Protein-primed and de novo initiation of RNA synthesis by norovirus 3Dpol

Abstract

Noroviruses (Caliciviridae) are RNA viruses with a single-stranded, positive-oriented polyadenylated genome. To date, little is known about the replication strategy of norovirus, a so-far noncultivable virus. We have examined the initiation of replication of the norovirus genome in vitro, using the active norovirus RNA-dependent RNA polymerase (3D(pol)), homopolymeric templates, and synthetic subgenomic or antisubgenomic RNA. Initiation of RNA synthesis on homopolymeric templates as well as replication of subgenomic polyadenylated RNA was strictly primer dependent. In this context and as observed for other enteric RNA viruses, i.e., poliovirus, a protein-primed initiation of RNA synthesis after elongation of the VPg by norovirus 3D(pol) was postulated. To address this question, norovirus VPg was expressed in Escherichia coli and purified. Incubation of VPg with norovirus 3D(pol) generated VPg-poly(U), which primed the replication of subgenomic polyadenylated RNA. In contrast, replication of antisubgenomic RNA was not primer dependent, nor did it depend on a leader sequence, as evidenced by deletion analysis of the 3' termini of subgenomic and antisubgenomic RNA. On nonpolyadenylated RNA, i.e., antisubgenomic RNA, norovirus 3D(pol) initiated RNA synthesis de novo and terminated RNA synthesis by a poly(C) stretch. Interestingly, on poly(C) RNA templates, norovirus 3D(pol) initiated RNA synthesis de novo in the presence of high concentrations of GTP. We propose a novel model for initiation of replication of the norovirus genome by 3D(pol), with a VPg-protein-primed initiation of replication of polyadenylated genomic RNA and a de novo initiation of replication of antigenomic RNA.

FIG. 1.

Expression and purification of norovirus 3D^pol and VPg in E. coli. (A) SDS-PAGE analysis of the expression products. Lane 1, wild-type norovirus 3D^pol; lane 2, norovirus VPg. M, molecular mass marker (kDa). (B) Western blot analysis of the expression products. Lane 1, wild-type norovirus 3D^pol; lane 2, norovirus VPg.

FIG. 2.

Primer-dependent initiation of RNA synthesis on homopolymeric templates. RNA synthesis was performed in the presence (black bars) or in the absence (gray bars) of a cRNA oligonucleotide primer. Incorporation of [α-³²P]CMP, [α-³²P]AMP, [α-³²P]GMP, or [α-³²P]UMP was measured after trichloroacetic acid precipitation and collection on G/C glass fiber filters. Incorporation values are indicated.

FIG. 3.

De novo initiation of RNA synthesis on homopolymeric templates. (A) Reaction products were analyzed on formaldehyde-agarose gel and visualized by autoradiography. RNA synthesis was performed in the presence or absence of 50 μM cold CTP, ATP, GTP, or UTP for poly(G) RNA, poly(U) RNA, poly(C) RNA, or poly(A) RNA templates, respectively. (B) RNA synthesis was performed in the presence (black bars) or in the absence (gray bars) of 50 μM cold CTP, ATP, GTP, or UTP for poly(G) RNA, poly(U) RNA, poly(C) RNA, or poly(A) RNA templates, respectively. Incorporation of [α-³²P]CMP, [α-³²P]AMP, [α-³²P]GMP, or [α-³²P]UMP was measured after trichloroacetic acid precipitation and collection on G/C glass fiber filters. Incorporation values are indicated.

FIG. 4.

Primer-dependent replication of full-length subgenomic polyadenylated RNA by norovirus 3D^pol. In all reactions, synthetic subgenomic polyadenylated RNA was used as a template. Reaction products were analyzed on formaldehyde-agarose gels and visualized by autoradiography. (A) The reaction was performed in the presence of an oligo(U)₂₀ RNA primer as indicated (mM). M, marker (in vitro transcribed subgenomic polyadenylated RNA). (B) The reaction was performed in the presence of an RNA oligonucleotide complementary to the sequence contiguous to the poly(A) tail. RNA oligonucleotide primer concentrations (mM) are indicated.

FIG. 5.

Uridylylation and elongation of VPg by norovirus 3D^pol. Uridylylation and elongation reactions were performed in the presence of poly(A) RNA. Products were analyzed on 12% SDS-polyacrylamide gel or formaldehyde-agarose gel and visualized by autoradiography. (A) Uridylylation of VPg by norovirus 3D^pol and analysis of the uridylylation reaction by SDS-PAGE. Uridylylation of VPg (1 μg) was performed in the presence of UTP (10 μM) or in the presence or absence of poly(A) RNA (1 μg), 3D^pol, mutated 3D^pol (m3D^pol), or a bacterial cell lysate, as indicated. (B) Analysis of the specificity of uridylylation of VPg by norovirus 3D^pol and analysis of the reaction by SDS-PAGE. Uridylylation of VPg (1 μg) was performed in the presence of UTP (10 μM), in the presence or absence of poly(A) RNA (1 μg) or 3D^pol, and in the presence of [γ-³²P]UTP, as indicated. As a control, the uridylylation reaction was performed in the presence of [α-³²P]UTP, as described above. (C) Elongation of VPg by norovirus 3D^pol and analysis of the elongation reaction by SDS-PAGE. Elongation of VPg (1 μg) was performed in the presence of UTP (100 μM) or in the presence or absence of poly(A) RNA (1 μg), 3D^pol, m3D^pol, or a bacterial cell lysate, as indicated. (D) Analysis of the specificity of the elongation of VPg by norovirus 3D^pol and analysis of the reaction by SDS-PAGE. Elongation of VPg (1 μg) was performed in the presence of UTP (100 μM), poly(A) RNA (1 μg), and 3D^pol, as indicated. The elongated VPg-poly(U) was then treated with a mixture of RNase A and RNase V1 for 2 h at 37°C, as indicated. The reaction products were visualized on SDS-PAGE gels. (E) Protein-dependent initiation of replication by VPg. The subgenomic polyadenylated RNA was incubated in the presence of an oligo(U)₂₀ RNA primer and wild-type 3D^pol and 0.4 mM of ATP, GTP, CTP, and UTP, yielding a replicated antisubgenomic RNA. In contrast, incubation of the subgenomic polyadenylated RNA [SG-poly(A)] with oligo(U)₂₀ RNA in the presence of m3D^pol or with 3D^pol but without an oligo(U)₂₀ RNA primer did not yield a replication product. Incubation of the subgenomic RNA with VPg (1 μg) and 3D^pol as well as UTP (100 μM), but without ATP, GTP, or CTP, allowed initiation of RNA synthesis.

FIG. 5.

Uridylylation and elongation of VPg by norovirus 3D^pol. Uridylylation and elongation reactions were performed in the presence of poly(A) RNA. Products were analyzed on 12% SDS-polyacrylamide gel or formaldehyde-agarose gel and visualized by autoradiography. (A) Uridylylation of VPg by norovirus 3D^pol and analysis of the uridylylation reaction by SDS-PAGE. Uridylylation of VPg (1 μg) was performed in the presence of UTP (10 μM) or in the presence or absence of poly(A) RNA (1 μg), 3D^pol, mutated 3D^pol (m3D^pol), or a bacterial cell lysate, as indicated. (B) Analysis of the specificity of uridylylation of VPg by norovirus 3D^pol and analysis of the reaction by SDS-PAGE. Uridylylation of VPg (1 μg) was performed in the presence of UTP (10 μM), in the presence or absence of poly(A) RNA (1 μg) or 3D^pol, and in the presence of [γ-³²P]UTP, as indicated. As a control, the uridylylation reaction was performed in the presence of [α-³²P]UTP, as described above. (C) Elongation of VPg by norovirus 3D^pol and analysis of the elongation reaction by SDS-PAGE. Elongation of VPg (1 μg) was performed in the presence of UTP (100 μM) or in the presence or absence of poly(A) RNA (1 μg), 3D^pol, m3D^pol, or a bacterial cell lysate, as indicated. (D) Analysis of the specificity of the elongation of VPg by norovirus 3D^pol and analysis of the reaction by SDS-PAGE. Elongation of VPg (1 μg) was performed in the presence of UTP (100 μM), poly(A) RNA (1 μg), and 3D^pol, as indicated. The elongated VPg-poly(U) was then treated with a mixture of RNase A and RNase V1 for 2 h at 37°C, as indicated. The reaction products were visualized on SDS-PAGE gels. (E) Protein-dependent initiation of replication by VPg. The subgenomic polyadenylated RNA was incubated in the presence of an oligo(U)₂₀ RNA primer and wild-type 3D^pol and 0.4 mM of ATP, GTP, CTP, and UTP, yielding a replicated antisubgenomic RNA. In contrast, incubation of the subgenomic polyadenylated RNA [SG-poly(A)] with oligo(U)₂₀ RNA in the presence of m3D^pol or with 3D^pol but without an oligo(U)₂₀ RNA primer did not yield a replication product. Incubation of the subgenomic RNA with VPg (1 μg) and 3D^pol as well as UTP (100 μM), but without ATP, GTP, or CTP, allowed initiation of RNA synthesis.

FIG. 6.

Primer extension analysis at the 5′ terminus of the replication product of norovirus 3D^pol. In all reactions, synthetic subgenomic RNA was used as a template for the replication reaction, yielding a replicated RNA corresponding to the antisubgenomic RNA. (A) Lanes 1 and 2, reverse transcription and amplification of the 5′ terminus of the replicated RNA by RNA ligase-mediated rapid amplification of cDNA ends. Amplified cDNA was visualized on 2% agarose gel by UV transillumination after ethidium bromide staining. (B) Sequence analysis of the amplification product. The sequences of the template (3′ terminus of norovirus subgenomic RNA) and the 3D^pol replication product (5′ terminus) are shown.

FIG. 7.

CapFinder analysis of the 3′ terminus of the replication product of norovirus 3D^pol. In all reactions, synthetic subgenomic RNA was used as a template for the replication reaction, yielding a replicated RNA corresponding to the antisubgenomic RNA. (A) Amplification of the 3′ terminus of replicated RNA by CapFinder analysis. Amplified cDNA was visualized on 2% agarose gel by UV transillumination after ethidium bromide staining. Lane 1, reverse transcription and amplification of the 3′ terminus, with the CapFinder-poly(dCTP) oligonucleotide used as the reverse primer; lane 2, reverse transcription and amplification of the 3′ terminus, with the CapFinder-poly(dGTP) oligonucleotide used as the reverse primer. (B) Sequence analysis of the amplification product. The sequences of the template (3′ terminus of the 3D^pol replication product) and the CapFinder product (5′ terminus) are shown. The 3′ end of the CapFinder-d(GTP) oligonucleotide, the corresponding poly(C) stretch, and the predicted start codon of the subgenomic RNA (located at the 3′ end of the replicated RNA) are highlighted.

FIG. 8.

De novo initiation of RNA synthesis on norovirus antisubgenomic RNA. In all reactions, synthetic antisubgenomic RNA was used as a template for the replication reaction. RNA reaction products were analyzed on native agarose gels and visualized by UV transillumination after ethidium bromide staining. (A) Lanes 1 to 3, RNA synthesis in the presence of wild-type 3D^pol, in the presence of mutated 3D^pol, and in the absence of 3D^pol, respectively. Lanes 4 to 6, RNA synthesis in the presence of wild-type 3D^pol and a 3′ terminus cRNA oligonucleotide, in the presence of mutated 3D^pol and a 3′ terminus cRNA oligonucleotide, and with a 3′ terminus cRNA oligonucleotide but without NV 3D^pol, respectively. Lanes 7 to 9, RNA synthesis in the presence of wild-type 3D^pol and a 3′ terminus cDNA-oligonucleotide, in the presence of mutated 3D^pol and a 3′ terminus cDNA-oligonucleotide, and with a 3′ terminus cDNA-oligonucleotide but without NV 3D^pol, respectively. T, template RNA. R, replication product. M, RNA molecular size marker (kb). (B) Strand separation analysis of the replication products. Reaction products were analyzed on formaldehyde-agarose gels and visualized by UV transillumination after ethidium bromide staining. Lanes 1 and 2, template (antisubgenomic RNA) and norovirus 3D^pol replication products, respectively.

FIG. 9.

Deletion analysis of the 3′ terminus of the subgenomic RNA template. (A) Schematic representation of deleted subgenomic RNA used as a template. Sizes of deleted fragments are indicated (nt). The ultimate nucleotides at the template's 3′ ends are indicated. (B) In vitro transcription of subgenomic RNA. Reaction products were analyzed on formaldehyde-agarose gel and visualized by UV transillumination after ethidium bromide staining. Lane 1, undeleted subgenomic RNA; lanes 2 to 4, subgenomic RNAs with successive deletions at their 3′ termini. M, RNA molecular size marker (kb). (C) Replication of 3′ terminus-deleted subgenomic RNAs. Reaction products were analyzed on formaldehyde-agarose gel and visualized by autoradiography. Lanes 2 to 4, replication of synthetic subgenomic RNAs with successive deletions at their 3′ termini. T, template RNA; R, replication product.

FIG. 10.

Deletion analysis of the 3′ terminus of antisubgenomic RNA. (A) Schematic representation of deleted antisubgenomic RNA used as a template. Sizes of deleted fragments are indicated (nt). The ultimate nucleotides at the template's 3′ ends are indicated. (B) In vitro transcription of antisubgenomic RNA. Reaction products were analyzed on formaldehyde-agarose gel and visualized by UV transillumination after ethidium bromide staining. Lane 1, undeleted antisubgenomic RNA; lanes 2 to 7, antisubgenomic RNA with successive deletions at their 3′ termini. M, RNA molecular size marker (kb). (C) Replication of 3′ terminus-deleted antisubgenomic RNA. Reaction products were analyzed on formaldehyde-agarose gel and visualized by autoradiography. Lanes 2 to 7, replication of antisubgenomic RNAs with successive deletions at their 3′ termini. T, template RNA; R, replication product.