the inoculum (15). This phenomenon was called genome activation and was long considered to compensate for the lack of a tRNA-like structure (TLS) at the 3Ј end of their genomic RNAs, a prominent feature of bromo-and cucumovirus RNAs (3). However, in 1999 we demonstrated that the 3Ј end of AMV RNAs can adopt an alternative conformation that shows many structural similarities to the TLS of other Bromoviridae, although it could not be charged with an amino acid (20). The tRNA-like (TL) conformation (Fig. 1) turned out to be the replicative form of the 3Ј termini (19,20), whereas the other, coat protein binding (CPB), conformer was subsequently shown to be required for translation (16)(17)(18). Although other models have been forwarded (9), we have proposed that ...

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A structural element was identified in the 59-proximal sequence of the bamboo mosaic virus (BaMV) RNA. Mutational analysis of the hairpin showed that disruptions of the secondary structure or substitutions of the loop sequences resulted in reduced accumulation of BaMV genomic RNA. Phylogenetic analysis further suggested the presence of structural homologues of this hairpin in all other potexviruses. In addition, remarkable structural homology was discovered between the BaMV hairpin and a stem-loop in the 59untranslated region of satellite RNAs responsible for attenuation of BaMV in co-infected plants. The role of this homology in the helpersatellite interaction is discussed. Bamboo mosaic virus (BaMV) is a plant virus of the genusPotexvirus with a positive-sense RNA genome of 6366 nt, comprising five open reading frames (ORFs) . Infections by BaMV are frequently associated with the presence of satellite (sat) RNAs, some of which may reduce accumulation of BaMV RNA and attenuate BaMVinduced symptoms in co-infected plants (Hsu et al., 1998).The 140 nt 39untranslated region (UTR) of BaMV genomic RNA (gRNA) is composed of a pseudoknot structure involving the poly-A tail, a stem-loop structure harbouring a conserved hexamer motif and a cloverleaf-like structure formed by three adjacent hairpins Tsai et al., 1999). These structural elements have been implicated in the initiation of minus-strand synthesis by the viral RNAdependent RNA polymerase (Huang et al., 2001;Cheng et al., 2002). However, structural elements in the 59UTR of BaMV gRNA that potentially play roles in replication and/or translation have not been investigated in detail, although two stem-loop structures in the corresponding region of the negative-strand RNA have been suggested to be involved in positive-strand RNA synthesis (Lin et al., 2005).Using Mfold (Zuker, 2003), a large stem-loop structure was predicted in the 59 117 nt of BaMV positive-strand RNA (Fig. 1a). To verify the secondary structure of the predicted hairpin, in particular the upper part (nt 53-99), a transcript of approximately 1000 nt was synthesized by in vitro transcription of a DNA template obtained by PCR amplification of a full-length cDNA of BaMV-S (Lin et al., 2004). Purified transcripts were probed with enzymes and chemicals, and analysed by denaturing gel-electrophoresis using an RNA sequencing ladder as reference, as described previously (Chen et al., 2007b). Fig. 1(b) shows that nt C80, C77, A71, A70, C69, A68, C52, A51, A50 and A41 are strongly reactive to dimethyl sulfate (DMS) while A42, A57, A58, C86 and A90 are weakly reactive. Interestingly, the absence of strong modification on C63, C64, C89 and A90 (Fig. 1b) could indicate that the C : C and the C : A mismatches in stem-II (Fig. 1a) potentially form non-Watson-Crick base pairs, as had been found in the turnip yellow mosaic virus (TYMV) 59 hairpins (Hellendoorn et al., 1997;Bink et al., 2002). Nuclease S1 recognized nt A68-U73 in internal loop (IL)-I and U78 in the apical loop (AL). To some extent, U60 and U62 in st...