- Short report
- Open Access
Permissive human cytomegalovirus infection of a first trimester extravillous cytotrophoblast cell line
Virology Journalvolume 1, Article number: 8 (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].
The lytic replication cycle of HCMV is a temporally regulated cascade of events that is initiated when the virus binds to host cell receptors. Upon entry into the cell, the viral DNA translocates to the nucleus where viral gene expression occurs in a stepwise fashion beginning with the expression of immediate early (IE) genes (reviewed in ). To initiate studies of HCMV infection in the SGHPL-4 cell line, placental CTBs and human foreskin fibroblasts (HFFs) were infected with HCMV and the nuclear HCMV IE proteins (IE 1/2; Chemicon, Temecula, CA) were examined by immunofluorescence at various intervals after viral infection. At 3 h p.i., IE 1/2 was present in SGHPL-4 cells in similar numbers to that of HFFs. In fact, the percentages of IE-positive cells initially did not differ between CTBs and HFFs, suggesting that viral entry into the cells and IE transcription occurred at similar rates between the cell types (Figure 1A,1B,1C). Characteristic cytopathic effects of HCMV infection including swollen cells with nuclear inclusions were observed in both SGHPL-4 and HFF cells by 48 h p.i. (data not shown), and throughout a 6 day culture period, the numbers of IE-positive cells increased continuously in both cell types (Figure 1A). Interestingly, the rate of IE 1/2 protein expression in SGHPL-4 cells as compared to HFFs appeared to differ beginning at 72 h p.i. By 72 h p.i., there was a 40% increase in the percentage of IE-positive HFFs over SGHPL-4 cells. While nearly 100% of HFFs stained positive for IE 1/2 120 h (5 days) p.i., the maximum fraction of IE-positive SGHPL-4 cells did not exceed 60% (Figure 1A,1D,1E), suggesting that subsequent viral gene expression and thus cell-to-cell viral spread may be kinetically delayed. These findings are consistent with other reports demonstrating delayed kinetics of viral gene expression in primary CTBs as compared to primary fibroblasts .
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.
human foreskin fibroblasts
multiplicity of infection
plaque forming unit
4', 6-diamidino-2-phenylindole, dihydrochloride
Stagno S, Pass RF, Dworsky ME, Henderson RE, Moore EG, Walton PD, Alford CA: Congenital cytomegalovirus infection: The relative importance of primary and recurrent maternal infection. N Engl J Med 1982, 306: 945-949.
Stagno S, Pass RF, Cloud G, Britt WJ, Henderson RE, Walton PD, Veren DA, Page F, Alford CA: Primary cytomegalovirus infection in pregnancy. Incidence, transmission to fetus, and clinical outcome. Jama 1986, 256: 1904-1908. 10.1001/jama.256.14.1904
Britt WJ: Congenital cytomegalovirus infection. In Sexually transmitted diseases and adverse outcomes of pregnancy. Edited by: Hitchcock PJ MKHTWJN. Washington D.C., ASM Press; 1999:269-281.
Benirschke K, Mendoza GR, Bazeley PL: Placental and fetal manifestations of cytomegalovirus infection. Virchows Arch B Cell Pathol 1974, 16: 121-139.
Hayes K, Gibas H: Placental cytomegalovirus infection without fetal involvement following primary infection in pregnancy. J Pediatr 1971, 79: 401-405.
Mostoufi-zadeh M, Driscoll SG, Biano SA, Kundsin RB: Placental evidence of cytomegalovirus infection of the fetus and neonate. Arch Pathol Lab Med 1984, 108: 403-406.
Fisher S, Genbacev O, Maidji E, Pereira L: Human cytomegalovirus infection of placental cytotrophoblasts in vitro and in utero: implications for transmission and pathogenesis. J Virol 2000, 74: 6808-6820. 10.1128/JVI.74.15.6808-6820.2000
Maidji E, Percivalle E, Gerna G, Fisher S, Pereira L: Transmission of human cytomegalovirus from infected uterine microvascular endothelial cells to differentiating/invasive placental cytotrophoblasts. Virology 2002, 304: 53-69. 10.1006/viro.2002.1661
Terauchi M, Koi H, Hayano C, Toyama-Sorimachi N, Karasuyama H, Yamanashi Y, Aso T, Shirakata M: Placental extravillous cytotrophoblasts persistently express class I major histocompatibility complex molecules after human cytomegalovirus infection. J Virol 2003, 77: 8187-8195. 10.1128/JVI.77.15.8187-8195.2003
Choy MY, Manyonda IT: The phagocytic activity of human first trimester extravillous trophoblast. Hum Reprod 1998, 13: 2941-2949.
Cartwright JE, Holden DP, Whitley GS: Hepatocyte growth factor regulates human trophoblast motility and invasion: a role for nitric oxide. Br J Pharmacol 1999, 128: 181-189.
Cartwright JE, Tse WK, Whitley GS: Hepatocyte growth factor induced human trophoblast motility involves phosphatidylinositol-3-kinase, mitogen-activated protein kinase, and inducible nitric oxide synthase. Exp Cell Res 2002, 279: 219-226. 10.1006/excr.2002.5616
Fortunato EA, McElroy AK, Sanchez I, Spector DH: Exploitation of cellular signaling and regulatory pathways by human cytomegalovirus. Trends Microbiol 2000, 8: 111-119. 10.1016/S0966-842X(00)01699-1
Halwachs-Baumann G, Wilders-Truschnig M, Desoye G, Hahn T, Kiesel L, Klingel K, Rieger P, Jahn G, Sinzger C: Human trophoblast cells are permissive to the complete replicative cycle of human cytomegalovirus. J Virol 1998, 72: 7598-7602.
Amirhessami-Aghili N, Manalo P, Hall MR, Tibbitts FD, Ort CA, Afsari A: Human cytomegalovirus infection of human placental explants in culture: histologic and immunohistochemical studies. Am J Obstet Gynecol 1987, 156: 1365-1374.
Hemmings DG, Kilani R, Nykiforuk C, Preiksaitis J, Guilbert LJ: Permissive cytomegalovirus infection of primary villous term and first trimester trophoblasts. J Virol 1998, 72: 4970-4979.
Isomura H, Stinski MF: The human cytomegalovirus major immediate-early enhancer determines the efficiency of immediate-early gene transcription and viral replication in permissive cells at low multiplicity of infection. J Virol 2003, 77: 3602-3614. 10.1128/JVI.77.6.3602-3614.2003
Sainz BJ, Halford WP: Alpha/Beta interferon and gamma interferon synergize to inhibit the replication of herpes simplex virus type 1. J Virol 2002, 76: 11541-11550. 10.1128/JVI.76.22.11541-11550.2002
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.).
The authors declare that they have no competing interests.
HL participated in the experimental design, performed all experiments and drafted the manuscript. BS participated in the experimental design and assisted with viral propagation and viral replication assays. CM conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.