1. Unconventional Mechanism of mRNA Capping by the RNA-Dependent RNA Polymerase of Vesicular Stomatitis Virus 
Tomoaki Ogino, Amiya K. Banerjee 
Molecular Cell 
Volume 25, Issue 1, Pages (January 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
Establishment of an In Vitro RNA Capping Assay with the VSV RNP
(A) The VSV RdRp complex transcribes the negative-strand genomic RNA into the leader RNA (∼47 nt) and 5′-capped and 3′-polyadenylated mRNAs. The mRNA- and leader RNA-start sequences are shown.
(B) 5′-triphosphorylated oligo-RNAs with the mRNA- and leader RNA-start sequences were synthesized by T7 RNA polymerase from synthetic oligo-DNA templates.
(C and D) The oligo-RNAs ([C], AACAG; [D], ACGAA), labeled with [γ-32P]ATP at their 5′ ends, were purified from the transcription mixtures containing prematurely terminated RNAs (lane 1). The purified RNAs (lane 2) were incubated in the presence or absence of RNase T1 (lane 3) or RNase A (lane 4) and analyzed by urea-20% PAGE followed by autoradiography. Marker (M) lanes indicate poly(A) RNAs with 5–8 nucleotides (pp[pA]5–8), labeled with [γ-32P]ATP at their 5′ ends, and a mixture of [γ-32P]ATP and poly(A) RNAs with 2–5 nucleotides (pp[pA]1–5). The positions of the origin (ori.), xylene cyanol FF (XC), bromophenol blue (BPB), and oligo-RNAs with indicated sequences (asterisk denotes 32P) are shown on the right.
(E) The purified VSV virion (5 μg of protein) and RNP (3 μg of protein) were analyzed by SDS-10% PAGE followed by staining with Coomassie Brilliant Blue. The positions of marker proteins and viral proteins are shown on the left and right, respectively.
(F) The VSV RNP (3 μg of protein) was incubated with the indicated 5′-triphosphorylated oligo-RNA and [α-32P]GTP. The RNA products were analyzed by urea-20% PAGE followed by autoradiography. Lane 1 indicates no RNP. M lanes indicate [32P]pp(pA)1–5 and a mixture of [32P]pppApApCpApG and [32P]GpppApApCpApG, which was made from pppApApCpApG and [α-32P]GTP by the vaccinia virus CE, ([G]pppAACAG).
(G) The VSV RNP (3 μg of protein) or the vaccinia virus (Vac) CE (2.5 units) was incubated with the indicated 5′-triphosphorylated oligo-RNA and [α-, β-, or γ-32P]GTP. Nuclease P1 digests of the RNA products were analyzed by PEI-cellulose TLC followed by autoradiography. Lanes 1, 4, 7, 10, and 13 indicate no enzyme. The positions of the origin (ori.) and standard marker compounds, visualized under UV light at wavelength of 254 nm, are indicated on the right.
Molecular Cell  , 85-97DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2
The VSV CE Uses the 5′-Triphosphorylated RNA and GDP Derived from GTP as Substrates
(A and B) 5′-di- (lane 2, pp-) and mono- (lane 3, p-) phosphorylated AACAG RNAs, labeled with [α-32P]GMP, were generated from the 5′-triphosphorylated one (lane 1, ppp-) with RTPase of the vaccinia virus CE and TAP, respectively, and then analyzed by urea-20% PAGE (A) or PEI-cellulose TLC (B) followed by autoradiography. In (B), the positions of marker compounds and the AACAG RNAs (asterisk denotes 32P) are shown on the left and right, respectively.
(C) The vaccinia virus (Vac) CE (2.5 units) or the VSV RNP (3 μg of protein) was incubated with ppp-, pp-, or p-ApApCpApG and [α-32P]GTP. The cap structures formed were analyzed as in Figure 1G. Lanes 1 and 5 indicate no enzyme.
(D) [α-32P]GDP (lane 2) and [α-32P]GMP (lane 3) were generated from [α-32P]GTP (lane 1) with nucleoside 5′-triphosphatase of the vaccinia virus CE and TAP, respectively, and then analyzed by PEI-cellulose TLC followed by autoradiography. The positions of marker compounds are indicated on the left.
(E) The vaccinia virus (Vac) CE (2.5 units) or the VSV RNP (3 μg of protein) was incubated with pppApApCpApG and [α-32P]GTP, GDP, or GMP. The cap structures formed were analyzed as in Figure 1G. Lanes 1 and 5 indicate no enzyme.
(F) The VSV RNP (3 μg of protein) was incubated with [γ- or α-32P]GTP, and then products were analyzed by PEI-cellulose TLC followed by autoradiography. Lanes 1 and 3 indicate no RNP. Marker (M) lane indicates a digest of [γ-32P]GTP with calf intestine alkaline phosphatase (CIAP) to denote the position of inorganic phosphate (Pi).
Molecular Cell  , 85-97DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3 The VSV L Protein Forms a Possible Enzyme-RNA Intermediate
(A and B) The vaccinia virus (Vac) CE ([A], 0.25 unit) or the VSV RNP ([B], 3 μg of protein) was incubated with the indicated mono- or polyribonucleotide labeled with 32P at the indicated position(s) (shown in red) and then analyzed by SDS-10% PAGE followed by autoradiography. Note that all ribonucleotide probes had the same specific radioactivities in each experiment. Lanes 1, 4, 7, and 10 indicate no enzyme. The positions of marker proteins and the vaccinia virus D1 protein (A) or the VSV L protein (B) are shown on the left and right, respectively. In (B) (lane 13), the VSV RNP (5 μg of protein) was analyzed by SDS-10% PAGE followed by staining with Coomassie Brilliant Blue (CBB) to locate the viral proteins.
(C) The VSV RNP (8 μg of protein) was preincubated with pppApApCpApG labeled with 32P at the indicated position(s) (shown in red) and then purified by ultracentrifugation. The L-[32P]RNA complex in the purified RNP was treated with or without calf intestine alkaline phosphatase (CIAP) or RNase A and then analyzed as in (B). Lanes 1 and 5 indicate no RNP.
(D) The L-RNA complex formation was performed with the VSV RNP (3 μg of protein) and 5 μM ppp∗Ap∗ApCp∗ApG (asterisk indicates 32P) in the presence or absence of the indicated compound at the indicated concentration. Pi and PPi indicate inorganic phosphate and pyrophosphate, respectively. The L-[32P]RNA complex was analyzed as in (B). Lanes 1 and 9 indicate no RNP.
Molecular Cell  , 85-97DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4 The VSV L Protein Alone Catalyzes the Capping Reaction
(A) The VSV RNP (5 μg of protein), N-RNA (3 μg of protein), and L-P (1 μg) complexes and recombinant P (r P, 0.25 μg) and L (r L, 0.75 μg) proteins were analyzed by SDS-10% PAGE followed by staining with Coomassie Brilliant Blue. The positions of marker proteins and viral proteins are shown on the left and right, respectively.
(B–E) One-fifth amounts of the samples shown in (A) were subjected to the whole capping reaction with pppApApCpApG and [α-32P]GTP (B), the GTPase reaction with [α-32P]GTP (C), the capping reaction with pppApApCpApG and [α-32P]GDP (D), or the enzyme-RNA complex (E-RNA) formation with ppp∗Ap∗ApCp∗ApG (asterisk denotes 32P) (E), as described in Figures 1–3. Lane 1 indicates no enzyme.
Molecular Cell  , 85-97DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5
RNA Substrate Specificity of the Recombinant L Protein in the Capping Reaction
(A and B) The recombinant L protein (r L, 50 ng) was incubated with [α-32P]GDP and the indicated 5′-tri- or diphosphorylated oligo-RNA. Aliquots of the RNA products were analyzed by urea-20% PAGE (A) as in Figure 1F, and other aliquots treated with nuclease P1 were analyzed by PEI-cellulose TLC (B) as in Figure 1G. Lane 1 indicates no enzyme.
(C and D) 5′-triphosphorylated RNAs with indicated sequences were subjected to capping reactions with the recombinant L protein (r L, 50 ng) in the presence of [α-32P]GDP (C) or the vaccinia virus (Vac) CE (2.5 units) in the presence of [α-32P]GTP (D). The cap structures formed were analyzed as in Figure 1G. Lane 1 indicates no enzyme.
Molecular Cell  , 85-97DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6 Stability of the Linkage between the L Protein and RNA
(A) The L-[32P]RNA complex (L-RNA, lanes 2–5) or the vaccinia virus CE-(lysyl-N)-[32P]GMP complex (Vac CE-pG, lanes 7–10), formed as described in Experimental Procedures, was incubated with 0.1 M Tris-HCl buffer (pH 7.5) containing 1% SDS at indicated temperature for 5 min and then analyzed by SDS-10% PAGE followed by autoradiography. Lanes 1 and 6 show input complexes. The positions of marker proteins and the L and D1 proteins are shown on the left and right, respectively.
(B and C) The L-RNA (lanes 2–4) or the Vac CE-pG (lanes 6–8) was incubated with 0.1 M Tris-HCl (pH 7.5), 0.1 M HCl, 0.1 M NaOH, or 0.1 or 1 M hydroxylamine (pH 7.5) in the presence of 1% SDS at 37°C for 45 min and then analyzed as in (A). Lanes 1 and 5 indicate input complexes.
(D) The ppp∗Ap∗ApCp∗ApG RNA (asterisk indicates 32P) was treated with indicated reagents as in (B) and (C) and then analyzed by urea-20% PAGE followed by autoradiography. Lane 1 shows input RNA. Arrowheads indicate degraded RNAs. M lane shows the marker RNAs (see Figure 1C).
Molecular Cell  , 85-97DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7 Strategies of mRNA Cap Formation
A possible mechanism of VSV mRNA cap formation (A) is compared with the known mechanism for eukaryotes and some DNA and double-strand RNA viruses (B). In (A), the VSV proteins required for synthesis of the 5′-capped mRNAs are shown in green. The 5′-phosphate groups derived from GTP and nascent mRNAs are shown in red and blue, respectively. For details, see text.
Molecular Cell  , 85-97DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions