Generation of infectious and transmissible virions from a GB virus B full-length consensus clone in tamarins

Sbardellati, Andrea; Scarselli, Elisa; Verschoor, Ernst J.; Tomassi, Amedeo De; Lazzaro, Domenico; Traboni, Cinzia

doi:10.1099/0022-1317-82-10-2437

Cited by 37 publications

(31 citation statements)

References 41 publications

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“…4C). These titers are comparable with those obtained for the wild-type infectious GBV-B clone, which gave values ranging from 1-3 ϫ 10 9 ge/ml for data collected from four tamarins (15,17). 4 At weeks 6, 12, 14, and 16 post-inoculation, the genomic sequence was determined from viral RNA isolated from serum.…”

Section: Maturation Of Core By Spp Is Essential For Productive Gbv-b supporting

confidence: 67%

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Signal Peptide Peptidase Cleavage of GB Virus B Core Protein Is Required for Productive Infection in Vivo

Targett-Adams

Schaller

Hope

et al. 2006

Journal of Biological Chemistry

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Chronic infection by hepatitis C virus (HCV) is a leading cause of liver disease for which better therapies are urgently needed. Because a clearer understanding of the viral life cycle may suggest novel anti-viral approaches, we studied the role of host signal peptide peptidase (SPP) in viral infection. This intramembrane protease cleaves within a C-terminal signal sequence in the viral core protein, but the molecular determinants of cleavage and whether it is required for infection in vivo are unknown. To answer these questions, we studied SPP processing in GB virus B (GBV-B) infection. GBV-B is the closest phylogenetic relative of HCV and offers an accurate surrogate model for HCV infection. We demonstrate that SPP also processes GBV-B core protein and that a serine residue in the hydrophobic region of the signal sequence (present also in HCV) is critical for efficient SPP cleavage. The small size of the serine side chain combined with its ability to form intra-and interhelical hydrogen bonds likely contributes to recognition of the signal sequence as a substrate for SPP. By introducing mutations with differing effects on SPP processing into an infectious GBV-B molecular clone, we demonstrate that SPP processing of the core protein is required for productive infection in primates. These results broaden our understanding of the mechanism and requirements for SPP cleavage and reveal a functional role in vivo for intramembrane proteolysis in host-pathogen interactions. Moreover, they identify SPP as a potential therapeutic target for reducing the impact of HCV infection.Intramembrane-cleaving proteases are a family of enzymes that target transmembrane domain sequences of proteins and thereby modulate cellular signaling, lipid biosynthesis, and the unfolded protein response (1-3). Signal peptide peptidase (SPP) 2 is an intramembrane-cleaving protease that cleaves certain signal sequences following proteolysis by signal peptidase (4, 5). Studies on the biological functions associated with SPP cleavage are very limited. In higher eukaryotes, cell-based analyses have indicated that disrupting the SPP processing of HLA-E epitopes blocks their presentation on the cell surface (6, 7). In the lower eukaryotes, such as Caenorhabditis elegans (8) and Drosophila melanogaster (9), inhibition of SPP activity by either RNAi or mutation impairs embryonic and larval development. SPP is necessary for maturation of the hepatitis C virus (HCV) core protein (10, 11). Core protein forms the virus capsid and is released from the viral polyprotein by signal peptidase and SPP through two coordinated cleavage events at a signal sequence that separates core from the E1 glycoprotein (4). Cleavage by SPP is essential for transfer of core from the endoplasmic reticulum (ER) membrane to lipid droplets, which are cytosolic storage organelles (10). However, because of the limited availability of animal models, it has not been possible to demonstrate that SPP cleavage of core is necessary for the production of virus progeny in vivo.GB virus B ...

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Section: Maturation Of Core By Spp Is Essential For Productive Gbv-b supporting

confidence: 67%

“…GB virus B (GBV-B) can act as a surrogate model of HCV infection, because the two viruses have identical genome organizations and share significant sequence similarity (12)(13)(14)(15)(16)(17)(18)(19). GBV-B establishes an acute infection in several species of New World monkeys including tamarins (Saguinus sp.)…”

mentioning

confidence: 99%

Signal Peptide Peptidase Cleavage of GB Virus B Core Protein Is Required for Productive Infection in Vivo

Targett-Adams

Schaller

Hope

et al. 2006

Journal of Biological Chemistry

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“…Another possibility is that the transmission mode influences the outcome, since both animals were transfected from molecular clones. However, five animals that we transfected with the wild-type clone all had acute resolving infections, as did two animals transfected with another molecular GBV-B clone (21). Lanford et al (13) observed that transient immunosuppression of four tamarins could prolong GBV-B viremia in some cases, but it did not lead to persistent infections.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast to HCV infection, which frequently progresses to chronicity in humans and in experimentally infected chimpanzees (9), all reported GBV-B infections in tamarins initiated with wild-type virus have resolved during the acute phase (1,3,4,21,23). Recently, however, it was reported that an animal transfected with RNA transcripts from a full-length GBV-B clone had viremia for more than 2 years before it cleared the infection (15).…”

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confidence: 99%

In Vivo Analysis of the 3′ Untranslated Region of GB Virus B after In Vitro Mutagenesis of an Infectious cDNA Clone: Persistent Infection in a Transfected Tamarin

Nam

Faulk

Engle

et al. 2004

J Virol

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GB virus B (GBV-B), the virus most closely related to hepatitis C virus (HCV), infects tamarins and causes acute hepatitis. The 3 untranslated region (UTR) of an infectious GBV-B clone (pGBB) has a proximal short sequence followed by a poly(U) tract and a 3 terminal sequence. Our investigators previously demonstrated that the 3 terminal sequence was critical for in vivo infectivity. Here, we tested the effect of deleting the short sequence and/or the poly(U) tract from pGBB; infectivity of each mutant was tested by intrahepatic transfection of two tamarins with transcribed RNA. A mutant lacking both regions was not viable. However, mutants lacking either the short sequence or the poly(U) tract were viable. All four tamarins had a wild-type-like acute infection and developed acute hepatitis. Whereas we found that five tamarins transfected with the wild-type clone pGBB had acute resolving infection, one tamarin transfected with the poly(U) deletion mutant became persistently infected. This animal had viremia and hepatitis until its death at week 90. The genomes recovered at weeks 2, 7, 15, 20, 60, and 90 lacked the poly(U) stretch. Eight amino acid changes were identified at week 90. One change, in the putative p7 protein, was dominant at week 15. Thus, persistence of GBV-B, like persistence of HCV, was associated with the emergence of virus variants. Four tamarins inoculated with serum collected at weeks 2 and 90 from the tamarin with persistent infection had an acute resolving infection. Nonetheless, the demonstration that GBV-B can persist in tamarins strengthens its relevance as a surrogate model for the study of HCV.The study of GB virus B (GBV-B) has generated considerable interest because it is the virus most closely related to hepatitis C virus (HCV) (16,18). In humans, infection with HCV is one of the leading causes of chronic liver disease, which in many cases will progress to liver cirrhosis and liver cancer (9). The natural host of GBV-B remains unknown, but it was demonstrated that this virus does not infect chimpanzees, a surrogate of humans, suggesting that it is not a human virus (3). However, experimental infection of tamarins with GBV-B causes acute hepatitis. Thus, experimental GBV-B infection of tamarins could potentially serve as a surrogate model for the study of HCV, which can only be studied in chimpanzees.The GBV-B virus contains a positive-sense, single-stranded RNA genome of about 9.5 kb (4, 24). Like the other members of the Flaviviridae virus family, it has a single long open reading frame (ORF), bracketed by 5Ј and 3Ј untranslated regions (UTR). Based on hydropathy plots and known motifs, it was predicted to encode the same kinds of proteins as HCV (16). However, it should be emphasized that the overall homology of the predicted polyproteins between GBV-B and HCV is only about 25 to 30% (16). Significant homology was observed between GBV-B and HCV in the NS3 serine protease, the NS3 RNA helicase, and the NS5 RNA-dependent RNA polymerase regions (16,34,35). Furthermore, functional simi...

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“…Upon intrahepatic inoculation of tamarins (Saguinus spp. ), only genomes with the complete 3Ј NTR were viable, showing that this region is essential for infectivity (8,51). For pestivirus BVDV, it was shown that mutations within the 3Ј- 3 and 22 to 24).…”

Section: Vol 76 2002mentioning

confidence: 99%

Genetic Analysis of Sequences in the 3′ Nontranslated Region of Hepatitis C Virus That Are Important for RNA Replication

Friebe

Bartenschlager

2002

J Virol

249

250

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The genome of the hepatitis C virus (HCV) is a plus-strand RNA molecule that carries a single long open reading frame. It is flanked at either end by highly conserved nontranslated regions (NTRs) that mediate crucial steps in the viral life cycle. The 3 NTR of HCV has a tripartite structure composed of an about 40-nucleotide variable region, a poly(U/UC) tract that has a heterogeneous length, and a highly conserved 98-nucleotide 3-terminal sequence designated the X tail or 3X. Conflicting data as to the role the sequences in the 3 NTR play in RNA replication have been reported. By using the HCV replicon system, which is based on the self-replication of subgenomic HCV RNAs in human hepatoma cell line Huh-7, we mapped in this study the sequences in the 3 NTR required for RNA replication. We found that a mutant with a complete deletion of the variable region is viable but that replication is reduced significantly. Only replicons in which the poly(U/UC) tract was replaced by a homouridine stretch of at least 26 nucleotides were able to replicate, whereas RNAs with homopolymeric guanine, adenine, or cytosine sequences were inactive. Deletions of individual or all stem-loop structures in 3X were not tolerated, demonstrating that this region is most crucial for efficient RNA replication. Finally, we found that none of these deletions or substitutions within the 3 NTR affected RNA stability or translation, demonstrating that the primary effect of the mutations was on RNA replication. These data represent the first detailed mapping of sequences in the 3 NTR assumed to act as a promoter for initiation of minus-strand RNA synthesis.Hepatitis C virus (HCV), the etiologic agent of non-A, non-B hepatitis, is an enveloped plus-strand RNA virus that belongs to the family Flaviviridae (46). One of the hallmarks of HCV infection is the high frequency of persistence. About 80% of infected individuals are unable to eliminate the virus, and these patients are at high risk to develop chronic liver disease, including liver cirrhosis and hepatocellular carcinoma (32). In spite of recent improvements in therapy of chronic hepatitis C with ribivirin and alpha interferon conjugated with polyethyleneglycol, only about one-half of the patients develop a sustained response, and nonresponders and relapses are common (40). Therefore, more-effective therapies are certainly required.Like those of other members of the Flaviviridae family, the HCV genome carries a single long open reading frame (ORF), which encodes a polyprotein that is cleaved co-and posttranslationally into a series of products (for reviews see references 4 and 47). For HCV, the cleavage products are arranged in the following order (from the amino to the carboxy terminus): core, envelope protein 1 (E1), E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Structural proteins core, E1, and E2 are the main constituents of the virus particle whereas the NS3, NS4B, NS5A, and NS5B proteins are essential and sufficient for RNA replication (36). NS3 is composed of two domains that carry disti...

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Generation of infectious and transmissible virions from a GB virus B full-length consensus clone in tamarins

Cited by 37 publications

References 41 publications

Signal Peptide Peptidase Cleavage of GB Virus B Core Protein Is Required for Productive Infection in Vivo

Signal Peptide Peptidase Cleavage of GB Virus B Core Protein Is Required for Productive Infection in Vivo

In Vivo Analysis of the 3′ Untranslated Region of GB Virus B after In Vitro Mutagenesis of an Infectious cDNA Clone: Persistent Infection in a Transfected Tamarin

Genetic Analysis of Sequences in the 3′ Nontranslated Region of Hepatitis C Virus That Are Important for RNA Replication

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