Cell autonomous regulation of herpes and influenza virus infection by the circadian clock

Abstract

Viruses are intracellular pathogens that hijack host cell machinery and resources to replicate. Rather than being constant, host physiology is rhythmic, undergoing circadian (∼24 h) oscillations in many virus-relevant pathways, but whether daily rhythms impact on viral replication is unknown. We find that the time of day of host infection regulates virus progression in live mice and individual cells. Furthermore, we demonstrate that herpes and influenza A virus infections are enhanced when host circadian rhythms are abolished by disrupting the key clock gene transcription factor Bmal1. Intracellular trafficking, biosynthetic processes, protein synthesis, and chromatin assembly all contribute to circadian regulation of virus infection. Moreover, herpesviruses differentially target components of the molecular circadian clockwork. Our work demonstrates that viruses exploit the clockwork for their own gain and that the clock represents a novel target for modulating viral replication that extends beyond any single family of these ubiquitous pathogens.

Keywords: circadian; clock; herpes; influenza; virus.

Fig. 1.

Herpesvirus infection in mice is regulated by the circadian clock. (A) WT female mice were intranasally infected with M3:luciferase Murid Herpesvirus 4 (M3:luc MuHV-4) at Zeitgeber Time 0 (ZT0) (lights on; n = 6) or at ZT10 (n = 6). Schematic illustrates Bmal1 mRNA levels and active (genome-bound) BMAL1 protein over the day and night. Infection was monitored by bioluminescence imaging. Primary infection in the nose is higher in mice inoculated at the onset of the resting phase (ZT0) compared with infection before the active phase (ZT10) [mean ± SEM; two-way ANOVA (ZT of infection × time postinfection): ZT of infection effect, P = 0.0021; post hoc t tests, *P < 0.05]. See also Fig. S1A. (B) Female Bmal1^−/− mice were infected with M3:luc MuHV-4 at either ZT0 (n = 5) or ZT10 (n = 6) and infection monitored as for A [mean ± SEM; two-way ANOVA (ZT of infection × time postinfection): ZT of infection effect, P > 0.05; NS = not significant). See also Fig. S1B.

Fig. S1.

M3:luc MuHV-4 infection in WT and Bmal1^−/− mice infected at ZT0 vs. ZT10. (A) Individual subject plots from Fig. 1A. WT mice show higher levels of MuHV-4 infection at ZT0 vs. ZT10 (mean ± SEM; n = 6). (B) Individual subject plots from Fig. 1B. No significant difference in MuHV-4 pathogenesis is observed in Bmal1^−/− mice infected at ZT0 (n = 5) and ZT10 (n = 6) (mean ± SEM). (C) No significant difference in MuHV-4 intranasal infection is observed between WT and Bmal1^−/− mice infected at ZT0 [mean ± SEM; n = 5 (Bmal1^−/− group), n = 6 (WT group); maximum radiance two-way ANOVA (genotype × time postinfection): genotype effect, P > 0.05; NS = not significant]. (D) MuHV-4 intranasal infection is significantly greater in Bmal1^−/− mice vs. WT mice infected at ZT10 [mean ± SEM; n = 6; maximum radiance two-way ANOVA (genotype × time postinfection): genotype effect, ***P < 0.001; post hoc t test: **P < 0.01 ***P < 0.001]. (E) No significant difference in MuHV-4 infection of the SCLNs is observed between WT mice infected at ZT0 vs. ZT10 or between Bmal1^−/− mice infected at ZT0 vs. ZT10 [maximum radiance two-way ANOVA (time of infection × time postinfection): time of infection effect, P > 0.05, NS = not significant]. (F) MuHV-4 SCLN infection in WT and Bmal1^−/− mice infected at ZT0 and ZT10. SCLN infection is significantly higher in Bmal1^−/− mice vs. WT infected at ZT10 on day 9 after infection (post hoc t test, *P < 0.05).

Fig. 2.

Herpesvirus infection is augmented in arrhythmic Bmal^−/− mice. (A) WT (n = 6) and Bmal1^−/− (n = 5) female mice were intranasally infected with M3:luc MuHV-4 at ZT7. Extent and spread of infection was monitored by bioluminescence imaging. Representative images are shown with overlaid bioluminescence radiance measurements. (B) M3:luc MuHV-4 progressively disseminates from the nose to the SCLNs and is significantly higher in Bmal1^−/− mice [mean ± SEM; nose two-way ANOVA (genotype × time postinfection): genotype effect, P = 0.0031; SCLN two-way ANOVA (genotype × time postinfection): genotype effect, P = 0.0348; post hoc t tests: *P < 0.05, **P < 0.01, ***P < 0.001]. See also Fig. S2A. (C) Male WT (n = 5) and Bmal1^−/− (n = 6) mice were infected with CMV:luciferase (CMV:luc) herpes simplex virus 1 (HSV-1) by scarification of the left ear at ZT7. Extent and spread of infection was monitored and images presented as for A. (D) CMV:luc HSV-1 progressively disseminates from the left ear to the head and right ear and is significantly higher in Bmal1^−/− mice [mean ± SEM; left ear two-way ANOVA (genotype × time postinfection): genotype effect, P = 0.0004; right ear two-way ANOVA (genotype × time postinfection): genotype effect, P = 0.0054; post hoc t tests: *P < 0.05, **P < 0.01]. See also Fig. S2E.

Fig. S2.

M3:luc MuHV-4 and CMV:luc HSV-1 primary and latent infection in WT and Bmal1^−/− mice. (A) Individual subject plots from Fig. 2B. During primary infection, MuHV-4 progressively spreads from the nose to SCLNs [mean ± SEM; n = 5 (Bmal1^−/− group), n = 6 (WT group)]. Infection in the nose and SCLNs is significantly higher in Bmal1^−/− mice vs. WT [nose maximum radiance two-way ANOVA (genotype × time postinfection): genotype effect, P = 0.0031; SCLN maximum radiance two-way ANOVA (genotype × time postinfection): genotype effect, P = 0.0348]. (B) Twenty-four days after infection, mice were culled. Latent viral genome loads in the spleen were analyzed by qPCR, which compares MuHV-4 M2 gene copy number with cellular APRT gene copy number (1000xM2/APRT) [mean ± SEM; n = 5 (Bmal1^−/− group), n = 6 (WT group); two-tailed t-test P > 0.05; F-test, ***P = 0.0001]. See Methods for further details. (C) Reactivation of latent MuHV-4 in the spleen was assessed by the number infectious centers (plaques) on cell monolayers cocultured with ex vivo splenocytes [mean ± SEM; n = 5 (Bmal1^−/− group), n = 6 (WT group); two-tailed t-test, P > 0.05; F-test, *P = 0.0228]. Thus, no statistically significant difference between mean values of MuHV-4 latent infection was observed by either infectivity assay. (D) Dissemination of HSV-1 infection from the left ear to the chest is significantly increased in Bmal1^−/− mice vs. WT [chest maximum radiance two-way ANOVA (genotype × time postinfection): genotype effect, P = 0.0037]. (E) Individual subject plots from Fig. 2D. During primary infection, HSV-1 progressively spreads from the left ear to the right ear and chest [mean ± SEM; n = 5 (WT group); n = 6 (Bmal1^−/− group)]. Infection in the left ear is significantly higher in Bmal1^−/− mice vs. WT [maximum radiance two-way ANOVA (genotype × time postinfection): genotype effect P = 0.0004]. HSV-1 spreads to secondary sites more effectively in Bmal1^−/− mice vs. WT: n = 4 of 6 Bmal1^−/− mice showed substantial infection in the right ear, whereas this is evident in only n = 1 of 5 WT mice. Similarly, n = 5 of 6 Bmal1^−/− mice showed dissemination of HSV-1 to the chest, vs. n = 3 of 5 WT mice. Virus infection in the chest is significantly increased in Bmal1^−/− mice compared with WT [maximum radiance two-way ANOVA (genotype × time postinfection): genotype effect, P = 0.0037]. (F) Twenty-four days after infection, mice were culled. Viral genome loads in the dorsal root ganglion were analyzed by qPCR, which compares HSV-1 ICP0 gene copy number with cellular APRT gene copy number (1000xICP0/APRT). See Methods for further details. No statistically significant difference in HSV-1 latent genome load was observed (two-tailed t-test, P > 0.05).

Fig. 3.

Circadian rhythms modulate herpesvirus replication in cells. (A) Bioluminescence recordings from control (uninfected) temperature-synchronized Bmal1:luciferase (Bmal1:luc) and Per2:luciferase (Per2:luc) circadian reporter NIH 3T3 cells (mean ± SEM; n = 3). Peak Bmal1:luc bioluminescence is designated Circadian Time 24 (CT24). Colored arrows indicate circadian times (CT) at which parallel cultures of synchronized NIH 3T3 cells were infected with M3:luc MuHV-4. (B) Representative bioluminescence recording and kinetic analysis parameters of M3:luc MuHV-4 replication using asymmetrical sigmoidal nonlinear regression. See Fig. S4 A and B for raw bioluminescence recordings obtained from cells infected at different CTs and R² regression coefficients. (C) Amount of MuHV-4 replication varies significantly depending on the circadian time of infection (mean ± SEM; n = 3; one-way ANOVA: total bioluminescence, P = 0.0178; multiple comparisons, *P < 0.05). Total bioluminescence calculated by the area under curve method (AUC) and normalized (0% = baseline total bioluminescence between 0 and 1 h after fection, 100% = maximum total bioluminescence value), with variation across different CTs presented as (% total bioluminescence – mean % total bioluminescence across all experimental CTs). See Fig. S4C for correlation analysis of total bioluminescence and infectious particle production (log₁₀ pfu). Open arrowheads highlight CT18/24 (higher infection) and solid arrowheads highlight CT30/36 (lower infection). (D) The rate of viral gene expression varies significantly depending on the circadian time of infection (one-way ANOVA: Hill slope, P < 0.0001; post hoc multiple comparisons: **P < 0.01, ***P < 0.001).

Fig. S3.

Replicative activity of confluent cell monolayers after synchronization. (A) Confluent NIH 3T3 cell monolayers stably transduced with dual FUCCI reporters amCyan::Geminin and mCherry::Cdt1 were trypsinized, stained with DNA dye DRAQ5, and analyzed by flow cytometry. mCherry::Cdt1 is expressed during G1 phase (2n DNA content), whereas amCyan::Geminin is expressed during S/G2 phase (2 < n ≤ 4 DNA content). (B) Representative images of confluent FUCCI reporter NIH 3T3 cell monolayers at different circadian times after synchronization (red indicates mCherry::Cdt1; blue indicates amCyan::Geminin; Movie S1). (C) Confluent FUCCI reporter NIH 3T3, primary WT, and Bmal1^−/− fibroblast monolayers were synchronized and imaged between CT0–66 h (Movie S1). Cells expressing either amCyan::Geminin or mCherry::Cdt1 were counted at the stated CTs (n = 5 fields of view for each cell type; >300 cells observed per time point). Across all CTs, G2 phase amCyan::Geminin-positive cells accounted for 5.60 ± 1.4%, 1.70 ± 0.31%, and 3.35 ± 0.79% (mean ± SEM) of 3T3s, WT, and Bmal1^−/− monolayers, respectively. Linear regression analysis shows a significant negative correlation between time after synchronization and % G2 phase amCyan::Geminin-positive cells for NIH 3T3 and Bmal1^−/− fibroblasts, but not for WT fibroblasts (3T3s: R² = 0.9223, Pearson r = −0.960, P < 0.001; Bmal1^−/−: R² = 0.965, Pearson r = −0.9780, P < 0.001; WT: R² = 0.317, Pearson r = −0.563, P = 0.1145). Critically, for all three cell types, we could detect no circadian oscillation in the ratio of G1 to G2 phase cells. Damped sine wave modeling (nonlinear regression) yields best-fit period values >50 h (not within circadian range 18–30 h) and two-way ANOVA (cell cycle phase × circadian time): circadian time effect, P > 0.05. Additionally, comparison of cell cycle phase markers between WT and Bmal1^−/− cell types at each circadian time by multiple two-tailed t tests revealed no significant results (FDR, Q = 1%).

Fig. S4.

Kinetics and total amount of MuHV-4 single-cycle replication are a function of the circadian time at which cells are infected. (A) Raw bioluminescence recordings from temperature-synchronized NIH 3T3 cells infected with M3:luc MuHV-4 at 6-h intervals from CT42 to CT66 (mean; n = 3). cps = counts per second. (B) Coefficients of determination (R²) for asymmetric sigmoidal nonlinear regression of data from Fig. 3. (C) Parallel cultures of primary WT fibroblasts were incubated with M3:luc MuHV-4 at different MOIs between 0.001 and 2 pfu per cell. After 2 h, cells were acid-washed to remove the input virus. Real-time bioluminescence was recorded and the amount of infectious MuHV-4 particles produced at 0, 12, 24, 48, and 96 h after infection was determined by plaque assay (mean ± SEM; n = 3). Over this range of MOI, total bioluminescence during exponential growth (AUC) linearly correlates with log₁₀ pfu (linear regression analysis: R² = 0.677, P < 0.0001; Pearson’s r = 0.823; a 23.56% difference in total bioluminescence, ∼10-fold change pfu). (D) Time to 50% peak infection and 50% decrease in peak infection varies significantly depending on the circadian time of infection (mean ± SEM; n = 3; one-way ANOVA: time to 50% peak infection P = 0.0002; one-way ANOVA: time to 50% decrease in peak infection, P < 0.0001; post hoc multiple comparisons: **P < 0.01 ***P < 0.001). Over each circadian cycle, there is a significant linear correlation between the time to 50% peak infection and the time to 50% decrease in peak infection [Pearson’s r = 0.999 (first cycle) P = 0.022; or r = 0.982 (second cycle) P = 0.006]. Infection is sustained less robustly at circadian times that yield more rapid viral gene expression initially, with the entire kinetic profile of infection depending on the circadian time of infection. (E) Parallel cultures of NIH 3T3 fibroblasts were incubated with CMV:luc HSV-1 at different MOIs between 0.001 and 10 pfu per cell. After 1 h, cells were acid-washed to remove the input virus. Real-time bioluminescence was recorded and the amount of infectious MuHV-4 particles produced at 0, 8, 24, 48, and 72 h after infection was determined by plaque assay (mean ± SEM; n = 3). Over this range of MOIs, total bioluminescence during exponential growth (AUC) linearly correlates with log₁₀ pfu (linear regression analysis: R² = 0.706, P = 0.0002; Pearson’s r = 0.840; 15.9% difference in total bioluminescence, ∼10-fold change pfu).

Fig. 4.

Herpesvirus replication is enhanced in Bmal1^−/− cells. (A) Pseudocolored bioluminescence image of WT and Bmal1^−/− primary cells infected with M3:luc MuHV-4. See also Movie S2. (B) Representative bioluminescence recordings of synchronized WT and Bmal1^−/− primary cells infected with M3:luc MuHV-4 (mean ± SEM; n = 3). (C) Synchronized WT and Bmal1^−/− primary cells were infected with M3:luc MuHV-4 at either CT18 or CT30. MuHV-4 replication is significantly increased in Bmal1^−/− cells compared with WT cells (mean ± SEM; n = 3) [total bioluminescence (AUC) normalized as for Fig. 3C; two-way ANOVA (genotype × CT of infection): genotype effect, P < 0.0001]. Time-of-day effect on viral replication is observed in WT cells, but not Bmal1^−/− cells [total bioluminescence two-way ANOVA (genotype × CT of infection): post hoc multiple comparisons: NS = not significant, *P < 0.05). See Fig. S5 for circadian reporter controls and M3:luc MuHV-4 kinetic analysis. (D) Pseudocolored bioluminescence image of WT and Bmal1^−/− primary cells infected with CMV:luc HSV-1. See also Movie S3. (E) CMV:luc HSV-1 replication is significantly increased in Bmal1^−/− cells compared with WT cells (mean ± SEM; n = 3). Total bioluminescence (AUC) normalized as for Fig. 3C (two-tailed t test: ***P < 0.001). See Fig. S4E for correlation analysis of total bioluminescence and infectious particle production (log₁₀ pfu).

Fig. S5.

Circadian time effect on MuHV-4 kinetics in WT but not Bmal1^−/− cells. (A) Dexamethasone-synchronized mPeriod2:luciferase (Per2:luc) and Bmal1: luciferase (Bmal1:luc) circadian reporter fibroblasts (mean ± SEM; n = 3). Circadian controls for synchronization protocol used in Figs. 4C and 6 C and D. In Fig. 4C, dexamethasone-synchronized WT and Bmal1^−/− primary cells were infected with M3:luc MuHV-4 at either CT18 (open arrowhead) or CT30 (solid arrowhead). (B) Kinetic analysis of experiment described in Fig. 4C. Kinetic analysis was performed as shown in Fig. 3B (R² regression coefficients: WT CT18 = 0.9782, WT CT30 = 0.9932, Bmal1^−/− CT18 = 0.9668, Bmal1^−/− CT30 = 0.9768). Time to 50% peak infection is significantly decreased in Bmal1^−/− cells compared with WT cells [two-way ANOVA (genotype × circadian time of infection): genotype effect, P < 0.0001]. Time-of-day effect on viral replication is observed in WT cells, but not Bmal1^−/− cells [time to 50% peak infection two-way ANOVA (genotype × circadian time of infection): post hoc multiple comparisons: NS = not significant, ***P < 0.001].

Fig. 5.

Virus infection differentially affects clock gene expression. (A) Bioluminescence recordings from synchronized Bmal1:luciferase (Bmal1:luc) circadian reporter NIH 3T3 cells either mock infected or infected with MuHV-4 at CT18 (open arrowhead) and CT30 (solid arrowhead). Mean baseline-subtracted (detrended) bioluminescence (n = 3 per group) shown. Infection at CT18 induced an additional peak in Bmal1:luc expression, disrupting the circadian rhythm. Infection at CT30 induced Bmal1:luc that synergizes with circadian Bmal1:luc expression and preserves rhythms. (B) Peak bioluminescence from synchronized Bmal1:luc cells either mock infected or infected with MuHV-4 at 3-h intervals from CT18 to CT39 (mean ± SEM; n = 3). Bmal1:luc expression is significantly increased, irrespective of the circadian time of infection (one-way ANOVA P < 0.0001; post hoc multiple comparisons: *P < 0.05, **P < 0.01, ***P < 0.001). For raw bioluminescence recordings and error boundaries, see Fig. S6A. (C) Baseline-subtracted (detrended) bioluminescence traces from synchronized mCryptochrome1:luciferase (Cry1:luc) circadian reporter NIH 3T3 cells (mean; n = 3). (Inset) Raw bioluminescence traces (mean ± SEM; n = 3). Cry1:luc is significantly decreased during MuHV-4 infection (postinfection peak bioluminescence two-tailed t test, *P = 0.0188). (D) Bioluminescence recording from synchronized Bmal1:luc cells mock infected or infected with HSV-1 at CT36 (solid arrowhead) (mean ± SEM; n = 3). Bmal1:luc expression is significantly increased during HSV-1 infection (postinfection peak bioluminescence two-tailed t test, ***P < 0.001).

Fig. S6.

MuHV-4 infection rapidly induces Bmal1 expression. (A) Raw and detrended (baseline-subtracted) bioluminescence recordings from synchronized Bmal1:luc circadian reporter NIH 3T3 cells either mock infected or infected with MuHV-4 at 3-h intervals from CT = 18 h to CT = 39 h. Gray lines indicate CT of infection. (Top) Raw Bmal1:luc bioluminescence recordings (counts per second) (mean ± SEM boundaries; n = 3). (Bottom) Detrended Bmal1:luc bioluminescence analysis (moving-average subtracted; mean± SEM boundaries; n = 3). Selected data are presented in Fig. 5A (infection at CT = 18 and 30 h), and peak Bmal1:luc bioluminescence data are summarized in Fig 5B. (B) Bioluminescence traces from synchronized Per2:luc circadian reporter NIH 3T3 cells (mean; n = 3) either mock infected or infected with MuHV-4 (gray line indicates CT of infection). (Inset) Raw bioluminescence traces (mean ± SEM).

Fig. S7.

Bmal1 expression is induced in cells overexpressing herpesvirus transcriptional activators. (A) Synchronized NIH 3T3 cells expressing Bmal1:luc transcriptional reporter were either mock-infected or infected with WT MuHV-4 or M50 MHV-68, a recombinant virus that overexpresses ORF50, which encodes the main viral transcriptional transactivator. Bmal1:luc bioluminescence is significantly increased during M50 MuHV-4 infection compared with WT MuHV-4 or mock-infected controls (mean ± SEM; n = 3; one-way ANOVA: P = 0.0049; post hoc multiple comparisons: *P < 0.05, **P < 0.01). (B) An adenoviral Tet-On system was used to investigate whether the HSV-1 viral transactivator ICP0 can initiate Bmal1 transcription. Synchronized NIH 3T3 cells expressing the Bmal1:luc transcriptional reporter were infected with adenoviral constructs expressing rtTA from the HCMV IE promoter (Ad.CMV.rtTA), ICP0 under the control of a TRE promoter (Ad.TRE.ICP0), a nonfunctional RING-finger deletion mutant (FXE) of ICP0 under the control of a TRE promoter (Ad.TRE.FXE), or a combination thereof. Doxycycline (Dox) was added 46 h after infection to enable transcription from the TRE promoter if rtTA is present. ICP0 significantly increases Bmal1:luc (% change 3 h pre-Dox vs. 3 h post-Dox addition) compared with controls (mean ± SEM; n = 3; one-way ANOVA: P = 0.0038; post hoc multiple comparisons: **P < 0.01, ***P < 0.001).

Fig. 6.

Global proteomic comparison of WT and Bmal1^−/− cells reveals clock-regulated pathways that impact on viral replication. (A) Influenza A viral protein expression was enhanced in Bmal1^−/− cells. WT and Bmal1^−/− cells were infected with PB2::GLUC (Gaussia luciferase) influenza A virus (IAV) and luciferase activity quantified at stated intervals. Rate of PB2 expression was increased in Bmal1^−/− compared with WT cells [mean ± SEM; n = 3; two-way ANOVA (genotype × time postinfection): genotype effect, P = 0.0004; interaction, P < 0.0001; post hoc multiple comparisons: *P < 0.05, **P < 0.01, ***P < 0.001), as was total PB2 expression (sigmoidal nonlinear regression: WT R² = 0.9902, Bmal^−/− R² = 0.9836; total PB2::GLUC bioluminescence (AUC) two-tailed Student t test: **P < 0.0019]. (B) Single-cycle IAV growth was enhanced in Bmal1^−/− cells. IAV-infected cells were harvested and amount of infectious IAV particles determined by plaque assay [two-way ANOVA (genotype × time postinfection): genotype effect, *P = 0.0102]. (C) Synchronized WT and Bmal1^−/− primary cells were harvested at CT18 and CT30 and global proteomics performed by LC coupled to MS (n = 3). DAVID functional annotation clustering analysis of proteins that significantly differed at CT18 vs. CT30, and significantly increased in Bmal1^−/− cells compared with WT cells at both CT18 and CT30. Protein number represented by node size and cluster P value by node grayscale. Annotations were prescribed by the Markov cluster algorithm. Number of nodes per group represented by label size. See Fig. S8A for heat map analysis and Table S1 for enrichment scores. (D) Proteomics analysis performed as in C. DAVID functional annotation clustering analysis of proteins that significantly differed at CT18 vs. CT30 and significantly decreased in Bmal1^−/− cells compared with WT cells at both CT18 and CT30. Proteins are represented as in C. See Fig. S8B for heat map analysis and Table S2 for enrichment scores.

Fig. S8.

Proteins that show significantly different expression levels at between WT and Bmal1^−/− cells. (A) Proteins whose abundance significantly changes at CT18 vs. CT30 and is significantly increased in Bmal1^−/− cells compared with WT cells at both CT18 and CT30. See Fig. 6C and Table S1 for DAVID functional annotation clustering analysis. (B) Proteins whose abundance significantly changes at CT18 vs. CT30 and is significantly decreased in Bmal1^−/− cells compared with WT cells at both CT18h and CT30. See Fig. 6D and Table S2 for DAVID functional annotation clustering analysis.