Single-Cell Analysis of the Impact of Host Cell Heterogeneity on Infection with Foot-and-Mouth Disease Virus

ABSTRACT

Viral infection and replication are affected by host cell heterogeneity, but the mechanisms underlying the effects remain unclear. Using single-cell analysis, we investigated the effects of host cell heterogeneity, including cell size, inclusion, and cell cycle, on foot-and-mouth disease virus (FMDV) infection (acute and persistent infections) and replication. We detected various viral genome replication levels in FMDV-infected cells. Large cells and cells with a high number of inclusions generated more viral RNA copies and viral protein and a higher proportion of infectious cells than other cells. Additionally, we found that the viral titer was 10- to 100-fold higher in cells in G₂/M than those in other cell cycle phases and identified a strong correlation between cell size, inclusion, and cell cycle heterogeneity, which all affected the infection and replication of FMDV. Furthermore, we demonstrated that host cell heterogeneity influenced the adsorption of FMDV due to differences in the levels of FMDV integrin receptors expression. Collectively, these results further our understanding of the evolution of a virus in a single host cell.

IMPORTANCE It is important to understand how host cell heterogeneity affects viral infection and replication. Using single-cell analysis, we found that viral genome replication levels exhibited dramatic variability in foot-and-mouth disease virus (FMDV)-infected cells. We also found a strong correlation between heterogeneity in cell size, inclusion number, and cell cycle status and that all of these characteristics affect the infection and replication of FMDV. Moreover, we found that host cell heterogeneity influenced the viral adsorption as differences in the levels of FMDV integrin receptors' expression. This study provided new ideas for the studies of correlation between FMDV infection mechanisms and host cells.

INTRODUCTION

Cellular heterogeneity within a population of cells is apparent even when the cells possess the same genotype or are derived from the same clone, as evidenced by morphological differences in single-celled organisms, cell polarity differentiation in higher organisms, phenotypic differences in in vitro cell culture, and differences in growth and the cell cycle (1–3). Intrinsic factors, such as random mutations during transcription and translation or cell switching controlled by genotype and epigenetics, or external factors, such as adaptive transformation caused by environmental changes, can induce cellular heterogeneity (4, 5).

Cellular heterogeneity occurs in mixed cell populations exhibiting different functional phenotypes that exist in a dynamic balance and undergo phenotypic transformation among different states (6). The switch between functional phenotypes directly regulates the interaction of cells with viruses. It has been suggested that fluctuations in viral protein expression result in the generation of small subpopulations of latent cells during human immunodeficiency virus (HIV) replication. The existence of these heterogeneous cell subpopulations hinders drug efficacy, contributing to long-term viral transmission and persistent infection (7). Moreover, persistent hepatitis C virus (HCV) and HIV infections significantly reduce the number of cells in the G₁ and S phases but increase the number of G₂/M phase cells (8, 9). Differences in cellular characteristics, such as size and cell cycle, also result in significant differences in the number of viral progeny in vesicular stomatitis virus (VSV)-infected cells (10, 11). Early studies showed that host cells produce at least six different phenotypes during the course of persistent infection with foot-and-mouth disease virus (FMDV) and that these altered phenotypes were caused by inheritable cell modifications that were selected during virus persistence (12). Similarly, we found that FMDV-infected BHK-2l cells exhibit morphological heterogeneities that are different from those of normal BHK-2l cells (13, 14). Thus, studying the mechanisms of cellular heterogeneity and their role in viral infection could impact the development of antiviral strategies.

However, studies on the occurrence, development, and completion of the viral infection cycle have been confined to whole populations of infected cells, yielding only the average response of the cellular population, and few studies have focused on a single infected cell. Although all host cells can be infected simultaneously, viral replication kinetics are different in each cell due to cellular heterogeneity (15, 16), which is attributed to a variety of factors, such as cell size, inclusion, and cell cycle heterogeneity in normal host cells (17–19).

FMDV, a positive-strand RNA virus in the Picornaviridae family (20), causes acute and persistent infections in host cells and cloven-hoofed animals (21–23). Cells coexist with virus without obvious cytopathic effects (CPE) and produce infectious virions during serial passage of BHK-21 cells persistently infected with FMDV (14, 24). We sorted single cells using fluorescence-activated cell sorting (FACS) and determined viral RNA copy numbers using single-cell reverse transcriptase quantitative PCR (RT-qPCR) to determine intercell replication differences. The results revealed marked variability in the positive- and negative-strand viral RNA levels in FMDV-infected cells, ranging from below the detection limit to millions. We next investigated the effects of host cell heterogeneity, including cell size, number of inclusions, and cell cycle status, on FMDV infection (acute and persistent) and replication. We evaluated viral proteins, RNA, and infectious particles from heterogeneous cells and found that the viral outcome depends on cell size and number of inclusions. Furthermore, we demonstrated that heterogeneity in cell size and inclusion number also affects the adsorption of FMDV by altering the expression of FMDV integrin receptors. Cells in the G₂/M phase were more amenable to viral infection. Finally, we found correlations between heterogeneity in cell size, number of inclusions, and cell cycle status, which all affect the infection and replication of FMDV. Thus, our results advance our current understanding of the evolution of virus in a single host cell and provide new insights into the correlation between viral infection and cellular heterogeneity.

RESULTS

Replication of FMDV varies significantly between individual infected cells.

Although viral replication levels vary in virus-infected cells within cell populations, it is unclear if differences in viral replication occur among single cells. Thus, we used single-cell quantitative assays to examine the replication of FMDV in virus-infected cells (Fig. 1A). Single BHK cells infected with FMDV were sorted, and RNA levels were assessed. We found >10⁶-fold differences in the number of positive-strand viral RNAs per single cell and average positive-strand RNA copy number values of 43,400, 69,700, and 139,000 for the three multiplicity of infection values (MOIs) of 0.0001, 0.001, and 3, respectively (Fig. 1B). The two very low MOIs were used to truly reflect viral dynamics, as the virus was cytocidal and induced rapid cell death (15), and the high MOI was used to maximize the likelihood that every cell was infected by at least one virus particle (95% on a Poisson distribution). To confirm that the viral RNAs were not nonspecifically lost before the quantification procedure, we compared single-cell- and population-derived measurements (25). For both approaches, intracellular viral RNA levels were in the same order of magnitude (Fig. 1C). The number of negative-strand viral RNAs in 48 cells varied from 707 to 324,761 at an MOI of 0.001. They varied from 1,393 to 156,400 in cells infected at an MOI of 3 (Fig. 1D). Thus, the distribution of viral RNA levels in single cells was very wide, indicating that viral replication levels vary significantly in FMDV-infected cells. The ratios of positive- to negative-strand viral RNA in single cells infected at an MOI of 0.001 were primarily between 10 and 40, ranged from 5 to 200, and averaged 16. A similar distribution was observed in FMDV-infected cells at an MOI of 3 (Fig. 1E), which indicated significant variability in RNA synthesis. Collectively, these results show that viral replication levels vary significantly in single cells infected at different MOIs, and these differences may be related to host cell heterogeneity.

FIG 1.

Single-cell analysis and distribution of viral RNA levels in FMDV-infected cells. (A) Schematic of single-cell isolation and measurement. A population of adherent BHK-21 cells was infected with FMDV at MOIs of 0.0001 and 0.001 for 24 h or at an MOI of 3 for 6 h and then trypsinized to obtain a cell suspension. The cells were sorted into 96-well PCR plates by FACS, and intracellular viral RNAs were quantified using RT-qPCR. (B) Positive-strand RNA distributions in cells infected at different MOIs (n_0.0001 = 84; n_0.001 = 84; and n₃ = 168). (C) Comparison of single-cell- and population-based experiments. Means of single-cell- and population-derived measurements of virus RNA levels for indicated MOIs. Means of single-cell-derived measurements were calculated from data shown in Fig. 1B. Three independent experiments were performed for population-derived measurements. Error bars represent standard deviations. (D) Negative-strand RNA distributions from cells infected at MOIs of 0.001 and 3 (n_0.001 = 48, and n₃ = 66). (E) Ratio of positive-strand to negative-strand RNA distributions in cells infected at MOIs of 0.001 and 3 (n_0.001 = 48, and n₃ = 66).

Heterogeneity in host cell size affects FMDV infection.

To examine if cell heterogeneity affects acute FMDV infection, cells were divided into three groups based on their forward scatter (FSC) intensity and pulse width, which reflect cell size. We sorted the cells into high FSC intensity and pulse width (here “FSC-high”), FSC-medium, and FSC-low cell populations using FACS (Fig. 2A) and confirmed that FSC correlated with cell size using a Scepter cell counter (Fig. 2B). The percentages of FMDV protein 3D-positive cells as determined by FACS in the FSC-high, -medium, and -low cell populations were 83.1%, 34.4%, and 18.9%, respectively (Fig. 2C), suggesting that a larger cell size corresponded to a higher viral positive rate. Similar results were obtained for cells infected at MOIs of 0.001 and 0.0001 (Fig. 2C). The viral RNA copies in single cells infected at an MOI of 0.0001 were further analyzed 24 h postinfection (hpi). Viral RNA copy numbers were much lower in FSC-low single cells than in FSC-high single cells (Fig. 2D). Similar results were obtained in BHK-21 cells infected at an MOI of 3 (Fig. 2E), suggesting that larger cells contained more copies of viral RNA than did smaller cells.

FIG 2.

Larger cells have greater virus replication in acute FMDV infection than do smaller cells. (A) Infected cells were divided into three groups based on their FSC intensity and pulse width. FSC-high, -medium, and -low cells accounted for 25%, 50%, and 25% of the cells, respectively. (B) The size distribution of sorted cells was determined using a Scepter cell counter and Scepter Software 1.2. The mean diameter for each group is shown. (C) Percentages of FMDV 3D-positive cells from FSC-high, -medium, and -low cells. BHK-21 cells were infected with FMDV at MOIs of 0.01, 0.001, and 0.0001. At 24 hpi, the cells were collected, stained with anti-3D antibodies, and analyzed by flow cytometry for viral gene expression. (D) Viral RNA distributions in individual FSC-high, -medium, and -low infected cells. The cells were infected at an MOI of 0.0001, and their intracellular viral RNA content at 24 hpi was analyzed via RT-qPCR; n = 84. Friedman test, χ² = 145.31, df = 2, P < 0.001; FSC-low versus FSC-high: ***, P < 0.001; FSC-medium versus FSC-high: ***, P < 0.001. (E) Viral RNA distributions from FSC-high, -medium, and -low single cells. Single cells infected at an MOI of 3 were analyzed for viral RNA content at 6 hpi via RT-qPCR; n = 56. Friedman test, χ2 = 78.804, df = 2, P < 0.001; FSC-low versus -high: ***, P < 0.001; FSC-medium versus FSC-high: ***, P < 0.001. (F) Percentages of infectivity-positive cells in FSC-high, -medium, and -low cells. The cells were infected at an MOI of 0.0001, and single cells were isolated 24 hpi and placed in a 96-well plate (one cell per well) containing confluent normal cells. Morphological alterations were observed under a microscope at the indicated times; n = 96. (G) Percentages of infectivity-positive cells at an MOI of 3 for 6 hpi in FSC-high, -medium, and -low cells; n = 64.

To evaluate the effect of heterogeneity in host cell size on the infectivity of progeny virions, BHK-21 cells were infected with FMDV at an MOI of 0.0001 and sorted into FSC-high, -medium, and -low single cells at 24 hpi. We then cocultured the cells with normal BHK-21 cells in 96-well culture plates and counted the wells with CPE at different time points postinfection. Ideally, we believe that the earlier the cell CPE occurs, the higher the progeny virus yield. The results showed that the ratio of infectivity-positive cells in the FSC-high population at 24 hpi was 34%, which was higher than the ratios in the FSC-medium and -low cells (24% and 23%, respectively). A similar trend was observed at 120 hpi (Fig. 2F), and comparable results were obtained in the cells infected with FMDV at an MOI of 3 (Fig. 2G), suggesting that FSC-high cells contained a higher ratio of infectivity-positive cells and that cell size might influence FMDV infection.

Having shown that cell size affects acute FMDV infection, we sought to determine if cell size also influences viral infection in persistently FMDV-infected cells. Thus, cells persistently infected with FMDV were sorted at passage 31 (BHK-21-Op31) into three single-cell components comprising FSC-high, -medium, and -low populations, and the viral RNA copy numbers were examined. The results were similar to those obtained in acutely FMDV-infected cells. The percentage of cells with more than 50 RNA copies was 53.1% in FSC-high persistently infected cells but was 18.75% in the FSC-medium and -low single cells (Fig. 3A). We also sorted BHK-21-Op51 cell populations to detect infectious virions released from FSC-high, -medium, and -low cells after different amounts of time in culture. The results showed that persistently infected FSC-high cells continuously released virions following prolonged incubation, whereas FSC-medium and -low cells released a small number of virions during the early culture period and no virions at the later stage (Fig. 3B). Similarly, CPE was observed in FSC-high cells at 168 h postculture (Fig. 3C), suggesting that large, persistently infected cells contained more infectious virions than smaller cells. Collectively, our results showed that the host cell size influences both acute and persistent FMDV infections.

FIG 3.

Large cells are beneficial to virus replication in persistently FMDV-infected cells. (A) Viral RNA distributions from individual FSC-high, -medium, and -low cells in persistently FMDV-infected cells. BHK-21-Op31 were analyzed for their intracellular viral RNA content via RT-qPCR; n = 32. Friedman test, χ² = 32.71, df = 2, P < 0.001; FSC-low versus FSC-high: ***, P < 0.001; FSC-medium versus FSC-high: **, P = 0.002. (B) Viral yields in supernatants from FSC-high, -medium, and -low cells sorted from BHK-21-Op51 cells were determined by performing plaque assays at each time point. BHK-21-Op51 cells were divided into FSC-high, -medium, and -low populations, and cells from each group were sorted into a 24-well plate at 5,000 cells per well with 1 ml of medium for the plaque assay. (C) Morphological observation of FSC-high, -medium, and -low cells sorted from BHK-21-Op51 cells at 168 h.

Heterogeneity of host cell inclusion number affects infection of FMDV.

Cell growth heterogeneity is a common feature associated with an increase or decrease in inclusions. Thus, we sought to determine if heterogeneity in the number of inclusions also influences FMDV infection. To this end, we divided FMDV-infected BHK-21 cells into three groups based on their side scatter (SSC) and pulse width, which reflect inclusion number, referred to here as SSC-high, -medium, and -low cell populations (Fig. 4A). We confirmed that the expression levels of calnexin and GM130 in SSC-high cells were higher than levels in SSC-medium and -low cells using flow cytometry (Fig. 4B). The results suggested that cell populations with higher SSC intensity had more inclusions (more organelles). The percentage of 3D-positive cells in each group was analyzed using FACS. The results revealed that the percentages of 3D-positive cells in SSC-high, -medium, and -low cells were 69.4%, 39.9%, and 24.5%, respectively (Fig. 4C), suggesting that a greater number of inclusions in cell populations may result in higher percentages of 3D-positive cells. Moreover, the fluorescence intensity (greater than 10²) of SSC-high cells was more intense than that of SSC-medium and -low cells, indicating that the viral protein 3D levels were higher in cells with more inclusions. Similar results were obtained in cells infected with FMDV at MOIs of 0.0001 and 0.001 (Fig. 4C).

FIG 4.

Viral replication depends upon inclusion number in acute FMDV infection. (A) Isolation of SSC-high, -medium, and -low infected cells. Infected cells were divided into three groups based on their SSC intensity and pulse width. SSC-high, -medium, and -low cell populations accounted for 25%, 50%, and 25% of the cells, respectively. (B) Flow cytometry analysis of calnexin and GM130 expressed in SSC-high, -medium, and -low cells. BHK-21 cells were incubated with (open histogram) or without (solid histogram) the indicated antibody, followed by incubation with an FITC-conjugated goat anti-rabbit isotype-specific secondary antibody. The mean fluorescence intensity is shown for each antibody. (C) Percentages of FMDV 3D-positive cells from SSC-high, -medium, and -low cells. BHK-21 cells were infected with FMDV at MOIs of 0.01, 0.001, and 0.0001. At 24 hpi, the cells were collected, stained with anti-3D antibodies, and analyzed by flow cytometry for viral gene expression. (D) Viral RNA distributions in individual SSC-high, -medium, and -low infected cells. The cells were infected at an MOI of 0.0001, and intracellular viral RNA content 24 hpi was analyzed via RT-qPCR; n = 32. Friedman test, χ² = 30.489, df = 2, P < 0.001; SSC-low versus SSC-high: **, P = 0.005; SSC-medium versus SSC-high: *, P = 0.034. (E) Viral RNA distributions from SSC-high, -medium, and -low single cells. Single cells infected at an MOI of 3 were analyzed for viral RNA at 6 hpi via RT-qPCR; n = 56. Friedman test, χ² = 73.374, df = 2, P < 0.001; SSC-low versus SSC-high: ***, P < 0.001; SSC-medium versus SSC-high: P = 0.12. (F) Percentages of infectivity-positive cells in individual SSC-high, -medium, and -low infected cells. The cells were infected at an MOI of 0.0001, and single cells were isolated 24 hpi and put into a 96-well plate (one cell per well) containing confluent normal cells. Morphological alterations were observed under a microscope at the indicated times; n = 96. (G) Percentages of infectivity-positive cells at an MOI of 3 at 6 hpi in SSC-high, -medium, and -low cells; n = 64.

We also found that viral RNA copy numbers were higher in single SSC-high cells than in single SSC-medium and -low cells. The percentage of cells with more than 100 RNA copies was 68.75% in single SSC-high cells but only 37.5% and 31.25% in the single SSC-medium and -low cells, respectively, revealing that SSC-high cells contained more viral RNA copies (Fig. 4D). Next, we infected BHK-21 cells with FMDV (MOI, 3) and found that the number of viral RNAs in 56 SSC-high and -medium cells ranged from 10³ to 10⁷, whereas the viral RNA copies in 56 SSC-low cells were below the detection limit in 26 cells and ranged from 10³ to 10⁵ in the remaining 30 cells (Fig. 4E). Thus, we found that the number of inclusions was also positively correlated with protein 3D levels and viral RNA copies during FMDV infection. To clarify whether cells containing more inclusions produced more infectious virions, single SSC-high, -medium, and -low cells infected with FMDV were sorted and cocultured with BHK-21 cells. The ratio of infectivity-positive cells was highest in SSC-high cells, with a ratio of 47% at 120 hpi, which was higher than the ratios in the SSC-medium, and -low cells (Fig. 4F), and comparable results were obtained in the cells infected with FMDV at an MOI of 3 (Fig. 4G). The results suggested that the number of inclusions in host cells was closely related to FMDV infection during acute infections. We also found that persistently FMDV-infected cells with more inclusions contained more viral RNA and released more infectious virions than those with fewer inclusions (Fig. 5A to C). Collectively, these results demonstrated that heterogeneity of host cell inclusion number also affects acute and persistent FMDV infections.

FIG 5.

Heterogeneity of host cell inclusion number affects persistent infection by FMDV. (A) Viral RNA distributions from SSC-high, -medium, and -low individual cells in persistently FMDV-infected cells. The intracellular viral RNA content of BHK-21-Op cells was analyzed via RT-qPCR; n = 96. Friedman test, χ² = 76.984, df = 2, P < 0.001; SSC-low versus SSC-high: ***, P < 0.001; SSC-medium versus SSC-high: ***, P < 0.001. (B) Viral yields of SSC-high, -medium, and -low cells sorted from BHK-21-Op51 cells were determined from supernatants at each time point by plaque assay. BHK-21-Op51 cells were divided into SSC-high, -medium and -low populations, and cells from each group were sorted into a 24-well plate at 5,000 cells per well with 1 ml of medium. (C) Morphological observation of SSC-high, -medium, and -low cells sorted from BHK-21-Op51 cells at 168 h.

Effects of host cell cycle heterogeneity on FMDV infection.

Because many viral infections interfere with the host cell cycle (8, 9, 26, 27), we sought to determine if cell cycle heterogeneity affected FMDV infection by investigating the effects of cell cycle on FMDV replication. The percentage of 3D-positive FMDV-infected BHK-21 cells in each cell cycle phase was analyzed using FACS. Cells in the G₂/M phase had the greatest number of 3D-positive cells (Fig. 6A and B). Single cells in different cell cycle phases were sorted, and viral RNA copies in each cell were detected. The results showed that the mean numbers of copies of viral RNA/cell following infection at an MOI of 0.0001 were 2,609 in the G₀/G₁ phase, 14,104 in the S phase, and 46,257 in the G₂/M phase. The peak numbers of copies/cell in the G₂/M phase ranged from 10³ to 10⁴, whereas the peak G₀/G₁ and S phase values ranged from 10² to 10³ (Fig. 6C). Similarly, we found that the peak viral RNA copies were 10- to 100-fold higher in the G₂/M phase than in the S and G₀/G₁ phases following infection with FMDV at an MOI of 3 for 6 h (Fig. 6D), suggesting that G₂/M phase cells contained more viral RNA. To our surprise, we found that FMDV infection had no significant effects on cell cycle (Fig. 6E and F). Collectively, these results demonstrated that host cell cycle heterogeneity influences FMDV infection.

FIG 6.

FMDV replication was higher in the G₂/M phase than in other phases. (A) Cells were infected at MOIs of 0.01, 0.001, and 0.0001. At 24 hpi, the cells were collected and stained with propidium iodide (PI), and the numbers of cells in the G₀/G₁ (R6), S (R7), and G₂/M (R8) phases are shown. (B) Percentages of FMDV 3D-positive cells after infection at an MOI of 0.01 in different cell cycle phases. (C, D) Cells were infected at MOIs of 0.0001 and 3. At 24 and 6 hpi, the cells were collected and stained with Vybrant DyeCycle orange stain (5 μM). The cell cycle profiles are shown. Subsequently, single cells in different cell cycle phases were sorted to 96-well PCR plates using FACS, and intracellular viral RNAs were quantified using RT-qPCR. (C) Distributions of viral RNA from individual cells infected at an MOI of 0.0001 in different cell cycle phases; n = 32. Friedman test, χ² = 46.941, df = 2, P < 0.001; G₀/G₁ versus G₂/M: ***, P < 0.001; S versus G₂/M: ***, P < 0.001; G₀/G₁ versus S: ***, P < 0.001. (D) Distributions of viral RNA from individual cells infected at an MOI of 3 in different cell cycle phases; n = 56. Friedman test, χ² = 97.879, df = 2, P < 0.001; G₀/G₁ versus G₂/M: ***, P < 0.001; S versus G₂/M: ***, P < 0.001; G₀/G₁ versus S: ***, P < 0.001. (E, F) FMDV infection had no significant effects on cell cycle. BHK-21 cells were mock infected or infected with FMDV at different MOIs. At 24 hpi, the cells were collected and analyzed by flow cytometry. The error bars represent standard deviations of triplicates (ns, P > 0.05).

Effects of host cell size and inclusion number heterogeneity on FMDV adsorption.

The development and occurrence of viral infection are the result of virus-host interactions. Viral infection can trigger the expression of several genes, which can differ between cells and increase cellular heterogeneity. To determine if host cell heterogeneity affects FMDV infection or if viral infection alters host cell heterogeneity, we sorted normal BHK-21 cells into FSC and SSC-high, -medium, and -low fractions using FACS. For each fraction, 100,000 cells were cultured in 24-well plates for 20 h and infected with FMDV at an MOI of 3. At 6 hpi, the cells were freeze-thawed repeatedly, and the viral titer of each fraction was calculated. The results showed that the viral titers were higher in FSC-high cells than in FSC-medium and -low cells (Fig. 7A) and that viral titers were higher in SSC-high cells than in SSC-medium and -low cells (Fig. 7B). These results suggested that heterogeneity in host cell size and inclusion number affected FMDV infection.

FIG 7.

Effects of cell size and inclusion number on viral yield and adsorption. (A and B) BHK-21 cells were divided into FSC (A) and SSC (B) high, medium, and low populations. Cells from each group were sorted into a 24-well plate at 100,000 cells per well with 1 ml of medium. After 24 h, cells were infected with FMDV at an MOI of 3. At 6 hpi, the viral titers in the cell lysis were determined using a TCID₅₀ assay. (C and D) BHK-21 cells were divided into FSC (C) and SSC (D) high, medium, and low populations. Cells from each group were sorted into a 96-well plate at 10,000 cells per well with 0.1 ml of medium. After 24 h, cells were infected with FMDV at an MOI of 3. At 1 hpi at 4°C, the cells were analyzed using RT-qPCR for the detection of viral RNA; n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student's t test. Error bars indicate standard deviations. (E) Flow cytometry of FMDV integrin receptors (αvβ1, αvβ3, αvβ6) expressed on FSC- and SSC-high, -medium, and -low cells. BHK-21 cells were incubated with (open histogram) or without (solid histogram) the indicated anti-integrin antibody, followed by incubation with an FITC-conjugated goat anti-rabbit isotype-specific secondary antibody.

To further verify if host cell heterogeneity influenced viral entry, we performed a viral adsorption assay using 10,000 cells from the FSC- and SSC-high, -medium, and -low fractions infected with FMDV at an MOI of 3. The results showed that the adsorption of viral RNA was higher in FSC- and SSC-high cells than in FSC- and SSC-low cells (Fig. 7C and D), indicating that large cells and cells with more inclusions, respectively, were more favorable to viral adsorption. Finally, we used flow cytometry to determine the expression profiles of integrin receptors (αVβ1, αVβ3, αVβ6) in FSC- and SSC-high, -medium, and -low cells, as these receptors promote adsorption of FMDV. The results showed that BHK-21 cells express αVβ1, αVβ3, and αVβ6 as their RGD-binding integrins and that the expression of αVβ6 was significantly higher than those of αVβ1 and αVβ3. As expected, the expression of FMDV integrin receptors was higher in FSC- and SSC-high cells than in the FSC- and SSC-medium and -low cells (Fig. 7E). Interestingly, the expression of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and β-actin exhibited similar trends, suggesting that larger cells or those with more inclusions contain more protein (Fig. 7E). Collectively, these results demonstrated that heterogeneity in host cell size and inclusion number influenced early-stage FMDV infection by altering the expression of FMDV integrin receptors.

Cell cycle arrest at the G₂/M phase facilitates progeny virus production.

After discovering that FMDV replication was increased in the G₂/M phase, we determined whether host cell cycle heterogeneity directly contributes to FMDV infection. Thus, we examined viral titers in cells that arrested in different cell cycle phases and determined that the viral titer in G₂/M phase cells was 10 to 100 times higher than titers in G₁/S and G₀ phase cells (Fig. 8A and B). Similar results were obtained in persistently infected cells (Fig. 8C), suggesting that cell cycle arrest at the G₂/M phase promoted efficient viral proliferation. In addition, we used flow cytometry to confirm the FMDV integrin receptor (αVβ6) expression profiles in cells in different cell cycle phases and found that cells in the G₂/M phase had the highest αVβ6 expression levels (Fig. 8D).

FIG 8.

Effects of cell cycle on viral yield. (A) BHK-21 cell cycle profiles were tested following treatment for 16 h with the indicated cell cycle inhibitor (mimosine [Mim], hydroxyurea [HyU], or nocodazole [Noc]). To ensure that the cell cycle inhibitors were effective, the inhibitor-treated cells were stained with PI and analyzed by flow cytometry. (B) Production of infectious FMDV is cell cycle dependent. BHK-21 cells were grown to 70% confluence in complete medium and then for 16 h in the presence of the indicated cell cycle inhibitors. G₀ cells were obtained by growing cells in medium without FBS. Cells were infected with FMDV at an MOI of 0.0001 for 1 h, washed with saline solution, and given the appropriate medium to maintain their cell cycle arrest. At 20 h, viral titers were determined using cell lysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student's t test. Error bars indicate standard deviations. (C) BHK-21-Op cells were grown to 70% confluence in complete medium and then for 20 h in the presence of the indicated cell cycle inhibitors. G₀ cells were obtained by growing cells in medium without FBS. Viral titers were determined using cell lysis. (D) Flow cytometry of FMDV integrin receptors (αvβ6) expressed on G₀/G₁, S, and G₂/M cells. BHK-21 cells were incubated with (open histogram) or without (solid histogram) the indicated anti-integrin antibody, followed by incubation with an FITC-conjugated goat anti-rabbit isotype-specific secondary antibody.

Correlation of host cell size, inclusion number, and cell cycle heterogeneity.

Next, we sought to examine potential correlations among cell size, inclusion, and cell cycle heterogeneity. We analyzed the correlation among the three heterogeneous cell populations and found that among FSC- and SSC-high cells, approximately 50% of cells were in the G₂/M phase, and fewer G₂/M phase cells were noted in FSC- and SSC-low cell populations (Fig. 9A). The mean values for FSC and SSC in G₂/M phase cells were higher than in other phases (Fig. 9B and C). Similarly, the mean FSC value was higher in SSC-high cells and vice versa (Fig. 9D and E), indicating a correlation between the three types of heterogeneous cells that affected FMDV infection and replication.

FIG 9.

Correlation between cell cycle, cell size, and number of inclusions. (A) Distribution of cells in different cell cycle phases. BHK-21 cells were stained with PI, divided into groups (FSC- or SSC-high, -medium, and -low), and the cell cycle profiles of each group were determined by flow cytometry. (B and C) Changes in the FSC (B) or SSC (C) of cells in the G₀/G₁, S, and G₂/M phases. (D and E) Changes in the SSC (D) or FSC (E) of cells in high, medium, and low groups.

DISCUSSION

In this study, we evaluated single cells to quantify viral RNA copy numbers and viral replication and to determine intercell replication differences and the effects of host cell heterogeneity on FMDV infection. Our results showed that significant variability existed in positive- and negative-strand RNA replication levels in single FMDV-infected cells (Fig. 1) and that this variability was related to host cell heterogeneity. The effects of cell heterogeneity on viral infection are not limited to FMDV-infected cells but are common to all virus-infected cells. Viral RNA levels in different single cells can span 1 to 2 orders of magnitude in poliovirus-infected cells (28). Recently, Frank et al. studied differences in influenza A virus (IAV) replication using single cells and found that intracellular viral RNA levels can span 3 orders of magnitude and differ in single-cell viral genome segments (25).

We also investigated the effects of heterogeneity in cell size, number of intracellular inclusions, and cell cycle status on acute and persistent FMDV infections and determined that large cells and cells with more inclusions contained more viral proteins and viral RNA copies than did smaller cells and those with fewer inclusions, as well as a high ratio of infectivity-positive cells. Similar results were obtained for VSV-infected cells, whereby larger cells produced more viral progeny than smaller cells (11). Larger cells or cells with more inclusions may have added resources for conducting biological reactions that are beneficial to viral infection and replication. Further, the levels of most proteins and mRNAs and the size or number of organelles reportedly increase with cell size (29–31), and viruses need these resources to replicate. It has also been suggested that cell heterogeneity is caused mainly by intrinsic and external noise and that these factors might be amplified at the early IAV infection stage to further increase cell heterogeneity. However, unlike FMDV infection, cell size had no effect on viral replication of IAV during the early stage of infection, possibly due to replication differences between segmented and nonsegmented viruses such as FMDV and VSV (25).

The intensity of SSC, an important parameter of flow cytometry, is determined by cellular composition and complexity, including cytoplasmic granules, cell vesicles, organelles, and membrane structures (32), which are related to the synthesis and transport of intracellular nucleic acids, proteins, and other biological resources. Here, we used SSC to characterize cells and found that cells with high SSC contained more viral proteins and viral RNA copies and a higher ratio of infectivity-positive cells than cells with medium or low SSC, and this result is consistent with a previous study showing that viral titers increased with increased SSC in baculovirus-infected insect cells (33). Correlations between virus and host cell cycle have also been studied. Some RNA viruses interfere with or cause cells to arrest in a specific cell cycle phase. Infectious bronchitis virus and HCV induce G₂/M cell cycle arrest, which is favorable for viral replication, whereas murine hepatitis virus and some severe acute respiratory syndrome coronavirus (SARS-CoV) proteins induce G₀/G₁ phase arrest (8, 9, 26, 27). To our surprise, we found that FMDV infection had no significant effects on cell cycle (Fig. 6E and F). Thus, we investigated the single-cell FMDV RNA content and the percentage of 3D-positive cells and found variation in the distribution of FMDV RNA in different cell cycle phases. Cells in the G₂/M phase contained more viral RNAs and a higher percentage of 3D-positive cells than cells in other phases. Furthermore, we used drugs to arrest the cell cycle in different phases and found that cells arrested in the G₂/M phase had increased replication efficiency and favored progeny virus production (Fig. 8B and C). Dove et al. used nocodazole to arrest the cell cycle in G₂/M phase and found that infectious bronchitis virus (IBV)-infected G₂/M phase-synchronized cells exhibited increased viral protein production (34). Similar results were reported, i.e., that cell cycle influenced VSV yields and that cells arrested in the G₂/M phase had the highest viral yield (11).

We demonstrated that heterogeneity in cell size, inclusion number, and cell cycle status existed in the host cell populations before and after FMDV infection. We infected heterogeneous cells with FMDV and found that viral titers in FSC- and SSC-high cells were higher than those in FSC- and SSC-low cells, demonstrating that heterogeneity in cell size and cell inclusion number influenced FMDV infection. The FMDV life cycle includes adsorption, entry, virus uncoating, genome replication and translation, assembly, and release (35). Thus, it is important to determine which stage of the FMDV life cycle is affected by host cell heterogeneity. In this study, the viral adsorption experiment showed that the absorbed FMDV RNA content in large cells and cells with high numbers of inclusions was greater than in cells that were smaller and had fewer inclusions, suggesting that cell size and inclusions affected the FMDV adsorption processes. Recent studies have shown that FMDV adsorbs to host cells by binding to four integrin receptors (αvβ1, αvβ3, αvβ6, and αvβ8) that contain highly conserved RGD sequences and then enters the cells through clathrin-dependent endocytosis (36, 37). We hypothesized that cell size and number of inclusions might be related to FMDV receptor expression, and we found increased expression of FMDV integrin receptors in FSC- and SSC-high cells. These results demonstrated that host cell heterogeneity influenced the adsorption of FMDV due to differences in the levels of FMDV integrin receptor expression. Similarly, Berend et al. found that the cellular environment (dense or sparse population environment) could influence the heterogeneity of endocytosis and viral infection in cells (38).

Our studies also demonstrated a correlation between cell size, inclusion, and cell cycle heterogeneity. We found that in the population of cells with large size and a high number of inclusions, approximately 50% of cells were in G₂/M phase. In addition, the mean values of FSC and SSC in cells in G₂/M phase were higher than those in other phases, consistent with the cell growth results (39). For successful replication, cells must produce enough protein, DNA, membrane structures, and organelles prior to division, expand the cellular volume to contain these components, and distribute them equally to the two daughter cells (40, 41). Thus, the cells are larger and contain more inclusions in the G₂/M phase prior to division than in other phases (39). We also found that the mean SSC value was higher in FSC-high cells and vice versa, but there are some cells with very low SSC values among FSC-high cells, and vice versa (Fig. 9D and E). Therefore, these results demonstrated a correlation between larger cells and cells with higher inclusions, and they were not the same group of cells. This study showed that cell size, inclusion number, and cell cycle heterogeneity affect the viral life cycle and that cells with larger size or increased inclusions as well as cells in the G₂/M phase are more conducive to viral infection, likely due to the presence of sufficient resources. The expression of GAPDH and β-actin were higher in FSC- and SSC-high cells, supporting the notion that cells with larger size or more inclusions contained more protein (Fig. 7E), which is consistent with a prior report that the majority of proteins and mRNAs and the size and number of organelles increase with cell size (29–31).

In summary, we studied the effects of host cell heterogeneity on acute and persistent FMDV infections and found that heterogeneity in cell size, inclusion number, and cell cycle phase had a significant effect on FMDV infection and replication. We propose that direct correlations exist between FMDV infection and host cell heterogeneity and that control of host cell heterogeneity factors is conducive to viral infection and replication, offering new ideas for studies of the correlation between FMDV infection mechanisms and host cells.

MATERIALS AND METHODS

Cells and viruses.

FMDV serotype O viral strains (Akesu/58/2002, GenBank accession no. AF511039) were supplied by the Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences. BHK-21 cells were provided by the China Center for Type Culture Collection. Persistently FMDV-infected BHK-21 cells were previously described (14). The cells were maintained in minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/ml), and streptomycin (0.1 mg/ml) at 37°C with 5% CO₂. Cells were used to propagate viral stocks and to measure viral titers in plaque and 50% tissue culture infectious dose (TCID₅₀) assays. The MOI was calculated based on the PFU titer.

Isolation of individual infected cells.

The isolation of individual infected cells was performed as previously described (28). Briefly, BHK-21 cells were plated in a 6-well plate (1 × 10⁶ cells/well) and infected with 200 μl of virus at an MOI of 0.0001, 0.001, or 3 for 1 h at 37°C. The inoculum was removed, and cells were washed twice and overlaid with 2 ml of 2% FBS medium. Cells were trypsinized at 37°C for 5 min at the designated time postinfection, resuspended in cold 2% FBS medium, and incubated on ice. The cells were sorted via light scattering using a MoFlo XDP flow cytometer (Beckman Coulter) to one cell per well in a 96-well PCR plate containing 5 μl 0.9% NaCl solution and 10 U of RNase inhibitor (TaKaRa) (15) per well. The individual isolated cells were then frozen on dry ice in the 96-well PCR plates.

The cells were lysed as previously described (15). Briefly, the frozen cells were heat denatured at 98°C for 3 min to disrupt cell membranes, frozen immediately on dry ice, and thawed at 37°C for 1 min. This cycle was repeated twice, and samples were stored at −80°C until quantitative real-time PCR (qPCR) analysis.

Cell sorting and size measurements.

BHK-21 cells infected with FMDV at an MOI of 0.0001 or 3 were divided into three groups based on their FSC intensity and pulse width or their SSC and pulse width. The FSC-high, -medium, and -low populations accounted for 25, 50, and 25% of gated cells, respectively. Similarly, the SSC-high, -medium, and -low populations accounted for 25, 50, and 25% of gated cells, respectively (11, 32). Cells from each group were sorted into a 96-well PCR plate or cell culture plate, and the sizes of sorted cells were determined using a Scepter cell counter and Scepter Software 1.2 (Millipore) (32).

Single-cell RT-qPCR.

The intracellular viral RNA was quantified using single-cell RT-qPCR as previously described (15). The cDNA was synthesized from total RNA using the Rever TraAce qPCR RT kit (Toyobo) and 125 nM strand-specific reverse transcription primers (positive-strand RT, 5′-CATATCTTTGCCAATCAACATCAG-3′, and negative-strand RT, 5′-GAACACATTCTTTACACCAGGAT-3′) in a 5-μl reaction volume. RT reactions were performed for positive- and negative-strand RNAs for each sample. Strand-specific qPCR was performed based on a published protocol (15). The reaction mixture contained 5× Thunderbird Probe qPCR mix (Toyobo), 250 nM primers (forward primer [FP], 5′-GAACACATTCTTTACACCAGGAT-3′, and reverse primer [RP], 5′-CATATCTTTGCCAATCAACATCAG-3′), and 250 nM probe (5′-FAM-ACAACCTACCGCCGAGCCAATTC-TAMRA-3′) (42) in a 10-μl reaction volume. A 10-fold dilution series of in vitro-transcribed positive- and negative-strand RNA standards were analyzed concurrently and used to construct a standard curve.

The RT-qPCRs were performed using a Bio-Rad CFX96 instrument. Based on the manufacturer's protocol, cDNA was synthesized at 37°C for 15 min. The PCR profile consisted of 95°C for 1 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Data analyses were performed using Rotor-Gene 4.6.

Viral infectivity assay (cocultivation).

Monolayer BHK-21 cells were infected with FMDV (at an MOI of 0.0001 or 3) and incubated in MEM supplemented with 2% FBS. At designated times postinfection, single cells were isolated and placed in a 96-well plate (one cell per well) containing confluent normal cells. Morphological changes were observed under a microscope (15).

Immunofluorescence staining.

A total of 2 × 10⁶ cells were collected after infection and resuspended in 4% paraformaldehyde fixative and processed for immunofluorescence staining as previously described (43). Briefly, after removing the fixative, the cells were permeabilized with 0.5% Triton X-100 for 10 min and incubated in blocking buffer (phosphate-buffered saline [PBS], 2% bovine serum albumin) for 1 h at room temperature. The cells were then incubated with the primary antibodies overnight at 4°C, washed with PBS, and incubated with the appropriate secondary antibodies conjugated with fluorescein isothiocyanate (FITC) (Thermo Fisher) for 1 h at room temperature. The cells were then analyzed by flow cytometry using FACS (CyAn ADP; Beckman Coulter). The anti-3D polymerase of FMDV polyclonal antibody was provided by B. Yuan (44). The anti-calnexin (endoplasmic reticulum marker) antibody, anti-GM130 (Golgi matrix protein) antibody, anti-integrin polyclonal antibodies against β1, β3, and β6, and monoclonal antibodies against GAPDH and β-actin were purchased from Proteintech. The anti-αVβ6 polyclonal antibody was purchased from Huabio.

Synchronization of cells.

Subconfluent BHK-21 cells were synchronized in the G₀ phase using serum deprivation. Approximately 2 × 10⁵ cells were plated in a 12-well plate and maintained in medium containing no serum for 24 h. For mitotic arrest, BHK-21 cells were treated with cell cycle inhibitor (300 μM mimosine, 1 mM hydroxyurea, 60 mg/ml nocodazole) for 16 h (27). All inhibitors (and control wells) contained dimethyl sulfoxide (DMSO) at a final concentration of 0.1%. The cells were infected with FMDV (MOI, 0.0001) for 1 h and washed with PBS, and then the appropriate medium was added to maintain the arrested cell cycle status. The viral titers were determined 20 h later using thawed lysates and a TCID₅₀ assay.

Evaluation of cell cycle by PI staining.

Propidium iodide (PI) staining and flow cytometry were used to confirm that cell cycle inhibitors or serum starvation were effective. BHK-21 cells were treated with cell cycle inhibitors in complete medium for 16 h, as described above. The cells were trypsinized, washed twice with PBS, fixed in 1 ml of cold 70% ethanol overnight at 4°C, and resuspended in staining buffer (50 μg/ml PI [Sigma], 20 μg/ml RNase in PBS) for 20 min at room temperature. The PI-stained cells were then analyzed by FACS (CyAn ADP), and at least 2 × 10⁴ cells from each sample were counted. Data analysis was performed using Flowjo 7.6.1.

RNA extraction.

The BHK-21 cells were centrifuged and homogenized with 400 μl TRIzol reagent (Life Technologies) in 1.5-ml Eppendorf polypropylene tubes. Total RNA was extracted according to the manufacturer's instructions.

Plaque assays and TCID₅₀ assays.

Viral titers were determined using a standard plaque assay and TCID₅₀ assay (14).

Adsorption and internalization assays.

Viral adsorption and internalization in BHK-21 cells infected with FMDV (MOI, 3) were analyzed as previously described (14). Briefly, cell samples were collected at 4°C 1 h postadsorption and analyzed by RT-qPCR to detect viral RNA.

Statistical analysis.

Data are presented as means ± standard deviations. All pairwise comparisons were tested for statistical significance using Student's t test or Friedman and pairwise Wilcoxon rank sum tests with a Bonferroni correction (45). Significant values are indicated with asterisks: *, P < 0.05; **, P < 0.01; ***, P < 0.001.