1. Introduction: MS2 Phage, Lysis Regulation, and Host Resistance

Bacteriophage MS2, a canonical member of the Leviviridae family, is a positive-sense single-stranded RNA virus renowned for its role as a model system in molecular biology. It infects Escherichia coli strains expressing the conjugative F-pilus, using it as the port of entry for its compact ~3.5kb RNA genome. This genome, famously the first RNA genome to be fully sequenced (by Walter Fiers and colleagues in 1976), provided fundamental insights into gene organization, overlapping reading frames, and translational control.

Despite its simplicity, the MS2 genome encodes four essential proteins through a marvel of genetic economy, utilizing overlapping genes and sophisticated translational regulation:

  1. Maturation Protein (A protein): Involved in attaching to the F-pilus, potentially aiding genome entry, and incorporated into new virions during assembly (one copy per phage).
  2. Coat Protein (CP): The primary structural protein, forming the icosahedral capsid shell (180 copies per phage). It also plays a key regulatory role, acting as a translational repressor of the Replicase gene later in infection.
  3. Lysis Protein (L protein): A small protein, encoded in a +1 reading frame overlapping the end of the Coat gene and the start of the Replicase gene. It is directly responsible for lysing the host cell by disrupting the inner membrane.
  4. Replicase Protein (Rep protein): The catalytic β-subunit of the viral RNA-dependent RNA polymerase (RdRP). It recruits host proteins (ribosomal protein S1, elongation factors Tu and Ts) to form the active holoenzyme that replicates the viral RNA genome.

The MS2 infection cycle begins with attachment and genome injection. The viral (+)ssRNA immediately functions as mRNA. Translation is tightly regulated: Coat protein is made abundantly first; Replicase synthesis is translationally coupled to Coat synthesis but later repressed by Coat protein dimers binding an operator hairpin on the RNA; Maturation protein is made at very low levels due to its inaccessible start codon except on nascent replicating RNA; and Lysis protein is also produced at low levels from its overlapping frame. The Replicase complex copies the (+)RNA into (-)RNA intermediates, which then serve as templates for synthesizing vast numbers of progeny (+)RNA genomes. These new genomes are both translated and packaged into capsids assembling with Coat and Maturation proteins.

The crucial final step is host cell lysis, orchestrated by the L protein. Accumulating at the inner membrane, L protein requires interaction with the host chaperone DnaJ to execute its function. However, functional studies, particularly the key observation that truncated L protein (lacking the N-terminal DnaJ-interaction domain, L_sol) causes much faster lysis, strongly suggest that DnaJ's primary role is not simple activation but rather inhibition or timing. We hypothesize that DnaJ binding normally acts as a crucial 'timer', preventing the L protein's transmembrane domain (L_TMD) from disrupting the membrane prematurely. This programmed delay is likely essential for maximizing phage yield, allowing sufficient time for genome replication and virion assembly before the host cell is destroyed.

This dependence on DnaJ, however, creates a significant vulnerability. Evidence indicates that E. coli can readily evolve resistance through single mutations in its own DnaJ gene. Such mutations can disrupt the precise DnaJ-L protein interaction, effectively blocking the final lysis step and rendering the phage incapable of completing its infection cycle, even if viral replication and assembly occur flawlessly. This represents a critical bottleneck, potentially limiting the long-term effectiveness of MS2 in applications like phage therapy or biocontrol.

Many phage engineering efforts focus on increasing general phage 'lethality' (e.g., faster replication, more potent enzymes). However, we believe that unless specific resistance bottlenecks like the DnaJ dependency are addressed, such efforts may be undermined by rapid host adaptation. Simply making the phage kill faster won't help if the host can easily evolve a way to block the killing mechanism entirely. Furthermore, if the DnaJ interaction is indeed a carefully evolved delay mechanism, simply removing it (like truncation) might lead to suboptimal phage yields due to premature lysis.

With that in mind, our project aims to engineer robustness against this specific resistance pathway. We propose to introduce a 'Plan B' or 'time bomb' mechanism for L protein activation – one that can function alongside, or as a backup to, the potentially vulnerable DnaJ pathway, while also offering the possibility to fine-tune the lysis timing.

2. Project Goal

To engineer the MS2 L protein to create a modified lysis phenotype that is:

  1. More Robust: Less dependent on the specific DnaJ interaction, providing resistance against host DnaJ mutations.
  2. Tunable: Allowing modulation of lysis timing (potentially faster than wild-type but slower than immediate lysis from truncated L protein) to optimize phage propagation.

3. Proposed Solution: Engineered L Protein with Protease-Sensitive Linker

Our core strategy is to insert an engineered, flexible linker sequence between the L protein's N-terminal cytoplasmic, DnaJ-interacting domain (L_sol) and its C-terminal transmembrane domain (L_TMD). This linker is designed to be susceptible to cleavage by endogenous E. coli cytoplasmic proteases. The linker will present repeats of known cleavable sites by endogenous machinery increasing the chances of cleavage and allowing for activation of the L protein independently of the DnaJ interaction.

4. Design Rationale & Strategy