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Influenza A viruses (IAV) are responsible for seasonal epidemics, and pandemics can arise from novel zoonotic influenza A viruses transmitting to humans 1,2 . IAV contain a segmented negative sense RNA genome that is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the PB1, PB2, and PA subunits [3][4][5] . Although the high-resolution crystal structure of bat IAV polymerase (FluPol A ) has been reported 6 , there are no complete structures available for human and avian FluPol A . Furthermore, the molecular mechanisms of viral RNA (vRNA) replication, which proceeds through a complementary RNA (cRNA) replicative intermediate and requires polymerase oligomerisation 7-10 , remain largely unknown. Here we Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Aquatic birds represent a vast reservoir from which novel pandemic influenza A viruses can emerge 1 . Influenza viruses contain a negative-sense segmented RNA genome which is transcribed and replicated by the viral heterotrimeric RNA polymerase (FluPol) in the context of viral ribonucleoprotein (vRNP) complexes 2 , 3 . RNA polymerases of avian influenza A viruses (FluPol A ) replicate viral RNA poorly in human cells because of species-specific differences in acidic nuclear phosphoprotein 32 (ANP32), a family of essential host proteins for FluPol activity 4 . Interestingly, host adaptive mutations, particularly a glutamic acid to lysine mutation at amino acid residue 627 (E627K) in the 627 domain of the PB2 subunit (PB2 627 ), allow avian FluPol A to overcome this restriction and efficiently replicate viral RNA in the presence of human ANP32 proteins. However, the molecular mechanisms of genome replication and the interplay with ANP32 proteins remain largely unknown. Here, we report cryo-EM structures of influenza C virus polymerase (FluPol C ) in complex with human and chicken ANP32A. In both structures, two FluPol C molecules form an asymmetric dimer bridged by the N-terminal leucine-rich repeat domain (LRR) of ANP32A. The C-terminal low complexity acidic region (LCAR) of ANP32A inserts between the two juxtaposed PB2 627 domains of the asymmetric FluPolA dimer, providing insight into the mechanism behind the PB2E 627K adaptive mutation in mammalian hosts. We propose that this complex represents a replication platform for the viral RNA genome, in which one of the FluPol molecules acts as a replicase while the other initiates the assembly of the nascent replication product into a vRNP.

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The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14–nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14–nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14–nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3′-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14–nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12–nsp7–nsp8 (nsp12–7–8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14–nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14–nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.

In prokaryotes, RNA polymerase and ribosomes can bind concurrently to the same RNA transcript, leading to the functional coupling of transcription and translation. The interactions between RNA polymerase and ribosomes are crucial for the coordination of transcription with translation. Here, we report that RNA polymerase directly binds ribosomes and isolated large and small ribosomal subunits. RNA polymerase and ribosomes form a one-to-one complex with a micromolar dissociation constant. The formation of the complex is modulated by the conformational and functional states of RNA polymerase and the ribosome. The binding interface on the large ribosomal subunit is buried by the small subunit during protein synthesis, whereas that on the small subunit remains solvent-accessible. The RNA polymerase binding site on the ribosome includes that of the isolated small ribosomal subunit. This direct interaction between RNA polymerase and ribosomes may contribute to the coupling of transcription to translation.

Background: The PDZ protease Deg2 is involved in chloroplast protein quality control through a yet unknown molecular mechanism.Results: A novel PDZ domain with an internal ligand mediates hexamer formation and locks Deg2 into the resting state.Conclusion: Formation of the resting hexamer may be a common strategy in a Deg protease subfamily.Significance: We provide structural insights into the PDZ domain-mediated regulation of Deg proteases.