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Home , Influenza C virus, Metapneumovirus, Pneumoviridae, Rabies virus

COMMENTARY

Insight into the multifunctional RNA synthesis machine of COMMENTARY Ervin Fodora,1

Rabies virus (RABV) is the causative agent of a fatal Until recently, negative-strand RNA virus polymer- neurological disease in and animals. It is ases were refractory to structural analyses, hampering transmitted to humans from infected animals, mainly the understanding of how these transcribe and domestic dogs, through their saliva by biting or by replicate their RNA and the development of scratching. Rabies is controlled by vaccination of antiviral drugs targeting this group of viral enzymes. domestic dogs and cats, but RABV nevertheless kills This lack of progress was primarily caused by the more than 50,000 people annually, especially in de- difficulties of expressing sufficient quantities of these veloping countries where vaccination rates in domes- large ∼250-kDa single-polypeptide or, in some cases tic dogs are lower. In humans, rabies is preventable by multisubunit, polymerases in a recombinant form and vaccination prior to or immediately after exposure, but difficulties with their crystallization. However, improve- there are no specific antiviral drugs available that ments in expression technologies and the recent rev- target the virus directly (1). In PNAS, Horwitz et al. (2) olution in electron cryomicroscopy has dramatically present a high-resolution electron cryomicroscopy changed this. High-resolution structures have been structure of the RNA synthesis machine of RABV, pro- described for the polymerases of A, B, C, viding valuable mechanistic insight into its activities and D viruses of the family (5–9), and opening up the way toward developing antiviral La Crosse virus (LACV) of the Peribunyaviridea family approaches for this fatal virus. (10), RSV and (HMPV) of the RABV belongs to the group of negative-strand family (11, 12), vesicular stomatitis vi- RNA viruses that includes many human pathogens rus (VSV) of the family (13, 14), and such as the influenza viruses, respiratory syncytial virus now, as reported by Horwitz et al. (2), for RABV, also (RSV), virus, and measles virus. The of of the Rhabdoviridae family. These structures provide these viruses consist of one or more single-stranded, an unprecedented insight into the molecular archi- negative-sense RNA molecules that are always assem- tecture of these RNA synthesis machines as well as bled with multiple copies of viral (N) their activities. into megadalton-sized complexes (3). In order to initiate The genome of RABV is a single-stranded RNA , viruses such as RABV must first transcribe their molecule of ∼11.9 kb, coated along its length with negative-sense RNA into mRNA and therefore must multiple copies of the viral N. It is composed of five carry an RNA-dependent RNA polymerase within their genes encoding N, phospho- (P), matrix (M), glyco- (G), infectious particle. These viral polymerases are multi- and large (L) proteins that are flanked by the noncoding functional machines that not only transcribe the 3′-leader and 5′-trailer regions. When RABV infects a negative-strand RNA into mRNA but also replicate cell, the viral genome is released from the virus particle the genome through a complementary replicative in- into the cell cytoplasm where RABV forms liquid-like termediate, the antigenome. In addition to catalyzing inclusion bodies. It is in these inclusion bodies that RNA synthesis, these polymerases also ensure that the viral polymerase transcribes the viral genome to viral transcripts are protected with a 5′-cap structure. produce mRNAs required for viral protein synthesis, This is achieved either by a cap-snatching mechanism and replicates it to produce new viral genomes to that involves stealing caps from host capped be packaged into progeny virions (1). To transcribe through cap-binding and endonuclease functions, or the genome, the polymerase initiates at a promoter at de novo synthesis of a 5′-cap structure using capping the 3′ end of the genome, sequentially transcribing the and methyltransferase enzymes, which are encoded leader region and five internal genes (N, P, M, G, and L) in the same polypeptide as the polymerase domains into the leader RNA and five monocistronic mRNAs (Fig.1)(4). with a 5′-cap-1 structure and a 3′-poly(A) tail by using

aSir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom Author contributions: E.F. wrote the paper. The author declares no competing interest. Published under the PNAS license. See companion article on page 2099 in issue 4 of volume 117. 1Email: [email protected]. First published January 28, 2020.

www.pnas.org/cgi/doi/10.1073/pnas.2000120117 PNAS | February 25, 2020 | vol. 117 | no. 8 | 3895–3897 Downloaded by guest on September 29, 2021 RABV RdRp CAP CD MT CTD

LACV Endonuclease RdRp Cap-binding

IAV PA PB1 PB2 Fig. 1. Domain structures of cap-synthesizing and cap-snatching polymerases of negative-strand RNA viruses. Nonsegmented negative- strand RNA viruses such as (RABV) encode single-polypeptide cap-synthesizing polymerases, which are composed of the RNA-dependent RNA polymerase (RdRp) domain, capping (CAP) domain, connector domain (CD), methyltransferase (MT) domain, and C-terminal domain (CTD). Segmented negative-strand RNA viruses such as La Crosse virus (LACV) and (IAV) encode single-polypeptide (LACV) or multisubunit (IAV) cap-snatching polymerases with RdRp, cap-binding, and endonuclease activities. The IAV polymerase is composed of polymerase acidic (PA) (endonuclease), polymerase basic 1 (PB1) (RdRp), and polymerase basic 2 (PB2) (cap-binding) subunits.

a stop–start mechanism. This results in the generation residue, capable of forming base-stacking interactions with the of a gradient of mRNA abundance with N mRNA being the most initiating nucleoside triphosphates, emerges from the CAP do- and L mRNA being the least abundant (N > P > M > G > L). main, and protrudes into the active site of the RdRp domain, Initiation and termination are regulated by cis-acting start–stop sig- suggesting that the captured structure represents the polymer- nals that flank each gene. To replicate the genome, the polymerase ase in a preinitiation state. This is similar to the active-site ar- initiates at the 3′ promoter and produces a full-length copy of the rangement of the VSV L-P structure, but contrasts with the RSV genome, ignoring the internal start–stop signals. The resulting anti- and HMPV L-P structures in which this loop is retracted from the genome is neither capped nor polyadenylated, and is coreplica- RdRp active site and is more integrated with the CAP domain. tively assembled into a ribonucleprotein complex by associating The retraction of the priming loop is consistent with elongation with N. The replicative intermediate antigenome acts as a tem- during RNA synthesis, suggesting that the different structures plate for further rounds of replication to generate genomic RNA represent the polymerase in different functional modes. Retrac- for progeny virions. Remarkably, all enzymatic activities required tion of the priming loop also creates a continuous cavity shared for viral transcription and genome replication of RABV are per- by the RdRp and CAP catalytic sites, which allows the nascent formed by a complex of just two viral proteins, the large L poly- transcript to enter the catalytic cavity of the CAP domain. Cap- merase and P phosphoprotein. ping occurs by an unconventional capping mechanism that in- In a breakthrough achievement, Horwitz et al. (2) present the volves the covalent attachment of the monophosphorylated structure of the RABV L in complex with the N-terminal 91 amino 5′ terminus of the nascent transcript to a histidine side chain in acids of P. This exciting advance builds on a number of recent the CAP active site, followed by the transfer of the 5′ monophos- discoveries. In 2015, the three-dimensional structure of the related phate of the nascent transcript onto a GDP acceptor (4). The prim- VSV L protein was described, followed by a more recent study of the ing loop also plays an important functional role during capping, L-P complex of VSV (13, 14). In 2017, recombinant RABV L was leading to the suggestion that the priming loop acts as a dual- expressed and purified in a form that was transcriptionally active functional priming–capping loop (16). in vitro (15). The addition of full-length recombinant P protein en- Comparison of the RABV L-P structure with the structures of hanced initiation and processivity of L and an N-terminal fragment VSV, RSV, and HMPV also suggests how transcription and replica- of P was found to be sufficient for its stimulatory activity. Com- tion might be regulated in this group of polymerases. Horwitz et al. parison with the closely related structure of the VSV L-P protein, (2) propose an elegant mechanism for switching between a replica- together with biochemical data and mapping studies of function- tion mode, in which full-length products acquire an N protein coat, ally important amino acid residues in L and P of RABV (13–16), and transcription mode, in which an mRNA acquires a 5′-cap-1 struc- allowed the authors to interpret their structure and propose a ture. Specifically, they suggest that the covalent attachment of the model for how transcription and replication are performed by 5′ end of the nascent transcript to the CAP active-site cavity during the RABV L-P complex. capping and the force generated as a result of nascent RNA filling The domain arrangement of the RABV L is very similar to that the cavity could result in the release of the CD-MT-CTD module of the previously reported VSV L protein. The 2,127-amino acid from the RdRp-CAP core module. This would expose the MT cata- polypeptide folds into three catalytic and two structural do- lytic site for the subsequent N7 and 2′-O methylation of the 5′ cap mains. The core of the L protein structure is formed by the RNA- after the 5′ end of the nascent transcript has been transferred onto a dependent RNA polymerase (RdRp) domain and the capping GDP and released from CAP. On the other hand, during replication, domain (CAP) that possesses polyribonucleotidyl transferase the RNA product likely exits without the displacement of CD-MT- (PRNTase) activity. The third catalytic domain, the dual-specificity CTD at a site proximal to the N-terminal end of P. The N-terminal methyltransferase (MT), is linked at its N terminus to the CAP of the end of P is known to associate with monomeric N, and hence its RdRp-CAP module through the connector domain (CD), and its location close to the exit for replication products could facilitate the C terminus to the C-terminal domain (CTD) (Fig. 1). In contrast to delivery of N protein to the growing RNA chain, ensuring its core- the L-P structures of RSV and HMPV, the CD, MT, and CTD are plicative encapsidation. To reveal the detailed movements of poly- fully resolved in RABV L-P, similarly to VSV L-P. In both RABV L-P merase domains, it will be necessary to obtain further snapshots of and VSV L-P structures, P makes clear interactions with the CTD, transcribing and replicating polymerases with bound template RNA CD, and the RdRp domains, locking CD, MT, and CTD in a fixed and nascent product RNA as has recently been reported for influ- arrangement with respect to the large RdRp-CAP module. A enza viruses (5, 17). central cavity in the RdRp domain represents the polymerization The RABV L-P polymerase structure reported by Horwitz et al. active site. A priming loop containing a priming amino acid (2) is a major breakthrough that will significantly advance drug

3896 | www.pnas.org/cgi/doi/10.1073/pnas.2000120117 Fodor Downloaded by guest on September 29, 2021 development efforts to help combat this important human path- structure represents a significant step forward in the field of ogen. The structure also provides fascinating insights into how a negative-strand RNA viruses. complex of just two viral proteins is able to perform a set of Acknowledgments complex catalytic reactions, resulting in different types of viral I thank Aartjan te Velthuis, Alex Walker, and Jeremy Keown for helpful RNAs requiring different modes of initiation as well as termina- comments on the draft. E.F.’s research is supported by the UK Medical Research tion. Many questions remain, but the determination of this Council (Programme Grant MR/R009945/1).

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