Although chronic HCV infection is associated with cancer, the impact of HCV infection on the hepatocyte cell cycle is poorly characterized. Prior overexpression studies have demonstrated both pro- and antiproliferative activities of HCV proteins, but these studies have questionable relevance to the situation existing within infected cells in which multiple viral proteins are expressed and interact with each other as well as numerous host proteins to promote amplification of the genome and production of infectious virus. Data presented here show that Huh7.5 cells infected with HCV proliferate more slowly than uninfected cells, accumulate in the G2 phase of the cell cycle, and fail to enter M phase. The degree of cell cycle arrest correlates with the level of viral protein accumulation.
In common with other studies, we demonstrated here that HCV-infected Huh7.5 cells undergo apoptosis, but we also showed that the frequency with which this occurs varies among different HCV strains. In particular, the genotype 1 strain H77Sv3 and the genotype 1/2 chimera HJ3-5 show a reduced potential to induce apoptosis compared to JFH1. In support of these findings, previous studies demonstrated that an intragenotypic (type 2) chimera, J6/JFH1, is a more potent inducer of apoptosis than the parent JFH1 virus (22
). Both J6/JFH1 and HJ3-5 consist of JFH1 sequence with the coding sequence of core to p7 (and NS2 in the case of HJ3-5) of other viruses. Thus, it is likely that sequences that determine the potency of apoptosis induction lie within this region.
Our finding that HCV slows proliferation of Huh7.5 cells is in agreement with a previous study that reported a doubling time of 32 h for naïve Huh7.5 cells compared to 34 h for JFH1-infected cells (36
). Here, we show that the rate of proliferation in HCV-infected cells inversely correlates with viral antigen levels.
Previous studies that examined the effects of HCV infection on the cell cycle have suggested that infection with the JFH1 strain of HCV results in a G1
-phase-specific cell cycle arrest (21
). Walters et al. (38
) argued that JFH1 infection results in accumulation of cells in G1
phase, based on the observation that fewer infected cells accumulate in S phase, as determined by immunofluorescence analysis of EdU-labeled cells (38
). However, our more detailed analyses demonstrate that the decrease in the proportion of JFH1-infected cells in S phase is accompanied by increases in the proportions of cells in both G1
phases. Moreover, in cells transfected with genotype 1a H77S RNA, we observed significant decreases in G1
accompanied by a slight increase in cells in S phase (). It is noteworthy that both H77Sv3 and HJ3-5 show different effects on the host cell cycle compared to JFH1. Genotype 1 infections are most frequently associated with cancer, likely reflecting the higher prevalence of genotype 1 infection (17
). Furthermore, studies of HCV pathogenesis that use the genotype 2a JFH1 virus may not be representative of other HCV strains, particularly since this viral RNA replicates to such high levels within the cell. Levels of viral antigen are much lower in infected liver (15
). Thus, it is likely that observations made with the genotype 1a H77Sv3 virus may more closely approximate events in vivo
Few studies have specifically examined the effect of HCV infection at initiation of mitosis. In contrast to our findings, one report suggested that expression of HCV core protein in HepG2 cells promotes nuclear import of cyclin B1, thus promoting mitotic initiation (34
). However, the physiological relevance of this observation is questionable, since core protein was overexpressed in the absence of RNA replication and other viral proteins.
Very little is known about the effects of HCV infection on cell cycle regulation within hepatocytes in situ
. One study examined cell cycle markers in liver biopsies from HCV-infected individuals and demonstrated accumulation of cells in G1
). Greater numbers of cells were actively cycling in HCV-infected livers than in uninfected liver, which was almost entirely quiescent (G0
-specific markers were elevated, but there were reduced numbers of cells with S-, G2
- and M-phase-specific markers in liver samples from HCV-positive patients compared to samples from regenerating liver following ischemic reperfusion. However, it is difficult to interpret these results, since multiphoton microscopy suggests that only a minority of hepatocytes (typically 5 to 20%) are infected with HCV (15
). Thus, it is uncertain whether the cells identified by Marshall et al. (21
) that displayed G1
-phase markers were infected. Hepatocytes are also subject in vivo
to the effects of interferon, either produced by the host or administered therapeutically as antiviral treatment (30
). Interferon is known to cause G1
-phase-specific cell cycle arrest (5
What difference does it make to the host hepatocyte whether HCV causes arrest in the G1 or G2 phase of the cell cycle? Apoptosis could result from either, but the cell will replicate its chromosomal DNA prior to arrest in the case of G2-phase arrest. This will occur in an environment of oxidative stress and, through decades of chronic infection, it is possible that the occasional infected hepatocyte may acquire one or more mutations that allow it to escape G2 arrest and apoptosis and proceed through the cell cycle, providing an apoptosis-resistant clone of cells with altered cell cycle regulation.
The changes we observed in Mad2 expression ( and ) may also be relevant to the development of cancer. Unscheduled overexpression of Mad2 can lead to defects in the spindle checkpoint, aneuploidy, and cancer (10
). A recent study suggested that HCV core protein can cause transcriptional downregulation of Rb, leading to unscheduled overexpression of Mad2 and aneuploidy (19
). Contrary to this report, our data are clear in suggesting that Mad2 protein levels are downregulated by HCV infection ( and B to E), along with several other proteins that are involved in mitosis, such as phospho-histone H3(Ser10). Furthermore, our data indicate that Mad2 is downregulated posttranscriptionally (), despite increases in Mad2 transcript levels, consistent with NS5B-mediated degradation of Rb (27
). Reduced Mad2 expression has also been linked to defects in the mitotic spindle checkpoint in Mad2 haploinsufficient mice (26
). However, since our data suggest that HCV-infected cells do not enter mitosis, any defects in the mitotic spindle checkpoint that result from the activities of HCV proteins are unlikely to lead to aneuploidy and cancer, since heritable changes would not be passed to daughter cells. An important caveat to this assertion is that the current study was performed in derivatives of the human hepatoma cell line Huh7, which expresses high levels of a mutant p53 protein (3
) and may differ significantly in aspects of cell cycle regulation compared to primary hepatocytes. Future studies should be aimed at understanding HCV disturbances of cell cycle regulation in primary hepatocytes.