Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 273

Protein priming. A protein-linked oligonucleotide serves as a primer for RNA synthesis by RdRPs of members of the Picornaviridae and Caliciviridae. Protein priming also occurs during DNA replication of adenoviruses, certain DNA-containing bacteriophages (Chapter 10), and hepatitis B virus (Chapter 7). A terminal protein provides a hydroxyl group (in a tyrosine or serine residue) to which the first (priming) oligonucleotide can be linked by viral polymerases, via a phosphodiester bond. The primer is then elongated.

Polioviral genomic RNA, as well as newly synthesized (+) and (−) strand RNAs, are covalently linked at their 5′ ends to the 22-amino-acid protein VPg (Fig. 6.9A), initially suggesting that VPg might function as a primer for RNA synthesis. This hypothesis was supported by the discovery of a uridylylated form of the protein, VPg-pUpU, in infected cells. VPg can be uridylylated in vitro by 3D^pol and can then prime the synthesis of VPg-linked poly(U) from a poly(A) template. The template for uridylylation of VPg is either the 3′ poly(A) on (+) strand RNA [during synthesis of (−) strand RNA] (Fig. 6.10) or an RNA hairpin, the cis-acting replication element (cre), located in the coding region [during synthesis of (+) strand RNA] (Fig. 6.9B and C).

Figure 6.8 Mechanism of de novo initiation. (A) Ribbon diagram of RdRP of hepatitis C virus. Fingers, palm, and thumb domain are colored blue, green, and magenta, respectively. The C-terminal loop that blocks the active site is shown in brown. Active-site residues are yellow (PDB file 4WTM). (B) Swinging-gate model of initiation. With the RNA template (green) in the active site of the enzyme, a short β-loop (red) provides a platform on which the first complementary nucleotide (light green) is added to the template (left). The second nucleotide is then added, producing a dinucleotide primer for RNA synthesis (middle). At this point, nothing further can happen because the priming platform blocks the exit of the RNA product from the enzyme. The solution to this problem is that the polymerase undergoes a conformational change that moves the priming platform out of the way and allows the newly synthesized complementary RNA (right) to exit as the enzyme moves along the template strand.

Structures of the RdRPs of different picornaviruses and caliciviruses indicate that the active site is more accessible than in polymerases with a de novo mechanism of initiation. The small thumb domains of these polymerases leave a wide central cavity that can accommodate the template and the protein primer.

Figure 6.9 Uridylylation of VPg. (A) Linkage of VPg to polioviral genomic RNA. Polioviral RNA is linked to the 22-amino-acid VPg (orange) via an O4-(5′-uridylyl)-tyrosine linkage. This phosphodiester bond is cleaved at the indicated site by a cellular enzyme to produce the viral mRNA containing a 5′-terminal pU. (B) Structure of the poliovirus (+) strand RNA template, showing the 5′ cloverleaf structure, the internal cre (cis-acting replication element) sequence, and the 3′ pseudoknot. (C) Model for assembly of the VPg uridylylation complex. Two molecules of 3CD bind to cre. The 3C dimer melts part of the stem. 3D^pol binds to the complex by interactions between the back of the thumb domain and the surface of 3C. VPg then binds the complex and is linked to two U moieties in a reaction templated by the cre sequence.

Biochemical and structural studies have identified three different VPg binding sites on 3D^pol. Uridylylation of foot- and-mouth disease virus VPg can be achieved in a reaction containing 3D^pol, a template (rA)₁₀, UTP, and Mg²⁺ and Mn²⁺. Crystallographic analysis of 3D^pol carrying out uridylyation reveals that VPg-pU is bound in the template-binding channel, with the N terminus of VPg in the NTP entry channel and the C terminus pointing toward the template-binding channel. The hydroxyl group of a tyrosine in VPg is covalently linked to the α-phosphate of UMP and interacts with a divalent metal ion that binds an Asp of the Gly-Asp-Asp motif in the active site. This arrangement of VPg is similar to that of the primer terminus in the nucleotidyl transfer reaction, demonstrating that 3D^pol catalyzes VPg uridylylation using the same two-metal mechanism as the nucleotidyl transfer reaction. A second binding site for VPg has been located on the base of the thumb subdomain of the polymerase of Coxsackievirus B3, in a position that does not allow uridylylation in cis. It has been suggested that uridylylation of VPg might be accomplished in trans by another 3D^pol molecule. A third binding site on the 3D^pol of enterovirus 71 is at the base of the palm and also would require uridylylation by another polymerase molecule.

When VPg uridylylation begins at the 3′-poly(A) tail of the (+) strand template, the polymerase continues nucleotidyl transfer reactions and copies the entire genome. However, when uridylylation of VPg takes place on the cre, the protein must dissociate and transfer to the 3′ end of the RNA. How this process is accomplished is not known (Fig. 6.10).

Protein priming by the birnavirus RdRP VP1 is unusual because the primer is the polymerase, not a separate protein. Even in the absence of a template, VP1 has self-guanylylation activity that is dependent on divalent metal ions. The guanylylation site is a serine located approximately 23 Å from the catalytic site of the polymerase. The long distance between these sites suggests that guanylylation may be carried out at a second active site. The finding that some altered polymerases that are inactive in RNA synthesis retain self-guanylylation activity supports this hypothesis. After two G residues are added to VP1, it binds to a conserved CC sequence at the terminus of the viral RNA template to initiate RNA synthesis. The 5′ ends of mRNAs and genomic double-stranded RNAs produced by this reaction are therefore linked to a VP1 molecule.

Priming by capped RNA fragments. Influenza virus mRNA synthesis is blocked by treatment of cells with the fungal toxin α-amanitin at concentrations that inhibit cellular DNA-dependent RNA polymerase II. This surprising finding demonstrated that the viral RNA polymerase is dependent on host cell RNA polymerase II. Inhibition by α-amanitin is explained by a requirement for newly synthesized cellular transcripts made by this enzyme to provide primers for influenza viral mRNA synthesis (Fig. 6.11). Presumably, these cellular transcripts must be made continuously because they are exported rapidly from the nucleus once processed. Such transcripts are cleaved in the nucleus by an influenza virus-encoded, cap-dependent endonuclease that is part of the RdRP (Fig. 6.12). The resulting 10- to 13-nucleotide capped fragments serve as primers for the initiation of viral mRNA synthesis.

Figure 6.10 Poliovirus (−) strand RNA synthesis. The precursor of VPg, 3AB, contains a hydrophobic domain and is a membrane-bound donor of VPg. A ribonucleoprotein complex is formed when poly(rC)-binding protein 2 (PCBP2) and 3CD^pro bind the cloverleaf structure located within the first 108 nucleotides of (+) strand RNA. The ribonucleoprotein complex interacts with poly(A)-binding protein 1 (PAbp1), which is bound to the 3′ poly(A) sequence, bringing the ends of the genome into close proximity. Protease 3CD^pro cleaves membrane-bound 3AB, releasing VPg and 3A. VPg-pUpU is synthesized by 3D^pol using the 3′ poly(A) sequence as a template, and comprises the primer for RNA synthesis. Modified from Paul AV. 2002. p 227–246, in Semler BL, Wimmer E (ed), Molecular Biology of Picornaviruses (ASM Press, Washington, DC).

Figure 6.11 Influenza virus RNA synthesis. (A) Viral (−) strand genomes are templates for the production of either subgenomic mRNAs or full-length (+) strand RNAs. The switch from viral mRNA synthesis to genomic RNA replication is regulated by both the number of nucleocapsid (NP) protein molecules and the acquisition by the viral RdRP of the ability to catalyze initiation without a primer. Binding of the NP protein to elongating (+) strands enables the polymerase to read to the 5′ end of genomic RNA. (B) Capped RNA-primed initiation of influenza virus mRNA synthesis. Capped RNA fragments cleaved from the 5′ ends of cellular nuclear RNAs serve as primers for viral mRNA synthesis. The 10 to 13 nucleotides in these primers do not need to hydrogen bond to the common sequence found at the 3′ ends of the influenza virus genomic RNA segments. The first nucleotide added to the primer is a G residue templated by the penultimate C residue of the genomic RNA segment; this is followed by elongation of the mRNA chains. The terminal U residue of the genomic RNA segment does not direct the incorporation of an A residue. The 5′ ends of the viral mRNAs therefore comprise 10 to 13 nucleotides plus a cap structure snatched from host nuclear pre-mRNAs. Adapted from Plotch SJ et al. 1981. Cell 23:847–858, with permission.

Bunyaviral mRNA synthesis is also primed with capped fragments of cellular RNAs. In contrast to that of influenza virus, bunyaviral mRNA synthesis is not inhibited by α-amanitin because it occurs in the cytoplasm, where capped cellular pre-mRNAs are abundant.

The influenza virus RdRP is a heterotrimer composed of PA, PB1, and PB2 proteins (Fig. 6.12). The PB1 protein is the RNA polymerase, the PB2 subunit binds capped host mRNAs, and the PA protein harbors endonuclease activity. The influenza RdRP binds to the C-terminal domain of RNA polymerase II, an interaction that activates the viral enzyme and allows the capture of capped RNA primers from nascent host mRNAs. In contrast, acquisition of caps by bunyavirus is accomplished by a single protein, the RdRP (L). The N-terminal domains of influenza PA and bunyavirus L have endonuclease activities that participate in such cap snatching. The structures of endonuclease domains from these viruses reveal the presence of a common nuclease fold.