Structures of Qß virions, virus-like particles, and the Qß-MurA complex reveal internal coat proteins and the mechanism of host lysis.

In single-stranded RNA bacteriophages (ssRNA phages) a single copy of the maturation protein binds the genomic RNA (gRNA) and is required for attachment of the phage to the host pilus. For the canonical Allolevivirus Qß the maturation protein, A2, has an additional role as the lysis protein, by its ability to bind and inhibit MurA, which is involved in peptidoglycan biosynthesis. Here, we determined structures of Qß virions, virus-like particles, and the Qß-MurA complex using single-particle cryoelectron microscopy, at 4.7-Å, 3.3-Å, and 6.1-Å resolutions, respectively. We identified the outer surface of the ß-region in A2 as the MurA-binding interface. Moreover, the pattern of MurA mutations that block Qß lysis and the conformational changes of MurA that facilitate A2 binding were found to be due to the intimate fit between A2 and the region encompassing the closed catalytic cleft of substrate-liganded MurA. Additionally, by comparing the Qß virion with Qß virus-like particles that lack a maturation protein, we observed a structural rearrangement in the capsid coat proteins that is required to package the viral gRNA in its dominant conformation. Unexpectedly, we found a coat protein dimer sequestered in the interior of the virion. This coat protein dimer binds to the gRNA and interacts with the buried α-region of A2, suggesting that it is sequestered during the early stage of capsid formation to promote the gRNA condensation required for genome packaging. These internalized coat proteins are the most asymmetrically arranged major capsid proteins yet observed in virus structures.

A(2) expression and assembly regulates lysis in Qß infections.

The capsids of ssRNA phages comprise a single copy of an ~45 kDa maturation protein that serves to recognize the conjugative pilus as receptor, to protect the ends of the viral RNA and also to escort the genomic RNA into the host cytoplasm. In the Alloleviviridae, represented by the canonical phage Qß, the maturation protein A(2) also causes lysis. This is achieved by inhibiting the activity of MurA, which catalyses the first committed step of murein biosynthesis. Previously, it was shown that Qß virions, with a single copy of A(2), inhibit MurA activity. This led to a model for lysis timing in which, during phage infection, A(2) is not active as a MurA inhibitor until assembled into virion particles, thus preventing premature lysis before a sufficient yield of viable progeny has accumulated. Here we report that MurA inactivates purified Qß particles, casting doubt on the notion that A(2) must assemble into particles prior to MurA inhibition. Furthermore, quantification of A(2) protein induced from a plasmid indicated that lysis is entrained when the amount of the lysis protein is approximately equimolar to that of cellular MurA. Qß por mutants, isolated as suppressors that overcome a murA(rat) mutation that reduces the affinity of MurA for A(2), were shown to be missense mutations in A(2) that increase the translation of the maturation protein. Because of the increased production of A(2), the por mutants have an attenuated infection cycle and reduced burst size, indicating that a delicate balance between assembled and unassembled A(2) levels regulates lysis timing.