Principles of Virology. Jane Flint

to the NP-RNA.

      The genomes of many (+) strand RNA viruses encode helicases that serve functions similar to that of the nucleoproteins of (−) strand RNA viruses (see “Unwinding the RNA Template” below). In addition to its enzymatic activity, the poliovirus RdRP (3D^pol) is a cooperative single-stranded-RNA-binding protein and can unwind RNA duplexes without the hydrolysis of ATP, as is characteristic of helicase-mediated unwinding.

Figure 6.1 Strategies for replication and mRNA synthesis of RNA virus genomes are shown for representative virus families. Picornaviral genomic RNA is linked to VPg at the 5′ end. The (+) genomic RNA of some flaviviruses does not contain poly(A). Only one RNA segment is shown for segmented (−) strand RNA viruses.

The RNA Synthesis Machinery

      Identification of RNA-Dependent RNA Polymerases

      The first evidence for a viral RdRP emerged in the early 1960s from studies of mengovirus and poliovirus, both (+) strand RNA viruses. In these experiments, extracts were prepared from virus-infected cells and incubated with the four ribonucleoside triphosphates, one of which was radioactively labeled. The incorporation of nucleoside monophosphate into RNA was then measured. Infection with mengovirus or poliovirus led to the appearance of a cytoplasmic enzyme that could synthesize viral RNA in the presence of actinomycin D, a drug that was known to inhibit cellular DNA-directed RNA synthesis by intercalation into the double-stranded template. Lack of sensitivity to the drug suggested that the enzyme was virus specific and could copy RNA from an RNA template. This enzyme was presumed to be an RdRP. Similar assays later demonstrated that the particles of (−) strand viruses and of double-stranded RNA viruses contain an RdRP that synthesizes mRNAs from the (−) strand RNA present in the particles.

Figure 6.2 RNA secondary structure. (A) Schematic of different structural motifs in RNA. Red bars indicate base pairs; green bars indicate unpaired nucleotides. (B) Schematic of a pseudoknot. (Top) Stem 1 (S₁) is formed by base pairing in the stem-loop structure, and stem 2 (S₂) is formed by base pairing of nucleotides in the loop with nucleotides outside the loop. (Middle) A different view of the formation of stems S₁ and S₂. (Bottom) Coaxial stacking of S₁ and S₂ resulting in a quasicontinuous double helix. (C) Structure of a pseudoknot as determined by X-ray crystallography. The sugar backbone is highlighted with a green tube. Stacking of the bases in the areas of S₁ and S₂ can be seen (PDB file 1L2x). Adapted from Pleij CW. 1990. Trends Biochem Sci 15:143–147, with permission.

Figure 6.3 Structure of viral ribonucleoproteins. (A) Space-filling model of vesicular stomatitis virus partial helical filament formed by the nucleoprotein-RNA complex. Protein is colored blue and RNA green; a single nucleoprotein subunit is colored dark blue (PDB file 2WYY). (B) Ribbon diagram of vesicular stomatitis virus N protein monomer bound to RNA (colored green) (PDB file 2WYY). (C) Space-filling model of Ebolavirus partial helical filament formed by the nucleoprotein-RNA complex. Protein is colored blue and RNA green; a single nucleoprotein subunit is colored dark blue (PDB file 6NUT). (D) Ribbon diagram of Ebolavirus nucleoprotein (blue) monomer bound to RNA (green) (PDB file 6NUT). (E) Space-filling model of helical influenza virus NP bound to RNA, viewed from one end down the central axis. One NP monomer is colored dark blue (PDB file 4BBL). (F) Space-filling model of helical influenza virus NP bound to RNA, viewed from the side. One NP monomer is colored dark blue. (PDB file 4BBL). (G) Ribbon diagram of influenza virus nucleoprotein monomer (blue) bound to RNA (green) (PDB file 4BBL).

The initial discovery of a putative RdRP in poliovirus-infected cells was followed by attempts to purify the enzyme and show that it can copy viral RNA. Because polioviral genomic RNA contains a 3′ poly(A) sequence, polymerase activity was measured with a poly(A) template and an oligo(U) primer. A poly(U) polymerase was purified from infected cells and shown to copy polioviral genomic RNA in the presence of this primer. Poly(U) polymerase activity coincided with a single polypeptide, now known to be the polioviral RdRP 3D^pol (see Appendix, Fig. 21, for a description of this nomenclature). Purified 3D^pol cannot copy polioviral genomic RNA in the absence of a primer.

      Such assays for RdRP activity have been used to detect the presence of virus-specific enzymes in virus particles or in extracts of cells infected with a wide variety of RNA viruses. Amino acid sequence alignments can be used to identify viral proteins with motifs characteristic of RdRPs. These approaches were applied in identification of the L proteins of paramyxoviruses and bunyaviruses, the PB1 protein of influenza viruses, and the nsP4 protein of alphaviruses as candidate RdRPs. When the genes encoding these polymerases are expressed in cells, the proteins that are produced can copy viral RNA templates.

RNA-directed RNA synthesis obeys a set of universal rules. RNA synthesis is catalyzed by virus-encoded polymerases and initiates and terminates at specific sites in the template, but viral accessory proteins and even host cell proteins may also be required. Some RdRPs can initiate RNA synthesis de novo. Others require a primer with a free 3′-OH end to which nucleotides complementary to the template are added. Some RNA primers are protein linked, while others bear a 5′ cap structure (the cap structure is described in Chapter 8). A comparison of the structures and sequences of polynucleotide polymerases has led to the generality that all DNA and RNA polymerases catalyze synthesis by a mechanism that requires two metals (Box 6.2). RNA is usually synthesized by template-directed, stepwise incorporation of ribodeoxynucleoside monophosphates (NMPs) into the 3′-OH end of the growing RNA chain, which undergoes elongation in the 5′ → 3′ direction.

Three-Dimensional Structures of RNA-Dependent RNA Polymerases

Viral RdRPs show the highest degree of conservation of all viral proteins and are the only proteins common to all RNA viruses. Seven conserved sequence motifs (A to G) that contain amino acid residues crucial for enzymatic function have been identified in all RdRPs (Fig. 6.4). Some of the conserved sequence motifs are shared among all four classes of nucleic acid polymerases (Fig. 6.4).

BOX 6.2

      BACKGROUND

       Two-metal mechanism of catalysis by polymerases

      All polynucleotide polymerases are thought to catalyze synthesis by a two-metal mechanism that requires two conserved aspartic acid residues (illustrated in the figure for a DNA polymerase). The carboxylate groups of these amino acids coordinate two divalent metal ions, shown as Mg²⁺ in the figure. One metal ion promotes deprotonation of the 3′-OH group of the nascent strand, and the other ion stabilizes the transition state at the α-phosphate of the NTP substrate and facilitates the release of pyrophosphate (PP_i).