Samji: Influenza A: understanding the viral life cycle 155 TRANSCRIPTION AND REPLICATION OF THE VIRAL GENOME The influenza viral genome is made up of negative sense strands of RNA. In order for the genome to be transcribed, it first must be converted into a positive sense RNA to serve as a template for the production of viral RNAs. Replication of the genome does not re- quire a primer; instead, the viral RNA de- pendent RNA polymerase (RdRp) initiates RNA synthesis internally on viral RNA. This is possible, as the extreme 5’ and 3’ ends of the genome exhibit partial inverse complementarity and, hence, are able to base pair with one another to form various corkscrew configurations. It appears that a great number of di-nucleotide base pairs form, although the full mechanism of viral genome replication is still yet to be under- stood [13-16]. Given that the influenza A virus only encodes for 11 proteins, it has generated many sophisticated methods of utilizing the host cell’s machinery for its own purposes. Through understanding viral transcription, we have learned of a unique mechanism whereby the virus hijacks the host’s tran- scription machinery for its own benefits. Mature cellular messenger RNAs (mRNAs) have a 5’ methylated cap and a poly(A) tail. It is known that the vRNPs have poly(A) tails but no 5’ caps. It was confusing when the influenza community discovered that the viral mRNAs did have a 5’ methy- lated cap and a poly(A) tail, but the 5’ cap was not found in the viral genome [17,18]. Much study went into this problem, and soon it was determined that the 5’ methylated caps of the viral mRNAs actually belonged to the cellular mRNAs. That discovery lead to the formulation of the “cap-snatching” mecha- nism [19-26]. The viral RdRp is made up of three viral proteins: PB1, PB2, and PA. PB2 has endonuclease activity. It binds to the 5’ methylated caps of cellular mRNAs and cleaves the cellular mRNAs’ 10 to 15 nu- cleotides 3’ to the cap structure. This cellular capped RNA fragment is used by the viral RdRp to prime viral transcription [27]. Cellular RNA Polymerase II (Pol II) binds to DNA and starts transcription. Dur- ing transcription initiation, serine 5 on the C-terminal repeat domain (CTD) of Pol II is phosphorylated, leading to the activation of cellular cap synthesis complex. The in- fluenza RdRp has been shown to bind pref- erentially to this form of Pol II, indicating that this could be the point at which “cap snatching” could occur [28]. Six but two of the viral segments en- code for one protein. Segments 7 and 8 en- code for two proteins each due to splicing. Segment 7 encodes for M1 and M2; whereas, segment 8 encodes for NS1 and NEP. M2 and NEP are the spliced products and generally are found in much lower abundance than NS1 and M1 [29]. The virus uses the host cell’s splicing machinery to ex- press both of these proteins [30]. Despite in- fluenza’s need for the cellular splicing machinery, it prevents the host cell from using its own splicing machinery for pro- cessing the host cell mRNAs. NS1 binds to U6 small nuclear RNAs (snRNAs) [31,32] and other splicing components, causing them to re-localize to the nucleus of infected cells [33]. In this way, influenza is able to inhibit splicing of cellular mRNAs. It also has been shown to bind to a novel protein called NS1 binding protein (NS1-BP), caus- ing it to re-localize to the nucleus in infected cells. The function of NS1-BP is unknown, although it is predicted to be involved in splicing given its co-localization with SC35, a spliceosome assembly factor [34]. NP also has been shown to interact with UAP56, a splicing factor involved in spliceosomal for- mation and mRNA nuclear export, although the importance of NP’s binding to UAP56 is yet to be established [35]. The mechanism of polyadenylation of viral mRNAs is very unusual. Cellular mRNAs are polyadenylated through cleav- age at the polyadenylation signal (AAUAAA) by cleavage and polyadenyla- tion specificity factor (CPSF) and subse- quent addition of a poly(A) tail at the 3’ end of the mRNA. Viral mRNAs do not contain this sequence; instead, the viral RdRp re- mains bound to the 5’ end of the template