Acquisition of susceptibility to hepatitis C virus replication in HepG2 cells by fusion with primary human hepatocytes: establishment of a quantitative assay for hepatitis C virus infectivity in a cell culture system.

Hepatitis C virus (HCV) replicates in human and chimpanzee hepatocytes. To characterize the nature of HCV and evaluate antiviral agents, the development of an HCV replication system in a cell culture is essential. We developed a cell line derived from human hepatocytes by fusing them with a hepatoblastoma cell line, HepG2, and obtained several clones. When we tested the clones for their ability to support HCV replication by nested RT-PCR, we found 1 clone (IMY-N9) that was more susceptible to HCV replication than HepG2. The negative-strand HCV RNA was detected in IMY-N9 by strand-specific RT-PCR, and viral RNA was identified in culture supernatant during the culture. Then we monitored HCV RNA titers in IMY-N9 and HepG2, respectively, by real-time detection PCR throughout the culture. A significant increase in the HCV RNA titer was observed only in IMY-N9. Serial passages of HCV culture supernatant were shown in the culture system. Furthermore, we tested several infectious materials for viral infectivity by monitoring HCV RNA titers and/or 50% tissue culture infectious dose (TCID50) of HCV on IMY-N9. In each material, HCV showed various growth patterns and a different TCID50 even though the PCR titer in each material was identical. The results showed that HCV in each material served various growth patterns and different TCID50 even though PCR titer in each material was identical. This cell line is useful for estimating viral activity and for studying cellular factors that may be necessary to HCV replication in human hepatocytes.

Assembly of JC virus-like particles in COS7 cells.

JC virus lacks an appropriate cell line to support virus replication. The establishment of a JC pseudovirus assembly system could play an alternative role for a virus culture system. COS7 cells and a transfer vector, pcDL-SR alpha 296, were used to express JC viral structural genes. VP231-SR alpha, which encodes VP2/VP3 and VP1, but lacks 137 bp of the 5'-terminus of agnogene, showed both efficient nuclear migration and quantitative expression of the major capsid protein VP1. JC pseudovirus assembly was observed in the nucleus of VP231-SR alpha transfected cells. Evidence of JC pseudovirus assembly is presented. The further utilization of this system, which includes a study for the viral morphogenesis, serological diagnosis, as well as the potential application for gene transfer vector, is discussed.

Cultivation of hepatitis C virus in primary hepatocyte culture from patients with chronic hepatitis C results in release of high titre infectious virus.

To investigate the viral replication cycle and genomic heterogeneity of hepatitis C virus (HCV), we established an HCV cultivation system by using a primary hepatocyte culture from patients with chronic hepatitis C. Liver tissue was obtained by needle biopsy or surgery, then hepatocytes were isolated by collagenase digestion. After several weeks, we determined the HCV RNA titre of the cultured cells and supernatant by a competitive polymerase chain reaction (PCR) method. A significant amount of HCV RNA was observed in the cells and supernatant during cultivation. Negative-strand RNA, regarded as a marker of viral replication, could be detected by a strand-specific reverse transcription PCR method and the HCV core protein could be detected by immunofluorescence microscopy. Many HCV particles released into the supernatant were infectious. In addition, we compared the nucleotide sequences in the E2/NS1 region of pre- and post-cultivation hepatocytes for 8 weeks. At the beginning of the culture period, three major HCV types containing two subtypes were isolated. Following cultivation, the same types were isolated from the cultured hepatocytes in the same ratio as prior to cultivation. We could detect the same clones in this patient's serum, but in vivo we observed genetic variability over a 6 month interval. One clone detected throughout the 6 month period mutated extensively in the hypervariable region. These results indicated that HCV can replicate in cultured hepatocytes, and that infectious virions are released into the supernatant. This cultivation system should facilitate the study of HCV genomic heterogeneity, infection and replication.

Involvement of the influenza A virus PB2 protein in the regulation of viral gene expression.

To determine the function(s) of the PB2 protein of influenza A virus, six temperature-sensitive (ts) mutants of A/Udorn/72 (H3N2) virus, each carrying a ts mutation in the PB2 gene, were analysed for virus RNA and protein synthesis. One of the mutants, ICRC27, exhibited unique phenotypes and was characterized in detail. At the non-permissive temperature, 40 degrees C, the accumulation of mRNA for each genome segment was reduced severely, leading to delayed and reduced synthesis of viral proteins, complementary and viral RNAs (cRNAs and vRNAs). At the permissive temperature, 34 degrees C, the mutant virus produced several-fold greater concentrations of both mRNAs and cRNAs of PB2, PB1 and PA segments than wild-type virus. The synthesis of the three polymerase proteins and the induction of RNA polymerase activity were also greatly increased. By contrast, the expression of the haemagglutinin (HA) gene was severely suppressed. The over-production of the polymerase mRNAs was not observed during primary transcription, i.e. in the presence of cycloheximide. The ts+ revertants of ICRC27 did not exhibit the ts defects and also lost most of the non-ts phenotypes at 34 degrees C. These observations indicate that the PB2 protein participates not only in the synthesis of viral RNAs, but also in the regulation of viral gene expression, i.e. in the down-regulation of the three polymerase genes and the up-regulation of the HA gene during secondary transcription.

Two signals mediate nuclear localization of influenza virus (A/WSN/33) polymerase basic protein 2.

Polymerase basic protein 2 (PB2), a component of the influenza virus polymerase complex, when expressed alone from cloned cDNA in the absence of other influenza virus proteins, is transported into the nucleus. In this study, we have examined the nuclear translocation signal of PB2 by making deletions and mutations in the PB2 sequence. Our studies showed that two distant regions in the polypeptide sequence were involved in the nuclear translocation of PB2. In one region, four basic residues (K-736 R K R) played a critical role in the nuclear translocation of PB2, since the deletion or mutation of these residues rendered the protein totally cytoplasmic. However, seven residues (M K R K R N S) of this region, including the four basic residues, failed to translocate a cytoplasmic reporter protein into the nucleus, suggesting that these sequences were necessary but not sufficient for nuclear translocation. Deletion of another region (amino acids 449 to 495) resulted in a mutant protein which was cytoplasmic with a perinuclear distribution. This novel phenotype suggests that a perinuclear binding step was involved prior to translocation of PB2 across the nuclear pore and that a signal might be involved in perinuclear binding. Possible involvement of these two signal sequences in the nuclear localization of PB2 is discussed.

Purification and molecular structure of RNA polymerase from influenza virus A/PR8.

The RNA-dependent RNA polymerase of influenza virus A/PR/8 was isolated from virus particles by stepwise centrifugation in cesium salts. First, RNP (viral RNA-NP-P proteins) complexes were isolated by glycerol gradient centrifugation of detergent-treated viruses and subsequently NP was dissociated from RNP by cesium chloride gradient centrifugation. The P-RNA (P proteins-viral RNA) complexes were further dissociated into P proteins and viral RNA by cesium trifluoroacetate (CsTFA) gradient centrifugation. The nature of P proteins was further analyzed by glycerol gradient centrifugation and immunoblotting using monospecific antibodies against each P protein. The three P proteins, PB1, PB2, and PA, sedimented altogether as fast as the marker protein with the molecular weight of about 250,000 Da. Upon addition of the template vRNA, the RNA-free P protein complex exhibited the activities of capped RNA cleavage and limited RNA synthesis. When a cell line stably expressing cDNAs for three P proteins and NP protein was examined, the three P proteins were found to be co-precipitated by antibodies against the individual P proteins. These results indicate that the influenza virus RNA-dependent RNA polymerase is a heterocomplex composed of one each of the three P proteins and that the RNA-free RNA polymerase can be isolated in an active form from virus particles. Furthermore, the three P proteins form a complex in the absence of vRNA.

Control of influenza virus gene expression: quantitative analysis of each viral RNA species in infected cells.

We established a quantitative hybridization system by which three types of influenza virus RNAs (vRNA, mRNA, and cRNA) for the 8 genome segments were measured individually. As the hybridization probes, 32P-labeled RNAs of both plus and minus polarity were produced employing an SP-6 transcription system and used in a large molar excess, sufficient to overcome complementary RNAs present in the viral RNA samples. Employing the system, we studied the control of the synthesis of each viral RNA species in MDCK cells infected with A/Udorn/72 (H3N2). Our new observations were as follows. 1) Segment-specific transcription was observed at the primary transcription. 2) Replication of the virus genome began simultaneously for all segments. No delay was observed in the replication of the segments carrying late genes. 3) In addition to control at the transcriptional levels, the expression of viral late genes was regulated at some post-transcriptional step(s). These results are not compatible with the concepts reported previously, and lead us to propose unique regulations operating on the expression of the viral late genes.

Inhibition of transcriptase activity of influenza A virus in vitro by anti-haemagglutinin antibodies.

An investigation was made of inhibition of transcriptase activity of influenza viruses in vitro by binding of antibody to the surface of the virion. Eight monoclonal antibodies which were directed against at least four non-overlapping antigenic regions of the haemagglutinin protein of A/Aichi/68 virus were tested for inhibitory effect. One of the antibodies directed against the B antigenic site, 22/1, inhibited transcriptase activity, while the other seven antibodies did not. Antibody from a hyperimmune rabbit serum to A/Udorn/72 (H3N2) virions inhibited the transcriptase activity of A/Udorn/72 and A/Aichi/68 (H3N2) viruses but not that of A/WSN/33 (H1N1). The antibody did not cause irreversible inactivation of the transcriptase since full activity was recovered by isolating ribonucleoprotein (RNP) cores from the inhibited virions using NP-40 treatment and subsequent centrifugation in a caesium sulphate density gradient. The antibody did not inhibit transcriptase activity of isolated RNP cores. The virion transcriptase activity was not inhibited by addition of the antiserum after the detergent treatment which is necessary for the activation of the transcriptase activity in vitro. These results suggest that the antibody blocks the activation process of the transcriptase by detergent treatment.