The circadian clock coordinates our physiology. Circadian disruption, as occurs during shift work, increases the risk of chronic diseases. For infectious diseases, circadian regulation of systemic immunity seems to underpin “time-of-day” differences in responses to extracellular pathogens. However, circadian rhythms are cell autonomous, and their interaction with intracellular pathogens, such as viruses, is poorly understood. We demonstrate that time of day of virus infection has a major impact on disease progression, in cellular models as well as in animals, highlighting the key role that cellular clocks play in this phenomenon. Clock disruption leads to increased virus replication and dissemination, indicating that severity of acute infections is influenced by circadian timekeeping.

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.

Diverse behavioral, physiological, and cellular processes exhibit daily (circadian) rhythms, which persist without external timing cues. Cell autonomous biological clocks drive circadian rhythms observed at the whole organism level, enabling adaptation to the 24-h cycle produced by the Earth’s rotation (1). At the molecular level, circadian oscillations are thought to be generated by genetic feedback loops involving the activating transcription factors BMAL1 (ARNTL/Mop3), NPAS, and CLOCK. These drive transcription of repressor proteins CRYPTOCHROME1/2 (CRY1/2) and PERIOD1/2 (PER1/2) that feedback to repress their own transcription, additionally regulated by myriad posttranslational processes (2⇓–4).

Circadian clocks confer competitive advantages to organisms. Their disruption incurs fitness costs, and they influence many aspects of human health and disease including sleep/wake cycles and immune function (5, 6). Indeed, many innate and adaptive immune responses are clock regulated. The immune response undergoes regeneration and repair as the host transitions to the resting phase of the daily cycle, but is primed for pathogen attack at the onset of the active phase (5, 6). Although changes in host responses to bacterial endotoxin or infection at different times of day have been reported (7, 8), the influence of host circadian clocks on progression of viral diseases is unknown. Here, we demonstrate dynamic host–virus interactions over the 24-h day and also show that genetic clock disruption augments virus replication in mice and cells.

Results

Viruses are obligate intracellular pathogens and require host organisms to proliferate. Over the course of a day, viruses may encounter host environments that are more or less conducive to replication and dissemination (5, 9, 10). We hypothesized that the time of day of infection would influence viral replication. To test this, we infected WT mice intranasally with a recombinant luciferase-expressing virus, Murid Herpesvirus 4 (M3:luc MuHV-4), at two times of day (Fig. 1A and Fig. S1A). As a rodent pathogen, this virus elicits natural host immune responses and implements evasion strategies in laboratory mice (11, 12), which allow it to establish latent (or quiescent) infection after primary infection. WT mice infected intranasally at the onset of resting phase [Zeitgeber Time 0 (ZT0); lights on], exhibited 10-fold higher viral replication than mice infected just before their active phase (ZT10) (Fig. 1A). This time-of-day effect required a functional clock because Bmal1–/– mice, which have no overt circadian rhythms (2), showed no difference when infected at different times (Fig. 1B and Fig. S1B). Furthermore, Bmal1–/– mice exhibited high levels of MuHV-4 infection when inoculated at either time of day (Fig. S1 C–F). Together, these results indicate that the timing of infection in relation the circadian cycle has major effects on herpesvirus pathogenesis.

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).

Because infection of Bmal1–/– mice resulted in high levels of virus replication in vivo (Fig. S1 D and F), we hypothesized that its role in clock function was important in regulating virus propagation. We therefore tracked M3:luc MuHV-4 infection longitudinally in WT and Bmal1–/– mice, infecting intranasally at ZT7: the time when BMAL1 is maximally active at genomic sites in peripheral tissues (9). Strikingly, virus replication increased greater than threefold at days 5–7 in Bmal1–/– mice compared with WT mice (Fig. 2 A and B and Fig. S2A). We saw a similar pattern when the acute infection spread to the superficial cervical lymph nodes (SCLNs) (Fig. 2 A and B). By contrast, latent infection was established to a similar extent in WT and Bmal1–/– mice (Fig. S2 B and C).

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).

To exclude that elevated infection levels were specific to MuHV-4, we infected mice with a different herpesvirus, herpes simplex virus 1 (HSV-1), by scarification of the left ear. We tracked the progression and extent of HSV-1 infection using a recombinant virus encoding luciferase under the control of the cytomegalovirus immediate early gene promoter (CMV:luc HSV-1) (13). Acute HSV-1 infection was significantly enhanced in arrhythmic Bmal1–/– mice (Fig. 2 C and D and Fig. S2 D and E), as with MuHV-4. As infection progressed, Bmal1–/– mice failed to contain HSV-1 spread, which disseminated across the head to the right ear (Fig. 2 C and D). As with MuHV-4, although acute infection was more severe when circadian rhythms were disrupted, latent infection was established to a similar extent in both genotypes. Although apparent trends toward higher numbers of latent viral genomes in Bmal1–/– mice was noted, this did not reach statistical significance (Fig. S2F), suggesting that circadian rhythms principally modulate primary infection in vivo.

A more vigorous immune response to incoming virus at the onset of the active phase might oppose MuHV-4 infection at ZT10 in vivo. We therefore investigated virus replication at different circadian times in synchronized cell models, which display robust ∼24-h rhythms but are not subject to systemic immune regulation (Fig. 3A). For our in vitro cellular clock model, we used confluent monolayers in which there were limited numbers of dividing cells, and no detectable circadian rhythm in cell cycle activity after synchronization (Fig. S3 and Movie S1). We used high resolution real-time bioluminescence recording to monitor both M3:luc MuHV-4 replication kinetics and the amount of virus replication (measured by total bioluminescence) correlated with infectious particle production (Fig. 3B and Fig. S4 A–C). Strikingly, when cell populations were infected with MuHV4 at different times in vitro, the time-of-day effect on infection observed in mice was recapitulated (Fig. 3 C and D). Total bioluminescence was significantly increased in cells infected during the rising phase of Bmal1 expression (CT18–24, indicated by open arrowheads) compared with cells infected during decline of Bmal1 expression (CT30–36, indicated by solid arrowheads) (Fig. 3C). Moreover, MuHV-4 infection at different times significantly altered the rate of virus replication (Fig. 3D). Indeed, the entire kinetic profile of infection depended on the circadian phase the virus encountered, such that slower initial replication rates were associated with prolonged viral gene expression [Fig. 3D and Fig. S4D; Pearson’s r = 0.999 (first cycle) or r = 0.982 (second cycle), P < 0.01].

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 (log 10 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 log 10 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 log 10 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).

Moreover, in agreement with our in vivo observations, MuHV-4 infection was significantly increased in primary Bmal1–/– fibroblasts compared with WT cells (Fig. 4 A and B and Movie S2). When synchronized WT and Bmal1–/– fibroblasts were infected at different circadian times (Fig. S5A; CT of infection indicated by open and solid arrowheads), the time-of-day effect on MuHV-4 infection in WT cells was abolished in those from Bmal1–/– mice (Fig. 4C and Fig. S5B). Additionally, HSV-1 replication was significantly enhanced in Bmal1–/– cells compared with WT cells (Fig. 4 D and E and Movie S3). Thus, the cellular circadian clock exerts a major effect on herpesvirus infection, indicating that our observations in live mice do not simply result from circadian modulation of immune cell function.

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 (log 10 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].

Given that cellular circadian rhythms impact on virus replication, we speculated that herpesviruses may manipulate the molecular clockwork during infection. To assess this, we infected mouse NIH 3T3 cells, expressing luciferase under the control of the Bmal1 promoter (Bmal:luc), with MuHV-4 at different circadian times (Fig. 5A and Fig. S6A). Interestingly, MuHV-4 acutely induced Bmal1 expression from ∼6 h after infection, irrespective of the circadian phase at which the cells were infected (one-way ANOVA: peak Bmal1:luc, P < 0.0001). The subsequent cellular circadian rhythms during viral infection depended on the time at which cells were infected. Virus-mediated Bmal1 induction during the endogenous fall in Bmal1 transcription generated a Bmal1 peak and disrupted circadian reporter expression (infection at CT18-24, indicated by open arrowhead in Fig. 5A and Fig. S5). In contrast, viral induction at other times (infection at CT30–36; indicated by solid arrowhead in Fig. 5A) enhanced the usual rise in Bmal1 transcription, and cellular rhythms remained robust for three cycles afterward (Fig. S6A). These findings strongly suggest that induction of Bmal1 expression by herpesviruses has different consequences for clock function depending on when in the circadian cycle infection occurs.

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).

Analogous to arrhythmic Bmal1–/– in vivo and cellular models, enhanced viral replication was observed in cells infected at circadian times when endogenous circadian rhythms were subsequently disrupted (Figs. 3 D and E and 5 A and B; indicated by open arrowheads). In mouse peripheral tissues that support herpesvirus replication, cellular CT24 corresponds to onset of the rest (light) period (14), where rapid, higher levels of initial replication would maximize the chance of transmission during the subsequent active (dark) phase 12–24 h later. Cellular CT36 corresponds to the onset of the active period, when slower, lower levels of replication would permit efficient transmission in the following active phase 24–36 h later and perhaps reduce detection at a time when the immune system is primed for pathogen attack.

Critically, expression of repressive clock genes, such as mCryptochrome1 (mCry1) and mPeriod2 (mPer2), was not induced during viral infection (Fig. 5C and Fig. S6B), but a significant, rapid reduction was seen when cells were infected at CT18 (Fig. 5C). These findings are consistent with MuHV-4 infection ushering cells from a repressive circadian phase to one where BMAL1 is active, via sustaining Bmal1 expression and relieving CRYPTOCHROME-mediated repression. Furthermore, HSV-1 infection also acutely up-regulated Bmal1 (Fig. 5D), even more so than MuHV-4, suggesting that Bmal1 is specifically targeted by both α- and γ-herpesvirus families. In support of this, Bmal1 expression is induced in cells overexpressing viral transcriptional activators from either herpesviruses (Fig. S7), and interactions between BMAL1/CLOCK and several HSV-1 transcriptional activators in vitro have been reported previously (15, 16).

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).

Herpesviruses co-opt cellular transcriptional mechanisms to replicate and target clock transcription factors (Fig. 5). We next asked if the impact of BMAL1 ablation on viral infection extended beyond direct transcriptional regulation, to the global changes in cellular physiology that occur when circadian rhythms are disrupted. To investigate this, we infected WT and Bmal1−/−cells with the orthomyxovirus, influenza A (IAV) (Fig. 6 A and B). IAV replicates within the nucleus but encodes its own RNA-dependent RNA polymerase and therefore does not directly use the host cell’s transcriptional machinery for viral gene expression, in contrast to herpesviruses. Remarkably, loss of BMAL1 also significantly augmented IAV protein expression and replication (PB2::GLUC bioluminescence two-way ANOVA: genotype effect, P = 0.0004; single-cycle growth two-way ANOVA: genotype effect, P = 0.0102). The similar impact of cellular arrhythmicity on two disparate, clinically relevant virus families implies a broader influence of circadian clocks, and specific components such as BMAL1, on viral infection.

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.

To determine which cellular systems underpin the time-of-day effect on viral replication, we first identified proteins that exhibit changes in abundance between opposite circadian phases (CT18 vs. CT30) in WT cells, when viral replication in WT, but not in Bmal1−/− cells, was significantly different (Fig. 4C). Given that virus infection is augmented in Bmal1−/− cells at both time points (Fig. 4C), we then focused on the subset of proteins within this group whose abundance was either increased or decreased at both of these times in Bmal1−/− cells compared with WT cells (Fig. 6 C and D and Fig. S8). Circadian-regulated proteins expressed at higher levels in Bmal1−/− cells were enriched for those involved in protein biosynthesis (Fig. 6C, Fig. S8A, and Table S1), including amino acid biosynthesis, ribosome structure, translation, and protein folding clusters. Additionally, proteins involved in endoplasmic reticulum function, protein localization, and intracellular vesicle trafficking were significantly enriched. These results indicate that enhanced capability for viral protein biosynthesis, assembly, and egress contribute to clock control of virus replication. Conversely, circadian-regulated proteins expressed at lower levels in Bmal1−/− cells were enriched for those involved in organization of the cortical actin cytoskeleton and chromatin assembly (Fig. 6D, Fig. S8B, and Table S2), suggesting that virus particle uncoating, genome trafficking, and histone association contribute to clock control of virus replication. Thus, clock-mediated effects on viral infection in cells can be ascribed to discrete functional categories of protein effectors targeting specific aspects of the virus replication cycle.

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.

Table S1. Enrichment cluster scores and P values of DAVID enrichment analysis