Analysis of functional differences between hepatitis C virus NS5A of genotypes 1-7 in infectious cell culture systems
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Analysis of functional differences between hepatitis C virus NS5A of genotypes 1-7 in infectious cell culture systems. / Scheel, Troels K H; Prentoe, Jannick; Carlsen, Thomas H R; Mikkelsen, Lotte S; Gottwein, Judith M; Bukh, Jens.
I: P L o S Pathogens, Bind 8, Nr. 5, e1002696, 2012, s. 1-16.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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T1 - Analysis of functional differences between hepatitis C virus NS5A of genotypes 1-7 in infectious cell culture systems
AU - Scheel, Troels K H
AU - Prentoe, Jannick
AU - Carlsen, Thomas H R
AU - Mikkelsen, Lotte S
AU - Gottwein, Judith M
AU - Bukh, Jens
PY - 2012
Y1 - 2012
N2 - Hepatitis C virus (HCV) is an important cause of chronic liver disease. Several highly diverse HCV genotypes exist with potential key functional differences. The HCV NS5A protein was associated with response to interferon (IFN)-α based therapy, and is a primary target of currently developed directly-acting antiviral compounds. NS5A is important for replication and virus production, but has not been studied for most HCV genotypes. We studied the function of NS5A using infectious NS5A genotype 1-7 cell culture systems, and through reverse genetics demonstrated a universal importance of the amphipathic alpha-helix, domain I and II and the low-complexity sequence (LCS) I for HCV replication; the replicon-enhancing LCSI mutation S225P attenuated all genotypes. Mutation of conserved prolines in LCSII led to minor reductions in virus production for the JFH1(genotype 2a) NS5A recombinant, but had greater effects on other isolates; replication was highly attenuated for ED43(4a) and QC69(7a) recombinants. Deletion of the conserved residues 414-428 in domain III reduced virus production for most recombinants but not JFH1(2a). Reduced virus production was linked to attenuated replication in all cases, but ED43(4a) and SA13(5a) also displayed impaired particle assembly. Compared to the original H77C(1a) NS5A recombinant, the changes in LCSII and domain III reduced the amounts of NS5A present. For H77C(1a) and TN(1a) NS5A recombinants, we observed a genetic linkage between NS5A and p7, since introduced changes in NS5A led to changes in p7 and vice versa. Finally, NS5A function depended on genotype-specific residues in domain I, as changing genotype 2a-specific residues to genotype 1a sequence and vice versa led to highly attenuated mutants. In conclusion, this study identified NS5A genetic elements essential for all major HCV genotypes in infectious cell culture systems. Genotype- or isolate- specific NS5A functional differences were identified, which will be important for understanding of HCV NS5A function and therapeutic targeting.
AB - Hepatitis C virus (HCV) is an important cause of chronic liver disease. Several highly diverse HCV genotypes exist with potential key functional differences. The HCV NS5A protein was associated with response to interferon (IFN)-α based therapy, and is a primary target of currently developed directly-acting antiviral compounds. NS5A is important for replication and virus production, but has not been studied for most HCV genotypes. We studied the function of NS5A using infectious NS5A genotype 1-7 cell culture systems, and through reverse genetics demonstrated a universal importance of the amphipathic alpha-helix, domain I and II and the low-complexity sequence (LCS) I for HCV replication; the replicon-enhancing LCSI mutation S225P attenuated all genotypes. Mutation of conserved prolines in LCSII led to minor reductions in virus production for the JFH1(genotype 2a) NS5A recombinant, but had greater effects on other isolates; replication was highly attenuated for ED43(4a) and QC69(7a) recombinants. Deletion of the conserved residues 414-428 in domain III reduced virus production for most recombinants but not JFH1(2a). Reduced virus production was linked to attenuated replication in all cases, but ED43(4a) and SA13(5a) also displayed impaired particle assembly. Compared to the original H77C(1a) NS5A recombinant, the changes in LCSII and domain III reduced the amounts of NS5A present. For H77C(1a) and TN(1a) NS5A recombinants, we observed a genetic linkage between NS5A and p7, since introduced changes in NS5A led to changes in p7 and vice versa. Finally, NS5A function depended on genotype-specific residues in domain I, as changing genotype 2a-specific residues to genotype 1a sequence and vice versa led to highly attenuated mutants. In conclusion, this study identified NS5A genetic elements essential for all major HCV genotypes in infectious cell culture systems. Genotype- or isolate- specific NS5A functional differences were identified, which will be important for understanding of HCV NS5A function and therapeutic targeting.
KW - Base Sequence
KW - Cell Line, Tumor
KW - Genotype
KW - Hepacivirus
KW - Humans
KW - Protein Structure, Secondary
KW - RNA, Viral
KW - Sequence Analysis, RNA
KW - Viral Nonstructural Proteins
KW - Viral Proteins
KW - Virus Assembly
KW - Virus Replication
U2 - 10.1371/journal.ppat.1002696
DO - 10.1371/journal.ppat.1002696
M3 - Journal article
C2 - 22654662
VL - 8
SP - 1
EP - 16
JO - P L o S Pathogens
JF - P L o S Pathogens
SN - 1553-7366
IS - 5
M1 - e1002696
ER -
ID: 122663740