Human Cytomegalovirus Entry Research Articles

Differentiation-dependent redistribution of heparan sulfate in epithelial intestinal Caco-2 cells leads to basolateral entry of cytomegalovirus.

Human cytomegalovirus (HCMV) causes a broad spectrum of clinical manifestations in immunocompromised patients, including infection of the gastrointestinal tract. To investigate the role of epithelial cells in the gastrointestinal HCMV disease, we used the intestinal epithelial cell line Caco-2, which is permissive for HCMV replication. In differentiated Caco-2 cells, we showed previously that HCMV infection proceeds preferentially from the basolateral membrane, suggesting that receptors for HCMV may be contained predominantly in the basolateral membrane (A. Esclatine et al., 2000, J. Virol. 74, 513-517). Therefore, we examined expression and localization in Caco-2 cells of heparan sulfate (HS) proteoglycan and annexin II, previously implicated in initial events of HCMV infection. We observed that annexin II is expressed in Caco-2 cells, but is not essential for entry of HCMV. We showed that, during the differentiation process, HS, initially present on the entire surface of the membrane of undifferentiated cells, ultimately became sequestered at the basolateral cell surface of fully differentiated cells. We established by biochemical assays that membrane-associated HS proteoglycan mediates both viral attachment to, and subsequent infection of, Caco-2 cells, regardless of the cell differentiation state. Thus, the redistribution of HS is implicated in the basolateral entry of HCMV into differentiated Caco-2 cells.

Lactoferrin and cyclic lactoferricin inhibit the entry of human cytomegalovirus into human fibroblasts

Lactoferrin is mainly produced by polymorphonuclear leukocytes and has been demonstrated in mammalian milk and external secretions. Lactoferrin is an iron-binding, multifunctional protein and may play an important role in immune regulation and in defense mechanisms against bacteria, fungi and viruses. Lactoferricin is a potent antimicrobial peptide generated from the N-terminal part of lactoferrin by pepsin cleavage. We demonstrate that lactoferrins from different species and its N-terminal peptide lactoferricin (particularly the cyclic form) inhibit expression of early and late antigens, as well as production of infectious viral progeny during human cytomegalovirus (HCMV) infection in vitro. Iron-saturated lactoferrin did not affect HCMV antigen expression. Heparin had the same effects as iron-depleted lactoferrin. Yet, mixtures of lactoferrin and heparin did not inhibit HCMV multiplication i.e. lactoferrin and heparin seemed to mutually block each other's antiviral activities. HCMV-infected cells exposed to lactoferrin and cyclic lactoferricin contained less intracellular virus than unexposed cells. The antiviral activity of cyclic lactoferricin was more than seven-fold weaker than that of the maternal molecule. Lactoferrin and cyclic lactoferricin prevented HCMV entrance into the host cell.

Interference with annexin II has no effect on entry of human cytomegalovirus into fibroblast cells.

Annexin II has been identified as a human cytomegalovirus (HCMV)-binding protein, shown to be a component of purified virions and proposed as a cellular receptor for the virus. In addition, annexin II is capable of associating with the major HCMV envelope glycoprotein, gB (gpUL55). As one approach to examine the role of annexin II in virus entry, a high-titre polyclonal annexin II-specific antibody was produced and its effects in virus entry and cell-to-cell spread assays were tested. This anti-annexin II serum recognized virion and cell surface annexin II and annexin II-derived peptides. Recombinant annexin II, with characteristic calcium- and phospholipid-binding activities, was also examined. Pretreatment of cells, virions or both with polyclonal anti-annexin II serum, affinity-purified anti-annexin II antibodies or recombinant annexin II protein prior to infection was inconsequential to the entry of HCMV into fibroblasts. HCMV was also able to dose-dependently penetrate annexin II-deficient 293 cells. Furthermore, the spread of HCMV from cell to cell was not inhibited in the presence of polyclonal anti-annexin II antibodies or exogenous annexin II protein. These experiments do not support a direct role of annexin II in virus entry or spread. Alternative roles for the gB-annexin II interaction are proposed.

Receptor-binding properties of a soluble form of human cytomegalovirus glycoprotein B.

The human cytomegalovirus (HCMV) glycoprotein B (gB) (also known as gpUL55) homolog is an important mediator of virus entry and cell-to-cell dissemination of infection. To examine the potential ligand-binding properties of gB, a soluble form of gB (gB-S) was radiolabeled, purified, and tested in cell-binding experiments. Binding of gB-S to human fibroblast cells was found to occur in a dose-dependent, saturable, and specific manner. Scatchard analysis demonstrated a biphasic plot with the following estimated dissociation constants (Kd): Kd1, 4.96 x 10(-6) M; Kd2, 3.07 x 10(-7) M. Cell surface heparan sulfate proteoglycans (HSPGs) were determined to serve as one class of receptors able to facilitate gB-S binding. Both HSPG-deficient Chinese hamster ovary (CHO) cells and fibroblast cells with enzymatically removed HSPGs had 40% reductions in gB-S binding, whereas removal of chondroitin sulfate had no effect. However, a significant proportion of gB-S was able to associate with the cell surface in the absence of HSPGs via an undefined nonheparin component. Binding affinity analysis of gB-S binding to wild-type CHO-K1 cells demonstrated biphasic binding kinetics (Kd1, 9.85 x 10(-6) M; Kd2, 4.03 x 10(-8) M), whereas gB-S binding to HSPG-deficient CHO-677 cells exhibited single-component binding kinetics (Kd, 7.46 x 10(-6) M). Together, these data suggest that gB-S associates with two classes of cellular receptors. The interaction of gB with its receptors is physiologically relevant, as evidenced by an inhibitory effect on HCMV entry when cells were pretreated with purified gB-S. This inhibition was determined to be manifested at the level of virus attachment. We conclude that gB is a ligand for HCMV that mediates an interaction with a cellular receptor(s) during HCMV infection.