3 cells in a dose-dependent manner; however, amplification of the HCV replicon (indicated by NS3) was not affected, suggesting that hA3G was without effect on the HCV enzymes.

This agrees with the results from the hA3G stabilizers (Fig. 3A). Next, we investigated whether RN-5 (or IMB-26) treatment increases the incorporation of hA3G into HCV viral particles. In this experiment, HCV-infected Huh7.5 cells were cultured for 2 days in the presence or absence of the hA3G stabilizers. The resultant HCV viral particles in the culture PD-0332991 molecular weight were harvested with ultracentrifugation. The hA3G protein within the viral particles was measured using western blot. As compared to those from untreated cells, the hA3G protein significantly increased in the HCV particles produced from the RN-5-treated HCV-infected Huh7.5 cells (Fig. 3D). Similarly, the increase of

hA3G in HCV particles was also detectable after IMB-26 treatment (not shown). We assumed that the compounds might inhibit HCV through hA3G-mediated G/A viral mutation. To verify this, HCV genome sequencing was conducted for the supernatant viral particles released from HCV-infected Huh7.5 cells treated with RN-5 or IMB-26, as well as for the viruses that had already replicated in naïve Huh7.5 cells after infection with the HCV-containing supernatant mentioned above. The sequencing results, however, did not support our speculation. With respect to those from the cells without treatment, G/A mutation rate did not increase in the HCV viral particles generated from the RN-5 (or AZD2281 in vivo IMB-26)-treated Huh7.5 cells or in the newly P-type ATPase replicated HCV after entering into Huh7.5 cells (Table 1). To confirm this result, a sensitive technique was used in which denaturation temperature was set at levels below 95°C.22 The results agreed with those in Table 1. Our

studies show that hA3G might inhibit HCV replication through a mechanism different from that in HIV-1. As the antiviral mechanism of hA3G is complicated and varies among viruses,11, 23-28 more investigation is needed for the APOBEC superfamily in their action against HCV. To evaluate the in vivo safety in stabilizing hA3G, RN-5 was given once to normal healthy mice orally (po) or intraperitoneally (ip), followed by body weight monitoring and organ function examination. After 7 days follow-up we found that RN-5, at doses between 125 and 1,000 mg/kg for oral administration or 62.5 and 500 mg/kg for ip administration, did not cause animal death or body weight change (Fig. 4A). Blood samples were taken for liver and kidney function examination at the end of the 7-day experiment. As shown in Fig. 4B, abnormality was not found in blood glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), blood urea nitrogen (BUN), or creatine (CRE) after RN-5 administration by the oral or ip route at the maximum dose used in the experiment.