Although specific antibodies and cellular immune responses against core+1 antigens have been detected in HCV patients (2
), the biological importance of the core+1 ORF and protein(s) derived from it is still unknown. Previous studies with subgenomic replicons suggested that core+1 protein(s) is not required for viral replication (6
). However, cumulative data associating elevated levels of core+1 expression with the development of hepatocellular carcinoma in HCV patients (9
) and studies describing lower rates of HCV genome replication in tumor cells than in nontumor cells indicate that core+1 might exert a negative effect on viral replication. However, even under various experimental conditions of stable or transient coexpression, we did not observe such an effect, arguing that at least in cell culture, HCV replication is not affected by core+1 protein(s). In agreement with this observation, we found that insertion of a nonsense mutation predicted to terminate core+1 translation at codon 90 (mut 5) and in parallel disrupting one base pairing within the stem of SL248 did not affect viral-RNA replication, RNA stability, expression of viral proteins, and virus release. A termination codon at position 90 would abolish translation of the core+1 product initiated at the internal methionine codons 85/87 (53
), as well as expression of all core+1 proteins reported to initiate upstream of codon 90 (frameshift products associated with codons 8 to 11 [11
] and 42 [8
] and products arising from internal initiation at codon 26 [3
]). The data thus show that expression of core+1 is dispensable for virus production in cell culture. However, we could not exclude effects of core+1 protein(s) on host cell factors or conditions that contribute to HCV replication in vivo and that are not detectable in Huh7 cells. Moreover, not every inactivation of viral functions leads to a phenotype in cell culture, as shown, e.g., for herpes simplex virus (41
For these reasons, we studied the viability of the core+1 mutants in vivo by inoculating uPA-SCID mice xenografted with primary human hepatocytes. Consistent with our in vitro data, the JFH1 mut 5 virus exhibited replication kinetics like those of the wt in inoculated mice.
Apart from serving as an ORF for core and core+1, four highly conserved RNA stem-loop structures reside in the same coding region. By using a reverse-genetics approach, we found that disruption of the structural integrity of SL47 and SL87 inhibited RNA replication about 4- to 6-fold and reduced titers of infectious virus about 10- to 50-fold. In contrast, mutations affecting the more downstream RNA structures SL248 and SL443 had no effect. The difference in RNA replication between the wt and the SL47 or SL87 mutant were detectable only at early time points (24 h p.t.), whereas at later time points this difference vanished. This peculiar kinetics is due to the fact that RNA replication of JFH1 is most likely limited by the host cell (5
). For this reason, more subtle impairments of replication are detectable only at early time points, while later, when replication becomes limited by the host cell, RNA amounts from an impaired genome accumulate to wt levels. This property is a general limitation of the highly replication-competent JFH1 isolate that should be kept in mind when analyzing more subtle replication phenotypes with this HCV genome.
An analogous delay of viremia was observed when the mut 1-5 genome was inoculated into uPA SCID mice with human liver xenografts. Sequence analysis of genomes isolated 12 weeks postinoculation demonstrated that the mutations affecting the upper stem-loop structure of SL87 had reverted in such a way that base pairing of the stem was restored. Thus, SL87 plays a very crucial role in HCV replication, most likely by enhancing RNA translation. However, the mutation disrupting translation of all known forms of core+1 protein(s) (mut 5) was stable, clearly showing that this protein(s) is irrelevant for viral replication in vivo.
We note that during prolonged passage of mut 1-5 in cell culture, all mutations were stable and no reversion was found over a 4-week observation period. In contrast, in inoculated mice, mut 1-5 was strongly attenuated, and the slow increase in viremia is consistent with the emergence of revertants. This discrepancy between results in cell culture and in vivo is most likely due to the host cell environment. For instance, cell factors contributing to SL87-dependent RNA translation/replication may be more abundant in Huh7 cells than in xenografted human liver cells, and therefore, SL87 mutants would be less impaired in cell culture. Alternatively, inhibitory factors binding to SL87 might be expressed to a lower level in Huh7 cells. More detailed studies, especially of possible cellular interaction partners with SL87, are required to address this possibility.
While this study was ongoing, McMullan and colleagues reported the characterization of a series of nonsense mutations disrupting translation of the core+1 ORF and altering RNA structure in the core coding region (34
). These mutations correspond to mut 1-4 described in our study (codons 22/31/44/46 of the core+1 frame) and were tested in the context of an H77/JFH1 and a J6/JFH1 chimeric genome. However, mut 1 to 4 do not affect internal initiation of core+1 translation from codons 85/87. By including mut 5 in our study, we assessed the effect of a block of expression of all reported forms of the core+1 protein(s). Moreover, in contrast to the study by McMullan and coworkers, which focused on SL47 and SL87 only (designated SLV and SLVI, respectively, in their study), we also characterized the roles of the two downstream RNA elements SL248 and SL443 for HCV proliferation. In both studies, SL87 was found to be required for robust replication, but interestingly, our extensive substitution analysis revealed SL47 as an equally important RNA element. While we found that mutations disrupting SL47 and/or SL87 delay replication kinetics only transiently, McMullan et al. reported that mut 1 to 4 exhibited a dramatic and long-lasting defect in replication. During cell culture passage of the mutants, a mixture of reversions emerged in the case of the H77/JFH1 chimera concomitant with an increase in viral replication. Moreover, upon infection of a chimpanzee with this mutant, virus titers in the serum of the animal were low, the mutant appeared to be attenuated, and infection was controlled several weeks earlier than infection with a wt 1a isolate. In contrast, we found no evidence for adaptation in cell culture, and all mutations in mut 1-5 were conserved by week 4 p.t. This result is not too surprising, because in contrast to the study by McMullan et al., mut 1-5 was much less impaired in replication, and therefore, selective pressure was most likely not strong enough to enrich for revertants. However, upon infection of mice, mut 1-5 was clearly attenuated, and high-level viremia was delayed about 4 weeks compared to the wt. Thus, as in to the study by McMullan and coworkers, we also observed an attenuation of the mutant and a selection for revertants in vivo.
Our data cannot exclude the possibility that core+1 proteins perform a function that is not measurable in our cell culture or in vivo mouse model. The highly permissive nature of the Huh7.5 and Huh7-Lunet cells may be related to marked differences in the host environment relative to other liver cell lines and primary human hepatocytes that are naturally infected by HCV. Therefore, should core+1 proteins affect cellular genes that are already mutated in Huh7 subclones (and thus contribute to their permissiveness), core+1 mutants would behave like the wt in those cells. In addition, the JFH1 strain is unique among all HCV isolates and not necessarily representative, because it is the only isolate that replicates without requiring replication-enhancing mutations. JFH1 may contain genetic alterations that provide advantages for viral replication but at the same time make functions exerted by core+1 proteins obsolete. Finally, since the only in vivo model available to us is the xenografted Alb-uPA+/+
SCID mouse, our finding that lack of core+1 protein expression has no detectable effect on virus replication does not exclude the possibility that these proteins may have some other role, e.g., in immune evasion or pathogenesis. In fact, in the study by McMullan and colleagues, the mutant virus was clearly attenuated in the chimpanzee, with low-level viremia and rapid clearance (34
Interestingly, our data and those of McMullan and coworkers can explain the high degree of evolutionary constraint on sequence alterations in the core region encompassing RNA elements SL47 and SL87 (44
). These two stem-loops have been predicted by in silico analyses and supported by RNase cleavage studies. Most importantly, in this study, we show that SL47 and SL87 are not required for RNA stability but play important roles in RNA translation. The integrity of these RNA elements may be important for the regulation of HCV IRES activity either through a long-range RNA-RNA interaction(s) or through binding of a cellular protein(s) in the core encoding region. More specifically, both SL47 and SL87 may be involved in the stimulation of IRES function, e.g., by altering inhibitory interactions between the 5′ NTR and the core region (16
). In addition, a sequence at the 5′ end of SL87 (nt 428 to 442) anneals to nt 24 to 38 of the 5′ NTR, and this interaction restrains IRES-dependent translation (27
). SL47 may also be involved in interaction with host cell factors, such as nonstructural 1-associated protein 1 (NSAP1), which is highly homologous to human heterogeneous nuclear ribonucleoprotein and which has been shown to enhance IRES translation (26
). Finally, it is also possible that SL47 and SL87 are components of genome scale ordered RNA structure and may thus contribute to the conformation of the RNA genome, possibly modulating double-stranded RNA recognition that is associated with innate antiviral defense and virus persistence (43
). We note that simultaneous disruption of the structural integrity of SL47 and SL87 impairs RNA translation and replication to the same extent as disruption of only one of these stem-loops. Assuming that the stem-loops participate in RNA-RNA or RNA-protein interactions, the disruption of only one of the stem-loops may be sufficient to block this interaction, and thus, no further impairment of translation/replication will be observed with mutations disrupting the second stem-loop.
In conclusion, we have shown that expression of the core+1 ORF plays no role in HCV replication and virus production in cell culture and xenografted mice. We confirmed that the core structures SL47 and SL87 are important cis-acting RNA elements required for HCV genome translation. These two stem-loop elements are crucial determinants for robust virus proliferation in cell culture and in vivo, thus providing an explanation for their striking evolutionary conservation.