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.

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.

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.

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.

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. Hepatitis C virus (HCV) is now recognized as the principal agent in parentally transmitted non-A, non-B hepatitis. HCV is a single-stranded positive-sense RNA virus of about 9,500 nucleotides (nt) that encodes a large polyprotein, 1,2 which is processed into viral structural and nonstructural proteins. Recently, full-length HCV RNA transcripts from HCV cDNAs were shown to be infectious in chimpanzees. 3,4,5 Although our understanding of the molecular biology of HCV has progressed rapidly, little is known about its biological characteristics, because in vivo studies have been limited to the studies performed using chimpanzees that have been infected or transfected with HCV. The development of efficient animal cell culture systems for HCV or readily available animal models are priorities for the study of HCV. HCV replication has been described based on studies of liver tissue and peripheral blood mononuclear cells (PBMC) from infected patients. 6,7 Animal cell culture systems for HCV replication have been developed from human T and B cell lines, 8,9,10 human fetal liver cells, 11,12 and chimpanzee primary hepatocytes. 13 Efficient long-term viral replication, however, has not been reported in any system. HCV replication may be stringently restricted to differentiated hepatocytes, which may have receptors for HCV on their cell surface and specific factors to support viral growth. Recently, a host cellular factor, polypyrimidine-tract-binding protein (PTB), was reported to bind the HCV 3Ј-end, and was thought to be a cis-acting element for HCV replication. 14,15 PTB is also related to the mechanism of HCV translational regulation, 16,17 indicating that several cellular factors may be involved in the mechanism of HCV propagation. We previously reported that HCV replicated efficiently in primary cultured human hepatocytes, 18 and another group also confirmed our findings. 19 However, we still need to develop a more convenient cell-culture system because it is difficult to obtain a constant source of human hepatocytes. In this study, we fused human hepatocytes with a hepatoblastoma cell line, HepG2, and obtained hybrid cell lines. We then tested the ability of the hybrids to support HCV replication by monitoring HCV RNA titers in cultured cells using a real-time detection PCR (RTD-PCR). 20 The results showed that several hybrid cell lines were more suscept...