Permissive human cytomegalovirus infection of a first trimester extravillous cytotrophoblast cell line
© LaMarca et al; licensee BioMed Central Ltd. 2004
Received: 02 September 2004
Accepted: 17 November 2004
Published: 17 November 2004
Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection in the United States and Europe. Despite the significant morbidity associated with prenatal HCMV infection, little is known about how the virus infects the fetus during pregnancy. To date, primary human cytotrophoblasts (CTBs) have been utilized to study placental HCMV infection and replication; however, the minimal mitotic potential of these cells restricts experimentation to a few days, which may be problematic for mechanistic studies of the slow-replicating virus. The aim of this study was to determine whether the human first trimester CTB cell line SGHPL-4 was permissive for HCMV infection and therefore could overcome such limitations. HCMV immediate early (IE) protein expression was detected as early as 3 hours post-infection in SGHPL-4 cells and progressively increased as a function of time. HCMV growth assays revealed the presence of infectious virus in both cell lysates and culture supernatants, indicating that viral replication and the release of progeny virus occurred. Compared to human fibroblasts, viral replication was delayed in CTBs, consistent with previous studies reporting delayed viral kinetics in HCMV-infected primary CTBs. These results indicate that SGHPL-4 cells are fully permissive for the complete HCMV replicative cycle. Our findings suggest that these cells may serve as useful tools for future mechanistic studies of HCMV pathogenesis during early pregnancy.
Human cytomegalovirus (HCMV) is a ubiquitous beta-herpesvirus that is the leading cause of congenital viral infection in the United States and Europe. Intrauterine transmission of the virus occurs in approximately 40% of pregnant women with primary HCMV infection, and the incidence of congenital HCMV infection is an estimated 1% of newborns [1–3]. Although the pathogenesis of HCMV transmission to the fetus during pregnancy is unclear, the placenta has been implicated as an important determining factor [4–8]. Primary first trimester extravillous cytotrophoblasts (CTBs), which are specialized placental epithelial cells that invade and remodel the uterine wall during placentation, have been previously shown to be fully permissive for HCMV infection in vitro [7, 9]. Additionally, using an in vitro coculture system, Maidji and colleagues demonstrated that infected uterine microvascular endothelial cells transmit HCMV to differentiating invading CTBs, suggesting that placental HCMV infection can occur in a retrograde fashion that initiates in the maternal endothelium . Despite these reports, the minimal mitotic potential of primary CTBs restricts experimentation to a few days, which may be problematic for mechanistic studies of the slow-replicating virus. Alternatively, the utilization of trophoblast cell lines would provide an easily manipulative in vitro model for the study of HCMV infection of the placenta. In the present study, we used a first trimester human extravillous CTB cell line, termed SGHPL-4, to investigate HCMV replication. SGHPL-4 cells were derived from first trimester chorionic villous tissue and have been described previously. Importantly, these cells share many characteristics with isolated primary cells, including the expression of cytokeratin-7, HLA class I antigen, HLA-G, BC-1, CD9, human chorionic gonadotrophin, and human placental lactogen[10–12].
Although several studies have shown that first trimester primary trophoblasts can be permissively infected with HCMV, some reports have demonstrated that progression through the replicative cycle was slow and progeny virus remained predominantly cell associated [9, 15, 16]. To determine whether SGHPL-4 cells support productive HCMV replication, 9 day viral growth assays were performed (Figure 1F). SGHPL-4 and HFF cells were inoculated with HCMV at a MOI of 0.1 PFU per cell, and both culture lysates and supernatants were titered for infectious virus at various days p.i. While viral titers in infected HFFs were detectable as early as 2 days p.i., viral replication was undetectable or below the lower limit of detection of the assay in SGHPL-4 lysates up to 3 days p.i. However, at days 5–9 p.i., HCMV replicated to titers of ≥ 5000 and 3600 PFU/ml in SGHPL-4 cell lysates and supernatants, respectively. Relative to HFF-infected control cultures, viral titers recovered from SGHPL-4 culture lysates and supernatants were reduced by ~20- and ~200-fold, respectively (Figure 1F). While viral titers were decreased in infected SGHPL-4 cells as compared to infected HFFs, placental CTBs effectively supported productive viral replication as measured by infectious intracellular and extracellular virions. Moreover, when SGHPL-4 cells were infected with another laboratory-derived strain of HCMV (strain AD169), similar results were obtained (data not shown) suggesting that viral replication was not virus-strain specific. Collectively, these data indicate that SGHPL-4 cells support productive HCMV replication.
In the present study, we demonstrate that the first trimester extravillous CTB cell line SGHPL-4 is fully permissive for HCMV replication. The utilization of a CTB cell line, rather than primary CTBs and explant cultures that are short-lived cultures, may provide an experimental advantage for in vitro studies of placental HCMV infection. We propose that the permissiveness for HCMV replication in SGHPL-4 cells may allow for future studies in elucidating the molecular mechanism(s) of HCMV infection and pathogenesis at the maternal-fetal interface during early pregnancy.
List of abbreviations
human foreskin fibroblasts
multiplicity of infection
plaque forming unit
4', 6-diamidino-2-phenylindole, dihydrochloride
The authors would like to thank Dr. Mark Stinski at the University of Iowa for kindly supplying the virus strain used in these studies and Dr. Guy Whitley at St. George's Hospital Medical School in London for kindly providing the SGHPL-4 cell line and for critical review of this manuscript. This work was supported by the National Institutes of Health (HD045768; C.A.M.).
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