Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-11-29
2004-02-10
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S091100, C435S069300, C435S455000, C435S475000, C435S370000, C536S023720
Reexamination Certificate
active
06689559
ABSTRACT:
BACKGROUND
The hepatitis C virus (HCV) is the sole member of the genus Hepacivirus of the family Flaviviridae, which also includes the Flavivirus, yellow fever virus and the Pestivirus, bovine viral diarrhea virus. Since the discovery of HCV in 1989, the viral genome has been well characterized. The genome is a positive-sense single-stranded RNA of about 9.3 kb, that consists of a single open reading frame (ORF) and nontranslated regions (NTRs) at the 5′ and 3′ ends (Bartenschlager and Lohmann, 2000).
The 5′NTR is highly structured and contains an internal ribosomal entry site (IRES) that mediates cap-independent translation of the viral polyprotein. The 3′NTR is tripartite and is composed of a short variable region (~21-39 nucleotides), a poly (U) tract of variable length, and a highly conserved terminal sequence of 98 nucleotides.
The ORF of HCV is translated into a polyprotein (i.e., NH
2
-core-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) that is co-translationally and posttranslationally processed by host cell and viral proteases into at least 10 distinct products. The core and envelope (E1 and E2) proteins are the major viral constituents of the virus particle while the remainder, the non-structural (NS) proteins, are required for virus replication.
NS2 forms an autoprotease with the amino terminus of NS3. NS3 is a bifunctional molecule with serine proteinase and NTPase/helicase activities. NS4A is a cofactor of the NS3 proteinase. The functions of NS4B and p7 proteins are so far unknown. NS5B is identified as the RNA-dependent RNA polymerase (RdRp). NS5A is highly phosphorylated and contains the interferon sensitivity-determining region (ISDR), which appears to be involved in resistance of the virus to interferon treatment.
HCV replication involves the generation of antisense strand RNA intermediates, which serve as the template for synthesis of the genomic RNA. Replication is catalyzed by the cytoplasmic membrane-associated replicase complex, which is thought to contain NS5B and other NS proteins (Bartenschlager and Lohmann, 2000). However, despite the well-characterized viral genome of HCV, the individual steps underlying RNA replication are largely unknown.
RNA viruses, such as HCV, are quasispecies due to the error rate of RNA synthesis and absence of proof reading and editing functions. RNA viruses are genetic mixtures of which the “wild type” is the genotype replicating best under a fixed set of conditions. With most RNA viruses, the “wild type” efficiently outcompetes other genotypes (giving the impression of genetic purity). However, many genotypes can coexist in a host organism, giving rise to great genetic diversity. Different sets of genotypes occur in different geographical regions. Thus, a remarkable characteristic of HCV is its genetic heterogeneity.
Currently, at least six major genotypes (genotypes 1-6) each containing a number of subtypes (1a, 1b, 1c, 2a, 2b, 2c etc.) have been described (Zein and Persing et al., 1996). Subtypes 1a and 1b account for the majority of HCV infections in the United States, Europe, and Japan (Zein and Rakela et al., 1996). For example, HCV1a accounts for over 50% of HCV infections in the United States and approximately 30% of HCV infections in Europe and also in South America. In contrast, a low prevalence of infection with HCV1a occurs in Asia and Japan.
HCV subtype 1b accounts for about 70% of HCV infections in Asia and in Japan. Interestingly, HCV subtypes 1a and 1b do not appear to exist in HCV infections in the North African sub-continent.
The sequence similarity between 1a and 1b is approximately 81% based on the genomic variability in a 222-nucleotide segment of NS5 region. A number of studies have suggested that HCV genotype status may associate with the progression of liver disease, the outcome of HCV infection, and the response to interferon therapy (Bukh and Miller, 1995; Farci et al., 1997). Therefore, basic molecular studies would be valuable to define unique virological features for each of the HCV genotypes.
The recent availability of various infectious HCV cDNA clones provides a starting base for performing reverse genetics. However, due to a lack of an effective tissue culture system, the testing of mutant HCV clones has been restricted to the inoculation of chimpanzees. Numerous attempts have been undertaken to propagate HCV in cell culture systems including: (i) cultivation of hepatocytes from experimentally infected chimpanzees or from chronically infected patients; (ii) infection of primary cell lines with high-titer HCV-containing sera; (iii) transfection of human hepatocyte or non-hepatocyte cell lines with synthetic HCV RNA, etc. For a review of many of these attempts, see Bartenschlager and Lohmann, 2000.
The majority of these attempts have been unsuccessful. Further, even the few reports claiming sustained viral replication in cell culture systems showed poor reproducibility and a low level of replication (Dash et al., 1997). Detection of HCV RNA mostly depends on highly sensitive reverse-transcription RT-PCR assays, which is also error-prone due to carryover and sample-to-sample contamination.
Another approach was the establishment of a selectable HCV replicon derived from the HCV consensus genome of subtype 1b (Lohmann et al., 1999). This selectable HCV1b replicon is capable of autonomous replication in human hepatoma cell line Huh-7. This subgenomic HCV1b replicon lacks the structural regions and expresses the selectable marker neo, which confers resistance to neomycin sulfate (G418), which is toxic to normal cells (Southern, 1982). Resistance to neomycin sulfate is conferred by a phosphotransferase (neomycin phosphotransferase).
Upon transfection of the Huh-7 cells with the HCV1b replicon containing the neo selectable marker, a low number of colonies was obtained after selection with neomycin sulfate. Cell lines derived from these colonies contained actively replicating viral RNAs with 1000~5000 RNA molecules per cell. However, long-term follow-up studies showed that viral RNA replication relies on continuous selection and was strongly influenced by cell density and cell cycle. Furthermore, the RNA levels dropped significantly when the host cells reached confluency. This suggests that HCV RNA replication is tightly linked to host cell metabolism.
Although the HCV1b replicon developed by Lohmann et al. showed appreciable levels of RNA replication, this cell culture system was not efficient. The number of G418 resistance-colonies obtained after successful RNA transfection was consistently low and this low transduction efficiency limits its usefulness as a genetic tool.
To increase efficiency of the HCV1b replicon, Blight et al. (Blight et al., 2000), isolated and sequenced replicons from the Huh-7 cell clones and identified multiple independent adaptive mutations that cluster in the HCV NS5A ORF and bestowed increased replication competency in vitro. Transfection of the mutant HCV1b replicon conferred G418-resistant Huh-7 cells at a rate of 10%. The increase in the G418 transduction efficiency correlates with the level of replication measured with quantitative RT-PCR (Blight et al., 2000).
Due to the extensive genetic heterogeneity of HCV, it is desirable to develop self-replicating systems for more than one genotype. In an attempt to extend this system to other HCV genotypes, HCV genotype 1a-specific replicons were constructed according to the method used to provide HCV1b replicons. However, transfection of these HCV1a replicons failed to yield any G418-resistant colonies (Blight et al., 2000). Furthermore, engineering the most efficient adaptive mutations identified in the 1b replicon into the 1a-derived replicon did not yield detectable replication in Huh-7 cells.
To date, there have been no reports of an effective cell culture system for replication of HCV of subtype 1a.
Bukh's group at the N.I.H. (Yanagi, et al. 1997) constructed a stable full-length cDNA clone, H77C of HCV genotype 1a, strain H77. However, RNA derived from this construct is not an efficient replica
Hahm Bumsuk
Jang Sung Key
Liang Chengyu
Wimmer Eckard
Hoffman & Baron LLP
The Research Foundation of the State University of New York
Wortman Donna C.
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