Cis-active RNA elements (CREs) and picornavirus RNA replication

Abstract

Our understanding of picornavirus RNA replication has improved over the past 10 years, due in large part to the discovery of cis-active RNA elements (CREs) within picornavirus RNA genomes. CREs function as templates for the conversion of VPg, the Viral Protein of the genome, into VPgpUpU(OH). These so called CREs are different from the previously recognized cis-active RNA sequences and structures within the 5' and 3' NTRs of picornavirus genomes. Two adenosine residues in the loop of the CRE RNA structures allow the viral RNA-dependent RNA polymerase 3D(Pol) to add two uridine residues to the tyrosine residue of VPg. Because VPg and/or VPgpUpU(OH) prime the initiation of viral RNA replication, the asymmetric replication of viral RNA could not be explained without an understanding of the viral RNA template involved in the conversion of VPg into VPgpUpU(OH) primers. We review the growing body of knowledge regarding picornavirus CREs and discuss how CRE RNAs work coordinately with viral replication proteins and other cis-active RNAs in the 5' and 3' NTRs during RNA replication.

Figure 1. The Picornaviridae family

Figure 2. Virus in the genus Enterovirus: human enterovirus species A-D

Figure 3. Poliovirus and the poliovirus RNA genome

Poliovirus RNA genome (illustrated at the top of the figure) is packaged within virus particles (bottom left). The RNA genome is an mRNA encoding the capsid proteins and nonstructural proteins. Nonstructural proteins include the viral proteases and viral replicase proteins. Chemical formula for poliovirus from (Wimmer, 2006).

Figure 4. VPg and cis-active RNA sequences and structures required for poliovirus RNA replication

VPg, a 22 amino acid long viral protein, is covalently linked by the tyrosine hydroxyl to the 5′ terminus of viral RNA via a phosphodiester (Ambros and Baltimore, 1978). There are four distinct cis-active RNA sequences and structures required for RNA replication: a cloverleaf RNA sequence and structure at the 5′ terminus (5′ CL), a cis-replication element (CRE) in the open reading frame, a 3′ nontranslated region (3′ NTR), and a 3′ poly(A) tail.

Figure 5. Location and secondary structures of CREs

The location in the viral genome and the secondary structures of representative CREs. Nucleotide numbers from full-length RNA genome sequences. (A) PV1 CRE, representative of CREs in human enterovirus species A-D (Goodfellow et al., 2000; van Ooij et al., 2006) (B) Human rhinovirus 2 (HRV 2) CRE, representative of species A rhinoviruses (Gerber et al., 2001). (C) HRV 14 CRE, representative of species B rhinoviruses (McKnight and Lemon, 1998). (D) HRV A2 CRE, representative of species C rhinoviruses (Cordey et al., 2008). (E) EMCV CRE, representative of Cardioviruses (Lobert et al., 1999). (F) FMDV CRE, representative of Apthoviruses (Mason et al., 2002). The FMDV CRE is located 218 to 275 bases downstream from the variable length poly(C) tract in the 5′ NTR. (G) Human parechovirus 1 (HPeV1) CRE, representative of Parechoviruses (Al-Sunaidi et al., 2007). (H) Hepatitis A virus (HAV) CRE, representative of Hepatoviruses (Yang et al., 2008). Only sequences from the apex of the 110 nt long HAV CRE are presented due to space constraints.

Figure 5. Location and secondary structures of CREs

The location in the viral genome and the secondary structures of representative CREs. Nucleotide numbers from full-length RNA genome sequences. (A) PV1 CRE, representative of CREs in human enterovirus species A-D (Goodfellow et al., 2000; van Ooij et al., 2006) (B) Human rhinovirus 2 (HRV 2) CRE, representative of species A rhinoviruses (Gerber et al., 2001). (C) HRV 14 CRE, representative of species B rhinoviruses (McKnight and Lemon, 1998). (D) HRV A2 CRE, representative of species C rhinoviruses (Cordey et al., 2008). (E) EMCV CRE, representative of Cardioviruses (Lobert et al., 1999). (F) FMDV CRE, representative of Apthoviruses (Mason et al., 2002). The FMDV CRE is located 218 to 275 bases downstream from the variable length poly(C) tract in the 5′ NTR. (G) Human parechovirus 1 (HPeV1) CRE, representative of Parechoviruses (Al-Sunaidi et al., 2007). (H) Hepatitis A virus (HAV) CRE, representative of Hepatoviruses (Yang et al., 2008). Only sequences from the apex of the 110 nt long HAV CRE are presented due to space constraints.

Figure 6. CRE-dependent VPg uridylylation

VPg is converted into VPgpUpU_OH in defined reactions containing CRE RNA templates, 3D^Pol, VPg, and UTP (Paul et al., 2003a; Paul et al., 2000; Paul et al., 2003b). Viral protein 3CD stimulates these reactions (Nayak et al., 2005; Nayak et al., 2006; Pathak et al., 2007; Pathak et al., 2002; Yang et al., 2004; Yin et al., 2003).

Figure 7. CRE-dependent VPgpUpU_OH synthesis and translocation to the 3′ termini of viral RNA templates for RNA replication

3D^Pol and VPgpUpU_OH must translocate from the CRE portion of viral RNA templates to the 3′ ends of positive- and negative-strand RNA templates to prime the initiation of RNA synthesis. These processes are supported within viral RNA replication complexes formed within cell-free translation-replication reactions (Goodfellow et al., 2003b; Lyons et al., 2001; Morasco et al., 2003; Murray and Barton, 2003; van Ooij et al., 2006).