The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the envelope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these fulllength HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgi compartments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were detectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are important for virus particle assembly and/or release.The hepatitis C virus (HCV) was identified as the causative agent for most posttransfusion and sporadic non-A, non-B hepatitis cases (11,45). According to recent estimates, about 170 million individuals worldwide are infected. One striking characteristic of HCV is its strong propensity to persist in the infected host, which often leads to severe liver damage, ranging from chronic hepatitis to liver cirrhosis and even hepatocellular carcinoma (33). The possible immune evasion strategies that allow persistent viral replication in the presence of the host's immune response are not well understood, but the high variability of the virus appears to be a key determinant (38). As a consequence, HCV isolates exhibit marked sequence diversity and have been grouped according to phylogenetic analysis into six different genotypes which together form the genus Hepacivirus within the family Flaviviridae (60).HCV particles are enveloped, have a diameter of 55 to 65 nm, and harbor an ϳ9,600-nucleotide-long plus-strand RNA genome. It carries a s...

Subgenomic selectable RNAs of the hepatitis C virus (HCV) have recently been shown to self-replicate to high levels in the human hepatoma cell line Huh-7 (V. Lohmann, F. Körner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartenschlager, Science 285:110-113, 1999). Taking advantage of this cell culture system that allows analyses of the interplay between HCV replication and the host cell, in this study we characterized two replicon-harboring cell lines that have been cultivated for more than 1 year. During this time, we observed no signs of cytopathogenicity such as reduction of growth rates or ultrastructural changes. High levels of HCV RNAs were preserved in cells passaged under continuous selection. When selective pressure was omitted replicon levels dropped, but depending on culture conditions the RNAs persisted for more than 10 months. A tight coupling of the amounts of HCV RNA and proteins to host cell growth was observed. Highest levels were found in exponentially growing cells, followed by a sharp decline in resting cells, suggesting that cellular factors required for RNA replication and/or translation vary in abundance and become limiting in resting cells. Studies of polyprotein processing revealed rapid cleavages at the NS3/4A and NS5A/B sites resulting in a rather stable NS4AB5A precursor that was processed slowly into individual products. Half-lives (t 1/2 s) of mature proteins ranged from 10 to 16 h, with the exception of the hyperphosphorylated form of NS5A, which was less stable (t 1/2 ,~7 h). Results of immunoelectron microscopy revealed an association of the majority of viral proteins with membranes of the endoplasmic reticulum, suggesting that this is the site of RNA replication. In summary, repliconbearing cells are a good model for viral persistence, and they allow the study of various aspects of the HCV life cycle.The hepatitis C virus (HCV) is a major leading cause of chronic liver disease (reviewed in reference 45). Infections with HCV are usually subclinical, and most patients do not develop acute hepatitis or have only mild symptoms. However, most infected individuals are unable to eliminate the virus, resulting in a persistent infection in ϳ80% of all cases, and these patients are at high risk to develop liver fibrosis, liver cirrhosis, or hepatocellular carcinoma.HCV was classified as the distinct genus Hepacivirus in the family Flaviviridae (38). Other members of this family are the pestiviruses, to which the Classical swine fever virus (CSFV) belongs, and the flaviviruses, with the prototype member Yellow fever virus. These viruses have in common a plus-strand RNA genome carrying a single long open reading frame (ORF) that is flanked at both termini by nontranslated regions (NTRs). In case of HCV, the 5Ј NTR has a length of 341 nucleotides and carries an internal ribosome entry site (IRES) permitting the binding of ribosomes in close proximity of the start codon of the ORF (57, 58). The 3Ј NTR has a tripartite structure composed of a variable region following the stop codon of the OR...

Human immunodeficiency virus type 1 (HIV-1) does not replicate in murine cells. We investigated the basis of this block by infecting a murine NIH 3T3 reporter cell line that stably expressed human CD4, CCR5, and cyclin T1 and contained a transactivatable HIV-1 long terminal repeat (LTR)-green fluorescent protein (GFP) cassette. Although the virus entered efficiently, formed provirus, and was expressed at a level close to that in a highly permissive human cell line, the murine cells did not support M-tropic HIV-1 replication. To determine why the virus failed to replicate, the efficiency of each postentry step in the virus replication cycle was analyzed using vesicular stomatitis virus G pseudotypes. The murine cells supported reverse transcription and integration at levels comparable to those in the human osteosarcoma-derived cell line GHOST.R5, and human cyclin T1 restored provirus expression, consistent with earlier findings of others. The infected murine cells contained nearly as much virion protein as did the human cells but released less than 1/500 the amount of p24 gag into the culture medium. A small amount of p24 gag was released and was in the form of fully infectious virus. Electron microscopy suggested that aberrantly assembled virion protein had accumulated in cytoplasmic vesicular structures. Virions assembling at the cell membrane were observed but were rare. The entry of M-tropic JR.FL-pseudotyped reporter virus was moderately reduced in the murine cells, suggesting a minor reduction in coreceptor function. A small reduction in the abundance of full-length viral mRNA transcripts was also noted; however, the major block was at virion assembly. This could have been due to a failure of Gag to target to the cell membrane. This block must be overcome before a murine model for HIV-1 replication can be developed.Several murine models have been developed for studies of AIDS pathogenesis. Mice transgenic for either the entire or partial human immunodeficiency virus type 1 (HIV-1) genome develop symptoms with similarities to AIDS. In one model, mice expressing HIV-1 Nef developed a wasting syndrome characterized by the loss of CD4 ϩ cells (21, 45). In another model, SCID mice were reconstituted with human peripheral blood lymphocytes or fetal thymus and liver and then inoculated with HIV-1 (37). These have been useful for studies on mechanisms of CD4 ϩ cell depletion and for evaluation of therapeutic strategies.Current murine models lack a central feature of HIV-1-induced pathogenesis: virus replication. Inoculation of mice or rodents such as rats, hamsters, and guinea pigs with high-titer HIV-1 does not result in detectable viremia, nor does the virus replicate in murine cells in culture (37) or infect human-CD4 (hu-CD4) transgenic mice (33). Low levels of virus replication have been detected in experimentally infected rabbits (13,15,20,26,43) and cotton rats (30), but this does not induce pathogenesis. Development of a system in which HIV-1 could replicate in mice would allow the investigation of features...

Retroviruses are produced as immature particles containing structural polyproteins, which are subsequently cleaved by the viral proteinase (PR). Extracellular maturation leads to condensation of the spherical core to a capsid shell formed by the capsid (CA) protein, which encases the genomic RNA complexed with nucleocapsid (NC) proteins. CA and NC are separated by a short spacer peptide (spacer peptide 1 [SP1]) on the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein and released by sequential PR-mediated cleavages. To assess the role of individual cleavages in maturation, we constructed point mutations abolishing cleavage at these sites, either alone or in combination. When all three sites between CA and NC were mutated, immature particles containing stable CA-NC were observed, with no apparent effect on other cleavages. Delayed maturation with irregular morphology of the ribonucleoprotein core was observed when cleavage of SP1 from NC was prevented. Blocking the release of SP1 from CA, on the other hand, yielded normal condensation of the ribonucleoprotein core but prevented capsid condensation. A thin, electron-dense layer near the viral membrane was observed in this case, and mutant capsids were significantly less stable against detergent treatment than wild-type HIV-1. We suggest that HIV maturation is a sequential process controlled by the rate of cleavage at individual sites. Initial rapid cleavage at the C terminus of SP1 releases the RNA-binding NC protein and leads to condensation of the ribonucleoprotein core. Subsequently, CA is separated from the membrane by cleavage between the matrix protein and CA, and late release of SP1 from CA is required for capsid condensation.

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