Insight Into the Multifunctional RNA Synthesis Machine of Rabies Virus COMMENTARY Ervin Fodora,1
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COMMENTARY Insight into the multifunctional RNA synthesis machine of rabies virus COMMENTARY Ervin Fodora,1 Rabies virus (RABV) is the causative agent of a fatal Until recently, negative-strand RNA virus polymer- neurological disease in humans and animals. It is ases were refractory to structural analyses, hampering transmitted to humans from infected animals, mainly the understanding of how these viruses transcribe and domestic dogs, through their saliva by biting or by replicate their RNA genome 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 influenza A, B, C, viding valuable mechanistic insight into its activities and D viruses of the Orthomyxoviridae 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 human metapneumovirus (HMPV) of the RABV belongs to the group of negative-strand Pneumoviridae family (11, 12), vesicular stomatitis vi- RNA viruses that includes many human pathogens rus (VSV) of the Rhabdoviridae family (13, 14), and such as the influenza viruses, respiratory syncytial virus now, as reported by Horwitz et al. (2), for RABV, also (RSV), Ebola virus, and measles virus. The genomes 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 nucleoprotein (N) their activities. into megadalton-sized complexes (3). In order to initiate The genome of RABV is a single-stranded RNA infection, 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 RNAs 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 rabies virus (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 influenza A virus (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 transcription 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.