How does the polymerase of non-segmented negative strand RNA viruses commit to transcription or genome replication?

Abstract

The Mononegavirales, or non-segmented negative-sense RNA viruses (nsNSVs), includes significant human pathogens, such as respiratory syncytial virus, parainfluenza virus, measles virus, Ebola virus, and rabies virus. Although these viruses differ widely in their pathogenic properties, they are united by each having a genome consisting of a single strand of negative-sense RNA. Consistent with their shared genome structure, the nsNSVs have evolved similar ways to transcribe their genome into mRNAs and replicate it to produce new genomes. Importantly, both mRNA transcription and genome replication are performed by a single virus-encoded polymerase. A fundamental and intriguing question is: how does the nsNSV polymerase commit to being either an mRNA transcriptase or a replicase? The polymerase must become committed to one process or the other either before it interacts with the genome template or in its initial interactions with the promoter sequence at the 3´ end of the genomic RNA. This review examines the biochemical, molecular biology, and structural biology data regarding the first steps of transcription and RNA replication that have been gathered over several decades for different families of nsNSVs. These findings are discussed in relation to possible models that could explain how an nsNSV polymerase initiates and commits to either transcription or genome replication.

Keywords: RNA replication; RNA-dependent RNA polymerase; filovirus; paramyxovirus; pneumovirus; rhabdovirus; transcription.

Fig 1

Genome organization and arrangement of nsNSVs. Schematic diagrams of the genomes of the rhabdovirus, VSV, the pneumovirus, RSV, the paramyxovirus, SeV, and the filovirus, EBOV. The genomes are drawn based on Genbank numbers EF197793.1, U39661.1, DQ219803.1, and AF499101.1, respectively. They are drawn to scale, except for the cis-acting elements, and the gene overlaps. The white boxes represent a gs signal, blue boxes represent the labeled genes, and black boxes represent a ge signal. The 3´ le and 5´ tr are on the left and right of each genome, respectively. A representative intergenic region and/or gene overlap is shown for each virus. In the case of the RSV genome, the M2 and L genes overlap by 68 nt. Note that throughout the review, viral gene names and sequences containing cis-acting elements are italicized.

Fig 2

Transcription and replication by nsNSV polymerases. Schematic diagrams of transcription and replication by the nsNSV polymerases. The polymerase (L) is shown as an orange oval at different positions within a gene; the white boxes represent a gs signal, blue boxes represent the genes, and black boxes represent a ge signal. A U₄ tract within the ge signal is labeled. The positive-sense RNA product is shown as a black line coming out of the polymerase; mRNA with a 5´ cap and 3´ poly(A) tail for transcription, and antigenome that is encapsidated with N protein (blue circles) for replication.

Fig 3

The nsNSV polymerases (L) and phosphoproteins (P/VP35). (A) Schematic diagram of an nsNSV polymerase, based on the VSV polymerase structure. The domains are colored as follows: RdRp in cyan, PRNTase/capping domain in green, CD in yellow, MT in orange, and CTD in red. (B) Schematic diagram of the NDV phosphoprotein tetramer, showing the flexible N-terminal domain (P_NTD) and C-terminal domain (P_CTD) arms, and the ordered oligomerization domain (P_OD). (C) Representative structures of nsNSV polymerases from each family available: rhabdovirus VSV L-P (PDB: 6U1X), paramyxovirus PIV-5 L-P (PDB: 6V85), pneumovirus RSV L-P (PDB: 6PZK), and filovirus EBOV L-VP35 (PDB: 7YES). The L domain colors are the same as in A and the P tetramer is the same color as in B. Note that most of the flexible N- and C-terminal arms of PIV-5 and RSV P and EBOV VP35 are not visible in the cryo-EM structure, and only three small segments of the VSV P are visible. Comparison of the VSV and PIV-5 polymerase structures shows how the CD, MT, and CTD can be rearranged relative to the RdRp-PRNTase domain core.

Fig 4

Four nsNSV transcription initiation models. Schematic diagrams of four different transcription-replication initiation models. The negative-sense genome is shown with the 3´ leader (le), the gene start (gs) signal in white, and part of the first gene in blue. A putative nucleation encapsidation signal (not shown) is present at the 5´ end of the le(+) RNA and antigenome. Only when nucleoprotein (N, blue circles) is present at a sufficiently high concentration will the RNA become encapsidated. (A) Single promoter - single polymerase model: in this model, the polymerase (pol) begins transcription and replication at the 3´ end of the le region and becomes either a transcriptase or replicase depending on the levels of N⁰. If N⁰ is limiting, the polymerase produces a short le(+) transcript and then reinitiates RNA synthesis at the first gs signal. If N⁰ has reached a critical threshold, the RNA becomes encapsidated, and the polymerase can perform RNA replication. (B) Single promoter - replicase/transcriptase model: in this model, there are two pools of polymerase, replicase (rep) and transcriptase (trans), which have different functional capabilities but compete for binding to a single promoter and initiation site. (C) Dual promoter – single polymerase model: in this model, there is a single pool of polymerase that can either begin replication by initiating at the 3´ end of the le region or transcription by initiating directly at the first gs signal. It should be noted that according to this model, short le(+) transcripts might be synthesized as abortive replication products (at low N protein concentrations). (D) Dual promoter - replicase/transcriptase model: in this model, there are distinct pools of replicase and transcriptase (as described for B), which initiate directly at the 3´ end of the le region or the gs signal, respectively. In B and D, replicase and transcriptase and their respective RNA products are shown in purple or pink, respectively.

Fig 5

Structures of RSV and EBOV L proteins in complex with their cognate promoter RNAs. Structures of the (A) RSV L + nucleotides 1–10 of the le promoter (PDB: 8SNX), (B) RSV L + nucleotides 1–10 of the tr promoter (PDB: 8SNY), and (C) EBOV L + nucleotides 1–10 of the le promoter (PDB: 8JSM) complexes. For a better visual of the RNA, only the RdRp domains of L are shown in cyan. The GDN motif within the RdRp is shown as red spheres. Promoter RNA is colored in orange, except for the initiation sites colored in black (position 3C for RSV le 1–10, 1U for RSV tr 1–10, and 2C for EBOV le 1–10; note that the EBOV le RNA used for this analysis has the sequence 3´ GCCUGUGUGU). The distance between the initiation site of the RNA and the aspartic acid in the GDN motif is shown as a yellow dotted line.

Fig 6

The priming loops and channels of L. Structures of two domains are shown from the (A) VSV L (PDB: 6U1X) and (B and C) RSV L (PDB: 6PZK) proteins. The RdRp domains are colored in cyan, the PRNTase domains are colored in green, and in (C), the RSV phosphoprotein (P) is colored in purple. The GDN motif within the RdRp is shown as red spheres. The priming loop is colored in magenta, and the putative priming residues are shown as pink spheres (Trp1167 for VSV; Pro1261 and Trp1262 for RSV). (A) The Trp1167 residue on the VSV priming loop is located near the active site, suggesting an initiation conformation. (B) The RSV priming residues and priming loop are tucked away into the PRNTase domain, suggesting a non-initiation conformation. (C) A surface representation of the RSV L-P complex with a sliced cross-section (gray regions) is shown to indicate the putative channels for RNA templates, transcripts, and NTPs. The GDN motif in the RdRp active site and priming loop are overlaid onto the cross-section to show the rotation with respect to panel B. The nascent RNA transcript will exit toward the methyltransferase domain.