Analysis of Template Variations on RNA Synthesis by Respiratory Syncytial Virus Polymerase

Abstract

Respiratory syncytial virus (RSV) is a significant threat to infants and elderly individuals globally. Currently, there are no effective therapies or treatments for RSV infection because of an insufficient understanding of the RSV viral machinery. In this study, we investigated the effects of the template variations on RNA synthesis by the RSV polymerase through in vitro RNA synthesis assays. We confirmed the previously reported back-priming activity of the RSV polymerase, which is likely due to the secondary structure of the RNA template. We found that the expansion of the hairpin loop size of the RNA template abolishes the RSV polymerase back-priming activity. At the same time, it seemingly does not affect the de novo RNA synthesis activities of the RSV polymerase. Interestingly, our results show that the RSV polymerase also has a new primer-based terminal extension activity that adds nucleotides to the template and primer in a nonspecific manner. We also mapped the impact of the RNA 5' chemical group on its mobility in a urea-denaturing RNA gel shift assay. Overall, these results enhance our knowledge about the RNA synthesis processes of the RSV polymerase and may guide future therapeutic efforts to develop effective antiviral drugs for RSV treatment.

Keywords: RNA gel shift assay; RNA secondary structure; RNA-dependent RNA polymerase (RdRp); back-priming elongation; de novo RNA synthesis; primer-based elongation; respiratory syncytial virus (RSV); template variations.

Figure 1

Back-priming-based RNA elongation with different NTPs. (A) The RNA synthesis of the templates TrC14, TrC21, and TrC25 in the presence of different NTPs (NTPs, GTP only, GTP+ATP, GTP+UTP, or GTP+CTP) by RSV polymerase (5 μCi of [α-³²P] GTP was added in each reaction beside other NTPs). (B) The back-priming secondary structures of TrC25, TrC21, and TrC14 are predicted by mFold. The CTP in the wobble base pair A-C is underlined. (C) RNA synthesis starts at position 3 of the templates TrC25, TrC21, and TrC14 in the presence of GTP and ATP. Red G or GTP indicates radiolabeled GTP.

Figure 2

Impact of hairpin loop size on de novo RNA synthesis and back-priming-based 3′ terminal extension. (A) De novo RNA synthesis of TrC25-WT and mutants with different numbers of Us at the position 6-12 by the RSV polymerase with NTPs (ATP, CTP, and UTP each at 1.25 mM and GTP at 50 μM with 5 μCi of [α-³²P] GTP). (B) The 3′ terminal extension of wt and mutants of RNA templates TrC25 with GTP only. GTP at 50 μM with 5 μCi of [α-³²P] GTP was used in the reaction mixtures. (C) Sequences and secondary structures of the RSV wt and mutants of TrC25 RNA templates. Residues GDN are the catalytic residues in the RdRp domain that are responsible for RNA polymerization. The red cross indicates no activity towards TrC25+4U. Red G or GTP indicates radiolabeled GTP. The blue Us indicate additional UTP residues that were added to the wt TrC25 template, while the blue dashes indicate Us that were deleted from the wt TrC25 template.

Figure 3

The impact of RNA 5′ end modification on RNA mobility in Urea-denaturing polyacrylamide RNA gel. (A) Control: the RSV polymerase + NTPs and [α-³²P] GTP w/o T4 PNK treatment. (B) In vitro RNA synthesis of TrC21 by RSV polymerase in the presence of NTPs + [α-³²P] GTP with/without T4 PNK treatment. (C) In vitro RNA synthesis of the template TrC12 paired with primer Tr5 by RSV polymerase in the presence of NTPs + [α-³²P] ATP with/without T4 PNK treatment. (D) In vitro RNA synthesis of the template TrC12 paired with primer Tr4 by RSV polymerase in the presence of [α-³²P] GTP with/without T4 PNK treatment. Red GTP and ATP indicate radiolabeled GTP and ATP, respectively.

Figure 4

Terminal nucleotide(s) addition to the templates and primers by RSV polymerase. (A) Back-primer secondary-structure-based 3′ terminal extension of the templates from TrC14 to TrC21 using GTP only. GTP at 50 μM with 5 μCi of [α-32P] GTP was used in the reaction mixtures. (B) Primer-based terminal extension of the templates from TrC14 to TrC21 with primer Tr14 and GTP only. GTP at 50 μM with 5 μCi of [α-32P] GTP was used in the reaction mixtures. (C) Primer-based terminal extension of templates from TrC10 to TrC14 with primer Tr10 in the presence of ATP. ATP at 50 μM with 5 μCi of [α-32P] ATP was used in the reaction mixtures. (D) Primer-based terminal extension of templates from TrC12 to TrC21 with primer Tr12 in the presence of GTP. GTP at 50 μM with 5 μCi of [α-32P] GTP was used in the reaction mixtures. The 5′ hydroxyl RNA (RNA products extended from template or primer) migrates about 1 nt slower than 5′ monophosphate RNA (RNA ladders) when the RNA length is 12–24 nts (See Section 3.2 in Results), leading to the product from primer Tr12 at the length of 13 nts close to the ladder 14 nts. (E) Back-primer secondary-structure-based 3′ terminal extension of the templates from TrC15 to TrC21 using UTP only. UTP at 50 μM with 5 μCi of [α-32P] UTP was used in the reaction mixtures. Since U can not pair with C, no products were detected. (F) Primer-based terminal extension of the templates from TrC15 to TrC21 with primer Tr15 and UTP only. UTP at 50 μM with 5 μCi of [α-32P] UTP was used in the reaction mixtures. The UTP in the wobble base pair G-U is underlined. The superscript labels indicate that the band is generated from primer (^P) or template (^T). The red G or GTP, A or ATP, and U or UTP indicate radiolabeled GTP, ATP, and UTP, respectively.

Figure 5

Primer-based terminal extension activity of RSV polymerase is NTP nonspecific. (A) Primer-based RNA terminal extension of template TrC10 and TrC11 with primer Tr10 in the presence of [α-³²P] GTP or [α-³²P] ATP. (B) Primer-based RNA terminal extension of template TrC12 and TrC13 with primer Tr12 in the presence of [α-³²P] GTP or [α-³²P] ATP. (C) Primer-based RNA terminal extension of template TrC21 with primer Tr14 in the presence of [α-³²P] GTP or [α-³²P] ATP. The superscript labels indicate the band is generated from primer (^P) or template (^T). ATP at 50 μM with 5 μCi of [α-³²P] ATP and GTP at 50 μM with 5 μCi of [α-³²P] GTP were used in the reaction mixtures as indicated. The products from [α-³²P] GTP are slightly larger than that from [α-³²P] ATP. Red N represents [α-³²P] ATP or [α-³²P] GTP. The red GTP and ATP indicate radiolabeled GTP and ATP, respectively.

Figure 6

The models of back-priming-based and primer-based 3′ terminal extension to the template and primer. (A) The model of back-priming-based 3′ terminal extension to the template by RSV polymerase. It may incorporate a wobble base pair with low activity only when only a high concentration of nonspecific NTP was added to the reaction mixture. (B) The model of primer-based 3′ terminal extension to the primers. The incoming NTP will be specifically added to the sticky end when the template is longer than the primer. When the supplemented NTP is nonspecific, a wobble base pair may be introduced into the product by RSV polymerase with low activity. While the template is the same length as the primer, the RSV polymerase may add a nonspecific NTP to the blunt end of the double-strand RNA at the 3′ end of the primer. (C) The model of primer-based 3′ terminal extension to the templates. The double-strand RNA (template paired with the primer) may enter the active pocket to access the GDN catalyze motif in RdRp domain, allowing a nonspecific NTP addition to the 3′ blunt end of the template. GDN are the catalytic residues in the RdRp domain responsible for RNA polymerization. HR are the catalytic residues in the Cap domain responsible for mRNA capping.