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

Abstract

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 Qβ the maturation protein, A, 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 Qβ virions, virus-like particles, and the Qβ-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 β-region in A as the MurA-binding interface. Moreover, the pattern of MurA mutations that block Qβ lysis and the conformational changes of MurA that facilitate A binding were found to be due to the intimate fit between A and the region encompassing the closed catalytic cleft of substrate-liganded MurA. Additionally, by comparing the Qβ virion with Qβ 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 A, 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.

In Situ Structures of the Polymerase Complex and RNA Genome Show How Aquareovirus Transcription Machineries Respond to Uncoating

“…4B). Several dsRNA strands form stabilizing interactions with each TEC, allowing visualization of their major and minor grooves, just as they do for the stem-loops of single-stranded RNA (ssRNA) viruses (17)(18)(19). Three major dsRNA densities interact with the TEC and are labeled "bound," "side," and "back" RNA based on their locations relative to the above-described sides of the TEC (Fig.…”

Phage single-gene lysis: Finding the weak spot in the bacterial cell wall

“…The A 2 protein has multiple functions during Q␤ infection; it functions in virion assembly (63), is bound to and provides protection for the gRNA against RNase degradation (64), mediates interaction with the F-pilus, and is internalized into the host cytoplasm along with the genomic RNA (63-65). Remarkably, A 2 also functions as the Sgl protein.…”

The Single-Stranded RNA Bacteriophage Qβ Adapts Rapidly to High Temperatures: An Evolution Experiment

“…Here we found two amino acid substitutions, Ser401Arg and Phe411Cys, in two of three lines as well as two amino acid substitutions, Val132Ala and His385Arg, in one of three lines. Mapping these mutational positions on the A2 structure (Protein Data Bank 5MNT) reveals the following: (i) Ser401 is located near the position where coat and A2 interact [28] ( Figure S1a). (ii) Phe411 is located in α9 and is close to the RNA-binding region [29] ( Figure S1b).…”

“…(iii) Val132 and His385 are closely located at the protruding region ( Figure S1a). The region between the 30th and 120th amino acids of the N-terminus of A2 is in contact with MurA for cell lysis [28], and no mutations, except Glu30Asp, are observed in this region. In coat protein, we observed Val50Ile substitution in all three lines.…”