In the present study, we have identified cell surface nucleolin as a cofactor required for the efficient cellular entry of HPIV-3. The requirement of nucleolin during HPIV-3 infection and entry was evident from several observations, including (i) the binding of 35
S-HPIV-3 with nucleolin following VOPBA; (ii) the interaction of biotinylated HPIV-3 with nucleolin during cellular entry (adsorption stage); (iii) the specific interaction of HPIV-3 F, but not HN, with nucleolin; (iv) the inhibition of HPIV-3 infection and cell surface internalization by nucleolin antibodies; and (v) that preincubation of HPIV-3 with purified nucleolin reduced HPIV-3 infection and cell surface internalization. Moreover, the expression of nucleolin on the apical plasma membrane domain of polarized human lung epithelial cells, the domain preferentially utilized by HPIV-3 during entry (8
), additionally suggests the involvement of nucleolin during HPIV-3 entry.
Nucleolin is a major RNA binding protein of the nucleolus that shuttles between the nucleus and the cytoplasm (65
). Although nucleolin functions in the nucleus, it is now well established that a portion of intracellular nucleolin is also expressed on the surfaces of a variety of cells (11
). In addition, the present study has demonstrated that in human lung epithelial A549 cells, nucleolin is preferentially expressed on the apical plasma membrane domain. Cell surface nucleolin has been demonstrated to act as an adherence receptor for a bacterium, enterohemorrhagic Escherichia coli
), and as an entry cofactor for viruses like HIV-1 (33
) and coxsackie B virus (17
). For HIV-1, cell surface nucleolin was found to act as one of the coreceptors during virus entry. It is interesting that a recent study has reported that nucleolin may also be involved during the assembly of another retrovirus, murine leukemia virus (3
). In accordance with the previous studies on the role of cell surface nucleolin during cellular adherence and the entry of human pathogens, the present study has suggested similar involvement of nucleolin during HPIV-3 entry.
Although nucleolin is expressed on the cell surface, it does not possess a typical transmembrane domain. This observation led to the speculation that nucleolin may be localized on the cell surface as a peripheral membrane protein bound to an integral protein(s). Interestingly, a recent study has demonstrated that, like several secretory proteins (e.g., IL-1β, thioredoxin, and FGF), the cell surface nucleolin utilizes a nonclassical endoplasmic reticulum-Golgi pathway during its plasma membrane trafficking (53
). The physiological significance of cell surface nucleolin is borne out by previous observations that it functions as a receptor for several ligands. These ligands include cytokine midkine, lipoproteins, laminin, fructosyllysine, factor J, T-cell receptor, cell surface ectoprotein kinases, urokinase and its receptor, L-selectin, and heparan binding protein syndecans (11
). The interactions of cell surface nucleolin with these extracellular ligands were demonstrated to play an important role in cell proliferation, mitogenesis, differentiation, and immunogenic responses. Interestingly, a protein named NRP (nucleolin-related protein) bearing high (95%) homology to nucleolin was also reported to be expressed on the apical pole of polarized kidney epithelial cells (64
). In that context, a recent study has demonstrated that a bacterium, enterohemorrhagic E
O157:H7, that infects the epithelial cells from the luminal (apical) domain also utilizes nucleolin as one of its adherence receptors (63
Cellular entry of enveloped viruses constitutes a complex and orchestrated process whereby virus envelope proteins interact with an array of host proteins to facilitate efficient entry. The complexity of the viral entry mechanism is also evident from the observation that primarily two stages, attachment and fusion (internalization), comprise the entry process, whereby different cellular proteins are utilized during either of these steps. Furthermore, the interaction of envelope proteins with cell surface molecules at each step results in conformational changes, leading to their subsequent interaction with an additional primary and/or secondary receptor(s). These interactions have been well documented during the entry of HIV-1, which utilizes several attachment and fusion receptors and coreceptors (6
). For HPIV-3, it was demonstrated previously that SAR serves as the initial attachment receptor for the virus via its interaction with HN. It is postulated that this interaction induces conformational changes in the F protein (complexed with HN on the virus envelope), leading to the exposure of its fusogenic peptide and subsequent fusion of the host target membrane with the envelope. Interestingly, like HPIV-3, HIV-1 also initially attaches to the cell surface following the interaction of its envelope protein gp-160 with HS (58
). This event not only sequesters virus on the cell surface but also renders conformational changes in gp-160 that lead to its interaction with additional coreceptors that are required for efficient fusion. In that context, it is noteworthy that HIV-1 gp-160 shares a high degree of structural homology with paramyxovirus F protein (19
). Thus, it is possible that although HPIV-3 requires SAR during attachment, an additional cell surface cofactor(s) may be required for efficient internalization and/or fusion. Indeed, Sendai virus, an HPIV-3-related mouse parainfluenza virus that possesses both HN and F as its envelope proteins (42
) and requires SAR during entry (13
), also utilizes additional entry receptors, including glycophorins (72
), ganglioside GD1α (21
), and asialoglycoprotein receptor (44
), in a cell-specific manner. In that context, previous studies (9
) have demonstrated that both HS and SAR are required for efficient HPIV-3 entry into A549 cells, and in the present study we have shown that while cell surface nucleolin is not required for HPIV-3 attachment, it plays a role during postattachment internalization of the virus. Thus, we speculate that HPIV-3 utilizes SAR along with HS and nucleolin for efficient entry into human lung epithelial A549 cells. This hypothesis is further strengthened by our observation that nucleolin-blocking agents failed to completely abolish HPIV-3 infection and internalization. Similar utilization of SAR and/or HS and additional cell surface proteins during entry has been documented for several viruses (4
For example, rotavirus was shown to utilize both SAR and cell surface-expressed heat shock cognate protein 70 (hsc 70) during entry (27
). It is interesting that hsc 70, like nucleolin, is also a nucleocytoplasmic protein that does not possess a transmembrane domain and an endoplasmic reticulum-specific signal sequence. Moreover, like nucleolin during HPIV-3 entry, hsc 70 is not required for the attachment of rotaviruses but is required during postattachment events. The role of nucleolin during cellular entry of HPIV-3 could possibly be mediated by the chaperonin function of nucleolin, since nucleolin acts as a chaperone during the nuclear import or export of nuclear or cytoplasmic proteins (2
). It is well established that viral envelope proteins undergo major conformational changes during entry and the uncoating process as a result of their multiple contacts with various cell surface molecules. In that scenario, it could be speculated that chaperonin activity is required to obtain the conformationally favorable folding required for efficient entry. This phenomenon may also be true for rotaviruses, as suggested by a recent study (27
), since rotaviruses also utilize a nucleocytoplasmic chaperone protein hsc 70 for cellular entry.
Although, the present study has suggested a role of nucleolin in the entry of HPIV-3 into human lung epithelial cells, an additional cell surface molecule(s) is also required for HPIV-3 entry. This speculation is based on the observation that nucleolin-blocking agents failed to completely abolish HPIV-3 infection and internalization in A549 cells. In that scenario, nucleolin may act as a secondary cofactor during infection, since earlier studies have reported that the inhibition of primary receptors usually results in complete (95 to 100%) inhibition of virus infectivity (22, 28, 40, 66). Thus, the role of nucleolin during HPIV-3 entry may be limited to the augmentation of the efficiency of HPIV-3 entry into human lung epithelial A549 cells. Currently, studies are in progress to dissect the sequential and/or cooperative utilization of HS, nucleolin, and SAR by HPIV-3 envelope proteins during virus entry.
In summary, the present study has demonstrated that cell surface nucleolin serves as one of the cofactors required for the efficient entry of HPIV-3 into human lung epithelial cells. The role of nucleolin during this process is at the virus internalization stage but not at the attachment stage. However, based on the present study and previous observations, we speculate that an additional cell surface molecule(s) also plays an important role during HPIV-3 entry. Therefore, the identification and characterization of the function of this molecule(s) will provide novel insights into the mechanism of host-virus interactions and lead to the development of novel antiviral therapies.