It is well documented that TM sequences can play other roles than simply anchoring a protein in a membrane. HCV glycoproteins are good examples of additional functions performed by TMDs and represent an attractive model with which to understand the multifunctionality of such domains. We show here that mutation of charged residues located in the TMDs of HCV glycoproteins leads to an alteration in the processing, subcellular localization, and assembly of these envelope proteins. These data suggest that these charged residues play a key role in the formation of the viral envelope.
The first stretch of hydrophobic residues present in the TMDs of HCV glycoproteins E1 and E2 is not sufficient for an efficient arrest of translocation. A similar observation has been reported for a glycoprotein (prM) of another member of the family Flaviviridae
(dengue virus type 4) (44
). Natural stop-transfer sequences generally consist of more than 18 mainly hydrophobic amino acid residues and are followed by positive charges (62
). They have two functions: to interrupt the ongoing protein translocation and to anchor the final protein in the membrane. The inefficiency in membrane insertion in the absence of the second hydrophobic stretch (this work and reference 44
) might be due to the short length of the first hydrophobic sequence of the TMDs of the envelope proteins in the Flaviviridae
family. However, it has been shown experimentally that an artificial hydrophobic sequence as short as 8 leucine residues can cause an efficient stop of translocation (32
). In contrast, when a membrane-spanning sequence was made of alanine residues, up to 19 residues were necessary to obtain the same stop-transfer efficiency. With a 50:50 mixture of leucine and alanine, at least 11 residues were required. The first hydrophobic stretch of E2 contains 6 leucine residues which could compensate for its short length (11 residues). However, the charged residues located after this hydrophobic segment can also have some effect on the efficiency of a stop-transfer sequence. Indeed, positive charges after the hydrophobic segment are more favorable than negative charges (32
). For instance, in the case of prM of dengue virus type 4, an additional arginine residue introduced after the first stretch of hydrophobic residues has been shown to increase the efficiency of its membrane insertion (44
). It is likely that the short length of the first stretch of hydrophobic residues in the TMD of HCV glycoproteins and the lack of reinforcement by a positive charge make the first stretch of hydrophobic sequence an inefficient stop-transfer signal.
We demonstrate here that charged amino acid residues located in the segments connecting the two hydrophobic stretches of HCV glycoprotein TMDs play a major role in ER retention of these proteins. ER retention by TMDs has been reported for several TM proteins (2
), and a usual feature of membrane determinants for ER retention is the presence of one or several hydrophilic residues within the hydrophobic TMD. The introduction of charged residues in the hydrophobic segment of a plasma membrane protein is sufficient to cause its retention in the ER, and their effect is strongest when they are localized toward the middle of the TMD (2
). For HCV glycoproteins, there is no clear evidence that the charged residues are located in the middle of a single membrane-spanning segment. In the case in which the double-membrane-spanning topology is maintained, these charged residues should be exposed on the cytosolic face of the membrane. However, it cannot be excluded that a reorientation of the second stretch of hydrophobic residues occurs immediately after the signal sequence cleavage at its C terminus. If there is such a flipping of the second hydrophobic segment, the TMDs of HCV glycoproteins could form a single TM segment with the charged residues in the middle of the membrane-spanning sequence. The mechanism of ER localization by such a signal is poorly understood, but it has been proposed to be due to interactions with proteins involved in the ER retrieval machinery (36
). However, this does not explain how some proteins, like HCV glycoproteins (21
), are strictly retained in the ER by their TMD.
In the absence of any reorientation in the TMDs of HCV glycoproteins, the two membrane-spanning segments are expected to be short. Hydrophobic sequences which are shorter than the average of those of plasma membrane proteins have been shown to be involved in Golgi or ER retention (4
), and a lipid-based mechanism has been proposed for TMD-mediated retention (4
). The mixed lipid populations in the intracellular membrane would separate into lipid microdomains with distinct compositions, thicknesses, and degrees of structural perturbability. Proteins would selectively partition into one domain and so be prevented from entering transport vesicles comprising the other domain by virtue of physical properties of their TMDs. Whether the TM sequences of HCV glycoproteins fit into this model remains to be proven.
The charged amino acid residues located in the middle of the TMDs of HCV glycoproteins also play a major role in the assembly of HCV glycoproteins. These data suggest a direct interaction between the TMDs of HCV glycoproteins. This interaction might be due to a direct involvement of the charged residues of the TMDs of E1 and E2. This might explain the lack of interaction when these charged residues are replaced by an alanine. However, it is very likely that the interaction between the TMDs involves a direct contact between the hydrophobic segments of E1 and E2. Therefore, the lack of interaction after mutation of the charged residue(s) might be due to a conformational change in the structure of the TMDs of HCV envelope proteins.
Sequence analysis of the TMDs of envelope proteins of other Flaviviridae viruses revealed a similar organization for all the members of this viral family. Despite some differences observed for the members of the Flavivirus genus, the presence of at least one positively charged residue was systematically observed in the short segment connecting the two hydrophobic stretches. One can expect that the TMDs of the envelope proteins of the other Flaviviridae viruses should play functions similar to those reported here for HCV glycoproteins and that this charged residue might play a crucial role in these functions. However, when other charged or polar residues are present in the connecting segment, their concomitant mutation might also be required to modify the functions of these TMDs.
In addition to the functions described above, the TMDs of HCV glycoproteins might also play a direct role in the assembly of the particle. Viral envelope proteins can play a major role in virus budding (26
), which is a late stage of virus assembly corresponding to the acquisition of a membrane that surrounds the nucleocapsid (17
). Virus budding can occur at the plasma membrane or at an intracellular membrane along the secretory pathway. For the Flaviviridae
viruses, ultrastructural studies of virus-infected cells indicate that virion morphogenesis occurs in association with intracellular membranes believed to be derived from the ER (59
). It has generally been thought that budding is driven by interactions between the viral TM proteins and the nucleocapsid. This model fits very well for the alphaviruses (70
). However, it is now evident that enveloped viruses use various kinds of proteins for budding. Indeed, virus budding can also be driven by capsid or core protein only (retrovirus), by membrane protein only (coronavirus), or by matrix protein with the assistance of spikes and ribonucleoprotein (rhabdovirus and possibly paramyxovirus and orthomyxovirus) (26
). The mechanism of budding has not been specifically studied for members of the family Flaviviridae
, but flavivirus envelope proteins can exhibit an independent budding activity. Indeed, they can form capsid-free membrane particles by themselves (59
). This observation suggests that, at least for the genus Flavivirus
, the envelope proteins play a major role in budding. However, this does not exclude the possibility of a role played by the capsid. In this case, we would expect to have some interaction between the nucleocapsid and cytosolic residues of the envelope proteins. The charged residues which were shown to play an important role for the functions of the TMDs of HCV glycoproteins would be potential candidates for such interactions.
As shown in this work, the various functions played by the TMDs of HCV glycoproteins can be disrupted by mutating charged residues present in the membrane anchor of these proteins, indicating that these functions are tightly linked together. These charged residues probably play a crucial role in the structure of these domains. The analysis of the three-dimensional structure of these domains should allow further understanding of their multifunctionality. Such a knowledge would also help our understanding of some crucial events in the biogenesis of the envelope of HCV and other viruses of the Flaviviridae family.