In the present study we analyzed a series of basic residues at the N terminus of the HCV core protein to determine their potential involvement in HCV infectivity in the context of the HCVcc system (55
). Importantly, we identified four basic amino acid residues, R50, K51, R59, and R62, which are essential for the assembly of infectious viral particles. These residues are highly conserved in the HCV core protein (Fig. ).
As reported previously (9
), the N terminus of HCV core protein contains two highly conserved clusters of basic amino acid residues. These two clusters (aa 6 to 23 and 39 to 62) are separated by a neutral linker region. Moreover, the RNA sequence coding the first 62 residues of the core protein has been reported to contain highly conserved structures in the form of two stem-loops: SLV and SLVI (51
). These structures are important for RNA translation/replication (35
). Among the modifications introduced into the first basic cluster of the core protein, only the R17R18K23 mutant was concerned with the SLV structure. Nucleotide substitutions introduced into SLV decreased replication by about 3-fold and reduced infectious titer by about 5-fold. Indeed, disruption of SLV has been previously reported to reduce viral replication and infectious titer of HCV (54
), which can result from disruption of RNA-RNA or RNA-protein interactions.
In the first cluster of basic residues, mutants K6R9K10 and K12R13 did not disrupt SLV structure. The K6R9K10 mutant decreased viral replication by about 12-fold and the infectivity titer by about 7-fold, whereas the K12R13 mutant had replication and infectivity levels comparable to those of the wt. The data obtained from these three mutants suggest that the reduced infectivity could be due to the decreased levels of translation/replication. Moreover, a larger modification of basic residues as generated in the RK/8A mutant leads to a strict translation inhibition and to an unviable mutant. While most IRES sequences require only the 5′ NTR, the HCV IRES seems to be dependent on a sequence downstream of the initiating AUG codon (45
). Furthermore, a cellular RNA-binding protein has been identified that modulates HCV translation by interacting with the core protein-coding sequence. Indeed, modification of the NSAP1-binding site, which is composed of an adenosine-rich region, has been shown to reduce the HCV IRES activity in an in vitro
translation assay (24
). Replacing the basic residues with alanines led to modifications of the first two nucleotides of each codon in the RK/8A mutant, which changed several adenosines to cytosine and guanine. This probably prevents NSAP1 interaction with RNA and consequently reduces its translation. In cellulo
, the lower viral replication and production result consequently from a lower level of translation. Altogether, our data suggest that no specific residues or motifs affecting virus infectivity are present in the first basic cluster of core protein.
Some mutations of basic amino acid residues of the second cluster extend their nucleotide modifications to the SLVI RNA structure. The R39R40R43 and R47R50K51 mutants, which disrupt the upper and middle parts of SLVI, led to a decrease in replication by about 6- to 8-fold compared to the wt. R55R59 and R61R62 mutants had replication levels comparable to the level of the wt although R55 disrupted the lower part of SLVI. This is in agreement with previous reports (35
). However, despite stable core protein expression, these mutants produced neither intracellular nor extracellular infectious particles. Among the single mutants, only the R39 and R47 mutants decreased replication levels by about 6- to 8-fold, and these levels are comparable to those of the R39R40R43 and R47R50K51 mutants, respectively. Interestingly, in the predicted structure of SLVI, these modifications affect pairing bases at the same positions but in opposite strands (Fig. ). Altogether, our data indicate that only modifications in this part of SLVI affect RNA replication. The remaining mutants had replication levels comparable to the level of the wt.
Analyses of single mutants generated with mutations in the second cluster revealed that 6 out of 10 mutants (R39, R40, R43, R47, R55, and R61) were infectious with some attenuation. In contrast, the infectivity of the R50, K51, R59, and R62 mutants was abolished. No infectious viral particles were detected intracellularly or extracellularly. Our data suggest that the absence of infectious viral particles is not due to the instability of the core protein since this protein was detected at 72 h postelectroporation. In a study by Murray and coworkers, alanine scanning mutagenesis of HCV core protein revealed numerous residues essential for the production of infectious virus (40
). Mutants of J6/JFH1 with quadruple alanine substitutions in the core protein (C57/60A and C61/64A) could not be rescued by compensatory mutations located in the viral genome (40
). These mutants contained the basic residues R59 and R62, which were characterized in our study as crucial for HCV infectivity. In addition, as reported previously, amino acid insertions between aa 23 to 107 of the core protein were lethal (2
). Furthermore, these mutations did not disrupt the subcellular colocalization of the core protein with the LDs and NS5A protein, which have been reported to be essential for virus production (7
). Moreover, viral infectivity of a deleted core mutant, Δcore (62-160) could not be rescued by trans
-complementation with any of the four mutant core proteins. In contrast, the wt core protein could rescue these mutants, indicating that the effect is due only to amino acid changes and not to a change of nucleotides, which would be involved in RNA encapsidation.
Compared to the wt, analysis by sucrose gradient ultracentrifugation showed that all mutants were able to assemble into intermediate multimeric complexes, which might correspond to nucleocapsid-like structures. Previously reported data suggest that a deletion of 10 basic residues in cluster 2 reduces the ability of the capsid to assemble efficiently (25
). Furthermore, when the number of mutated basic residues was increased, a progressive decrease in the ability of core protein assembly was observed (25
). In our study, individual core mutations did not interfere with core assembly and multimerization. Moreover, all mutant nucleocapsids were able to protect the viral genome from the RNase treatment to a similar extent as the wt nucleocapsid, suggesting that core-RNA interactions were not altered. Therefore, this suggests that these mutations inhibit viral assembly at a stage downstream of the oligomerization of the core protein and the encapsidation of RNA. A recent report (1
) reveals that a cascade of core protein-membrane interactions is important in HCV morphogenesis. A deletion of aa 42 to 68 of the core protein, which contains the four residues identified in our study, decreased its envelopment by cellular membranes (1
). In our study, when core protein envelopment was assessed by a membrane protection assay, mutated core proteins were protected by membranes to a similar extent as the wt core protein, suggesting that these residues do not alter core protein envelopment by intracellular membranes.
In conclusion, a comprehensive mutagenesis study of basic amino acid residues located at the N terminus of HCV core protein allowed the identification of four amino acids, R50, K51, R59 and R62, that are essential for HCV morphogenesis. None of these amino acids affects core protein colocalization with NS5A and LDs, RNA encapsidation, core protein oligomerization, or envelopment by intracellular membranes. Understanding the detailed mechanism by which these mutations can modulate infectious virus production will help broaden our knowledge of viral particle assembly and further the identification of new potential therapeutic targets.