Competing pathways control host resistance to virus via tRNA modification and programmed ribosomal frameshifting

Abstract

Viral infection depends on a complex interplay between host and viral factors. Here, we link host susceptibility to viral infection to a network encompassing sulfur metabolism, tRNA modification, competitive binding, and programmed ribosomal frameshifting (PRF). We first demonstrate that the iron-sulfur cluster biosynthesis pathway in Escherichia coli exerts a protective effect during lambda phage infection, while a tRNA thiolation pathway enhances viral infection. We show that tRNA(Lys) uridine 34 modification inhibits PRF to influence the ratio of lambda phage proteins gpG and gpGT. Computational modeling and experiments suggest that the role of the iron-sulfur cluster biosynthesis pathway in infection is indirect, via competitive binding of the shared sulfur donor IscS. Based on the universality of many key components of this network, in both the host and the virus, we anticipate that these findings may have broad relevance to understanding other infections, including viral infection of humans.

Figure 1

Programmed ribosomal frameshifting. Schematic of a −1 programmed ribosomal frameshift. P-site tRNA slips in the −1 direction at the ‘slippery sequence.’

Figure 2

Dynamics of lambda phage infection. (A) A typical trace of a WT E. coli culture infected with lambda phage. Several hours after infection, lysis begins to outpace E. coli growth and absorbance begins to decrease. Regrowth is due to the lambda lysogen population. (B) Infection dynamics of infected ΔiscU and ΔtusA cultures were compared. (C) The displacement from the WT growth curve. In (C), the bars indicate the 95% confidence interval (CI). For (A) and (B), absorbance was recorded over the course of 16 h for three biological replicates with four technical replicates for each biological replicate (^**P<0.01, P-values were calculated using an independent two-sample t-test).

Figure 3

The effects of Fe-S cluster and tRNA thiolation deletions on lambda phage infection dynamics. (A) Cell-culture dynamics following infection overlay the uninfected control data (WT alone, gray). Small arrows in the cartoon indicate sulfur transfer and the large bent arrow indicates expression of the ISC operon. (B) Displacement from the WT strain for ISC operon members (^*P<0.05, ^**P<0.01, bars indicate 95% CI, P-values were calculated using independent two-sample t-test). (C) Comparison of the displacement from the WT strain for tRNA thiolation pathway members. Source data is available for this figure in the Supplementary Information.

Figure 4

Displacement vector comparisons for mutants in E. coli genes known to affect frameshifting. Comparison of the displacement from the WT for deletion mutants in genes known to affect frameshifting (^**P<0.01, bars indicate 95% CI, P-values were calculated using independent two-sample t-test). Green, reduced infectivity; yellow, no effect on infectivity. Source data is available for this figure in the Supplementary Information.

Figure 5

Lambda phage proteins gpG and frameshift product gpGT. (A) Schematic of lambda phage's frameshifting region in G and T. (B) Schematic of the arabinose-inducible region of the pBAD-λGT vector. (C) Immunoblotting of pBAD-λGT in BW25113, ΔiscU, ΔtusA, and ΔtusAΔiscU strains. We induced expression of the pBAD-λGT transcript with 0.2, 0.02, or 0.002% L-arabinose for 2 h and assessed the gpG and gpGT protein levels by immunoblotting against the Xpress Epitope tag. We found a decrease in gpGT levels in ΔiscU and an increase in ΔiscU and ΔtusAΔiscU.

Figure 6

Competitive binding of IscU and TusA to IscS: theory and experiment. (A) A schematic of our competitive binding model. IscS may bind and transfer sulfur to either IscU or TusA, leading to production of Fe-S cluster biosynthesis or thiolated tRNA, respectively. (B–I) Comparison of model predictions (top row) and experimental observations (bottom row) of infection time courses. The genetic perturbations include the deletion mutant strains ΔiscU (B, C) and ΔtusA (D, E), titration of expression from iscU (F, G) and the double deletion mutant strain ΔtusAΔiscU (H, I). (J) The host resistance metric calculated for the ΔtusA, ΔiscU, and ΔtusAΔiscU deletion mutant strains (^**P<0.01, bars indicate 95% CI, P-values were calculated using independent two-sample t-test). (K) The prediction of an alternate model—independent effect of IscU and TusA on viral biosynthesis—for an infection time course in the ΔtusAΔiscU background. Comparison of this plot with (H) and (I) indicates that the competitive binding model is better able to account for the experimental observations. Source data is available for this figure in the Supplementary Information.

Figure 7

APM northern blot of tRNA^Lys(UUU) in BW25113 and ΔiscU. tRNA northern blots for tRNA^Lys(UUU) in urea denaturing gels with and without APM. tRNA extracts were probed to determine the thiolated fraction of U34 in BW25113 and ΔiscU. A relative shift in the APM+ gel reflects the fraction of 2-thiolated U34 tRNA^Lys (tRNA^Lys s2U34). A small but detectable level of hypomodified tRNA^Lys (tRNA^Lys U34) was seen in BW25113 but was essentially undetectable in ΔiscU.

Figure 8

A network linking host resistance to viral infection to sulfur metabolism, tRNA modification, PRF, and competitive protein binding. IscS obtains sulfur from L-cysteine and passes it to either TusA or IscU. From TusA, the TUS pathway leads to thiolation by MnmA of tRNA^Lys (UUU) U34, which is also methylated by MnmE. During viral infection, the WT amount of modified tRNA leads to normal translation (and frameshifting) for the viral G and T genes, and consequently a favorable ratio of gpG:gpGT and virion production. Hypomodification of tRNA^Lys (UUU), whether by deletion of mnmE or the TUS pathway genes, or by overexpression of IscU, leads to increased frameshifting during translation of G and T. This increase in frameshifting leads to more gpGT expression and subsequently, a lower gpG:gpGT ratio and decreased virion production.