Exploring regulation of RNA synthesis by influenza virus polymerase: finding targets for the broad-spectrum inhibitors

Go to the profile of Yi Shi
Jun 17, 2019
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In our recent paper, "Structural insight into RNA synthesis by influenza D polymerase", we describe the cryo-EM structures of apo and promoter-bound influenza D polymerase and reveal that the binding conformations of RNA promoters are involved in the regulation of different RNA synthesis by influenza virus polymerase. We also show that a novel channel bound by the 3’RNA promoter can be the potential target for the broad-spectrum antiviral inhibitors.

When I was a Ph.D. student at Institute of Microbiology, Chinese Academy of Sciences (CAS), I came to the study of viruses due to the outbreak of 2009 H1N1 influenza pandemic. Since then, I have touched several kinds of emerging infectious viruses, including influenza viruses, coronaviruses, filoviruses, and flaviviruses. My mentor George F. Gao was highly interested in the molecular mechanisms of virus entry, and in his lab, I have focused on elucidating the molecular mechanism on the viral glycoprotein interacting with its receptors by structural and virological methods, which is one of the determinant factors for the interspecies transmission of virus. In September 2015, I was invited to give a talk on the topic “Host jump of emerging viruses: flu, MERS-CoV, and Ebola”, in the International Meeting on Respiratory Pathogens (IMRP) which was held in Singapore and organized by ISIRV (the International Society of Influenza and Respiratory Viruses). After my talk, I met Ruben O Donis who discovered the bat influenza virus, and he said, “You guys have done very well in the study of virus entry, why not touch the virus replication which is also very important for the interspecies transmission of virus”. By then, I became interested in the virus replication which is really a complex issue that involves both viral and host proteins.

Since March 2016, I set up my own laboratory in Institute of Microbiology, CAS. With the support of my former mentor George, I started to study the polymerases of emerging infectious viruses and chose influenza virus as the first subject. Influenza virus belongs to negative-strand RNA viruses (NSVs) that are responsible for a wide range of diseases in plants, animals and humans. NSVs can be broadly categorized as segmented and non-segmented viruses. For example, Orthomyxoviruses such as influenza virus contain six to eight RNA genomic segments, Bunyaviruses such as hantavirus contain three, and Arenaviruses such as Lassa virus contain two segmented RNAs, while non-segmented NSVs (Mononegavirales) include many deadly sporadic human pathogens such as Ebola and rabies viruses. By the efforts of the virologists, we have gained structural insights into the overall architecture of the polymerases of segmented NSVs including influenza A/B/C virus and La Crosse orthobunyavirus, and also non-segmented vesicular stomatitis virus (VSV). All the NSV polymerase would take a common two-step replication process, the first step is from negative-sense viral RNA (vRNA) genome to positive-sense complementary RNA (cRNA) genome, and the second step is synthesizing the vRNA genome using cRNA as template. In addition to replicating viral genome, NSV polymerases also transcribe the positive-sense viral mRNAs using the same vRNA template. Segmented NSVs will generate mRNA by a unique cap-snatching mechanism, while the non-segmented NSVs will generate capped mRNA by itself. However, the molecular mechanisms on regulation of RNA synthesis by these viral polymerases are yet elusive. 

To date, we know both viral and host factors are involved in the regulation of RNA synthesis by influenza virus polymerase, such as the viral NS1 and NP proteins, and also the host proteins including RNA pol II and ANP32A/ANP32B. How about the viral RNA genome itself? Are the RNA promoters involved in the regulation of RNA synthesis? Bearing this question in mind, we systematically solved the cryo-EM structures of apo influenza D virus polymerase, the vRNA-promoter-bound and cRNA-promoter-bound polymerase. Interestingly, we found that the vRNA promoter can bind in two conformations (mode A and B) where while the cRNA promoter could only bind in one of two conformations (mode B). Extensive functional experiments have revealed the critical role of the mode B conformation for vRNA synthesis via the intermediate cRNA but not for cRNA production, which is mainly regulated by the mode A conformation. Both conformations participate in the regulation of the transcription process. Further mutagenesis analysis has revealed that the key residues stabilizing the 3’RNA in mode B conformation are relatively conserved among the four influenza virus genera (influenza A, B, C and D).

We are happy that our work has confirmed that the RNA promoter binding conformation is involved in regulation of different RNA synthesis by influenza virus polymerase. Our finding has advanced our understanding of the regulatory mechanisms for the synthesis of different RNA species by influenza virus polymerase and opens new opportunities for broad-spectrum antiviral drug design. We are expecting to find more regulatory mechanisms for the RNA synthesis of influenza virus polymerase, and also to explore the functions and structures of polymerases from other NSVs, especially how the host proteins regulate the RNA synthesis. We hope to design broad-spectrum inhibitors based on the common features of these viral polymerases and prepare well for the outbreak of emerging NSVs.

References:

1. Pflug A et al. 2014. Structure of influenza A polymerase bound to the viral RNA promoter. Nature, 516(7531):355-60.

2. Reich S et al. 2014. Structural insight into cap-snatching and RNA synthesis by influenza polymerase. Nature, 516(7531):361-6.

3. Hengrung N et al. 2015. Crystal structure of the RNA-dependent RNA polymerase from influenza C virus. Nature, 527(7576):114-7.

4. Gerlach P et al. 2015. Structural insights into bunyavirus replication and its regulation by the vRNA promoter. Cell, 161(6):1267-79.

5. Liang B et al. 2015. Structure of the L protein of vesicular stomatitis virus from electron cryomicroscopy. Cell, 162(2):314-327.

Our manuscript in Nature Microbiology can be found here:

https://www.nature.com/articles/s41564-019-0487-5


Go to the profile of Yi Shi

Yi Shi

Professor, Institute of Microbiology, Chinese Academy of Sciences

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