Deep mutational scanning of influenza A virus NEP reveals pleiotropic mutations in its N-terminal domain.

The influenza A virus nuclear export protein (NEP) is a multifunctional protein that is essential for the viral life cycle and has very high sequence conservation. However, since the open reading frame of NEP largely overlaps with that of another influenza viral protein, non-structural protein 1, it is difficult to infer the functional constraints of NEP based on sequence conservation analysis. Besides, the N-terminal of NEP is structurally disordered, which further complicates the understanding of its function. Here, we systematically measured the replication fitness effects of >1,800 mutations of NEP. Our results show that the N-terminal domain has high mutational tolerance. Additional experiments demonstrate that N-terminal domain mutations pleiotropically affect viral transcription and replication dynamics, host cellular responses, and mammalian adaptation of avian influenza virus. Overall, our study not only advances the functional understanding of NEP, but also provides insights into its evolutionary constraints.

An adenine/thymidine-rich region is integral to RepL-mediated DNA replication.

The lytic replication of bacteriophage P1 requires RepL expression and the lytic stage origin, oriL, which is postulated to be located within repL gene sequence. The exact sequence of P1 oriL and the mechanism(s) of RepL-mediated DNA replication, however, are not fully understood. By using repL gene expression to induce DNA replication of a gfp and a rfp reporter plasmids, we demonstrated that synonymous base substitution in an adenine/thymidine-rich region of repL gene sequence, termed AT2, significantly inhibited the RepL-mediated signal amplification. Contrastingly, mutations in an IHF and two DnaA binding sites did not affect the RepL-mediated signal amplification significantly. A truncated repL sequence with the AT2 region allowed RepL-mediated signal amplification in trans therefore verifying a significant role of the AT2 region in RepL-mediated DNA replication. A combination of repL gene expression and a non-protein-coding copy of repL gene sequence (termed nc-repL) was able to amplify the output of an arsenic biosensor. Furthermore, mutation(s) at single or multiple positions within the AT2 region produced varying levels of RepL-mediated signal amplification. Overall, our results provide novel insights into the identity and location of P1 oriL as well as demonstrating the potential of using repL constructs to amplify and modulate the output of genetic biosensors.

Tail-Engineered Phage P2 Enables Delivery of Antimicrobials into Multiple Gut Pathogens.

Bacteriophages can be reprogrammed to deliver antimicrobials for therapeutic and biocontrol purposes and are a promising alternative treatment to antimicrobial-resistant bacteria. Here, we developed a bacteriophage P4 cosmid system for the delivery of a Cas9 antimicrobial into clinically relevant human gut pathogens Shigella flexneri and Escherichia coli O157:H7. Our P4 cosmid design produces a high titer of cosmid-transducing units without contamination by a helper phage. Further, we demonstrate that genetic engineering of the phage tail fiber improves the transduction efficiency of cosmid DNA in S. flexneri M90T as well as allows recognition of a nonnative host, E. coli O157:H7. We show that the transducing units with the chimeric tails enhanced the overall Cas9-mediated killing of both pathogens. This study demonstrates the potential of our P4 cas9 cosmid system as a DNA sequence-specific antimicrobial against clinically relevant gut pathogenic bacteria.