During infection of host cells by enveloped virus, the fusion of the viral envelope and the cellular membrane is facilitated by viral fusion proteins. The fusion mechanism shares common features in all viral families so far described, but the overall structure of the different fusion proteins evolved separately and they are characterized as three distinct classes 
. Fusion proteins of the Togaviridae
families belong to the class II 
, which most likely also contains the E2 protein of HCV 
. Class II fusion proteins share a common molecular architecture and need a “partner” subunit with which they fold and form heterodimers. The chaperone-like role of this second protein, whose synthesis precedes that of the class II proteins, has been demonstrated for Semliki Forest virus (SFV) and tick-borne encephalitis viruses (TBE) 
. Another aspect shared by these chaperone-like subunits is their ability to fold correctly once expressed individually. Indeed, both the p62 protein of SFV and the prM protein of TBE did not show any major difference in folding once expressed in the absence of the second subunit on the coding unit 
. Differently from both SFV p62 and TBE prM proteins, however, E1 does not undergo proteolytic processing prior to virus release. Furthermore, the resulted HCV infectious particles are resistant to acidic pH treatment 
. These data may suggest a different role of E1 during entry or fusion steps.
Previous data generated in our laboratory suggest that the HCV glycoprotein E1 could play a role analogous to p62 and PrM in viral maturation. In an in vitro
translation system, unescorted E1 was able to achieve a complete folding process once provided with a small region of E2 that ensured the correct topology of the C-terminus 
. Afterwards, folding/assembly analysis of E1E2 stably expressed in eukaryotic cells revealed that E1 oxidation preceded E2 maturation, a behavior consistent with a potential chaperone-like role for the E1 protein 
. The analysis performed here extends these previous reports by analyzing the mechanism of unescorted E1 folding in stable transfected CHO cells and its stability at longer time points post-synthesis. The folding pathways of individually expressed E2, and E1E2 as single coding unit, were evaluated in the same system.
In terms of its general features, the individually expressed E1 was indistinguishable from the E1 expressed in the control expression unit: it did not present high molecular weight aggregates, it received the proper number of carbohydrates chains and was retained in the ER. The oxidative kinetics of E1 expressed as a single protein showed a complete oxidation between 2 and 3 hours post-synthesis (t1/2
~45 min). This kinetics was slowed down by the presence of E2, requiring up to 4 hrs to be completed (t1/2
~80 min). This result is consistent with a chaperone-like role played by E2 whose co-expression delays folding kinetics. However, the establishment of a DTT-resistant conformation for the E1 protein was a slow process proceeding independently of the presence or absence of E2 and it required approximately 4 hours to be achieved. Indeed, the percentage of the DTT resistant species was roughly equivalent in the two samples, without any yield increase of fully oxidized E1 when co-expressed with E2. Our folding analysis was also extended to detect association of E1 with ER resident chaperones, however we were not able to identify these in our system. In particular, we were unable to reveal any transient association with ER chaperones and calnexin (data not shown). This observation was particularly surprising since in a previous study we were able to detect a transient association of this lectin-type chaperone with E1 expressed as the polyprotein 
. It could be possible that the accelerated oxidation of unescorted E1 does not require the association with calnexin or that their interaction is so accelerated that it is difficult to detect.
Although our primary focus was E1, we performed parallel analysis of individually expressed E2 folding. To this end, we used a series of monoclonal conformational antibodies that limited our study to the detection of partial or fully folded protein. In our hands, the oxidation pathway of individually expressed E2 was severely impaired as was the achievement of a DTT-resistant conformation. These observations confirm previous studies showing the requirement of E1 co-expression and are consistent with a chaperone-like role of E1 
. Recent work from Krey et al. further supports our conclusion on the relative role of E1 and E2 on their own folding 
. These authors obtained very interesting findings on the overall E2 structure by analysis of a purified soluble truncated form of E2 whose folding was assisted by co-expression of the full length E1.
The finding that individually expressed E1 was able to successfully achieve a complete folding pathway is consistent with assessed data on analogous proteins from related viruses, namely TBE preM or p62 from SFV. However, such proteins have been followed only for the time required to complete folding, which is 60 and 15 min post-synthesis, respectively 
. Our analysis, however, was extended to determine the stability of unescorted E1 inside the cell. This study revealed that the persistence of intracellular E1 was linked to the contemporary presence of E2, likely associated in a heterodimer. Since inhibition of mannose trimming stabilizes the substrate protein that is targeted for degradation, the recovery of a low amount of E1 in overnight kifunesine-treated cells suggests that endoplasmic reticulum associated degradation (ERAD), with the proteasome as a final destination, are involved in this clearing 
. Contrary to what was observed for the E1 folding process, E2 does play a role in stabilizing the native E1 conformation.
Overall, E1 and E2 appear to assist each other but via two different strategies. E1 is required for a correct oxidation pathway of E2, slowing down the oxidative kinetics, increasing yield and preventing aggregation for the time required to install productive intra-chain interactions and reshuffling of disulfide bonds. With respect to this aspect, E1 has a similar mechanism of action to that of chaperones belonging to the Hsp70 or the lectin-like family 
. On the other hands, E2 also slows down oxidation of E1 but it does not appear to interfere with the achievement of the mature conformation of E1 while protecting this molecule from cellular degradation. This behavior mimics the action of species belonging to the Hsp90 family that induce conformational changes of native species leading to their activation or stabilization 
. According to these conclusions, E2 would always require E1 co-expression in cis,
whereas a portion of E2 might be sufficient to stabilize full length E1 in a native conformation.
E2 carries the major antigenic epitopes and, for obvious reasons, it has been targeted as the most promising candidate for the development of a protective vaccine 
. E1 is the less studied molecule of the pair even though it could be an attractive vaccine candidate, in terms of sequence conservation, absence of hypervariable regions and the presence of monoclonal neutralizing anti-E1 antibodies 
. We believe that this report provides the experimental basis for generating a stable E1 molecule to be purified and investigated for its immunogenic properties.