The core protein of the alphavirus sindbis virus assembles into core-like nucleoproteins with the viral genome RNA and with other single-stranded nucleic acids in vitro

Significant advances have been made in understanding hepatitis C virus (HCV) replication through development of replicon systems. However, neither replicon systems nor standard cell culture systems support significant assembly of HCV capsids, leaving a large gap in our knowledge of HCV virion formation. Recently, we established a cell-free system in which over 60% of full-length HCV core protein synthesized de novo in cell extracts assembles into HCV capsids by biochemical and morphological criteria. Here we used mutational analysis to identify residues in HCV core that are important for capsid assembly in this highly reproducible cell-free system. We found that basic residues present in two clusters within the N-terminal 68 amino acids of HCV core played a critical role, while the uncharged linker domain between them was not. Furthermore, the aspartate at position 111, the region spanning amino acids 82 to 102, and three serines that are thought to be sites of phosphorylation do not appear to be critical for HCV capsid formation in this system. Mutation of prolines important for targeting of core to lipid droplets also failed to alter HCV capsid assembly in the cell-free system. In addition, wild-type HCV core did not rescue assembly-defective mutants. These data constitute the first systematic and quantitative analysis of the roles of specific residues and domains of HCV core in capsid formation.Hepatitis C virus (HCV) is a major public health concern worldwide. Two percent of the world's population is infected with the virus (54), which is the major cause of non-A, non-B hepatitis and which often leads to cirrhosis of the liver or hepatocellular carcinoma (42). While current treatments have improved, they are poorly tolerated, not completely efficacious, and not available in all settings (31). Development of better treatments and vaccines has been hampered by the lack of virus replication systems. Standard cell culture systems do not support HCV replication, and the chimpanzee is the only animal model capable of being infected and yielding high HCV titers (23). These limitations have been partly overcome by the development of HCV replicon systems, which support autonomous replication of HCV RNA (2,10,36,37). Replicon systems have allowed specific requirements of HCV RNA replication to be studied; however, HCV particles, or even nucleocapsids, are not produced in these systems (9,

Section: Discussionmentioning

confidence: 99%

Identification of Residues in the Hepatitis C Virus Core Protein That Are Critical for Capsid Assembly in a Cell-Free System

Klein

Dellos

Lingappa

2005

“…The in vitro assembly of alphavirus core-like particles has been shown both with C proteins isolated from virions as well as with recombinant proteins in combination with different single-stranded nucleic acids and even with nonnucleic acid polyanionic substances (38,40). Similar attempts at in vitro assembly of flavivirus CLPs by using C proteins from dengue and yellow fever virus expressed in E. coli have proven unsuccessful so far (14).…”

Section: Discussionmentioning

confidence: 97%

“…Using the purified C protein dimer (Fig. 4) and in vitro transcribed viral RNA as well as ssDNA oligonucleotides, we attempted to reconstitute CLPs in vitro under conditions comparable to those that had been applied for the in vitro assembly of alphavirus cores (38,40). The conditions of the in vitro assembly reactions are described in detail in Materials and Methods.…”

Section: Resultsmentioning

confidence: 99%

Isolation of Capsid Protein Dimers from the Tick-Borne Encephalitis Flavivirus and In Vitro Assembly of Capsid-Like Particles

Kiermayr

Kofler

Mandl

et al. 2004

Flaviviruses have a spherical capsid that is composed of multiple copies of a single capsid protein and, in contrast to the viral envelope, apparently does not have an icosahedral structure. So far, attempts to isolate distinct particulate capsids and soluble forms of the capsid protein from purified virions as well as to assemble capsid-like particles in vitro have been largely unsuccessful. Here we describe the isolation of nucleocapsids from tick-borne encephalitis (TBE) virus and their disintegration into a capsid protein dimer by high-salt treatment. Purified capsid protein dimers could be assembled in vitro into capsid-like particles when combined with in vitro transcribed viral RNA. Particulate structures could also be obtained when single-stranded DNA oligonucleotides were used. These data suggest that the dimeric capsid protein functions as a basic building block in the assembly process of flaviviruses.

“…In the context of viral assembly into the cell, this process might take place in the vicinity of a membrane ensuring the interaction of core protein with the lipid bilayer by its putative amphipathic C-terminal binding domain D2 (possibly including a part of the downstream signal peptide sequence) and with RNA by its basic N-terminal domain D1. Consequently, the assembly pathway of HCV virions might not include a step of core preassembly prior to membrane budding, as reported for alphaviruses (50,72,73,78,79), but might proceed via coassembly at endoplasmic reticulum-derived membrane. Interestingly, such a mechanism of coassembly on membranes has been proposed for various retroviruses (see, for instance, references 2, 22, 43, and 85).…”

Section: Discussionmentioning

confidence: 99%

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

Boulant

Vanbelle

Ebel

et al. 2005

129

156

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...