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Disease

Influenza A causes the infectious respiratory disease Influenza, more commonly known as the Flu.^[1] Influenza has several symptoms associated with it: fever, sore throat, runny nose, headache, muscle pain, and fatigue to severe and in some cases lethal pneumonia resultant from either influenza virus or bacterial infection in the lower respiratory tract.^[1] The CDC estimated in 2019-2020 that the overall burden of Influenza was 35 million Flu-related illnesses, 16 million Flu-related medical visits, 380,000 Flu-related hospitalizations, and 20,000 Flu-related deaths.^[2] The WHO estimated the global impact of annual Flu epidemics resulted in 3 to 5 million cases of severe illness, and about 290,000 to 650,000 respiratory deaths.^[3] Multiple types of vaccines against Influenza currently exist: inactivated influenza vaccines (IIV4s), recombinant influenza vaccine (RIV4), and live attenuated influenza vaccine (LAIV4).^[4]

Viral RNA Classification

Influenza A virus genome is carried on single-stranded, negative-sense RNA segments. In replication, these segments are bound by the heterotrimeric viral RNA-dependent RNA polymerase (RDRP) and multiple copies of nucleoprotein to form the viral ribonucleoprotein (vRNP) complex.^[5]

Function of RNA Dependent RNA Polymerases

RDRPs allow viruses to replicate their genome as well as carry out transcription. RDRPs catalyze RNA-template-dependent formation of phosphodiester bonds between ribonucleotides, initiating synthesis at the 3' end of the template through a primer-dependent or independent manner proceeding in the 5' to 3' direction.^[6] RDRPs lack proofreading exonuclease activity which allows for increased rates of mutation that can be selected under pressures from the host's defense mechanisms or other environmental factors.^[6] RDRPs are a good target for antiviral drugs, as viruses depend on them to transcribe and replicate their genome so that they can infect hosts and spread. A 2020 study on the anti-Flu capabilities of 1,2,4-triazolo[1,5-a]pyrimidine-2-carboxamid-based compounds found that such compounds were able to interfere with the PA-PB1 interactions.^[7] PA and PB1 are two of the subunits which make up the heterotrimeric Influenza A RDRP that are required for the RDRP to function properly and so the compounds showed promise; however, the study called for more research into such compounds to design drugs with even better anti-Flu properties.^[7]

Influenza A Basic Structure

Influenza A itself is made of three subunits: , , and . The PA subunit contains the endonuclease which is responsible for cleaving the transcript that had been pirated from the host 10-12 nucleotides downstream of its 5' cap.^[8] The PB1 subunit showcases the characteristic , , and domain of other RDRPs in addition to the central active site where the RNA synthesis occurs.^[8] The PB2 subunit is likely involved in separating the two strands of the template product within the active site and directs them into their respective exit tunnels.^[8]

Structural Features^[6]

The major structure of RDRPs is formed by the , , and subdomains, with an average length of the core domain being less than 500 amino acids. The three subdomains are involved in template binding, polymerization, and nucleoside triphosphate entry. The palm domain is structurally the most conserved for catalysis, and it serves as the junction of the fingers and thumb domains. The thumb subdomain contains residues involved in packing against the template RNA and stabilizing the initiating NTPs on the template. The fingers subdomain has the role of setting the geometry of the active site serving to hold the template RNA in place and facilitating polymerization. The channels within RDRPs are lined with positively charged residues which promote the binding of the template RNA, the primer, and the NTPs for catalysis. RDRPs also have a set of seven structural motifs labeled A to G, which characterize the conserved structural components of the RDRPs. Motif A houses the catalytic motive DX¬¬2-4D with the first aspartate conserved across various RDRPs. Motif B assists in binding the template RNA and acts as a flexible hinge to accommodate the conformational changes that must take place for template and substrate binding, and it has a conserved glycine residue at the junction of the loop and helix. Motif C contains the conserved GDD motif which is essential for binding metal ions which are required for catalysis within the active site. Motif D also has a conserved glycine which allows it to act as a pivot for conformational changes that are associated with the correct NTP binding. Motif E serves as the primer grip which aids in positioning the 3’ hydroxyl group of the primer for catalysis. Motif F is comprised of conserved positively charged residues which shield the negative charges of the incoming NTP phosphate groups. Motif G consists of a helix that interacts with the priming NTPs, and in Influenza A it is a component of the polymerase acidic (PA) subunit.

Viral RNA Transcription and Translation

Because Influenza A is a negative-sense RNA virus, it cannot be immediately translated by the host, and instead the viral RNA must first be copied so that the complementary strand runs in the proper 5' to 3' direction.^[9] Influenza A utilizes its viral polymerase to engage in cap-snatching in which it takes 5' capped RNA fragments from the host's capped RNAs.^[5] The cap binding site hosts several that recognize and orient the host RNA. Residues Q406, D361 and K376 (green) are able to recognize a methylated guanine base, which is then sandwiched by H357, F323 and F404 (red). Hydrogen bonding between the phosphates of the RNA backbone and residues H432, H357, K339 and N429 then orients the RNA in the active site.^[10] Afterwards, the cap-binding domain rotates to insert the 3' end of the capped RNA into the active site, and NTPs enter through the entry channel as the polymerase constructs a strand complementary to the viral RNA.^[5] Influenza A is also able to differentiate between RNA promoters, and it contains several that allow it to bind the correct cRNA promoter so that it can continue its viral life cycle.^[11]

Influenza A uses its trimer subunits to bind the template strand: the host capped RNA is bound by the PB2 cap-binding domain, followed by the cleavage of the PA/P3 endonuclease domain. ^[5] As mentioned before, the cap-binding domain then rotates allowing the insertion of the 3' end of the capped RNA, and then initiation begins once GTP is added to the 3' end of the capped primer which has become templated by the second residue in the viral RNA template. ^[5]

Nucleotides are guided into the polymerase through the entry channel, which is made of highly conserved basic amino acids and consists of all three Influenza A RDRP subunits.^[5] The priming loop is especially important, as it is a that protrudes from the PB1 thumb domain and has the role of supporting the sugar-base of the initiating nucleotide and it contains such as PRO651 and the catalytic ASP445-446.^[5] Additionally, the hairpin contributes to endonuclease activity, guides the duplex template/copy dsRNA out of the active site, and confers some selectivity of oligonucleotide primers via steric hindrance in the active site ^[12].

Conservation within Influenza A RDRP

The most highly conserved regions of the IAV RdRp are Motifs I-IV of the PB1 subunit, as they play a significant role in the replicative capabilities of the Influenza A RDRP ^[13]. Each motif is an approximately 10 residue-long sequence ^[14]. Click the respective scene links for close-ups of each motif (, , , IV).

Conclusion

The Influenza A viral RNA single-strand segments are negative-sense, and so they cannot be immediately read by the host cell, and therefore require transcription by the Influenza A RDRP into positive-sense RNA.^[15] Only after transcription to single-stranded positive-sense RNA, which is essentially mRNA, can the RNA be translated into viral proteins that carry on the viral lifecycle. Therefore, knowledge of the Influenza A RDRP is of utmost importance, as novel treatments can be designed that inhibit the RDRP and therefore prevent the virus from spreading after initial entry into the cell by preventing it from replicating its genome and producing viral proteins.