Intracellular effects of the Hepatitis C virus nucleoside polymerase inhibitor RO5855 (Mericitabine Parent) and Ribavirin in combination.

Mericitabine (RG7128) is the prodrug of a highly selective cytidine nucleoside analog inhibitor (RO5855) of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. This study evaluated the effects of combining RO5855 and ribavirin on HCV replication in the HCV subgenomic replicon by using two drug-drug interaction models. The effects of RO5855 and ribavirin on the intracellular metabolism of each compound, on interferon-stimulated gene (ISG) expression, and on the viability of hepatocyte-derived cells were also investigated. RO5855 and ribavirin had additive inhibitory activities against HCV subgenomic replicon replication in drug-drug interaction analyses. RO5855 did not affect the uptake or phosphorylation of ribavirin in primary human hepatocytes, human peripheral blood mononuclear cells, or genotype 1b (G1b) replicon cells. Similarly, ribavirin did not affect the concentrations of intracellular species derived from RO5855 in primary human hepatocytes or the formation of the triphosphorylated metabolites of RO5855. Ribavirin at concentrations of >40 µM significantly reduced the viability of primary hepatocytes but not of Huh7, the G1b replicon, or interferon-cured Huh7 cells. RO5855 alone or with ribavirin did not significantly alter the viability of Huh7 or G1b replicon cells, and it did not significantly affect the viability of primary hepatocytes when it was administered alone. The viability of primary hepatocytes was reduced when they were incubated with RO5855 and ribavirin, similar to the effects of ribavirin alone. RO5855 alone or with ribavirin had no effect on ISG mRNA levels in any of the cells tested. In conclusion, RO5855 did not show any unfavorable interactions with ribavirin in human hepatocytes or an HCV subgenomic replicon system.

In vitro resistance development for RO-0335, a novel diphenylether nonnucleoside reverse transcriptase inhibitor.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important components of current combination therapies for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance and serious side effects can compromise the benefits of the first generation compounds in this class (efavirenz and nevirapine). To study potential pathways leading to resistance against the novel diphenylether NNRTI, RO-0335, sequential passage experiments at low multiplicity of infection (MOI) were performed to solicit a stepwise selection of resistance mutations. Two pathways to loss of susceptibility to RO-0335 were observed, containing patterns of amino acid changes at either V106I/A plus F227C (with additional contributions from A98G, V108I, E138K, M230L and P236L) or V106I/Y188L (with a potential contribution from L100I, E138K and Y181C). Characterization of the observed mutations by site-directed mutagenesis in the isogenic HXB2D background demonstrated that a minimum of two or more mutations were required for significant loss of susceptibility, with the exception of Y188L, which requires a two-nucleotide change. Patterns containing F227C or quadruple mutations selected by RO-0335 showed a low relative fitness value when compared to wild-type HXB2D.

Incorporation of lysyl-tRNA synthetase into human immunodeficiency virus type 1.

During human immunodeficiency virus type 1 (HIV-1) assembly, tRNA(Lys) isoacceptors are selectively incorporated into virions and tRNA(Lys)3 is used as the primer for reverse transcription. We show herein that the tRNA(Lys)-binding protein, lysyl-tRNA synthetase (LysRS), is also selectively packaged into HIV-1. The viral precursor protein Pr55gag alone will package LysRS into Pr55gag particles, independently of tRNA(Lys). With the additional presence of the viral precursor protein Pr160gag-pol, tRNA(Lys) and LysRS are both packaged into the particle. While the predominant cytoplasmic LysRS has an apparent M(r) of 70,000, viral LysRS associated with tRNA(Lys) packaging is shorter, with an apparent M(r) of 63,000. The truncation occurs independently of viral protease and might be required to facilitate interactions involved in the selective packaging and genomic placement of primer tRNA.

Sequences within Pr160gag-pol affecting the selective packaging of primer tRNA(Lys3) into HIV-1.

The selective packaging of the primer tRNA(Lys3) into HIV-1 particles is dependent upon the viral incorporation of the Pr160gag-pol precursor protein. In order to map a tRNA(Lys3) binding site within this precursor, we have studied the effects of mutations in Pr160gag-pol upon the selective incorporation of tRNA(Lys3). Many of these mutations were placed in a protease-negative HIV-1 proviral DNA to prevent viral protease degradation of the mutant Gag-Pol protein. C-terminal deletions of protease-negative Gag-Pol that removed the entire integrase sequence and the RNase H and connection subdomains of reverse transcriptase did not inhibit the incorporation of either the truncated Gag-Pol or the tRNA(Lys3), indicating that these regions are not required for tRNA(Lys3) binding. On the other hand, larger C-terminal deletions, which also remove the thumb subdomain sequence, did prevent tRNA(Lys3) packaging, without inhibiting viral incorporation of the truncated Gag-Pol, indicating a possible interaction between thumb subdomain sequences and tRNA(Lys3). While point mutations K249E, K249Q, and R307E in the primer grip region of the thumb subdomain have been reported to inhibit the in vitro interaction of mature reverse transcriptase with the anticodon loop of tRNA(Lys3), we find that these mutations do not inhibit tRNA(Lys3) packaging into the virus, which supports other work indicating that the anticodon loop of tRNA(Lys3) is not involved in interactions with Pr160gag-pol during tRNA(Lys3) packaging.