Virus-host cell interactions during hepatitis C virus RNA replication: impact of polyprotein expression on the cellular transcriptome and cell cycle association with viral RNA synthesis

Abstract

Considerable controversy surrounds the impact of hepatitis C virus (HCV) protein expression on viability of host cells and regulation of the cell cycle. Both promotion of cellular proliferation and apoptosis have been observed in different experimental systems. To determine whether expression of the entire complement of HCV proteins in the context of ongoing viral RNA replication significantly alters the host cell transcriptome and cell cycle regulatory processes, we carried out high-density oligonucleotide microarray studies and analyzed cell cycle distributions and S-phase entry in Huh7 cell clones harboring selectable, full-length, replicating HCV RNAs that express the entire genotype 1b, HCV-N polyprotein, and clonally related cells in which all viral RNA was eliminated by prior treatment with alpha interferon. Oligonucleotide microarray analyses revealed only subtle, coordinated differences in the mRNA profiles of cells containing replicating viral RNA and their interferon-cured progeny, with variation between different cell clones having a greater influence on the cellular transcriptome than the presence or absence of replicating HCV RNA. Flow cytometric analysis demonstrated no significant differences in cell cycle distribution among populations of asynchronously growing cells of both types. Cell lines containing replicating viral RNA and their interferon-cured progeny were able to reenter the cell cycle similarly after transient G(1) arrest. In contrast, although viral protein expression and genome replication did not alter cell cycle control in these cells, HCV genome replication was highly dependent on cellular proliferation, with viral RNA synthesis strongly decreased in poorly proliferating, confluent, or serum-starved cells and substantially enhanced in the S phase of the cell cycle.

FIG. 1.

Elimination of replicating N-Neo/C5B RNAs by IFN-α treatment. (A) Diagram of full-length dicistronic selectable HCV RNA. The HCV IRES drives expression of the neomycin resistance gene (Neo) as a selectable marker. Expression of the full-length HCV polyprotein is under control of the encephalomyocarditis virus (EMCV) IRES. (B) Huh7 cells harboring dicistronic full-length replicating HCV RNAs (NNeo/C5B, clones 2-3 and 3) were cultured in the presence or absence of 100 U of IFN-α2b (IFN)/ml for 2 weeks. The RNA status of the cells was determined by Northern blot with a probe specific for NS5B (top panel). 28S rRNA is shown as a loading control (bottom panel). Cells treated with IFN-α had no detectable HCV RNA. (C) Absence of HCV protein expression after IFN-α treatment was verified by immunoblot with antibodies specific for E2 and the core protein. An immunoblot of β-actin is shown as a loading control.

FIG. 2.

HG-U133A Affymetrix high-density oligonucleotide microarray analyses comparing global mRNA transcript profiles in IFN-α-treated 2-3c cells that do not contain HCV RNA with clonally related 2-3 cells supporting replication of NNeo/C5B RNA (A) or 2-3c cells infected 20 h previously with SenV (B). Shown are the number of probe sets distributed across the range of differences in signal intensities between baseline 2-3c cells and the “experimental” data set: HCV RNA-containing clone 2-3 cells or SenV-infected 2-3c cells. Differences in signal intensities are expressed as the log₂ of the ratio of the experimental to baseline value. A positive value indicates an increase in transcript abundance in the experimental data set; a negative value indicates a decrease in abundance. Probe sets falling outside the dotted lines (log₂ signal ratio of >1 or <−1) represent transcripts with >2-fold differences in abundance from the baseline.

FIG. 3.

Affymetrix microarray profiling of mRNA transcripts in cells containing replicating NNeo/C5B RNA. Shown is an intensity matrix plot (“heat map”) produced by a hierarchical clustering analysis of those oligonucleotide probe sets representing mRNA transcripts identified by two-way ANOVA as being differentially regulated either as a result of cell clone (i.e., clone 2-3 versus clone 3) or the presence or absence of HCV RNA. Microarray analyses were carried out with the Affymetrix HG-U133A GeneChip with total cellular RNA isolated from two different HCV RNA-bearing Huh7 clones—2-3 and 3—and their IFN-treated, clonally related cell progeny (2-3c and 3c). After we eliminated probe sets marked “absent” in HCV-RNA-positive cells and transcripts with a difference in the hybridization intensity signal of <2-fold, the data from 557 probe sets were subjected to clustering analysis. The results demonstrate that the variation between the clone 2-3 and clone 3 Huh7 cell lines influences cellular transcription profiles much more than the presence or absence of replicating HCV RNA.

FIG. 4.

Flow cytometry determination of the S-phase distribution of asynchronously growing Huh7 2-3c cells (A) and clonally related 2-3 cells (B) containing replicating, full-length HCV RNA. Cells were labeled with BrdU for 30 min and then analyzed by flow cytometry after being stained for BrdU incorporation and DNA content. The percentage of BrdU-positive cells in the two cell lines is indicated.

FIG. 5.

Flow cytometry analysis of cell cycle entry in populations of Huh7 2-3c cells (A) and clonally related 2-3 cells (B) containing full-length, replicating NNeo/C5B RNA. Cells were arrested in the G₁ phase of the cell cycle with aphidicolin and released from the cell cycle block after 24 h. Prior to harvesting at the indicated time points, cells were labeled with BrdU for 30 min. BrdU incorporated into S-phase cells was stained with a monoclonal FITC-conjugated antibody, and the total DNA content was determined by staining with 7-AAD.

FIG. 6.

HCV RNA abundance declines in Huh7 2-3 cells as cells reach confluence. (A) Real-time RT-PCR quantitation of HCV RNA normalized to cellular GAPDH mRNA abundance. Cells were approximately 80% confluent on day 1 and 100% confluent on day 2. The cells were split on day 6 (arrow). The HCV RNA levels declined rapidly as cells grew more and more confluent and rebounded once logarithmic growth was resumed. (B) Northern blot detection of HCV RNA in a similar experiment with an NS5B-specific probe. Cells were near 100% confluence on day 1. HCV RNA levels decreased with increasing cell confluence, in agreement with the RT-PCR data.

FIG. 7.

HCV RNA abundance in 2-3 cells during serum starvation. (A) Northern blot detection of HCV RNA in clone 2-3 cells grown in medium containing the indicated percentage of serum for 3 days. Total RNA was extracted and hybridized to a probe specific for NS5B. HCV RNA abundance declined with decreasing concentrations in serum. (B) Analysis of HCV RNA synthesis during serum starvation by in vivo labeling. Normal Huh7 cells (lane 1) or clone 2-3 cells (lanes 2 to 5) were incubated in the presence (lanes 1 and 2) or absence of serum for 72 h (lanes 3 to 5). Serum was added back for 48 and 72 h, respectively, after the 72-h period of serum starvation for the samples shown in lanes 4 and 5. HCV RNA synthesis was monitored by incorporation of [³²P]orthophosphate for 15 h, followed by gel electrophoresis and autoradiography of the isolated RNA (top panel). Also shown is the 26S RNA band from the same gel visualized with ethidium bromide (bottom panel). (C) Results of densitometric quantitation of the 2-3 cell data shown in panel B, with values for HCV RNA normalized to the amount of 26S RNA and the values for cells growing in 10% serum set arbitrarily to 100%.

FIG. 8.

HCV RNA and protein abundances during cell cycle arrest and release. (A) Huh7 2-3 cells harboring replicating NNeo/C5B RNA were G₁ arrested with aphidicolin for 24 h and subsequently released from the cell cycle block. Cells were harvested at the indicated times and total RNA extracted and analyzed by real-time RT-PCR (top panel) and Northern blot (bottom panel). The levels of HCV RNA were not altered significantly between G₁ arrested and released cells and varied only slightly as the cells progressed though the cell cycle. (B) Immunoblot analysis of NS3 expression in clone 2-3 cells during and after aphidicolin release. Total cell lysates containing 50 μg of protein were analyzed by immunoblot with an MAb specific for NS3 (top panel) or β-actin (lower panel).

FIG. 9.

In vivo labeling of replicating full-length HCV RNA during cell cycle arrest and release. Huh7 2-3 cells were arrested in G₁ by treatment with aphidicolin for 24 h and subsequently released from the cell cycle block. At 4 h before being harvested, the cells were starved in phosphate-free medium for 1 h, followed by the addition of [³²P]orthophosphate at a concentration of 500 μCi/ml. Total RNA was extracted and analyzed by electrophoresis, and radioactively labeled, newly synthesized HCV RNA was quantitated by densitometry. There was a reproducible increase in HCV RNA synthesis as the cells progressed through S phase, with HCV RNA synthesis peaking approximately 8 h after release from the G₁ block.