Feedback-mediated signal conversion promotes viral fitness

Abstract

A fundamental signal-processing problem is how biological systems maintain phenotypic states (i.e., canalization) long after degradation of initial catalyst signals. For example, to efficiently replicate, herpesviruses (e.g., human cytomegalovirus, HCMV) rapidly counteract cell-mediated silencing using transactivators packaged in the tegument of the infecting virion particle. However, the activity of these tegument transactivators is inherently transient-they undergo immediate proteolysis but delayed synthesis-and how transient activation sustains lytic viral gene expression despite cell-mediated silencing is unclear. By constructing a two-color, conditional-feedback HCMV mutant, we find that positive feedback in HCMV's immediate-early 1 (IE1) protein is of sufficient strength to sustain HCMV lytic expression. Single-cell time-lapse imaging and mathematical modeling show that IE1 positive feedback converts transient transactivation signals from tegument pp71 proteins into sustained lytic expression, which is obligate for efficient viral replication, whereas attenuating feedback decreases fitness by promoting a reversible silenced state. Together, these results identify a regulatory mechanism enabling herpesviruses to sustain expression despite transient activation signals-akin to early electronic transistors-and expose a potential target for therapeutic intervention.

Keywords: feedback circuitry; mathematical model; single-cell imaging; virus.

Fig. 1.

Tegument-derived pp71 is transient in cells, but modeling predicts that positive feedback could sustain transactivation. (A) Schematic of initial regulatory events occurring during infection of cells with HCMV. (B) Time-lapse fluorescence microscopy of ARPE-19 cells synchronously infected with an EYFP-pp71 HCMV virus (AD169) at MOI of 1 and quantification of pp71 cellular levels over the first 11 h of infection (an average of 162 cells). (C and D) A mathematical model describing pp71 and IE1 dynamics. Eqs. 1 and 2 were solved numerically, using ODE45 solver (MATLAB; MathWorks). ST_I was calculated for every set of parameters as the ratio between IE1 expression at 24 h after infection and maximal IE1 expression. For parameters, see SI Appendix, Table S2.

Fig. 2.

IE1 positive feedback sustains expression from the HCMV immediate-early promoter. (A) Map of the MIE region of the recombinant HCMV TB40/E IE1 conditional-mutant dual-reporter virus (IE1-CMDR) showing the MIEP (black) and IE region exons 1–5 (gray), along with the recombinant fusions: Exon 2 is genetically fused at its N terminus to mCherry (mCh, red) and the FKBP degron tag (hashed) that destabilizes the IE1 protein in the absence of the small molecule Shield-1; exon 5 also contains a fusion to EYFP (green) at its C terminus. Translation typically begins at the 5′ end of exon 2 (now FKBP). See also SI Appendix, Fig. S3. (B) Schematic of the putative effects of Shield-1 on IE1-mediated positive feedback. (C) Degradation rate of IE1 in cells infected with IE1-CMDR virus as measured by flow cytometry. Protein synthesis was blocked with cycloheximide at 24 h after infection (time = 0) and the degradation rate was measured ±1 μM Shield-1. Shown is the average of two repeats, and error bars denote SD. Decay rate was calculated by fitting the data to an exponential decay model (solid lines). (D) Representative images from fluorescence time-lapse microscopy of IE-mCherry expression in cells infected with IE1-CMDR virus. Cells were cultured in medium containing 1 μM Shield-1 (Top) or without Shield-1 (Bottom). Cells were cultured in medium with 1 μM Shield-1 (Top, wild-type feedback) or without Shield-1 (Bottom, attenuated feedback) and tracked over time; “bg” represents background autofluorescence image. (E) IE-mCherry expression of cells infected with IE1-CMDR virus. Cells were cultured in medium with 1 μM Shield-1 (Left, wild-type feedback, averaged over 119 cells) or without Shield-1 (Right , attenuated feedback, averaged over 153 cells). Bold line denotes mean (i.e., general trend) of the population with gray shading showing SE. Cell trajectories were digitally synchronized to the first detection of mCherry signal. (Inset) The fraction of cells with sustained (S, ST_I > 0.5) or transient (T, ST_I < 0.5) IE-mCherry expression over three biological repeats. (F) HF-ACF of cells infected with IE1-CMDR virus in the presence of 1 μM Shield-1 (dark gray) or without Shield-1 (light gray). Shown is an average over 100 cells each; error bars denote SE. (G and H) IE-mCherry mean expression level and noise (CV) 15 h after infection. Dashed line is the expected value from change in IE1 half-life alone (Δ from τ_1/2), whereas the additional difference (Δ) is ascribed to the loss of IE1 positive feedback. (I) Relative IE-mCherry mRNA levels (from RT-qPCR) in cells infected with IE1-CMDR virus ±1 μM Shield-1 (P < 0.01, two-tailed t test). (J) Degradation rate of IE2-EYFP in cells infected with IE1-CMDR virus as measured by flow cytometry. Protein synthesis was blocked with cycloheximide at 24 h after infection (time = 0) and degradation rate was measured ±1 μM Shield-1. Shown is the average of two repeats, and error bars denote SD. Decay rate was calculated by fitting the data to an exponential decay model (solid lines). (K) Flow cytometry for IE2-EYFP levels in cells infected with IE1-CMDR virus. Cells were cultured in medium with 1 μM Shield-1 or without Shield-1. (Inset) Normalized mean fluorescence of IE2-EYFP. (L) Relative IE2-EYFP mRNA levels (from RT-qPCR) in cells infected with IE1-CMDR virus ±1 μM Shield-1 (P = 0.012, two-tailed t test).

Fig. 3.

Attenuated IE1 positive feedback decreases replication fitness, but feedback can be rescued to restore fitness. (A) Viral titers from cells infected with IE1-CMDR virus. Cells were cultured in media with 1 μM Shield-1 (black, wild-type feedback) or without Shield-1 (gray, attenuated feedback). Virus was harvested at different time points after infection. Viral titers were measured in medium containing 1 μM Shield-1; error bars denote SD. (B) IE-mCherry expression from time-lapse single-cell microscopy of cells infected with IE1-CMDR virus. Cells were cultured in medium without Shield-1 for 24 h and then supplemented with 1 μΜ Shield-1 for 12 h. Bold line denotes mean mCherry expression (67 cells); gray shading denotes SE. (C) Viral titers from cells infected with IE1-CMDR virus. Cells were cultured in medium with 1 μM Shield-1 (solid line), or Shield-1 was added to the medium after 24 h (dashed line). Virus was harvested at different time points after infection, titers were measured in media containing 1 μM Shield-1, and error bar denotes SD.

Fig. 4.

Increased pp71 abundance partially rescues expression and fitness of attenuated feedback virus. (A) Western blot analysis of pp71 in viral particles packaged on cells overexpressing pp71 (IE1-CMDR-pp71^hi) or on nonoverexpressing cells (IE1-CMDR-pp71^wt). (B) Representative time-lapse microscopy traces of IE-mCherry expression in cells infected with IE1-CMDR-pp71^hi or IE1-CMDR-pp71^wt. Cells were cultured without Shield-1 (attenuated feedback) and classified as sustained or transient based on expression kinetics as defined above. Bold line denotes mean mCherry signal of sustained or transient cells; shaded area denotes SE. Cell trajectories were digitally synchronized to the first detection of mCherry signal. (C) The fraction of cells with sustained IE-mCherry expression (ST_I > 0.5) after infection with IE1-CMDR-pp71^hi virus (116 cells) or IE1-CMDR-pp71^wt virus (243 cells). Cells were cultured without Shield-1 (attenuated feedback). P < 0.001 was calculated using a Fisher exact test. (D) High pp71 can partially compensate the fitness lost when feedback is attenuated. Viral titers from cells infected with IE1-CMDR-pp71^hi virus ± Sheild-1. Cells were infected in medium with 1 μM Shield-1 (black, wild-type feedback) or without Shield-1 (gray, attenuated feedback), virus was harvested at indicated times after infection, and titers were measured in medium supplemented with 1 μM Shield-1. Compare with Fig. 3A. (E) High pp71 partially compensates for fitness lost when feedback is attenuated. Relative single-round fitness (titer at day 4) for either IE1-CMDR-pp71^wt (Left) and IE1-CMDR-pp71^hi (Right) when feedback is attenuated (−Shield-1) normalized to viral titer for corresponding wild-type feedback (+Shield-1) case (P = 0.041, two-tailed t test).