Cis-acting RNA elements in human and animal plus-strand RNA viruses

Abstract

The RNA genomes of plus-strand RNA viruses have the ability to form secondary and higher-order structures that contribute to their stability and to their participation in inter- and intramolecular interactions. Those structures that are functionally important are called cis-acting RNA elements because their functions cannot be complemented in trans. They can be involved not only in RNA/RNA interactions but also in binding of viral and cellular proteins during the complex processes of translation, RNA replication and encapsidation. Most viral cis-acting RNA elements are located in the highly structured 5'- and 3'-nontranslated regions of the genomes but sometimes they also extend into the adjacent coding sequences. In addition, some cis-acting RNA elements are embedded within the coding sequences far away from the genomic ends. Although the functional importance of many of these structures has been confirmed by genetic and biochemical analyses, their precise roles are not yet fully understood. In this review we have summarized what is known about cis-acting RNA elements in nine families of human and animal plus-strand RNA viruses with an emphasis on the most thoroughly characterized virus families, the Picornaviridae and Flaviviridae.

Fig. 1

Cis-acting RNA elements in the genomes of PV and of HRV14 (Enterovirus, Picornaviridae). (A) PV. The 5′ NTR consists of the CL and the IRES. The ORF contains three domains, the structural (P1), and the nonstructural domains (P2 and P3). The cre is located in the coding sequence of protein 2C^ATPase. The ciRNA, inhibitor of RNAse L, is located in the 3C^pro coding sequence. The 3′ NTR contains two stem loops and is followed by a poly(A) tail. (B) Enlarged images of the PV CL, the c-rich region of the spacer (left), the cre(2C) hairpin (middle) and the 3′ NTR-poly(A) with a “kissing interaction” between stem loops X and Y. The PCBP2 binding site on SL-A of the CL is shown with bold letters and the tetra loop in SL-D is boxed. (C) HRV14. The 5′ NTR is similar to the genome of PV but the cre RNA is located in VP1 and the 3′ NTR has only one stem loop.

Fig. 2

Cis-acting RNA elements in the genomes of EMCV (Cardiovirus, Picornaviridae) and FMDV (Aphtovirus, Picornaviridae). (A) EMCV. The 5′ NTR contains a large hairpin and two pseudoknots followed by a poly(C) tract and the IRES. The cre element is located in the coding region of capsid protein VP2. The ORF contains a leader (L), one structural (P1), and two nonstructural domains (P2 and P3). The one stem loop of the 3′ NTR interacts with the poly(A) tail. (B) FMDV. The 5′ NTR contains the S fragment, a poly(C) tract, three to four pseudoknots, the cre element, and the IRES. The 3′ NTR has two stem loops, which interact both with the S fragment and the IRES.

Fig. 3

Cis-acting RNA elements in the genome of HAV (Hepatovirus, Picornaviridae). The genome of HAV contains a long 5′ NTR, which consists of three stem loops, a pyrimidine-rich tract, and IRES. The ORF contains one structural (P1) and two nonstructural domains (P2, P3). The cre element is located in the 3D^pol-coding region. The 3′ NTR contains a pseudoknot and a polyA) tail.

Fig. 4

Cis-acting RNA elements in the genomes of Aichi virus and Porcine kobuvirus (Kobuvirus, Picornaviridae) and of HpeV1 (Parechovirus, Picornaviridae). (A) Aichi virus (human kobuvirus) and Porcine kobuvirus. The 5′ NTR of Aichi virus consists of three stem loops (SL-A, B, C) and of a pseudoknot (Pk). At the 3′-terminal part of the 5′ NTR the putative IRES structure of Porcine kobuvirus is shown. The kobuvirus ORF contains a leader sequence (L), one structural (P1), and two nonstructural domains (P2, P3). The 3′ NTR (167 nt) of Porcine kobuvirus is predicted to form three hairpin structures (G. Reuter, unpublished data. Structure is shown with permission of G. Reuter). The genome is terminated with a poly(A) tail. (B) HpeV1. The 5′ NTR contains three stem loops (SL-A, B, C) and a pseudoknot, and the IRES. The ORF has a leader sequence, one structural (P1), and 2 nonstructural domains (P2, P3). The cre element is located in the VP0 coding sequence. The 3′ NTR consists of a stem loop, which is involved in a “kissing interaction” with the poly(A) tail.

Fig. 5

IRES elements of Picornaviruses. (A–C) The figure shows the type I IRES element of PV, the type II IRES of EMCV, and the type III IRES of HAV. (D–E) Type IV IRESes of Porcine kobuvirus and Porcine teschovirus, respectively. A–C are taken from Ehrenfeld et al., , with permission of the publisher. D is taken from Reuter et al., , with permission of the publisher. Figure E is taken from Chard et al., , with permission of the publisher.

Fig. 5

IRES elements of Picornaviruses. (A–C) The figure shows the type I IRES element of PV, the type II IRES of EMCV, and the type III IRES of HAV. (D–E) Type IV IRESes of Porcine kobuvirus and Porcine teschovirus, respectively. A–C are taken from Ehrenfeld et al., , with permission of the publisher. D is taken from Reuter et al., , with permission of the publisher. Figure E is taken from Chard et al., , with permission of the publisher.

Fig. 6

Cis-acting RNA elements in the genomes of HCV (Hepacivirus, Flaviviridae), BVDV (Pestivirus, Flavivirida), and DENV (Flavivirus, Flaviviridae). (A) HCV. The 5′ NTR contains one stem loop and the IRES, which extends into the core coding sequence. Stem loops II and III of the IRES are shown enlarged. (Figure of IRES is taken from Lukowsky et al. with permission of the publisher). The ORF contains a structural (SP) and a nonstructural (NSP) domain. The 3′ NTR contains the 3′X tail, a polyU/C tract, and a variable (VR) domain. There are four stem loops (3′SL-IV to 3′SL-VII) in the NS5B C-terminal coding sequence, one of which (3′SL-V) is involved in “kissing interactions” with stem loop 2 of the 3′ NTR and an unpaired sequence upstream in the NS5B coding sequence. (B) BVDV. The 5′ NTR contains 2 stem loops (1a, 1b) and the IRES (stem loops IIa, IIb, IIIa-IIId). The ORF contains a structural (SP) and a nonstructural (NSP) domain. The 3′ NTR contains three stem loops (SLI to SLIII). (C) DENV. The 5′ NTR contains two stem loops (SLA, B) with an additional small stem loop (cHP) in the capsid-coding region. SL-B contains a short sequence, UAR (upstream AUG region), which is complementary to a sequence in the 3′ NTR. Additional complementary sequences (CS) are located near the 5′- and 3-'terminus. The ORF contains a structural (SP) and a nonstructural (NSP) domain. The 3′ NTR consists of three domains with a total of five stem loops. The interaction between the 5′UAR/3′UAR and the 5′CS/3′CS are shown below. Fig. 6C is modified from two figures published in a review by Iglesias et al., , with permission of the publisher.

Fig. 7

Cis-acting RNA elements in the genome of CrPV (insect picorna-like virus, Dicistroviridae). (A) The genome structure of Cricket paralysis virus. The single-stranded RNA genome is linked to VPg at the 5′-end and contains two ORFs. The upstream ORF1 encodes the nonstructural proteins and the downstream ORF2 the capsid proteins. The two ORFs are separated by the intergenomic (IGR) IRES. Another IRES is located in the 5′ NTR. The 3′ NTR is polyadenylated. (B) Structure of the CrPV IGR-IRES element. Dotted lines indicate separation of domains. IRES nucleotides that are likely to interact with the ribosome are circled. Figure of IRES is taken from Schuler et al. , with permission of the publisher.

Fig. 8

Cis-acting RNA elements in the genomes of SIN (Alphavirus, Togaviridae) and of RV (Rubivirus, Togaviridae). (A) SIN. The 5′ NTR contains two stem loops (SL1 and SL2). Just downstream of SL2, in the adjacent nsp1 coding sequence, there are two stem loops comprising the CSE. Four additional stem loops, located further downstream, form an encapsidation signal. The ORF contains a nonstructural domain (NSP) and a structural domain (SP), which is translated from a subgenomic RNA (sg RNA). There is a short connecting segment (CSE) between the NSP and SP domains. The 3′ NTR contains repeated sequence elements, a short CSE segment and a poly(A) tail. (B) RV. The short 5′ NTR and the adjacent NSP coding sequence contain a single stem loop. The ORF consists of a nonstructural (NSP) and a structural domain (SP). A connecting region, J-UTR, contains two stem loops in the minus-strand (SLI and II). SLII is located within the subgenomic promoter. The 3′ NTR contains two stem loops (SL3 and SL4) followed by a poly(A) tail with two additional stem loops in the C-terminus of the E1 coding region (SL1 and SL2).

Fig. 9

Cis-acting RNA elements in the genomes of BCoV (Coronaviridae) and of mouse MHV (Coronaviridae). In BCoV the 5′ NTR contains 4 stem loops (SL-I to SL-IV) with two additional stem loops in the adjacent ORF1a. A packaging signal is located in ORF1b. The genome contains multiple ORFs. The 3′ NTRs of BCoV and MHV both contain a pseudoknot, a bulged stem loop, and a variable region (VAR). There is an interaction between the pseudoknot and the 3′ end of the MHV genome. The genome is terminated with a poly(A) tail. In SARS-CoV there is a slippery site and a pseudoknot (Pk) in the overlapping sequence of ORF1a and 1b.

Fig. 10

Cis-acting RNA elements in the genomes of EAV (Arteriviridae) and of PRRSV (Arteriviridae). (A) EAV. The 5′ NTR contains a leader sequence with 5 stem loops (SL-A to SL-E) and an additional five stem loops (SL-F-SL-J) of which the last two is located in the ORF1a coding sequence. The genome contains multiple ORFs. The 3′ NTR contains one stem loop (SL-V) and another stem loop (SL-IV) is located just upstream in ORF 7. The genome is terminated with a poly(A) tail. (B) PRRSV. In PRRSV there is a leader sequence in the 5′ NTR. The genome contains multiple ORFs. The 3′ NTR contains one stem loop, which interacts with another stem loop located in the coding sequence of ORF7. The genome is terminated with a poly(A) tail.

Fig. 11

Cis-acting RNA elements in the genome of MNV (Norovirus, Caliciviridae). The 5′ NTR contains one stem loop and an adjacent stem loop in the ORF1 coding sequence. The genome contains three ORFs. The 3′ NTR contains one large stem loop with an adjacent stem loop in the ORF3 coding sequence. The genome is terminated with a poly(A) tail. A minus-strand stem loop is located at the ORF1 and ORF2 junctions.

Fig. 12

Cis-acting RNA elements in the genome of Astrovirus (Astroviridae). The genome contains 3 ORFs (ORF1a, 1b and 2). The short 5′ NTR is not yet characterized. A ribosomal frame shift site is located at the junction of ORFs 1a and 1b, which is predicted to interact with a downstream sequence. A 120 nt long segment, containing three stem loops, overlaps the junction of ORF1b and ORF2. The 3′-terminal region (110 nt) contains two highly conserved stem loops.

Fig. 13

Cis-acting RNA elements in the genome of Flock House virus (Nodaviridae). The bipartite genome of FHV consists of RNA1 and RNA2, each of which encode a single ORF. The subgenomic RNA (RNA3) encodes two ORFs. minus-strand RNA synthesis requires the 3-proximal 108 and 50 nt of RNA 1 and RNA2, respectively. Plus-strand RNA synthesis requires only 3–14 nt at the 3′-end of minus-strands. The synthesis of RNA3 is governed by a long-range interaction between two cis-elements on RNA1 (DSCE and PSCE). A packaging signal (32 nt) is located in RNA2. Additional RNA elements are located on RNA1 and RNA2, as shown.