Figure 1 (Left) Schematic representation of a levivirus: the RNA inside the virion is highly structured. (Upper right) Escherichia coli bacterium with Enterobacteria phage MS2 (MS2) particles attached to its F-pili (courtesy A.B. Jacobson). The inset is a -pilus with phage- enlargement. (Courtesy R.I. Koning and H.K. Koerten.) (Lower right) Image reconstruction obtained from cryo-electron microscopy of MS2 phages. View from outside (left) and inside (right).
(Courtesy R.I. Koning and H.K. Koerten.)
Figure 2 General genetic map of a representative levivirus – Enterobacteria phage MS2 (MS2) – and an allolevivirus – Enterobacteria phage Qβ (Qβ). The maturation protein is also called A-protein. The lysis gene overlaps the replicase gene in a+1 frameshift. Arrows indicate repression of replicase translation by capsid protein binding to an RNA hairpin structure (the operator) present at the start of the gene. The UGA nonsense codon (nt 1743) is occasionally (ca. 6%) misread as tryptophan to produce the readthrough protein.
Figure 3 2D structures of operator hairpins in several RNA phages. Note the presence of a second, putative operator hairpin in all three phages. The structure shown for PRR1 has not been confirmed yet.
Figure 4 Genetic map of Acinetobacter phage AP205 (AP205). Note the location of the tentative lysis gene at the 5-terminus. AP205 is unusually long for a levivirus. This map corrects the one previously published (Klovins, J. et al. (2002). J. Gen. Virol. (2002), 83, 1523–1533). A: A-protein; CP: capsid protein; R: replicase; L: lysis.
Figure 5 Comparison of the RNA folding in the 3′UTR of Enterobacteria phage MS2 (MS2) and Enterobacteria phage GA (GA). GA lacks the three stem-loops U4, U5 and U6. In MS2 stem-loops V1 and V2 are part of the A-protein binding site. The other part of the protein’s binding site is located around nt 400.
Figure 6 RNA secondary structure for Enterobacteria phage Qβ (Qβ) RNA from nt 2966 to the 3′ end (nt 4217) marked as AOH. The UAA stop codon (nt 4119) of the replicase gene is boxed. Replicase Domain 2 (RD2) containing 1062 nt has been replaced by a dotted circle. Breaking two or three basepairs in the central pseudoknot (ldX) or ldVIII abolishes replication. However, breaking the pairs in ld IX, which buries the 3′-terminal nucleotides, stimulates replication. Production of minus strand is also inhibited by deletion of stem-loops U1, V1, V2 or U2. (R1 and R2 were not tested.)
Figure 7 RNA secondary structure in the 3′UTR of Pseudomonas phage PP7 (PP7) and Enterobacteria phage SP (SP). The folding of PP7 RNA is much more like that of SP RNA than that of either MS2 or GA (Figure 5). Compared to MS2 the stem-loops U3, U4, U5, U6 and one of the two V-loops are missing. The boxed sequence in the loop of hairpin U1 is conserved in all viruses of the family Leviviridae. The sequence is part of the central pseudoknot in Qβ. The pseudoknot is believed to exist also in the other phages.
Figure 8 Proposed phylogenetic tree for the family Leviviridae. Distances are arbitrary. The ancestor only has the three basic genes. Lysis is effected by the A-protein as it still is today in Qβ. Presumably, fitness of the ancestor was restricted by the double function of the A-protein (Bollback, J.P. and Huelsenbeck, J.P. (2001). J. Mol. Evol., 52, 117–128). The leviviruses solved the problem by evolving a separate lysis protein either encoded on a vacant region of the genome (AP205) or resulting from a ribosomal restart following translation termination at the end of the capsid gene (other leviviruses). Once restrictions on the A-protein were relaxed the gene could evolve in various directions to better fulfill its remaining function: virion maturation and infection. Two features of leviviruses can be explained by this scenario: first, lysis genes have variable startpoints (even between MS2 and fr or between GA and KU1) and secondly, of the three “old” genes, the A-protein gene shows the lowest sequence conservation. The alloleviviruses solved the dual-function problem by transferring part of the maturation and infection function to a new protein, readthrough, which arose by an insertion between coat and replicase genes. Presumably, this allowed A-protein to improve its lysis function. Such a scenario would provide a different reason why also in the alloleviviruses A-protein is least conserved of the “old” genes. Abbreviations of virus names are provided in the tables.