Monday, January 21, 2013

Hepatitis C - New Ways to Study Genotypes 3 and 4

New Ways to Study HCV, Genotypes 3 and 4

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Researchers can now study replication of Hepatitis C virus genotypes 3 and 4 in cultured cells, described in 2 articles in the January issue of Gastroenterology. These new tools will improve our understanding of how they cause liver disease, and could lead to new treatments.

HCV leads to chronic infection and advanced liver diseases in most infected adults. It is a positive-strand RNA virus that replicates its genome with the help of an RNA-dependent RNA polymerase.
There are 6 major HCV genotypes. Genotypes 1 and 2 are the most prevalent in North America, Europe, and Japan, and are the most highly studied. However, other genotypes have specific characteristics. Genotype 3a infection can cause hepatic steatosis, and is more resistant to treatment with telaprevir and boceprevir. Genotype 4 is prevalent in the Middle East and many African countries, and is becoming more common in central and northern Europe; it accounts for 93% of HCV infections in Egypt, and 5%–15% of infections in several European countries.

HCV replicons—almost-complete viral RNA sequences that can replicate autonomously in cells—are important for studying viral replication and were essential for the development of many current drugs against the infection. However, only replicons for HCV genotypes 1a, 1b, and 2a have been developed. The first efficient HCV cell culture system was based on genotype 1b-derived subgenomic replicons (see below figure).

Replicons comprise the HCV 5′ nontranslated region (NTR), the selection marker neomycin phosphotransferase (neo), the internal ribosome entry site of the encephalomycarditis virus (EMCV-IRES, which activates translation of the NS3 to NS5B coding sequence of HCV), and the 3′ NTR. Synthetic replicon RNA molecules are transfected into the human hepatoma cell line Huh7. Upon selection (with neomycin), only cells with efficient RNA replication will survive and form a stable replicon cell clone. Efficient RNA replication of genotype 1, 3, and 4 replicons depends on the acquisition of replication enhancing (adaptive) mutations. Cells that are not transfected or that do not support efficient RNA replication are eliminated.
Mohsan Saeed et al. recovered an almost complete HCV genotype 3a genome from the serum of a patient with post-transplantation recurrent HCV infection. The isolate, which they named S310, contained a 5′UTR (nucleoties 1−339), an open reading frame encoding 3021 amino acides (nucleotides 340−9402), and a 3′UTR (nucleotides 9403−9654). Only the last 44 nucleotides of the X-region (9611−9654) were not recovered.

They used this genome to construct a replicon, and showed that it replicated in a human liver cell line. Importantly, replicons in these cells accumulated adaptive, replication-enhancing mutations in nonstructural proteins (NS)3, NS5A, and NS5B—at similar or even identical positions as previously reported for HCV genotype 1b isolates.

When Saeed et al. tested the effects of different anti-HCV drugs, they found that interferon (IFN)-a caused a dose-dependent decreased in replication, as it does for all other HCV genotypes. The protease inhibitor BILN-2061 was more effective against replicons from genotypes 1b and 2a than 3a, and the non-nucleoside polymerase inhibitor JTK-109 was more potent against genotype 1b and 3a. However, the nucleoside polymerase inhibitor PSI-6130 equally inhibited all genotypes.

Betty Peng et al. synthesized and transcribed a genotype 4a subgenomic replicon and transfected new Huh-7–derived cell line. As for the genotype 3a replicon, the 4a replicon acquired adaptive mutations in the NS3, but also in 4A, which allowed the cells it to stably replicate in the cells.

In an editorial that accompanies the articles, Volker Lohmann and Ralf Bartenschlager explain that the availability of only a few HCV isolates capable of replicating in cell culture had been a serious limitation to studies on HCV infection and treatment. They say that now that we have functional genotype 3 and 4 replicons, it will now be possible to test anti-viral agents in culture.

Having the new replication systems could also help us to understand the molecular basis of genotype-specific differences in response to IFN. It is still not clear why patients infected with HCV genotypes 2 and 3 clear the virus more frequently than those infected with other genotypes.

Interestingly, the newly developed genotype 3 and 4 replicons are as sensitive to IFN-α as the genotype 1 and 2 isolates, indicating that IFN resistance is not a genotype-specific feature of the HCV nonstructural proteins.

Comparative analyses of the behaviors of HCV genotypes in culture can be used to study the molecular determinants of HCV pathogenesis.


Read the articles online.
Saeed M, Gondeau C, Hmwe S et al. Replication of hepatitis C virus genotype 3a in cultured cells. Gastroenterology 2013;144:56-58.e7.

Peng B, Yu M, Xu S, et al. Development of robust hepatitis C virus genotype 4 subgenomic replicons. Gastroenterology 2013;144: 59−61.e6.

Read the accompanying editorial.
Lohmann V, Bartenschlager R. Hepatitis C virus replicons volume 3 and 4. Gastroenterology 2013;144:13−15.

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