**RNA 5′-end Maturation: A Crucial Step in the Replication of Viral Genomes**

Frédéric Picard-Jean, Maude Tremblay-Létourneau, Elizabeth Serra, Christina Dimech, Helene Schulz, Mathilde Anselin, Vincent Dutilly and Martin Bisaillon

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56166

**1. Introduction**

Viruses are a vastly diverse group of infectious particles with many different structures, mechanisms of function and ingenious strategies of invading host organisms for their own proliferation. One of the key features that ties viruses together as an inclusive group, is the reliance on living cells for replication and propagation. On their own, viruses lack the cellular machinery necessary for many life-sustaining functions including protein translation and metabolism. Regardless of the organization of a viral genome or the type of nucleic acid, infection of a host cell and viral propagation is dependent on the transcription of viral mRNA and, in turn, the translation of viral proteins as well as genome replication. Because viruses are dependent on host cell machinery for most of these processes, they have driven an outstanding virus-host co-evolution. Viruses that rely on the replication machinery of the host cell become cell-cycle dependent in their own replication. Furthermore, just as viruses have evolved ways to hijack necessary cellular proteins, cells have evolved complex mechanisms for fighting infection by detection and degradation of foreign mRNA. In order for viral mRNA to utilize host cell machinery, begin translation and remain both stable and undetected in the cytoplasm, it must contain the post-translational modifications of a host cell mRNA including, but not limited to, a 5' cap structure. By disguising viral mRNA with the same structural elements found in host mRNA, the cellular defense mechanism can be evaded and protein translation may occur. The significance of the cap structure can be seen through the diversity of cap-synthesis pathways across vastly different viral families that all lead to the formation

© 2013 Picard-Jean et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of a ubiquitous RNA 5'-cap. The 5'→ 3' direction of nucleotide triphosphate (NTP) polymeri‐ zation during RNA synthesis creates a nascent mRNA molecule with a 5'-triphosphate moiety resulting from the initial NTP on the 5'-end. Through the processes involved in cap synthesis, the pppRNA structure is transformed into a basic, cap-0 RNA structure (m7GpppN). Further 2'-O-methylations of the first and second nucleotides of the RNA may occur.

eukaryotic viruses require strategies, such as RNA cap synthesis, in order to protect, replicate

RNA 5′-end Maturation: A Crucial Step in the Replication of Viral Genomes

http://dx.doi.org/10.5772/56166

29

**Figure 1. RNA 5'-cap structure**. The RNA 5'cap structure is composed of a 7-methylguanosine (blue) linked to the RNA (black) through a 5'-5' triphosphate bridge (blue and black). The N7 methylation of the guanosine (green) con‐ fers a positive charge to the cap structure. Additional 2'O-methylation (red) can be found on the first few nucleotides.

**3. Conventional and unconventional 5' RNA cap synthesis mechanism**

The importance of the cap structure in eukaryote metabolism has resulted in an evolutionary pressure for viruses to adopt a similar cap structure. A series of enzymatic reactions is required to synthesize a cap structure at the 5'-end of RNA. The most pervasive enzymatic pathway, also termed "conventional capping", consists of three sequential enzymatic activities that are required to generate a functional 7-methylguanosine 5'-5'-triphosphate bridged cap structure. As a result of the directional 5' to 3' polymerization of nucleotide triphosphates (NTP) during RNA synthesis, nascent RNA bear at their 5'-end a triphosphate moiety (originating from the initial NTP). This 5'-triphosphate end of the RNA is first converted into a 5'-diphosphate end by hydrolysis of the terminal phosphate, or γ-phosphate, by an RNA triphosphatase (RTPase). This is followed by a two-step reaction catalyzed by an RNA guanylyltransferase (GTase). The enzyme first specifically binds and hydrolyzes a GTP molecule to form a covalent enzyme-GMP intermediate, which then catalyzes the transfer of the GMP moiety onto the 5'-end of a diphosphorylated acceptor RNA (ppRNA) in the second step of GTase reaction. Lastly, an

and translate their genomes in eukaryotic hosts.

**3.1. Canonical cap synthesis by different viruses**

In this chapter, a number of processes used by viruses to synthesize, acquire or mimic a 5' cap are explored to highlight the similarities and differences in the enzymatic mechanisms that lead to the maturation of a 5'cap on viral RNA and its importance in viral genome replication within a host cell.

#### **2. Description of the RNA cap structure**

To understand the importance of an RNA cap structure for viruses, it is crucial to first understand why this structure is essential to their eukaryotic hosts. Prokaryotic RNA tran‐ scription and protein translation are coupled due to the spatial proximity between DNA and ribosomes. In eukaryotic cells however, newly synthesized RNA transcripts undergo several nuclear post-transcriptional modifications, known as RNA processing, before they are exported and translated in the cytoplasm. These eukaryotic pre-mRNA modifications include the addition of a cap structure at the 5'-end, the splicing out of introns, the editing of nucleo‐ bases and the addition of a poly(A) tail at the 3'-end. RNA capping is a co-transcriptional process that occurs when an RNA molecule is 20-30 nucleotides in length. The cap structure consists of a guanosine residue, harboring a methylation in the N-7 position, which is bound to the terminal 5'-end nucleotide with a peculiar 5'-5' triphosphate bridge (Fig. 1). This inverted link between the two nucleotides prevents RNA degradation by 5'-3' exonucleases. The second important feature of the cap structure is the presence of the methyl group on the guanosine, which confers a positive charge that plays an important role in its specific recognition by specialized proteins. The cap structure fulfills many roles which ultimately lead to mRNA translation. In the nucleus for instance, the cap structure of pre-mRNAs is recognized by the cap binding proteins (CBP20 and CBP80). This cap binding complex (CBC) protects mRNA from degradation and assists RNA transport from the nucleus to the cytoplasm. Once in the cytoplasm, ribosomes and translation factors must be recruited for translation of mRNAs into proteins. The eukaryotic translation initiation factor 4E (eIF4E) specifically binds to the RNA cap structure [1]. This association is mediated through stacking interactions between two aromatic residues of the eIF4E protein; the mRNA binding is further stabilized by specific hydrogen bonds between the positive charge of the 7-methylguanosine and an acidic residue [2]. Upon cap binding, eIF4E assembles with eIF4G (a scaffold protein) and eIF4A (an RNA helicase) into the eIF4F complex [3]. The scaffolding protein eIF4G recruits the small 40S ribosomal subunit through the eIF3 complex [4]. The translation initiation complex then scans the mRNA for the start codon before recruiting the larger subunit of the ribosome, and translation of the open reading frame (ORF) takes place [2]. Taken together, the roles fulfilled by the RNA cap structure are crucial for RNA stability and translation. Because of this, many eukaryotic viruses require strategies, such as RNA cap synthesis, in order to protect, replicate and translate their genomes in eukaryotic hosts.

**Figure 1. RNA 5'-cap structure**. The RNA 5'cap structure is composed of a 7-methylguanosine (blue) linked to the RNA (black) through a 5'-5' triphosphate bridge (blue and black). The N7 methylation of the guanosine (green) con‐ fers a positive charge to the cap structure. Additional 2'O-methylation (red) can be found on the first few nucleotides.
