The present work indicates the importance of GM1-rich microdomains (lipid rafts) and the requirement of cholesterol for RSV entry into normal human bronchial epithelial cells, which are considered to be one of the primary targets in natural infection. In addition, RSV hemifusion events appear to begin in the plasma membrane of NHBE cells.
The mixing of the outer membrane leaflets between the virus and the cell membrane seems to be triggered at the plasma membrane. This is supported by the fact that after the FRET release (indicated by the appearance of the green signal), there was a reduction in DiOC18-associated fluorescence intensity (A to C). This does not mean that DiOC18 is the only fluorophore that moves to the cell membrane. In order for the FRET release to happen, it is necessary that both R18 and DiOC18 fluorophores move away from the viral envelope into an area of larger volume such as the plasma membrane or an endosome. Regarding the loss of fluorescence, endosomal trafficking could not be the cause, since such traffic is expected to generate green endosomes due also to the FRET release. Moreover, based on what was previously reported by Sakai et al. (73
) in the context of tracking dually labeled influenza virus compared to what we found by tracking dually labeled VSV, there is a progression to the emergence of green endosomes in these viruses that are known to fuse in endosomes.
Our initial exploration using small-molecule inhibitors suggested that RSV infection may be facilitated by Pak1-driven rearrangements of the plasma membrane. Kolokoltsov et al. (41
) previously identified Pak1 as participating in RSV infection of HeLa cells. Pak1 activation is associated with actin phosphorylation and cytoskeletal reorganization (18
). However, Pak1 activation also accompanies endocytosis and macropinocytosis (17
). The intracellular trafficking of an endosome/macropinosome was not unambiguously identified by live-cell imaging in those experiments shown in . In a seminal paper describing the entry of human immunodeficiency virus by endocytosis, Miyauchi et al. (61
) labeled virions with both a lipophilic dye (DiD) and a diffusible content marker (NC-GFP). They found that viral markers were released (disappeared) sequentially in a subset of regular fusion events, often exhibiting a considerable delay between lipid and content transfer. They suggested that such events indicated a two-step fusion mechanism, in which the lipid transfer happens at the plasma membrane while the content is delivered from an endosome (61
). It is important to emphasize here that we have labeled only the virus envelope by using lipophilic dyes; we did not label the diffusible content marker, so we could not identify the site at which the content transfer occurred. However, in our case, the decline in DiOC18 after FRET release at the cell surface and the prevention of RSV infection by PAK1 inhibition suggest a two-step fusion phenotype for RSV, similar to the one described by Miyauchi et al. (61
). Moreover, they noted that the temporal separation of lipid and content transfer events suggests that the two-step fusion proceeds through a remarkably long-lived hemifusion intermediate.
On the other hand, RSV has been shown to inhibit the amiloride-sensitive sodium channels (ENaC) within minutes of contacting the bronchial epithelium, and such effect is reproduced by recombinant viral F protein (43
). Although amiloride and its analog 5-(N
-isopropyl)-amiloride (EIPA) are considered classical inhibitors of macropinocytosis, Karjalainen et al. (35
) have shown, using ruthenium red-based electron microscopy and fluorescence-based confocal microscopy, that EIPA does not block the internalization and formation of tubulovesicular structures inside the cell. However, EIPA does prevent the traffic of such structures from the peripheral cytoplasm. Consequently, it would be interesting to explore in a further study if RSV triggers the formation of intracellular tubulovesicular structures via Pak1 activation but prevents the trafficking of such structures from the peripheral cytoplasm via its amiloride-like effects. If this were the case, RSV would complete full fusion close to the cell plasma membrane, but possibly in an early endosome.
A requirement for clathrin-mediated endocytosis during RSV entry of HeLa cells was suggested by Kolokoltsov et al. (41
) based on the observation that RSV infection of HeLa cells was inhibited by knockdown of clathrin pathway-associated proteins. However, the fact that dynamin inhibition, using dynasore, did not prevent RSV entry implied that RSV may not be using clathrin-mediated endocytosis in NHBE cells. Dynamin is a large GTPase, with a remarkable ability to self-assemble into complex polymers that deform the membranes through a constriction process dependent on GTP hydrolysis. In association with other effector proteins, dynamin serves as a pinchase, releasing vesicles from donor membrane compartments (42
). Dynamin has been found to be a key player in clathrin-, caveola-, IL-2Rβ-, flotillin-, and circular dorsal ruffle-mediated endocytosis pathways as well as in phagocytosis (19
). Consequently, our finding that dynasore failed to prevent RSV infection suggests that RSV did not use dynamin-dependent endocytic mechanisms to enter NHBE cells. It is possible that HeLa cells, being an immortalized cell line, could behave differently from primary bronchial epithelial cells regarding the use of clathrin-mediated endocytosis for virus entry.
Although dynasore has been proven to be a very specific inhibitor of dynamin (1
), it will be important in future studies to determine if RSV localizes to clathrin-coated pits. On the other hand, the knockdown of those clathrin pathway-associated proteins identified in the siRNA screening by Kolokoltsov et al. (41
) may interfere with RSV infection through mechanisms different from clathrin-mediated endocytosis, such that inhibition of the clathrin pathway cannot distinguish between clathrin-mediated endocytosis and vesicular traffic from the trans-Golgi network (71
). Kolokoltsov et al. (41
) reported that the expression of a dominant negative version of Eps-15 inhibited viral infection of HeLa cells without affecting viral attachment and suggested that fusion, the only remaining step in entry, must be the target for this inhibition. However, this conclusion does not take into account the possibility that the F protein also has a receptor required for infection (13
) that may be sensitive to blockade of Eps-15 and, therefore, clathrin-mediated transport from the trans-Golgi network (9
). Overall, our data indicated that RSV hemifusion events in NHBE cells begin at the plasma membrane. It remains possible that the virus releases its contents from endosomes following the “two-step” scenario observed for HIV (61
), which begins in the plasma membrane but is completed in an endosome, or which it takes place very close to the cell membrane, as observed for poliovirus (4
Although it is possible that host cell proteins present in the RSV envelope could attach the virions to lipid rafts, it is tempting to speculate that the F protein mediates the viral attachment to such microdomains. Since cholesterol and sphingomyelin are the major constituents of the lipid rafts, it is interesting that the ectodomain of F protein has two cholesterol recognition amino acid consensus sites (CRACs)—[V/L-(X1-5
)-K/R, where X1-5
represents 1 to 5 of any amino acid] at the regions from 414 to 419 (VSCYGK) and from 452 to 461 (VGNTLYYVNK) (22
). These regions flank antigenic site IV, which is recognized by the neutralizing monoclonal antibodies 101F and MAb19 (3
). It could be that initially, or in any of the intermediary stages of the fusion process, the predicted CRACs bind to cholesterol present in the lipid rafts and that such a process could be sterically interrupted by a monoclonal antibody (56
The earliest stages of RSV entry were very sensitive to cholesterol depletion. Cholesterol may be a requirement for the entry of many viruses as demonstrated for Sindbis virus and Semliki Forest virus (36
). However, flavivirus penetration seems to be impaired by cholesterol (46
). We found that NHBE cultures depleted of cholesterol and subjected to cholesterol replenishment showed a clear increase in the percentage of RSV-infected cells to levels higher than those observed for control cultures. This observation is surprising, since HIV-1 and human herpesvirus 6, which both require cholesterol for successful entry, only partially recovered their infectivity with cholesterol replenishment (7
Although clathrin-mediated endocytosis also depends on the presence of cholesterol to facilitate the proper curvature at the cell membrane for endocytosis to ensue (70
), the sum of the observations reported here suggests that cholesterol is required for the formation of particular microdomains that enable RSV binding and fusion on the cell membrane of bronchial epithelial cells.
Zhang et al. (91
) reported that RSV preferentially targets the ciliated cells of the airway epithelium and that infection occurs exclusively via the apical surface. Interestingly, the lipid composition of the ciliary membrane is enriched in cholesterol-rich microdomains, consistent with lipid rafts having high liquid order, as determined by laurdan two-photon microscopy (21
). A classic study using electron microscopy and taking advantage of the ability of filipin to bind cholesterol found that filipin-sterol complexes appeared densely and uniformly distributed over most of the ciliary membrane (62
). In addition, both GM1 and GM3 gangliosides are found in the primary cilia (34
). Overall, the abundance of cholesterol-rich microdomains in the cilia may partially explain the preference of RSV to infect ciliated bronchial epithelial cells.
We have presented evidence that cholesterol-rich microdomains are of critical importance during the initial stages of attachment and fusion of RSV. The prophylactic and therapeutic implications of this finding might be exploited with cholesterol-tagged peptide inhibitors such as those developed by Porotto et al. (67
). The cholesterol moiety was found to target inhibitory peptides to the membrane site where fusion occurs, thereby increasing their antiviral potency to inhibit paramyxovirus (human parainfluenza virus type 3, Hendra virus, and Nipah virus) infection both in vitro
and in an animal model (67
Lipid rafts have also been reported to be the virion assembly site for RSV budding (55
). Targeting cholesterol-rich microdomains on the cell membrane might therefore impair both the production of infectious virions and their ability to spread. Gower and Graham (26
) reported that statins showed antiviral activity against RSV in vivo
. The pharmacological potential of cholesterol-lowering drugs to curb virus infection has also been proposed and evaluated in hepatitis C virus with promising results in which statins significantly improved the rapid antiviral response caused by pegylated interferon in an open-labeled randomized controlled study (60