Nucleosides for the treatment of respiratory RNA virus infections

Jordan, Paul C; Stevens, Sarah K; Deval, Jerome

Influenza virus is a (-)ssRNA virus and a member of the Orthomyxoviridae family. 5 The viral genome has eight segments in influenza A and B and seven segments in influenza C and D. In influenza A, these encode for 11 or 12 proteins. These are non-structural protein 1 (NS1), non-structural protein 2 (NS2), matrix protein M1 and ion channel protein M2, polymerase acidic (PA) protein, polymerase basic protein (PB1), polymerase basic protein 1-F2, polymerase basic protein 2 (PB2), nucleoprotein (NP), hemagglutinin (HA), and neuraminidase (NA). 44 Some viruses express the PB1-N40 protein. All four species of influenza adopt similar arrangements with the viral genomic segments forming viral ribonucleoprotein complexes associated with one heterotrimeric polymerase. Influenza A polymerase is composed of three subunits that yield a 270 kDa heterotrimeric complex. The longest viral RNA segments encode for the PA protein, PB1, and PB2, which assemble to form the influenza polymerase complex (see Figure 2 ). 45 The three subunits interact noncovalently to exert their polymerase activity. The polymerase transcribes viral RNA into messenger RNA (mRNA) and then replicates it using a complementary RNA intermediate. 5 The process of transcription includes cap snatching, where short-capped cellular RNA are bound by the PB2 subunit, cleaved from the PA endonuclease domain, and then utilized for priming mRNA synthesis by the PB1 domain. [46] [47] [48] [49] [50] [51] The PB2 subunit has an N-terminal domain (PB2-N) from residues 1 to 247 and a C-terminal domain (PB2-C) from residues 248 to 760. 52 PB2-N, including a lid domain, interacts with the C-terminal extension and thumb of PB1. 52 The PB2-C includes several notable structural features and subdomains such as the C-terminal nuclear localization signal, the PB2 627-domain, the PB2 cap-627 linker, the mid domain, and a cap binding domain. 46 Based on structural biology, the PB2 domain has a key exterior, positively charged residue at the 627 position within a flexible loop that partially wraps around an alpha helix to form what is known as a phi-loop. 53 Importantly, this residue is in the middle of a set of highly conserved, basic residues forming a net positive charge. A signature structural element is the conserved P[F/P]AAAPP motif on the N-terminal side of the 627 residue that is part of the alpha helix previously described. 53 Mutation of the P620 or F621 residue significantly decreased the ability of the virus to replicate, presumably by causing a slight kink in the alpha helix that alters the polymerase function. The exact role of the PB2 627 domain remains unclear, but recent evidence suggests it is not necessary for in vitro binding and transcription of viral RNA; this has not been proven true in a cell-based format. 54 The PB1 domain is at the center of the polymerase complex and within its center is a classic right-handed shape with the signature fingers, palm, and thumb subdomains (see Figure 2 ). 5 These subdomains are described as conserved RNA-dependent RNA polymerase (RdRp) motifs pre-A/F and A-E. 52 The pre-A/F motif describes the fingertips and a small loop, which spreads over to the thumb domain; the tip of this loop is stabilized by an alpha helix within the PA domain. Residues within the pre-A/F may guide and bind NTPs and the incoming template. In addition to these subdomains, N-terminal and C-terminal extensions interact with PA and PB2 domains. The fingers and palm are covered by