Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion.
The results of this study provide an explanation for several, apparently conflicting results from earlier studies with respect to the process of MHV cell entry, particularly also regarding the necessity of proteolytic cleavage of the CoV S protein. By using a replication-independent fusion assay, we confirmed that MHV entry requires clathrin-mediated endocytosis despite the well-known ability of the MHV S protein to cause cell-cell fusion at neutral pH. We demonstrate that MHV particles traffic to and fuse in lysosomes. Yet, MHV is much less sensitive to perturbation of the low pH in the endo-/lysosomal system than low pH-dependent control viruses VSV and IAV. Our results additionally indicate that, for fusion to occur, the S protein of MHV requires proteolytic cleavage immediately upstream of the FP, like other class I fusion proteins. Efficient inhibition of MHV entry was only observed using a pan-lysosomal protease inhibitor, and could not be achieved using more specific protease inhibitors. Introduction of an optimal furin cleavage site in the S protein immediately upstream of the FP abolished the requirement for trafficking of virions to lysosomes for fusion. However, this virus still required clathrin-mediated uptake for efficient entry. Consistent with a common mechanism for the entry of CoVs, FIPV, but not MERS-CoV, the latter of which contains a furin cleavage site immediately upstream of the FP, was shown to require trafficking to lysosomes and processing by lysosomal proteases for efficient entry. Based on these results we propose a model in which the cleavage site immediately upstream of the FP is an essential determinant of the intracellular site of CoV fusion
The importance of clathrin-mediated endocytosis and endosomal trafficking in the entry of MHV was revealed by several complementary approaches. One of these was siRNA-mediated gene silencing. Although - with the exception of RAB7A - knockdown was not monitored at the protein level, we believe this approach firmly demonstrates the importance of novel host factors for several reasons. Validated siRNAs were used and the experimental conditions were confirmed by analyzing the mRNA expression levels of several genes by quantitative RT-PCR. Furthermore, we made use of three independent siRNAs per target gene, and a target was only classified as a hit when at least two out three siRNAs showed the same phenotype. Importantly, our findings were strengthened by targeting multiple proteins per host cell pathway/complex, each time with very similar results. Moreover, hits obtained with the replication-dependent reporter assays were confirmed with our novel replication-independent enzyme complementation entry assay. Also the use of recombinant viruses differing only in their spike proteins enabled us to show that inhibition of virus infection upon siRNA transfection resulted from differences in virus entry and not virus replication. Finally, the results obtained were corroborated by using a large panel of inhibitors and by making use of haploid knockout cells, in which late endosome-to-lysosome trafficking was inhibited.
Our results demonstrate that MHV requires endocytic uptake for virus entry despite the S protein's ability to induce cell-cell fusion at neutral pH. Endocytic uptake is also required for a mutant virus carrying a S protein with a FCS immediately upstream of its FP, despite the relative insensitivity to high concentrations of BafA1. Therefore, the ability of a virus to infect cells in the presence of BafA1 does not necessarily imply virus entry to occur at the cell surface. Also a recombinant MHV carrying the spike protein of MHV-4 (MHV-JHM) was found to enter via clathrin-mediated endocytosis (MHV-S4;) despite its ability to cause extensive cell-cell fusion. The ability of MHV to cause cell-cell fusion at neutral pH while requiring endocytic uptake for virus-cell fusion suggests different requirements and triggers for these two fusion processes. Similarly, RSV was recently shown to enter cells after endocytic uptake despite the ability of this virus to cause cell-cell fusion.
The present study confirms and extends previous publications on MHV entry via clathrin-mediated endocytosis. Both siRNAs downregulating clathrin-mediated endocytosis-associated proteins, such as clathrin heavy chain (CLTC) and Dynamin 2 (DNM2), and agents affecting this uptake pathway (Chlopro, Dynasore, Dyngo-4a) were capable of inhibiting infection with MHV. Importantly, these findings could be confirmed in our novel replication-independent virus-cell fusion assay, thereby directly showing an involvement of clathrin-mediated endocytosis in entry of MHV. Analysis of several accessory factors of clathrin-mediated endocytosis showed that clathrin-mediated entry of MHV strain A59 depends on clathrin-adaptor DAB2, but not on EPS15 or AAK1. Previously, clathrin-mediated entry of MHV strain 2 was also shown to be independent of EPS15. Based on the use of inhibitors, it was earlier concluded that MHV entry depends on cholesterol and lipid-rafts, which may be indicative of caveolae-mediated endocytosis.
Although our replication-dependent assays indicate a requirement for caveolin 2 (CAV2) for infection, this protein was shown not to be involved in virus entry using our fusion assay. Also depletion of other proteins involved in caveolae-mediated endocytosis, including caveolin 1 (CAV1) and flotillins 1 and 2 (FLOT1 and FLOT2) did not affect MHV infection or fusion. Interestingly, fusion of MHV was severely inhibited by EIPA, an inhibitor of the Na+/H+ exchanger NHE1, which is regarded as a hallmark inhibitor of macropinocytosis. Apparently, inhibition of virus entry by EIPA does not prove by itself that a virus enters via this particular pathway. EIPA has been reported to affect several other cellular processes, including actin remodeling, internalization of lipid rafts, distribution of endosomes, and even clathrin-mediated endocytosis. Similar to the results obtained with the HeLa cells, also infection of murine LR7 cells was inhibited by compounds interfering with clathrin-mediated endocytosis.
MHV virions require trafficking through the endocytic pathway to lysosomes for efficient entry. Upon clathrin-mediated uptake these virions are temporarily associated with early endosomes as demonstrated by co-localization with RAB5 during live cell imaging. Furthermore, the importance of early endosomes for entry was indicated by siRNA-mediated downregulation of various proteins associated with early endosomes (EEA1, RAB5A, RAB5B, and RAB5C), which inhibited MHV infection, as well as virus-cell fusion. However, only very few MHV particles appeared to fuse in the early endosomes. Live cell imaging indicated fusion largely to occur in late endosomes and/or lysosomes. Consistently, depletion of host proteins associated with late endosome and late endosome-to-lysosome maturation (RAB7A, RAB7B, and the HOPS complex subunits VPS11, VPS33A, VPS39 and VPS41) or addition of U18666A, which blocks late endosome-to-lysosome trafficking, were shown to inhibit both infection and virus-cell fusion. The importance of lysosomes for entry was confirmed by using knockout cells lacking a functional HOPS complex (For a review on the HOPS complex ). Interestingly, in these cells lysosomes are clustered in a perinuclear region of the cell rather than dispersed throughout the cytoplasm. Complementation of the missing HOPS subunit restored the normal lysosome distribution and entry of MHV. The importance of late endosome-to-lysosome trafficking for efficient entry was also observed in murine cells and for MHV-S4 carrying the S protein of MHV-4.
Corroborating the importance of trafficking of MHV virions through the endocytic pathway, perturbation of endosome maturation by the addition of inhibitory agents, such as ammonium chloride, BafA1, Chloroquine, and Monensin inhibited infection and fusion of MHV. Also the importance of the actin and microtubule cytoskeleton - as demonstrated by the inhibition of MHV entry by downregulation of the Arp2/3 complex factors (ACTR2 and ACTR3), of the microtubule-associated transporter dynein (DYNC1H1 and DYNC2H1), or by addition of actin- or microtubule-affecting drugs - may be explained by the documented involvement of the cytoskeleton in endosome maturation. Indeed, entry of MHV-S2′FCS, which presumably fuses in early endosomes, was much less affected by actin-affecting drugs than that of MHV carrying wild type spike proteins. However, we cannot exclude that actin also plays a role in the clathrin-mediated uptake of MHV particles, as has been observed for VSV and other pathogens that depend on clathrin-mediated endocytosis.
MHV particles require trafficking to the low pH environment of lysosomes to achieve membrane fusion. Nevertheless, MHV was much less sensitive to elevation of pH in the endo-/lysosomal system by the addition of BafA1 than viruses known to fuse in early or late endosomes (VSV and IAV). BafA1, an inhibitor of vacuolar-type H+-ATPase was effective in blocking MHV entry only at high concentrations, which are known to prevent endosomal maturation in addition to the elevation of the pH. The absence of a functional HOPS complex, which is required for late endosome-to-lysosome maturation, did not affect infection of cells with VSV or IAV, while entry of MHV was severely reduced. Thus, the low pH trigger that mediates entry of VSV and IAV in the endosomal system of these cells, is not sufficient to induce fusion of MHV. Other environmental cues, present in lysosomes only, are apparently required to activate conformational changes in the S protein leading to fusion. Indeed, inhibition of the three major classes of proteases present in the lysosome by CPI effectively prevented MHV fusion. Infection of murine LR7 cells with MHV was also inhibited by CPI. Strikingly, other inhibitors that affect members of a single protease family had none or only little impact on MHV fusion. These results are in consistence with a functional redundancy of protease family members and may explain why previous studies using specific lysosome protease inhibitors failed to detect entry inhibition. Also, the inhibition of MHV entry by MG132 may be explained by the known ability of the proteasome inhibitor to negatively affect lysosomal proteases, although we cannot exclude that MG132 affects entry by its interference with lysosomal trafficking.
Our results indicate that cleavage of the S protein immediately upstream of the FP is essential for CoV entry and determines the intracellular site of fusion. Although we did not demonstrate cleavage of MHV S at the FP proximal position directly, a recent study found a cleaved form of the MHV S2 subunit to correspond with the fusion-active form. Furthermore, introduction of an optimal FCS at the FP proximal position abolished the entry inhibition by the pan-lysosomal protease inhibitor whilst introducing a dependency on furin-related enzymes. Consistent with the known presence of active furin in early endosomes the mutant virus no longer required trafficking to late endosomes/lysosomes for entry to occur. However, in the presence of furin inhibitor, entry of this mutant MHV was much more efficient in wild type cells than in cells lacking a functional HOPS complex, indicating that under certain circumstances lysosomal proteases may play a role in entry of this virus as well. Trafficking of virions to lysosomes was shown to be also important for entry of FIPV, but not of MERS-CoV, in agreement with the latter virus containing a putative FCS immediately upstream of the FP. Correspondingly, entry of FIPV was inhibited by the pan-lysosomal protease inhibitor CPI but not by furin inhibitor, while the reciprocal held true for MERS-CoV. The importance of S protein cleavage downstream of the S1/S2 boundary and upstream of the FP for infection has so far only been demonstrated for SARS-CoV and IBV.
Based on the present study and on the work of others, we conclude that cleavage at the FP proximal position is likely to be a general requirement for CoV entry. With the exception of possibly IBV, cleavage at this position does not appear to occur in the virion-producing cell as it is not observed in released virions, but in the target cell.This suggests that receptor binding or other environmental cues are necessary to render the cleavage site accessible for proteolysis in the intact virion. Also for several other viruses, including RSV and Ebola virus, cleavage of the fusion protein upon endocytosis has been shown to be required for entry. Our results furthermore show that cleavage at a FP-proximal position is an important determinant of the intracellular site of fusion. The question remains, however, why some CoVs evolved to fuse in early endosomal vesicles while others require trafficking to lysosomes. In view of the growing number of proteases that have been shown to cleave CoV spike proteins, this question should probably be studied in relation to the proteolytic enzymes available in the CoV target tissues and cells in vivo
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