Functional analysis of promoter element 2 within the viral polymerase gene of an emerging paramyxovirus, Sosuga virus

Abstract

Paramyxovirus genomes carry bipartite promoters at the 3' ends of both their genome and antigenome, thereby initiating RNA synthesis, which requires the viral polymerase to recognize two elements: the primary promoter element 1 (PE1) and the secondary promoter element 2 (PE2). We have previously shown that the antigenomic PE2 (agPE2) in many viruses in the Rubulavirinae subfamily is located within the coding region of the viral RNA polymerase L gene. Sosuga virus (SOSV), belonging to the Rubulavirinae subfamily, is highly pathogenic to humans, thus necessitating high-level containment facilities for infectious virus research. The use of a minigenome system permits studies of viral RNA synthesis at lower biosafety levels. Because minigenomes of negative-strand RNA viruses generally comprise only the untranslated regions, agPE2 within the L coding region-such as those found in Rubulavirinae like SOSV-is typically omitted. However, generating an SOSV minigenome that retains agPE2 led to a pronounced increase in activity, enabling a detailed examination of the role of agPE2 in SOSV replication. In many Rubulavirinae, the agPE2 not only acts as a promoter but also encodes part of the L protein, resulting in a distinct motif at the C-terminus of the L protein. We have further shown that this motif is preserved even in Rubulavirinae that no longer contain the agPE2 within the L gene.IMPORTANCEParamyxoviruses are classified into three major subfamilies: Orthoparamyxovirinae, Avulavirinae, and Rubulavirinae. All paramyxovirus genomes and antigenomes possess bipartite promoters, comprising two elements: promoter element 1 (PE1) at the 3' end and promoter element 2 (PE2) located internally. We previously revealed that, in many Rubulavirinae, the antigenomic PE2 lies within the coding region of the viral RNA polymerase L gene. In this study, we used Sosuga virus, a member of the Rubulavirinae subfamily, to elucidate the role of antigenomic PE2 in viral replication. Because the PE2 region encodes part of the L protein, its presence leads to a distinctive motif at the C-terminus of L protein. Notably, this motif is conserved in all Rubulavirinae, including those that do not harbor the antigenomic PE2 within their L gene, indicating its importance in viral propagation.

Keywords: RNA polymerases; negative-strand RNA virus; paramyxovirus; promoters; viral replication.

Fig 1

Establishment of Sosuga virus (SOSV) minigenome assay. (A) Helical structure of SOSV nucleocapsid. Promoter element 1 (PE1) and PE2 are shown. (B) Viruses in the Rubulavirinae subfamily are categorized into PE2 In-ORF and Out-ORF types. (C) Schematic diagram of SOSV minigenome construction. All viral genes in the genome are exchanged to be Nluc. Because the SOSV genome contains the PE2 within the L gene, two minigenomes with or without PE2 are created. The relative Nluc expression using the SOSV minigenome assay is shown. Data represent means and standard deviations from three different experiments. NP− indicates the values using an empty plasmid instead of SOSV NP.

Fig 2

Importance of CG repeats in PE2 for SOSV genome replication. Mutations in the PE2 sequences are shown in the left panel. The relative Nluc expressions using the SOSV minigenome assay system are shown in the right panel. The relative value when the value using an original minigenome is 1. Data represent means and standard deviations from three different experiments. NP− indicates the value using an empty plasmid instead of SOSV NP.

Fig 3

The replication of SOSV minigenome constructed by nucleotides not a multiple of six. (A) Effect of nucleotide addition in the 5′ or 3′ position of agPE2 for minigenome expression. One to five nucleotide additions (+1 to +5) at the indicated position and their effect for the shift between PE1 and PE2 are shown. (B) The relative value when the value using an original minigenome is 1. Data represent means and standard deviations from three different experiments.

Fig 4

Comparison of the agPE2 sequence of viruses in the subfamily Rubulavirinae. The agPE2 sequence of viruses in the genera Pararubulavirus and Orthorubulavirus are shown. Numbers in the circles indicate the numbers of the NP hexamer from the 3′ terminus. hPIV, human parainfluenza virus; Porcine, Porcine rubulavirus.

Fig 5

Amino acid sequences of PE2 encoding region in the RNA-dependent RNA polymerase gene. (A) Comparison of nucleotide and amino acid sequences of PE2 of viruses in the genera Pararubulavirus and Orthorubulavirus. (B) Homology of amino acid sequences of the L protein analyzed using WebLogo sequence logo generator. (C) Predicted model of P protein-bound SOSV L protein. RdRp, RNA-dependent RNA polymerase (colored in light blue); PRNTase, polyribonucleotidyltransferase (colored in green); CD, connecting domain (colored in orange); MTase, methyltransferase (colored in yellow); CTD, C-terminal domain (colored in blue). In an enlarged view of the C-terminal loop, carbon atoms of which are colored in pink, residues from Asp2266 to Asp2270 are shown in stick diagrams. P protein tetramer bound to L protein are colored purple. (D) The SOSV minigenome-2 system was used to study the effect of amino acid substitution in the D₂₂₆₅LAGD₂₂₆₉ of SOSV L. Minigenome-2 and helper plasmids containing SOSV NP, P, and L (normal or mutant) were transfected to BHK/T7-9 cells. The relative Nluc expression when L normal is set to 1 is shown. Data represent means and standard deviations from three different experiments. L− indicates the values using an empty plasmid instead of SOSV L.

Fig 6

Predicted evolutionary history of Rubulavirus. (A) Phylogenetic tree created from an alignment of the viral L protein of Rubulavirinae. (B) The length of genome nucleotides and L protein amino acids. The GenBank accession numbers used in this study are Mapuera (NC_009489.1), Porcine (NC_009640.1), mumps (NC_002200.1), PIV5 (KY685075.1), hPIV2 (AB176531.1), Alston (NC_055508.1), Menangle (AF326114.2), Sosuga (KF774436.1), Achimota (JX051319.1), Teviot (MH708896.1), Tioman (AF298895.2), and Tuhoko (GU128080.1). (C) Predicted evolutionary history of PE2 Out-ORF Rubulavirinae.