Data from this study indicate that MCAT-1-mediated MoMuLV entry can occur independent of the clathrin-coated-pit-mediated endocytic pathway. Based on immunofluorescence analysis, we have shown that clathrin is not involved in MoMuLV entry. Moreover, biochemical analysis demonstrated that the transduction ability of MoMuLV vector is not regulated by dynamin expression. In addition, disruption of a putative internalization signal in MCAT-1 did not affect the transduction ability of the MoMuLV vector.
The endocytic pathways of different ligands and cell surface molecules have been studied by indirect immunofluorescence labeling and confocal microscopy using antibodies that are specific to ligands, cell surface molecules, or cellular markers that reside in different stages of endocytic vesicles (14
). Since generating antibody specific to MCAT-1 or extracellular epitope-tagged receptor that retains wild-type receptor function has not been successful, we constructed GFP-tagged MCAT-1 (MCAT-1-GFP), which retains wild-type receptor function, to monitor MCAT-1 during MoMuLV entry. 293/MCAT-1-GFP cells showed bright green fluorescence primarily around the cell membrane and some inside the cell. Even though the green fluorescence inside the cell was not thoroughly analyzed, it has been shown by others (36a
) that some labeled receptor protein is in the Golgi compartment.
By indirect immunofluorescence labeling followed by confocal microscopy analysis, we observed that, in both 293/MCAT-1 and 293/MCAT-1-GFP cells, small portions of MoMuLV SU label started to appear inside the cell after a 5-min incubation at 37°C and then gradually increased. When the 37°C incubation time period was prolonged, we observed that complete disappearance of SU labels inside the cell and on the cell surface took 7 to 10 h, which is consistent with the results of previous studies by Andersen and Nexø (2
). They have shown that when lysates of 3T3 cells infected with B-tropic virus C57MC were separated by electrophoresis on SDS-polyacrylamide gel and analyzed by autoradiography, virus protein gp70 could be detected from the infected cells at up to 7 to 12 h of incubation at 37°C. In addition, in our study, colocalization of the receptor and the SU protein was also observed for the whole time (7 to 10 h), suggesting that the envelope protein, after binding, remains in contact with receptor inside the cell for an extended time and is recognized by specific antibody.
Even though the intracellular SU label appears to increase over the 37°C incubation time period, it is also true that substantial amounts of SU label colocalized with receptors is still present at the cell surface even after a 30-min incubation. This finding could be used to support the counterhypothesis that MoMuLV enters cells through direct membrane fusion. The significance of these populations of SU proteins in MoMuLV entry requires further investigation.
Immunofluorescence labeling and confocal microscopy analysis with purified virions (to exclude the staining of shed gp70 and membrane pieces containing envelope) gave the same results as the unpurified crude supernatants. A study by Yu et al. (75
) showed that the anti-SU antibody used in our immunofluorescence labeling (83A25) could not recognize purified gp70. Taken together, these data indicate that the SU labels we observed in our study are not from shed gp70.
For biochemical analysis of MoMuLV entry, we used HeLa cells (K44A HeLa) overexpressing a dominant-negative GTPase mutant of dynamin, K44A. Dynamin, a 100-kDa GTPase, is a component of clathrin-coated pits that helps the formation of constricted clathrin-coated vesicles. Dynamin is the mammalian homologue of the Drosophila shibire
gene product (9
). Mutations in shibire
cause a defect in endocytosis, leading to the accumulation of clathrin-coated pits on the cell membrane (29
). Similarly, invaginated clathrin-coated pits accumulate on the surfaces of K44A HeLa cells overexpressing a dominant-negative GTPase mutant of dynamin (12
), even though receptors on the cell surface can still bind to their ligands, establishing that dynamin is required for clathrin-coated-vesicle formation. In these HeLa cells, clathrin-coated-pit-mediated endocytosis of transferrin and EGF is blocked >80% and >60%, respectively (12
). Using K44A HeLa cells, adenovirus (70
) as well as ligands such as transferrin and EGF have been shown to utilize the clathrin-mediated endocytic pathway for receptor-mediated entry. In our study, both wild-type HeLa and K44A HeLa cells expressing MCAT-1 and MCAT-1-GFP demonstrated comparable binding and transduction ability by the MoMuLV vector, suggesting that dynamin is not critical for MCAT-1-mediated MoMuLV entry. Interestingly, while the mutant dynamin expression could significantly inhibit the transduction ability of VSV-G vector, the transduction ability was not completely abolished. Previous studies by Damke et al. (11
) and Wang et al. (70
) using the same HeLa cells to study the clathrin-mediated endocytosis of transferrin, EGF, and adenovirus infection demonstrated that ligand internalization and virus infection in these cells were not completely inhibited, suggesting the existence of an alternative pathway(s) (30
). Since the transduction ability of the MoMuLV vector did not give a significant difference in HeLa cells expressing wild-type or mutant dynamin, MoMuLV may utilize this alternative pathway(s) for infection. Further studies to characterize these alternative pathways and their possible role in MoMuLV entry are necessary. Finally, we cannot exclude the possibility of an incomplete inhibition of endogenous dynamin by the overexpressed dominant-negative mutant form of dynamin in HeLa cells.
For transmembrane receptors that are endocytosed by the clathrin-coated-pit-mediated pathway either constitutively or by ligand activation, the intracellular region or C terminus of the receptors has been shown to be important for recruiting receptors into the clathrin-coated pits and for subsequent sorting to different stages of cellular trafficking within the cell (34
). Mutagenesis studies of transferrin, EGF, and several G-protein-coupled multitransmembrane receptors support the idea of the existence of an internalization signal. Tyrosine-based motif structures are shown to bind in vitro with AP-2 protein (25
), a component of clathrin-coated pits. Separate domains of AP-2 protein can bind to clathrin and to some internalization signals, thereby allowing AP-2 protein to recruit transmembrane receptors and clathrin into the clathrin-coated pits. We constructed mutated receptors whose putative internalization signal for the clathrin-coated-pit-mediated endocytic pathway is disrupted. 293T cells expressing mutated receptors were able to be transduced by ecotropic retroviral vectors, as were cells expressing wild-type MCAT-1, demonstrating that the two tyrosine-based motifs in MCAT-1 do not play a significant role in MoMuLV transduction. These data further suggest that MoMuLV entry does not involve a clathrin-coated-pit-mediated endocytic pathway but the potential role of the two tyrosine-based motifs in MCAT-1 distribution or function requires further investigation. In addition, since we did not test every putative internalization signal of MCAT-1, further investigations will be needed to determine if there is an internalization signal in the intracellular region of MCAT-1 or the possibility of direct membrane fusion for MoMuLV entry.
Even though the clathrin-coated-pit-mediated endocytic pathway has been recognized as the best characterized adsorptive pathway so far, the discovery of non-clathrin-coated invaginations of the plasma membrane, caveolae, suggests that there is more than one pathway in the cell that can participate in cellular trafficking. Caveolae are specialized microdomains (55
) that can also be located by immunolabeling the marker protein caveolin (43
), and caveolae structures can be reconstituted in a cell-free system (55
). Originally, caveolae were postulated to play a role in transcytosis of molecules in polarized cells, but more recent work suggests that caveolae may also mediate receptor-mediated endocytosis. Caveolae appear to be involved in vesicular transport of toxins (44
), ligands bound to GPI-anchored protein (47
), viruses (24
), and seven-transmembrane surface proteins (55
). Interestingly, immunohistochemistry of porcine pulmonary artery endothelial cells demonstrates the colocalization of cationic amino acid transporter (CAT-1) with caveolar structures (41
). Therefore, although it is not clear whether CAT-1 and MCAT-1 utilize the same mechanism for amino acid transport and virus entry, respectively, it is still attractive to speculate that caveolae may play a role in ecotropic MuLV entry. However, a detectable amount of caveolin expression and morphologically identifiable caveolar structures are enriched only in certain cell types, such as lung endothelial and polarized cells (55
). In addition, endothelial cells change phenotypically when isolated and grown under culture conditions that lead to a 10-fold-lower level of caveolae in vitro (55
). Furthermore, it should be noted that not all non-clathrin-coated invaginations observed on the plasma membrane are caveolae. Non-clathrin-coated and non-caveola-coated invaginations that pinch off to form smooth vesicles carrying the fluid-phase marker horseradish peroxidase into cells have been extensively characterized in a number of cell types (30
Ecotropic MoMuLV-derived retroviral vectors have been engineered to target different cell surface molecules for use in gene therapy. Modifications of the SU region of MoMuLV have been introduced by insertion or replacement with a single-chain antibody or ligand that is specific to a cell surface antigen or receptor (10
). Most of these targeted virions retained wild-type binding ability for their specific receptor or cognizant cell surface molecule but demonstrated very low or no transduction ability, suggesting the existence of a postbinding block in the engineered vectors (10
). These data indicate that retroviruses require specific properties of cell surface molecules to allow the release of viral cores into the cytoplasm. Further investigations to elucidate the details of the MCAT-1-mediated MoMuLV entry pathway are needed. These strategies will be helpful in designing better MoMuLV-based targetable retroviral vectors for gene therapy.