Masanori Ikeda

Selectable Subgenomic and Genome-Length Dicistronic RNAs Derived from an Infectious Molecular Clone of the HCV-N Strain of Hepatitis C Virus Replicate Efficiently in Cultured Huh7 Cells

ABSTRACT Dicistronic, selectable subgenomic replicons derived from the Con1 strain of hepatitis C virus (HCV) are capable of autonomous replication in cultured Huh7 cells (Lohmann et al., Science 285:110-113, 1999). However, adaptive mutations in the NS3, NS5A, and/or NS5B proteins are required for efficient replication of these RNAs and increase by orders of magnitude the numbers of G418-resistant colonies selected following transfection of Huh7 cells. Here, we demonstrate that a subgenomic replicon (NNeo/3-5B) derived from an infectious molecular clone of a second genotype 1b virus, HCV-N (Beard et al., Hepatology 30:316-324, 1999) is also capable of efficient replication in Huh7 cells. G418-resistant cells selected following transfection with NNeo/3-5B RNA contained abundant NS5A antigen and HCV RNA detectable by Northern analysis. Replicon RNA in one of three clonally isolated cell lines contained no mutations in the NS3-NS5B polyprotein, confirming that adaptive mutations are not required for efficient replication in these cells. However, the deletion of a unique 4-amino-acid insertion that is present within the interferon sensitivity-determining region (ISDR) of the NS5A protein in wild-type HCV-N drastically decreased the number of G418-resistant colonies obtained following transfection of Huh7 cells. This effect could be reversed by inclusion of a previously described Con1 cell culture-adaptive mutation (S2005→I), confirming that this natural insertion has a controlling role in determining the replication capacity of wild-type HCV-N RNA in Huh7 cells. Additional selectable, dicistronic RNAs encoding NS2-NS5B, E1-NS5B, or the full-length HCV polyprotein were also capable of replication and gave rise to G418-resistant cell clones following transfection of Huh7 cells. We conclude that RNA derived from this documented infectious molecular clone has a unique capacity for replication in Huh7 cells in the absence of additional cell culture-adaptive mutations.

Characterization of antiviral activity of lactoferrin against hepatitis C virus infection in human cultured cells.

We recently found that bovine lactoferrin (bLF), a milk glycoprotein belonging to the iron transporter family, prevented hepatitis C virus (HCV) infection in human hepatocyte PH5CH8 cells, that are susceptible to HCV infection, and demonstrated that the anti-HCV activity of bLF was due to the interaction of bLF and HCV. In this study we further characterized the anti-HCV activity of bLF and the mechanism by which bLF prevents HCV infection. We found that bLF inhibited viral entry to the cells by interacting directly with HCV immediately after mixing of bLF and HCV inoculum. The anti-HCV activity of bLF was lost by heating at 65 degrees C, and other milk proteins (mucin, beta-lactoglobulin and casein) did not prevent HCV infection, indicating that bLF prevented HCV infection in a rather specific manner. Furthermore, we found that bovine lactoferricin, a basic N-terminal loop of bLF that is an important region for antibacterial activity, did not exhibit any anti-HCV activity, suggesting that some other region is involved in anti-HCV activity. We confirmed that prevention of HCV infection by bLF was a general phenomenon, because bLF inhibited HCV infection with all five inocula examined, and bLF inhibited HCV infection in human MT-2C T-cells, that were susceptible to HCV infection. In addition, infection with hepatitis G virus, which is distantly related to HCV, was prevented also by bLF. In conclusion, lactoferrin is a natural glycoprotein which effectively protects against HCV infection in hepatocytes and lymphocytes by neutralizing the virus.

Lactoferrin inhibits hepatitis C virus viremia in patients with chronic hepatitis C : A pilot study

Hepatitis C virus (HCV) is associated with the development of cirrhosis and hepatocellular carcinoma. We recently found that bovine lactoferrin, a milk protein belonging to the iron transporter family, effectively prevented HCV infection in cultured human hepatocytes (PH5CH8). We tested the hypothesis that lactoferrin inhibits HCV viremia in patients with chronic hepatitis C. Eleven patients with chronic hepatitis C received an 8‐week course of bovine lactoferrin (1.8 or 3.6 g/day). At the end of lactoferrin treatment, a decrease in serum alanine transaminase and HCV RNA concentrations was apparent in 3 (75%) of 4 patients with low pretreatment serum concentrations of HCV RNA. However, 7 patients with high pretreatment concentrations showed no significant changes in these indices. This pilot study suggests that lactoferrin is one potential candidate as an anti‐HCV reagent that may be effective for the treatment of patients with chronic hepatitis.

Human hepatocyte clonal cell lines that support persistent replication of hepatitis C virus

We previously found that a human T-cell leukemia virus type I infected T-cell line, MT-2, was susceptible to hepatitis C virus (HCV) infection, and that cloned MT-2C cells could support HCV replication more persistently than the parental MT-2 cells. Recently we found that a non-neoplastic hepatocyte line, PH5CH, showed good susceptibility to HCV infection. In this study, we cloned PH5CH cells to obtain cells that supported more persistent HCV replication, and consequently three clones (PH5CH1, PH5CH7 and PH5CH8) in which intracellular HCV RNA could be detected at least 25 days postinoculation (p.i.) were obtained. Semi-quantitative analysis of HCV RNA indicated that HCV replicated in these cloned PH5CH cells was released into the culture medium. Semi-quantitative analysis of internalized HCV RNA after treatment of cloned PH5CH cells and parental PH5CH cells with proteinase K immediately after virus inoculation revealed that PH5CH1, PH5CH7 and PH5CH8 cells contained 10-fold higher levels of HCV RNA than low susceptible cloned PH5CH or parental PH5CH cells. Furthermore, we demonstrated that HCV replication was maintained for 70-100 days in these three clonal lines when the temperature of cell culture after virus inoculation was reduced from 37 to 32 degrees C. Moreover, we demonstrated that interferon alpha had antiviral effect on HCV-infected PH5CH8 cells. The three PH5CH clones obtained in this study will provide a useful tool for the study of HCV replication and proliferation, and for development of an assay system for antiviral agents.

Nonstructural Protein Precursor NS4A/B from Hepatitis C Virus Alters Function and Ultrastructure of Host Secretory Apparatus

ABSTRACT The nonstructural proteins of hepatitis C virus (HCV) have been shown previously to localize to the endoplasmic reticulum (ER) when expressed singly or in the context of other HCV proteins. To determine whether the expression of HCV nonstructural proteins alters ER function, we tested the effect of expression of NS2/3/4A, NS4A, NS4B, NS4A/B, NS4B/5A, NS5A, and NS5B from genotype 1b HCV on anterograde traffic from the ER to the Golgi apparatus. Only the nominal precursor protein NS4A/B affected the rate of ER-to-Golgi traffic, slowing the rate of Golgi-specific modification of the vesicular stomatitis virus G protein expressed by transfection by approximately threefold. This inhibition of ER-to-Golgi traffic was not observed upon expression of the processed proteins NS4A and NS4B, singly or in combination. To determine whether secretion of other cargo proteins was inhibited by NS4A/B expression, we monitored the appearance of newly synthesized proteins on the cell surface in the presence and absence of NS4A/B expression; levels of all were reduced in the presence of NS4A/B. This reduction is also seen in cells that contain genome length HCV replicons: the rate of appearance of major histocompatibility complex class I (MHC-I) on the cell surface was reduced by three- to fivefold compared to that for a cured cell line. The inhibition of protein secretion caused by NS4A/B does not correlate with the ultrastructural changes leading to the formation a “membranous web” (D. Egger et al., J. Virol. 76:5974-5984, 2002), which can be caused by expression of NS4B alone. Inhibition of global ER-to-Golgi traffic could, by reducing cytokine secretion, MHC-I presentation, and transport of labile membrane proteins to the cell surface, have significant effects on the host immune response to HCV infection.

Contrast-enhanced, wide-band harmonic gray scale imaging of hepatocellular carcinoma correlation with helical computed tomographic findings

We evaluated the usefulness of contrast‐enhanced, wide‐band harmonic gray scale imaging for the diagnosis of hepatocellular carcinoma and compared it with helical computed tomography. Forty‐eight patients with 61 hepatocellular carcinoma lesions were scanned by contrast‐enhanced, wide‐band harmonic gray scale imaging after an intravenous bolus injection of the contrast agent Levovist. Fifty‐seven of the 61 hepatocellular carcinoma lesions showed hypervascular enhancement, and intratumoral vessels could be observed in 40 of the 57 lesions. Helical computed tomography revealed a high‐attenuation area in 54 of the 61 lesions, whereas the other lesions showed an equivocal‐attenuation area. Contrast‐enhanced, wide‐band harmonic gray scale imaging is a useful method for diagnosing the vascularity of hepatocellular carcinoma.

Lactoferrin inhibits hepatitis B virus infection in cultured human hepatocytes

We recently reported that lactoferrin (LF), a milk protein belonging to the iron transporter family, inhibits hepatitis C virus (HCV) infection in cultured human hepatocytes (PH5CH8) and that the interaction of LF with HCV is responsible for this inhibitory effect. As PH5CH8 cells were found to be a human hepatocyte line susceptible to hepatitis B virus (HBV) infection, we therefore examined if LF could effectively prevent HBV infection in PH5CH8 cells. Preincubation of the cell with bovine LF (bLF) or human LF (hLF) was required to prevent HBV infection of cells, and preincubation of HBV with bLF or hLF had no inhibitory effect on HBV infection. We further found that bovine transferrin, casein, and lactoalbumin had no anti-HBV activity. Our findings suggest that the interaction of LF with cells was important for its inhibitory effect, and that LF may well be among the candidates for an anti-HBV reagent that could prove effective in the treatment of patients with chronic hepatitis.

Comprehensive Analysis of the Effects of Ordinary Nutrients on Hepatitis C Virus RNA Replication in Cell Culture

ABSTRACT To date, only a limited number of studies have reported finding an influence of ordinary nutrients on hepatitis C virus (HCV) RNA replication. However, the effects of other nutrients on HCV RNA replication remain largely unknown. We recently developed a reporter assay system for genome-length HCV RNA replication in hepatoma-derived HuH-7 cells (OR6). Here, using this OR6 assay system, we comprehensively examined 46 nutrients from four nutrient groups: vitamins, amino acids, fatty acids, and salts. We found that three nutrients—β-carotene, vitamin D2, and linoleic acid—inhibited HCV RNA replication and that their combination caused additive and/or synergistic effects on HCV RNA replication. In addition, combined treatment with each of the three nutrients and interferon alpha or beta or fluvastatin inhibited HCV RNA replication in an additive manner, while combined treatment with cyclosporine synergistically inhibited HCV RNA replication. In contrast, we found that vitamin E enhanced HCV RNA replication and negated the effects of the three anti-HCV nutrients and cyclosporine but not those of interferon or fluvastatin. These results will provide useful information for the treatment of chronic hepatitis C patients who also take anti-HCV nutrients as an adjunctive therapy in combination with interferon. In conclusion, among the ordinary nutrients tested, β-carotene, vitamin D2, and linoleic acid possessed anti-HCV activity in a cell culture system, and these nutrients are therefore considered to be potential candidates for enhancing the effects of interferon therapy.

Genetic analysis of the hepatitis C virus (HCV) genome from HCV-infected human T cells

We recently established a cell culture system for the replication of hepatitis C virus (HCV) by using a human T cell leukaemia virus type I-infected cell line, MT-2, and showed that the quasi-species of the hypervariable region 1 observed in the original inoculum became homogeneous in MT-2 cells 10 days after inoculation of HCV. In this study, we obtained HCV cDNA clones covering the whole viral genome by RT-nested PCR using RNA from HCV-infected cloned MT-2C cells, which support viral replication more efficiently, at 12 days after inoculation. A total of 41 distinct HCV cDNA clones covering almost the whole viral genome were also isolated from a cDNA library derived from the original inoculum. Molecular evolutional analyses comparing the sequences of the HCV clones obtained from both sources revealed that the HCV populations became homogeneous in more than half of the compared regions. This finding suggests that limited HCV populations are able to replicate in MT-2C cells. In addition, we isolated cDNA clones containing a 3 X-tail sequence, which was recently identified as a bona fide 3 terminus of the HCV genome, in the HCV-infected MT-2C cells and confirmed that the nucleotide sequence of the 3 X-tail was highly conserved, suggesting its implication in HCV replication. Finally, on the basis of the sequences of HCV cDNA clones obtained from HCV-infected MT-2C cells, we determined the entire nucleotide sequence of the HCV genome containing the 3 X-tail as a candidate for an infectious HCV molecular clone.

Analysis of the cell tropism of HCV by using in vitro HCV-infected human lymphocytes and hepatocytes.

BACKGROUND/METHODSnWe recently established two hepatitis C virus (HCV) replication systems, using MT-2, a human T-cell leukemia virus type I-infected human T-cell line, and PH5CH, a non-neoplastic human hepatocyte line immortalized with simian virus 40 large T antigen. These HCV replication systems were used to assess the infective potencies of seven sera containing more than 10(6) HCV genomes per ml obtained from HCV-positive blood donors.nnnRESULTSnThe results showed that these sera had different infectivities for MT-2 and PH5CH cells. One of the seven sera, 1B-1, was more infective for MT-2 cells than PH5CH cells, whereas all the sera except serum 1B-1 were more infective for PH5CH cells than for MT-2 cells. Intracellular HCV RNA could be detected at least 30 days after inoculation with three of the sera. These findings suggested that the infective potency of each serum depends on the type of target cells. To further investigate HCV replication in these cells, we examined the hypervariable region 1 (HVR1) populations of HCV recovered from both MT-2 and PH5CH cells at 8 days postinoculation. The results revealed that the shift to limited HVR1 populations from the quasi-species of HVR1 populations in both cells usually occurred within 8 days after virus inoculation. Furthermore, in two of four sera, the predominant HVR1 populations in MT-2 and PH5CH cells appeared to be different.nnnCONCLUSIONnThese results suggest that HCV exhibits cell tropism.