Highly efficient random mutagenesis in transcription-reverse-transcription cycles by a hydrogen bond ambivalent nucleoside 5'-triphosphate analogue: potential candidates for a selective anti-retroviral therapy

The nucleoside P can base pair with both A and G. We evaluated the mutation frequency induced by the 5'-triphospbate of the ribonucleoside P (PTP) in an in vitro retroviral replication model. After 4 cycles of replication in the presence of PTP, the mutation frequency was raised to 3.8 x 10(-2) per nucleotide and C-to-U and U-to-C mutations were dominantly observed. These results suggest that ambivalent NTP analogues, like PTP, could induce mutations beyond the error threshold of retroviruses.     

Charged residues in hepatitis C virus NS4B are critical for multiple NS4B functions in RNA replication

The nonstructural 4B (NS4B) protein of hepatitis C virus (HCV) plays a central role in the formation of the HCV replication complex. To gain insight into the role of charged residues for NS4B function in HCV RNA replication, alanine substitutions were engineered in place of 28 charged residues residing in the N- and C-terminal cytoplasmic domains of the NS4B protein of the HCV genotype 1b strain Con1. Eleven single charged-to-alanine mutants were not viable, while the remaining mutants were replication competent, albeit to differing degrees. By selecting revertants, second-site mutations were identified for one of the lethal NS4B mutations. Second-site mutations mapped to NS4B and partially suppressed the lethal replication phenotype. Further analyses showed that three NS4B mutations disrupted the formation of putative replication complexes, one mutation altered the stability of the NS4B protein, and cleavage at the NS4B/5A junction was significantly delayed by another mutation. Individual charged-to-alanine mutations did not affect interactions between the NS4B and NS3-4A proteins. A triple charged-to-alanine mutation produced a temperature-sensitive replication phenotype with no detectable RNA replication at 39°C, demonstrating that conditional mutations can be obtained by altering the charge characteristics of NS4B. Finally, NS4B mutations dispensable for efficient Con1 RNA replication were tested in the context of the chimeric genotype 2a virus, but significant defects in infectious-virus production were not detected. Taken together, these findings highlight the importance of charged residues for multiple NS4B functions in HCV RNA replication, including the formation of a functional replication complex.     

Efficient rescue of hepatitis C virus RNA replication by trans-complementation with nonstructural protein 5A

Studies of Hepatitis C virus (HCV) RNA replication have become possible with the development of subgenomic replicons. This system allows the functional analysis of the essential components of the viral replication complex, which so far are poorly defined. In the present study we wanted to investigate whether lethal mutations in HCV nonstructural genes can be rescued by trans-complementation. Therefore, a series of replicon RNAs carrying mutations in NS3, NS4B, NS5A, and NS5B that abolish replication were transfected into Huh-7 hepatoma cells harboring autonomously replicating helper RNAs. Similar to data described for the Bovine viral diarrhea virus (C. W. Grassmann, O. Isken, N. Tautz, and S. E. Behrens, J. Virol. 75:7791-7802, 2001), we found that only NS5A mutants could be efficiently rescued. There was no evidence for RNA recombination between helper and mutant RNAs, and we did not observe reversions in the transfected mutants. Furthermore, we established a transient complementation assay based on the cotransfection of helper and mutant RNAs. Using this assay, we extended our results and demonstrated that (i) inactivating NS5A mutations affecting the amino-terminal amphipathic helix cannot be complemented in trans; (ii) replication of the helper RNA is not necessary to allow efficient trans-complementation; and (iii) the minimal sequence required for trans-complementation of lethal NS5A mutations is NS3 to -5A, whereas NS5A expressed alone does not restore RNA replication. In summary, our results provide the first insight into the functional organization of the HCV replication complex.     

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.