Assembly of Truncated HCV Core Antigen into Virus-like Particles in Escherichia coli

Lorenzo, Lazaro; Dueñas‐Carrera, Santiago; Falcón, Viviana; Acosta‐Rivero, Nelson; González, Ernesto; Rosa, Maria Cristina; Menéndez, Ivón; Morales, Juan

doi:10.1006/bbrc.2001.4449

Cited by 47 publications

(52 citation statements)

References 27 publications

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“…5E, which shows that C115 formed capsids that were 34 to 75 nm in diameter. The finding that C-terminal truncation mutants formed larger capsids is consistent with what was observed in studies of recombinant core encoding truncation mutations (30,41). Thus, these data indicate that the C terminus of core beyond residue 115 is not absolutely required for assembly but does affect the size of the capsids formed.…”

Section: Resultssupporting

confidence: 87%

“…In the presence of structured RNA, wild-type recombinant core assembles into particles with irregular shapes, while purified C-terminal truncation mutants assemble into regularly shaped capsids that more closely resemble HCV capsids from infected individuals (30). Similar results were obtained by assembling truncated core constructs in Escherichia coli (41). Together, these systems show that HCV core can assemble into capsid-like structures in the presence of RNA (30) and are useful for structural studies (31).…”

mentioning

confidence: 59%

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Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly

Klein

Polyak²,

Lingappa

2004

J Virol

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The assembly of hepatitis C virus (HCV) is poorly understood, largely due to the lack of mammalian cell culture systems that are easily manipulated and produce high titers of virus. This problem is highlighted by the inability of the recently established HCV replicon systems to support HCV capsid assembly despite high levels of structural protein synthesis. Here we demonstrate that up to 80% of HCV core protein synthesized de novo in cell-free systems containing rabbit reticulocyte lysate or wheat germ extracts assembles into HCV capsids. This contrasts with standard primate cell culture systems, in which almost no core assembles into capsids. Cell-free HCV capsids, which have a sedimentation value of Ϸ100S, have a buoyant density (1.28 g/ml) on cesium chloride similar to that of HCV capsids from other systems. Capsids produced in cell-free systems are also indistinguishable from capsids isolated from HCV-infected patient serum when analyzed by transmission electron microscopy. Using these cell-free systems, we show that HCV capsid assembly is independent of signal sequence cleavage, is dependent on the N terminus but not the C terminus of HCV core, proceeds at very low nascent chain concentrations, is independent of intact membrane surfaces, and is partially inhibited by cultured liver cell lysates. By allowing reproducible and quantitative assessment of viral and cellular requirements for capsid formation, these cell-free systems make a mechanistic dissection of HCV capsid assembly possible.Hepatitis C virus (HCV) affects about 2% of the world's population (53) and is the major cause of non-A, non-B hepatitis, which often leads to cirrhosis of the liver or hepatocellular carcinoma (44). Acute viremia develops into chronic infections in as many as 85% of cases (25), and current treatments are still poorly tolerated and not completely effective (34). Development of better drugs and vaccines has been hampered by the lack of an infectious culture system. Infectious HCV cannot be produced in routine cell culture systems, and the chimpanzee is the only animal model capable of being infected and yielding high HCV titers (18). As a result, the requirements for critical steps in virion formation, including capsid assembly, genome encapsidation, budding, and release, remain largely unknown.HCV is an enveloped, single-stranded, positive-sense RNA virus in the Flaviviridae family (4). The HCV genome has a single open reading frame that codes for a Ϸ3,000-amino-acid polyprotein. The core protein is the N-terminal cleavage product from the polyprotein. The polyprotein is targeted to the endoplasmic reticulum (ER) by an internal signal sequence (SS) that is cleaved by signal peptidase and signal peptide peptidase, releasing core into the cytoplasm (37, 45, 57). After release from the polyprotein, core assembles into capsids at the cytoplasmic face of the ER (7,8,48). Core is known to interact with the HCV envelope glycoprotein E1 at the ER (38), and assembled capsids are thought to acquire their envelopes by budding into the...

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Section: Resultssupporting

confidence: 87%

mentioning

confidence: 59%

Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly

Klein

Polyak²,

Lingappa

2004

J Virol

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“…Previous reports of nucleocapsid-like particles assembly from recombinant HCV core protein in vitro have shown that, in the presence of RNA with secondary structures such as the HCV internal ribosome entry site or tRNA, core protein assembles into particles that are heterogeneous in size and larger than the virus particles isolated from infected patients (34,38). Moreover, no particle formation was observed in the presence of the full-length HCV RNA genome.…”

Section: Discussionmentioning

confidence: 96%

Hepatitis C Virus Core Protein Is a Dimeric Alpha-Helical Protein Exhibiting Membrane Protein Features

Boulant

Vanbelle

Ebel

et al. 2005

J Virol

130

156

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The building block of hepatitis C virus (HCV) nucleocapsid, the core protein, together with viral RNA, is composed of different domains involved in RNA binding and homo-oligomerization. The HCV core protein 1-169 (C HCV 169) and its N-terminal region from positions 1 to 117 (C HCV 117) were expressed in Escherichia coli and purified to homogeneity suitable for biochemical and biophysical characterizations. The overall conformation and the oligomeric properties of the resulting proteins C HCV 169 and C HCV 117 were investigated by using analytical centrifugation, circular dichroism, intrinsic fluorescence measurements, and limited proteolysis. Altogether, our results show that core protein (C HCV 169) behaves as a membranous protein and forms heterogeneous soluble micelle-like aggregates of high molecular weight in the absence of detergent. In contrast, it behaves, in the presence of mild detergent, as a soluble, well-folded, noncovalent dimer. Similar to findings observed for core proteins of HCV-related flaviviruses, the HCV core protein is essentially composed of ␣-helices (50%). In contrast, C HCV 117 is soluble and monodispersed in the absence of detergent but is unfolded. It appears that the folding of the highly basic domain from positions 2 to 117 (2-117 domain) depends on the presence of the 117-169 hydrophobic domain, which contains the structural determinants ensuring the binding of core with cellular membranes. Finally, our findings provide valuable information for further investigations on isolated core protein, as well as for attempts to reconstitute nucleocapsid particles in vitro.Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. It is estimated that 1% of the general population in Western Europe and North America and 380 million people globally are infected with HCV worldwide (19). A protective vaccine does not exist to date, and therapeutic options are still limited. HCV has been classified in the Hepacivirus genus within the Flaviviridae family of viruses. It contains a 9.6-kb plus-strand RNA genome (10, 59, 70) composed of a 5Ј noncoding region (5Ј NCR), a long open reading frame (ORF) encoding a polyprotein precursor of about 3,000 amino acids (aa), and a 3Ј NCR. The HCV polyprotein precursor is co-and posttranslationally processed by cellular and viral proteases to yield the mature structural and nonstructural (NS) proteins. The structural proteins include the core protein, which forms the viral nucleocapsid, and the envelope glycoproteins E1 and E2. They are separated from the NS proteins by the viroporin p7. The NS proteins include the NS2-3 autoprotease and the NS3 serine protease, an NTPase/RNA helicase located in the Cterminal two-thirds of NS3, the NS4A cofactor of NS3, the NS4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase (for reviews, see references 55 and 58). Interestingly, alternative reading frames (ARF) were recently identified in the HCV core region that has the potential to encode proteins des...

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“…It is possible that these recombinant capsids could be empty, or that they could contain cellular RNA transcripts or the specific transcript. Although other systems have been used as well to study assembly of the HCV core protein [24,25,27,[53][54][55][56], the baculovirus expression system described here allows for the study of capsid assembly in a versatile, cell-based system and for the partial purification of the derived naked capsid-like particles in sufficient quantities, which might be useful for potential applications ranging from basic virological studies to developments in biomedicine.…”

Section: Discussionmentioning

confidence: 99%

Evidence for cellular uptake of recombinant hepatitis C virus non‐enveloped capsid‐like particles

et al. 2007

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Although the hepatitis C virus (HCV) is an enveloped virus, naked nucleocapsids have been reported in the serum of infected patients, and most recently novel HCV subgenomes with deletions of the envelope proteins have been identified. However the significance of these findings remains unclear. In this study, we used the baculovirus expression system to generate recombinant HCV capsid-like particles, and investigated their possible interactions with cells. We show that expression of HCV core in insect cells can sufficiently direct the formation of capsid-like particles in the absence of the HCV envelope glycoproteins and of the 5 0 untranslated region. By confocal microscopy analysis, we provide evidence that the naked capsid-like particles could be uptaken by human hepatoma cells. Moreover, our findings suggest that they have the potential to produce cell-signaling effects.

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Assembly of Truncated HCV Core Antigen into Virus-like Particles in Escherichia coli

Cited by 47 publications

References 27 publications

Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly

Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly

Hepatitis C Virus Core Protein Is a Dimeric Alpha-Helical Protein Exhibiting Membrane Protein Features

Evidence for cellular uptake of recombinant hepatitis C virus non‐enveloped capsid‐like particles

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