Human Cytomegalovirus Infects Caco-2 Intestinal Epithelial Cells Basolaterally Regardless of the Differentiation State

Esclatine, Audrey; Lemullois, Michel; Servin, Alain L.; Quero, A.M.; Géniteau-Legendre, M.

doi:10.1128/jvi.74.1.513-517.2000

Cited by 38 publications

(38 citation statements)

References 26 publications

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“…Several studies have determined that disruption of calciumdependent intercellular junctions by EGTA improves viral infection efficiency in various tissues and polarized cell cultures. 23,25,26,28 Previous evidence demonstrated that polarized apical membranes of normal human bronchial epithelial (NHBE) airway cells are resistant to lipofection. NHBE cells form multicellular islands similar to that of long-term primary rat hepatocyte cultures maintained in DMSO.…”

Section: Discussionmentioning

confidence: 99%

“…23 2,63 Infection of Caco-2 intestinal epithelial cell cultures by CMV was inefficient and limited to peripheral cells suggesting that CMV enters polarized Caco-2 cells preferentially through the basolateral membrane. 25 To overcome this limitation, the investigators pretreated the cultures with 100 mM EGTA in Krebs Ringer solution to deplete extracellular calcium stores, and therefore disrupt paracellular junctions and polarity, exposing the basolateral cell surface. Following cellular depolarization and disruption of paracellular junctions, efficiency of CMV infection increased and was homogeneous throughout the culture.…”

Section: Discussionmentioning

confidence: 99%

“…The cellular effects of calcium depletion were fully reversible and did not cause significant cytopathology to the Caco-2 cell cultures. 25 There are many advantages to the use of recombinant baculoviruses for gene therapy to hepatocytes in longterm DMSO culture. (i) Baculovirus can harbor very large pieces of DNA.…”

Section: Discussionmentioning

confidence: 99%

“…24 Several studies have determined that disruption of calcium-dependent paracellular junction complexes by EGTA improves viral infection efficiency in various tissues and polarized cell cultures. 14,23,[25][26][27][28] Transient depletion of extracellular calcium may overcome the inefficiency of baculovirus-mediated gene transfer by disassociating paracellular epithelial junction complexes and exposing the basolateral surface. We hypothesized that in normal medium, which contains calcium, the baculovirus binds and delivers its genome only to peripheral cells and baculovirus attachment, and entry to internal cells is prevented because paracellular junctions mask the baculovirus binding motif of these internal hepatocytes.…”

Section: Introductionmentioning

confidence: 99%

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Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

et al. 2003

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Gene delivery to differentiated hepatocytes is notoriously difficult. Hepatocytes plated on collagen-coated dishes and maintained in dimethyl sulfoxide (DMSO)-supplemented medium acquire paracellular junctions, arrange themselves in multicellular islands and are an excellent in vitro model for studying liver function. Baculovirus-mediated gene delivery to hepatocytes in this culture system is restricted to peripheral cells of the islands. However, this limitation can be overcome by transient calcium depletion of the cells prior to and during baculovirus infection. Examination of the mechanism underlying this process revealed that calcium depletion was accompanied by a transient loss of intercellular contacts and paracellular junction complex integrity, increased distance between adjoining cells, and internalization of the tight junction protein, zona occludens ZO-1.Internalization of ZO-1 was accompanied by baculovirus infection of internal cells of hepatocyte islands. When calcium levels were restored, paracellular junction complex integrity returned to normal by 12 h. No permanent alterations in hepatocyte ultrastructure and albumin mRNA, and protein expression were caused by this gene transfer method. Loss in paracellular junction complex integrity exposes the basolateral (sinusoidal) surface of hepatocytes resulting in homogeneous baculovirus-mediated gene delivery to approximately 75% of the cells in long-term DMSO culture. We conclude that the use of recombinant baculovirus as a vector in combination with transient calcium depletion is a highly efficient method for delivering exogenous genes to hepatocytes without loss of hepatic differentiation.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

et al. 2003

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“…The genetic integrity of UL128L determines the level of virus entry, which, in turn, regulates the infectivity of HCMV in these cells (Bodaghi et al, 1999;Ryckman et al, 2006;Wang & Shenk, 2005;Wang et al, 2007). However, it has been noted that the anatomical and tissue origin of both EP and endothelial cells affects HCMV infection efficiency and pathogenesis (Esclatine et al, 2000;Fish et al, 1998;Jarvis & Nelson, 2007;Jarvis et al, 1999;Millard et al, 2010;Tugizov et al, 1996). Both EP and endothelial cells are heterogeneous, exhibiting phenotypic differences and tissue-specific functions, while at the same time sharing common features like apico-basal polarity and tight junctions (TJ) (Elkouby-Naor & BenYosef, 2010;Gonzalez-Mariscal et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

The susceptibility of primary cultured rhesus macaque kidney epithelial cells to rhesus cytomegalovirus strains

Yue

Kaur

Lilja

et al. 2016

Journal of General Virology

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Kidney epithelial cells are common targets for human and rhesus cytomegalovirus (HCMV and RhCMV) in vivo, and represent an important reservoir for long-term CMV shedding in urine. To better understand the role of kidney epithelial cells in primate CMV natural history, primary cultures of rhesus macaque kidney epithelial cells (MKE) were established and tested for infectivity by five RhCMV strains, including two wild-type strains (UCD52 and UCD59) and three strains containing different coding contents in UL/b¢. The latter strains included 180.92 [containing an intact RhUL128-RhUL130-R hUL131 (RhUL128L) locus but deleted for the UL/b¢ RhUL148-rh167-loci], 68-1 (RhUL128L-defective and fibroblast-tropic) and BRh68-1.2 (the RhUL128L-repaired version of 68-1). As demonstrated by RhCMV cytopathic effect, plaque formation, growth kinetics and early virus entry, we showed that MKE were differentially susceptible to RhCMV infection, related to UL/b¢ coding contents of the different strains. UCD52 and UCD59 replicated vigorously in MKE, 68-1 replicated poorly, and 180.92 grew with intermediate kinetics. Reconstitution of RhUL128L in 68-1 (BRh68-1.2) restored its replication efficiency in MKE as compared to UCD52 and UCD59, consistent with the essential role of UL128L for HCMV epithelial tropism. Further analysis revealed that the UL/b¢ UL148-rh167-loci deletion in 180.92 impaired RhUL132 (rh160) expression. Given that 180.92 retains an intact RhUL128L, but genetically or functionally lacks genes from RhUL132 (rh160) to rh167 in UL/b¢, its attenuated infection efficiency indicated that, along with RhUL128L, an additional protein(s) encoded within the UL/b¢ RhUL132 (rh160)-rh167 region (potentially, RhUL132 and/or RhUL148) is indispensable for efficient replication in MKE.

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Hepatocyte polarization is essential for the productive entry of the hepatitis B virus

Mills²,

et al. 2011

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Human hepatitis B virus (HBV) is characterized by a high species specificity and a distinct liver tropism. Within the liver, HBV replication occurs in differentiated and polarized hepatocytes. Accordingly, the in vitro HBV infection of primary human hepatocytes (PHHs) and the human hepatoma cell line, HepaRG, is restricted to differentiated, hepatocyte-like cells. Though preparations of PHH contain up to 100% hepatic cells, cultures of differentiated HepaRG cells are a mixture of hepatocyte-like and biliary-like epithelial cells. We used PHH and HepaRG cells and compared the influence of virus inoculation dose, cell differentiation, and polarization on productive HBV infection. At multiplicities of genome equivalents (mge) >8,000, almost 100% of PHHs could be infected. In contrast, only a subset of HepaRG cells stained positive for HBcAg at comparable or even higher mge. Infection predominantly occurred at the edges of islands of hepatocyte-like HepaRG cells. This indicates a limited accessibility of the HBV receptor, possibly as a result of its polar sorting. Multidrug resistance protein 2 (MRP2), a marker selectively transported to the apical (i.e., canalicular) cell membrane, revealed two polarization phenotypes of HepaRG cells. HBV infection within the islands of hepatocyte-like HepaRG cells preferentially occurred in cells that resemble PHH, exhibiting canalicular structures. However, disruption of cell-cell junctions allowed the additional infection of cells that do not display a PHH-like polarization. Conclusion: HBV enters hepatocytes via the basolateral membrane. This model, at least partially, explains the difference of PHH and HepaRG cells in infection efficacy, provides insights into natural HBV infection, and establishes a basis for optimization of the HepaRG infection system. (HEPATOLOGY 2012;55:373-383) H uman hepatitis B virus (HBV), a small, enveloped DNA virus, is the prototypic member of the family, hepadnaviridae, causing acute and persistant liver infections.1 Currently, 360 million people are chronically infected with HBV and are, consequently, prone to developing progressive liver diseases, such as cirrhosis or hepatocellular carcinoma. Hepadnaviruses exhibit a pronounced liver tropism and a narrow host range.2 HBV predominantly infects and replicates in hepatocytes, but virus-specific antigens and nucleic acids have also been found in a number of nonhepatic tissues, including kidney, pancreas, and peripheral blood mononuclear cells.3 However, whether replication in extrahepatic tissues contributes to virus propagation is unclear.4,5 The preference for replication in the liver can only partially be explained by hepatocyte-specific transcription factors.6 Further liver specificity occurs at the stage of early infection, because the differentiation state of HepaRG cells and primary hepatocytes is a prerequisite for the expression of an HBVpreS1-specific receptor and subsequent infection (Meier et al., manuscript in preparation).

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Human Cytomegalovirus Infects Caco-2 Intestinal Epithelial Cells Basolaterally Regardless of the Differentiation State

Cited by 38 publications

References 26 publications

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

The susceptibility of primary cultured rhesus macaque kidney epithelial cells to rhesus cytomegalovirus strains

Hepatocyte polarization is essential for the productive entry of the hepatitis B virus

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