RdRp or RT, That is the Question

Abstract

The RNA-dependent RNA polymerase (RdRp) of all known double-stranded RNA viruses is located within the viral particle and is responsible for the transcription and replication of the viral genome. Through an RT-PCR assay, we determined that purified virions, in vitro translated RdRp proteins, and purified recombinant RdRp proteins of partitiviruses also have reverse transcriptase (RT) function. We show that partitivirus RdRps 1) synthesized DNA from homologous and heterologous dsRNA templates; 2) are active using both ssRNA and dsRNA templates; and 3) are active at lower temperatures compared to an optimal reaction temperature of commercial RT enzymes. This finding poses an intriguing question: why do partitiviruses, with dsRNA genomes, have a polymerase with RT functions? In comparison, 3Dpol, the RdRp of poliovirus, did not show any RT activity. Our findings lead us to propose a new evolutionary model for RNA viruses where the RdRp of dsRNA viruses could be the ancestor of RdRps.

Keywords: RNA-dependent RNA polymerase; partitivirus; polymerase origins; reverse transcriptase; virus evolution.

Fig. 1.

Reverse transcription activity of PCV1 virions using homologous and heterologous dsRNA templates. (A) A schematic representation of the assay using PCR to detect cDNA. (B) cDNA products of PCV1 dsRNA detected by PCR using RdRp primers; M: DNA marker, 1, MMuLV-RT used as enzyme, 2, PCV1 virions used as enzyme in RT reaction, 3, negative control (no enzyme), 4, negative control (no template), 5, negative control (isolated fraction from uninfected Jalapeño pepper used as enzyme in RT reaction). (C) RT-PCR analysis using ZMCV1 dsRNA as heterologous RNA template with ZMCV1-RdRp primers; M, DNA marker, 1, PCV1 virions used as enzyme, 2, negative control (no enzyme), 3, negative control (no template), 4, MMuLV used as enzyme in RT reaction. (D) RT-PCR analysis using PCV1 virions as the source of RNA template and PCV1-RdRp primers; M, DNA marker, 1, PCV1 virions were boiled and the RT reaction was without RT enzyme, 2, virions were boiled and then PCV1 virions were added to RT reaction as RT enzyme.

Fig. 2.

In vitro translated PCV 1 RdRp is active as an RT. (A) a schematic of in vitro translation of PCV1 RdRp and determination the RT activity of protein through RT-PCR. (B) RT-PCR analysis using PCV1 dsRNA as RNA template and PCV1-RdRp primers; M, DNA marker, 1, in vitro translated PCV1 RdRp as enzyme, 2, negative control (no enzyme), 3, negative control (no primer), 4, MMuLV used as enzyme. (C) RT-PCR analysis using PCV1 dsRNA as RNA template and PCV1-RdRp primers, RT reactions at 42°C; M, DNA marker, 1, negative control (no enzyme), 2, in vitro translated PCV1 RdRp used as RT enzyme, 3, MMuLV used as enzyme.

Fig. 3.

RT activity of various recombinant partitivirus RdRp enzymes. RT reactions were carried out using PCV2 transcript (ssRNA) as template. (A) AhV-RdRp as RT enzyme; M, DNA marker, 1, ProtoScript II used as enzyme, 2, negative control (no enzyme), 3, negative control (no template), 4, AhV-RdRp recombinant protein used as enzyme. (B) ACDMV-RdRp as RT enzyme; M, DNA marker, 1, ProtoScript II used as enzyme, 2, negative control (no enzyme), 3, negative control (no template), 4, ACDMV-RdRp recombinant protein used as enzyme. (C) HubeiPV53-RdRp as RT enzyme; M, DNA marker, 1, HubeiPV53-RdRp recombinant protein used as enzyme, 2, negative control (no enzyme), 3, negative control (no template), 4, ProtoScript II used as enzyme. (D) PdPV-RdRp and AfPV2 RdRp as RT enzymes; M, DNA marker, 1, negative control (no enzyme), 2, negative control (no template), 3, ProtoScript II used as enzyme, 4, PdPV-RdRp recombinant protein used as enzyme, 5, AfPV2-RdRp recombinant protein used as enzyme. (E) ClCV-RdRp as RT enzyme; M, DNA marker, 1, negative control (no enzyme), 2, ProtoScript II used as enzyme, 3, negative control (no template), 4, ClCV-RdRp recombinant protein used as enzyme. (F) PCV1-RdRp as RT enzyme; 1, PCV1-RdRp recombinant protein used as enzyme, M, DNA marker, 2, ProtoScript II used as enzyme, 3, negative control (no enzyme), 4, negative control (no template). (G) FpPV2-RdRp as RT enzyme; M, DNA marker, 1, ProtoScript II used as enzyme, 2, negative control (no enzyme), 3, negative control (no template), 4, FpPV2-RdRp recombinant protein used as enzyme. (H) PCV2-RdRp as RT enzyme; 1, negative control (no enzyme), 2, negative control (no template), M, DNA marker, 3, ProtoScript II used as enzyme, 4, PCV2-RdRp recombinant protein used as enzyme. (I) RT reaction was carried out using PCV1 dsRNA as template and AhV-RdRp as RT enzyme; M, DNA marker, 1, AhV-RdRp recombinant protein used as RT enzyme. All virus siglas are defined and GenBank accession numbers are given in supplementary table 2, Supplementary Material online.

Fig. 4.

Evaluation of the RT activity of recombinant ACDMV and HubeiPV53 RdRp proteins by RT-qPCR. (A,B) RT-qPCR analysis using Human T-cell leukemia total RNA as template and ACTB primer; (A) M, DNA marker, 1–3, ProtoScript II used as enzyme in RT reaction, 4–6, negative control (no enzyme), 7–9, negative control (no template), 10–12, ACDMV-RdRp recombinant protein used as enzyme in RT reaction. (B) M, DNA marker, 1–3, HubeiPV53-RdRp recombinant protein used as enzyme in RT reaction, 4–6, cDNA synthesized by LunaScript Super mix kit, 7–9, negative control (no primer). (C) Amplification profiles of several commercial RT enzymes (red), compared to ACDMV RdRp (green) and HubeiPV53 RdRp (brown) as RT enzyme and negative controls (blue). The shift in Cq when the amplifications included ACDMV RdRp and HubeiPV53 RdRp as RT enzyme indicate a low abundance of amplifiable cDNA.

Fig. 5.

Nucleotide selection and primer extension by 3D^pol. (A) Schematic representation of FAM labeled RNA and DNA primer extension by 3D^pol. RNA template is shown in orange, RNA primer is shown in pink and DNA primer in blue. FAM labels are shown as light purple stars. (B) CE results of the extension of FAM-RNA primer; reactions contained 3D^pol, RNA template, FAM labeled RNA primer, and NTPs or dNTPs. (C) CE results of the extension of FAM-DNA primer; reactions contained 3D^pol, RNA template, FAM labeled DNA primer, and NTPs or dNTPs. (D) RT-PCR analysis using PCV2 transcript (ssRNA) as template and recombinant protein of FpPV2-RdRp and 3D^pol as RT enzymes; 1, ProtoScript II used as enzyme in RT reaction, 2, negative control (no enzyme), 3, negative control (no template), 4, FpPV2-RdRp (1 μM) recombinant protein used as enzyme in RT reaction, 5, FpPV2-RdRp (2 μM) recombinant protein used as enzyme in RT reaction, 6, 3D^pol (1 μM) recombinant protein used as enzyme in RT reaction, 7, 3D^pol (2 μM) recombinant protein used as enzyme in RT reaction, 7, 3D^pol (3 μM) recombinant protein used as enzyme in RT reaction, M, DNA marker.