Hepatitis C genotype 1a replicon improved through introduction of fitness mutations

The use of subgenomic replicon systems has long been a valuable screening tool for the discovery of small molecule antivirals against Hepatitis C virus. While genotype 1a replicon systems have been widely used in stable systems, use in transient assays has been hampered by low signal. Here we describe the generation of a more robust genotype 1a (H77) replicon through the introduction of two fitness mutations, NS4A-K1691R and NS4B-E1726G, for use in transient transfections. While these mutations significantly improved the signal to noise ratio, leading to more robust data, they have no effect on the potency of tool compounds against various targets of HCV, thereby making this new system a powerful tool for screening of compounds against the genotype 1a replicon.     

Viral and cellular determinants of hepatitis C virus RNA replication in cell culture

Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i). mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii). mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself.     

Ribavirin resistance in hepatitis C virus replicon-containing cell lines conferred by changes in the cell line or mutations in the replicon RNA

Ribavirin (RBV), used in combination with alpha interferon to treat hepatitis C virus (HCV) infections, is a guanosine nucleotide analog that can increase the error rate of viral RNA-dependent RNA polymerases, imbalance intracellular nucleotide pools, and cause toxicity in many cell types. To determine potential mechanisms of RBV resistance during HCV RNA replication, we passaged HCV replicon-containing cell lines in the presence of increasing concentrations of RBV. RBV-resistant, HCV replicon-containing cell lines were generated, and the majority of RBV resistance was found to be conferred by changes in the cell lines. The resistant cell lines were defective in RBV import, as measured by [(3)H]RBV uptake experiments. These cell lines displayed reduced RBV toxicity and reduced error accumulation during infection with poliovirus, whose replication is known to be sensitive to RBV-induced error. For one RBV-resistant isolate, two mutations in the replicon RNA contributed to the observed phenotype. Two responsible mutations resided in the C-terminal region of NS5A, G404S, and E442G and were each sufficient for low-level RBV resistance. Therefore, RBV resistance in HCV replicon cell lines can be conferred by changes in the cell line or by mutations in the HCV replicon.     

Functional requirements of the yellow fever virus capsid protein

Although it is known that the flavivirus capsid protein is essential for genome packaging and formation of infectious particles, the minimal requirements of the dimeric capsid protein for virus assembly/disassembly have not been characterized. By use of a trans-packaging system that involved packaging a yellow fever virus (YFV) replicon into pseudo-infectious particles by supplying the YFV structural proteins using a Sindbis virus helper construct, the functional elements within the YFV capsid protein (YFC) were characterized. Various N- and C-terminal truncations, internal deletions, and point mutations of YFC were analyzed for their ability to package the YFV replicon. Consistent with previous reports on the tick-borne encephalitis virus capsid protein, YFC demonstrates remarkable functional flexibility. Nearly 40 residues of YFC could be removed from the N terminus while the ability to package replicon RNA was retained. Additionally, YFC containing a deletion of approximately 27 residues of the C terminus, including a complete deletion of C-terminal helix 4, was functional. Internal deletions encompassing the internal hydrophobic sequence in YFC were, in general, tolerated to a lesser extent. Site-directed mutagenesis of helix 4 residues predicted to be involved in intermonomeric interactions were also analyzed, and although single mutations did not affect packaging, a YFC with the double mutation of leucine 81 and valine 88 was nonfunctional. The effects of mutations in YFC on the viability of YFV infection were also analyzed, and these results were similar to those obtained using the replicon packaging system, thus underscoring the flexibility of YFC with respect to the requirements for its functioning.     

Genome-wide hypermutation in a subpopulation of stationary-phase cells underlies recombination-dependent adaptive mutation.

Stationary-phase mutation in microbes can produce selected (''adaptive'') mutants preferentially. In one system, this occurs via a distinct, recombination-dependent mechanism. Two points of controversy have surrounded these adaptive reversions of an Escherichia coli lac mutation. First, are the mutations directed preferentially to the selected gene in a Lamarckian manner? Second, is the adaptive mutation mechanism specific to the F plasmid replicon carrying lac? We report that lac adaptive mutations are associated with hypermutation in unselected genes, in all replicons in the cell. The associated mutations have a similar sequence spectrum to the adaptive reversions. Thus, the adaptive mutagenesis mechanism is not directed to the lac genes, in a Lamarckian manner, nor to the F'' replicon carrying lac. Hypermutation was not found in non-revertants exposed to selection. Therefore, the genome-wide hypermutation underlying adaptive mutation occurs in a differentiated subpopulation. The existence of mutable subpopulations in non-growing cells is important in bacterial evolution and could be relevant to the somatic mutations that give rise to cancers in multicellular organisms.     

Hep3B human hepatoma cells support replication of the wild-type and a 5'-end deletion mutant GB virus B replicon

Hepatitis C virus (HCV) and GB virus B (GBV-B) replicons have been reported to replicate only in Huh7 cells. Here we demonstrate that subpopulations of another human hepatoma cell line, Hep3B, are permissive for the GBV-B replicon, showing different levels of enhancement of replication from those of the unselected parental cell population. Adaptive mutations are not required for replication of the GBV-B replicon in these cells, as already demonstrated for Huh7 cells. Nonetheless, we identified a mutant replicon in one of the selected cell lines, which, although lacking the 5' end proximal stem-loop, is able to replicate in Hep3B cells as well as in Huh7 cells. This mutant indeed shows a higher replication efficiency than does wild-type replicon, especially in the Hep3B cell clone from which it was originally recovered. This indicates that the stem-loop Ia is not necessary for replication of the GBV-B replicon in human cells, unlike what occurs with HCV, and that its absence can even provide a selective advantage.     

Construction of a chimeric hepatitis C virus replicon based on a strain isolated from a chronic hepatitis C patient

Subgenomic replicons of hepatitis C virus (HCV) have been widely used for studying HCV replication. Here, we report a new subgenomic replicon based on a strain isolated from a chronically infected patient. The coding sequence of HCV was recovered from a Chinese chronic hepatitis C patient displaying high serum HCV copy numbers. A consensus sequence designated as CCH strain was constructed based on the sequences of five clones and this was classified by sequence alignment as belonging to genotype 2a. The subgenomic replicon of CCH was replication-deficient in cell culture, due to dysfunctions in NS3 and NS5B. Various JFH1/CCH chimeric replicons were constructed, and specific mutations were introduced. The introduction of mutations could partially restore the replication of chimeric replicons. A replication-competent chimeric construct was finally obtained by the introduction of NS3 from JFH1 into the backbone of the CCH strain.     

Rapid screening for dominant negative mutations in the beet necrotic yellow vein virus triple gene block proteins P13 and P15 using a viral replicon

Point mutations were introduced into the genes encoding the triple gene bock movement proteins P13 and P15 of beet necrotic yellow vein virus (BNYVV). Mutations which disabled viral cell-to-cell movement in Chenopodium quinoa were then tested for their ability to act as dominant negative inhibiters of movement of wild-type BNYVV when expressed from a co-inoculated BNYVV RNA 3-based replicon. For P13, three types of mutation inhibited the movement function: non-synomynous mutations in the N- and C-terminal hydrophobic domains, a mutation at the boundary between the N-terminal hydrophobic domain and the central hydrophilic domain (mutant P13-A12), and mutations in the conserved sequence motif in the central hydrophilic domain. However, only the boundary mutant P13-A12 strongly inhibited movement of wild-type virus when expressed from the co-inoculated replicon. Similar experiments with P15 detected four movement-defective mutants which strongly inhibited cell-to-cell movement of wild-type BNYVV when the mutants were expressed from a co-inoculated replicon. Beta vulgaris transformed with two of these P15 mutants were highly resistant to fungus-mediated infection with BNYVV.     

Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone

Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones.     

Distinct functions of NS5A in hepatitis C virus RNA replication uncovered by studies with the NS5A inhibitor BMS-790052

BMS-790052, targeting nonstructural protein 5A (NS5A), is the most potent hepatitis C virus (HCV) inhibitor described to date. It is highly effective against genotype 1 replicons and also displays robust genotype 1 anti-HCV activity in the clinic (M. Gao et al., Nature 465:96-100, 2010). BMS-790052 inhibits genotype 2a JFH1 replicon cells and cell culture infectious virus with 50% effective concentrations (EC(50)s) of 46.8 and 16.1 pM, respectively. Resistance selection studies with the JFH1 replicon and virus systems identified drug-induced mutations within the N-terminal region of NS5A. F28S, L31M, C92R, and Y93H were the major resistance mutations identified; the impact of these mutations on inhibitor sensitivity between the replicon and virus was very similar. The C92R and Y93H mutations negatively impacted fitness of the JFH1 virus. Second-site replacements at NS5A residue 30 (K30E/Q) restored efficient replication of the C92R viral variant, thus demonstrating a genetic interaction between NS5A residues 30 and 92. By using a trans-complementation assay with JFH1 replicons encoding inhibitor-sensitive and inhibitor-resistant NS5A proteins, we provide genetic evidence that NS5A performs the following two distinct functions in HCV RNA replication: a cis-acting function that likely occurs as part of the HCV replication complex and a trans-acting function that may occur outside the replication complex. The cis-acting function is likely performed by basally phosphorylated NS5A, while the trans-acting function likely requires hyperphosphorylation. Our data indicate that BMS-790052 blocks the cis-acting function of NS5A. Since BMS-790052 also impairs JFH1 NS5A hyperphosphorylation, it likely also blocks the trans-acting function.     

Increasing rate of cleavage at boundary between non-structural proteins 4B and 5A inhibits replication of hepatitis C virus

In hepatitis C virus, non-structural proteins are cleaved from the viral polyprotein by viral encoded proteases. Although proteolytic processing goes to completion, the rate of cleavage differs between different boundaries, primarily due to the sequence at these positions. However, it is not known whether slow cleavage is important for viral replication or a consequence of restrictions on sequences that can be tolerated at the cleaved ends of non-structural proteins. To address this question, mutations were introduced into the NS4B side of the NS4B5A boundary, and their effect on replication and polyprotein processing was examined in the context of a subgenomic replicon. Single mutations that modestly increased the rate of boundary processing were phenotypically silent, but a double mutation, which further increased the rate of boundary cleavage, was lethal. Rescue experiments relying on viral RNA polymerase-induced error failed to identify second site compensatory mutations. Use of a replicon library with codon degeneracy did allow identification of second site compensatory mutations, some of which fell exclusively within the NS5A side of the boundary. These mutations slowed boundary cleavage and only enhanced replication in the context of the original lethal NS4B double mutation. Overall, the data indicate that slow cleavage of the NS4B5A boundary is important and identify a previously unrecognized role for NS4B5A-containing precursors requiring them to exist for a minimum finite period of time.     

Analysis of the replicon region and identification of an rRNA operon on pBM400 of Bacillus megaterium QM B1551

An 18 633 bp region containing the replicon from the approximately 53 kb pBM400 plasmid of Bacillus megaterium QM B1551 has been sequenced and characterized. This region contained a complete rRNA operon plus 10 other potential open reading frames (ORFs). The replicon consisted of an upstream promoter and three contiguous genes (repM400, orfB and orfC) that could encode putative proteins of 428, 251 and 289 amino acids respectively. A 1.6 kb minimal replicon was defined and contained most of repM400. OrfB was shown to be required for stability. Three 12 bp identical tandem repeats were located within the coding region of repM400, and their presence on another plasmid caused incompatibility with their own cognate replicon. Nonsense, frameshift and deletion mutations in repM400 prevented replication, but each mutation could be complemented in trans. RepM400 had no significant similarity to sequences in the GenBank database, whereas five other ORFs had some similarity to gene products from other plasmids and the Bacillus genome. An rRNA operon was located upstream of the replication region and is the first rRNA operon to be sequenced from B. megaterium. Its unusual location on non-essential plasmid DNA has implications for systematics and evolutionary biology.     

Evasion of Superinfection Exclusion and Elimination of Primary Viral RNA by an Adapted Strain of Hepatitis C Virus

Cells that are productively infected by hepatitis C virus (HCV) are refractory to a second infection by HCV via a block in viral replication known as superinfection exclusion. The block occurs at a postentry step and likely involves translation or replication of the secondary viral RNA, but the mechanism is largely unknown. To characterize HCV superinfection exclusion, we selected for an HCV variant that could overcome the block. We produced a high-titer HC-J6/JFH1 (Jc1) viral genome with a fluorescent reporter inserted between NS5A and NS5B and used it to infect Huh7.5 cells containing a Jc1 replicon. With multiple passages of these infected cells, we isolated an HCV variant that can superinfect cells at high levels. Notably, the superinfectious virus rapidly cleared the primary replicon from superinfected cells. Viral competition experiments, using a novel strategy of sequence-barcoding viral strains, as well as superinfection of replicon cells demonstrated that mutations in E1, p7, NS5A, and the poly(U/UC) tract of the 3′ untranslated region were important for superinfection. Furthermore, these mutations dramatically increased the infectivity of the virus in naive cells. Interestingly, viruses with a shorter poly(U/UC) and an NS5A domain II mutation were most effective in overcoming the postentry block. Neither of these changes affected viral RNA translation, indicating that the major barrier to postentry exclusion occurs at viral RNA replication. The evolution of the ability to superinfect after less than a month in culture and the concomitant exclusion of the primary replicon suggest that superinfection exclusion dramatically affects viral fitness and dynamics in vivo.     

Comparative study on the replication of HCV1b genome between wild-type and cell culture-adaptive mutant in regard to sensitivities against anti-HCV drugs

The replicon system, which mimics viral genome replication in culture cells, has been widely used to analyze the genome replication of the hepatitis C virus (HCV). However, most HCV genomes used in the system include adaptive mutations (AMs) that are vital for replication in culture cells despite the nonexistence of such mutations in the genome of wild-type (WT) HCV in patients. In order to study the genome replications of WT HCV, new HCV subgenomic replicon (SGR) systems were established using Huh-7.5-derived cells producing Sec14-like protein 2 constitutively and SGR of KT9 (one of the HCV genotype 1b clones) with WT genome (SGR KT9WT) in this study. The replication efficiency and sensitivities of SGR KT9WT to anti-HCV drugs in the cloned cells permanently bearing replicon RNA, HS55-4 cells, were similar to those of reports using SGR, including AM. The SGR transient transfection system using SGR KT9WT and SGR KT9AM encoding secreted Nano-luciferase and HS55-4C cells established by the elimination of SGR KT9 RNA from HS55-4 cells, however, showed that the replication efficiency of SGR KT9WT was much lower than that of SGR KT9AM under a same condition. Furthermore, the sensitivities of SGR KT9WT to almost all tested anti-HCV reagents, except the inhibitor of miR-122, a cellular factor important for HCV replication, were quite low compared with SGR KT9AM. These results suggested that the new replicon systems might not only provide information about precise responses against new anti-HCV drugs but also reveal novel molecular mechanisms supporting negligent proliferation of HCV.