hepatitis c virus ( hcv ) infection is a major cause of chronic hepatitis , liver cirrhosis , and hepatocellular carcinoma . hcv is primarily transmitted by blood - borne routes , including shared needles and transfused blood products . the who estimates that a minimum of 23% of the world s population is chronically infected with hcv ( wasley and alter , 2000 ) . despite the fact that hcv is targeted by innate , cellular , and humoral immune mechanisms , it establishes long - standing persistent infection in a majority of the people that it infects ( pawlotsky , 2006 ) . the virus forms small round - shaped particles ranging from 50 to 80  nm in diameter . the mature hcv virion is thought to consist of a nucleocapsid , an outer envelope composed of e1 and e2 viral proteins , and a lipid membrane . density gradient analyses have shown that viral rna exists within both low- and high - density fractions ( andre et al . , 2005 ) , and that the low - density fractions contain lipoproteins that associate with apolipoprotein b ( apob ) , apolipoprotein e ( apoe ) , triglycerides , and cholesterol , as well as viral structural proteins ( thomssen et al . , 1992 ; andre et al . , 2002 ; maillard et al . , 2006 ; nielsen et al . , only the low - density fraction derived from hcv - positive human serum exhibits high infectivity in chimpanzees ( bradley et al . , 1991 ; beach et al . , 1992 ) . since the establishment of a robust tissue culture infection system using strain hcv jfh-1 ( lindenbach et al . , 2005 ; wakita et al . , 2005 , the entire hcv lifecycle has been studied and the biophysical properties of the viral particles produced using the hcvcc cell culture system have been characterized . most viral rna - containing particles secreted from hcv - infected cells are poorly infectious and fractionate at high densities such as 1.14  g / ml , while highly infectious hcvcc are found within low - density fractions of 1.10  g / ml ( lindenbach et al . , , we provide a general account of our current understanding of the hcv lifecycle and a review of recent studies focusing on the morphogenesis of hcv particles within cell culture systems . hepatitis c virus is a positive - stranded rna virus , and its 9.6-kb genome contains an open reading frame encoding a polyprotein of 3000 amino acids ( aa ) flanked by untranslated regions ( utrs ) at both ends . six genotypes have been reported based on hcv genome sequence variations ( simmonds , 1996 ) . the utrs are highly structured sequences encompassing critical cis - active rna elements essential for genome replication and translation . the 5 utr , which is 341 nucleotides ( nt ) in length , contains an internal ribosomal entry site , which is a prerequisite for cap - independent translation of viral rna , from which four highly structured domains ( i  iv ) are produced ( bukh et al . , 1992 ; tsukiyama - kohara et al . , 1992 ; wang et al . , 1993 ; honda et al . , 1996 ; friebe et al . , 2001 the 3 utr varies between 200 and 235  nt in length , including a short variable region , as well as a poly(u / uc ) tract with an average length of 80  nt which is considered crucial for rna replication , and a virtually invariant 98-nt x - tail region ( tanaka et al . , 1995 ; ito and lai , 1999 ; friebe and bartenschlager , 2002 ; yi and lemon , 2003 ) . the genome is translated into a single precursor polyprotein , which is processed by cellular and viral proteases into 10 structural and non - structural proteins ( core , e1 , e2 , p7 , ns2 , ns3 , ns4a , ns4b , ns5a , and ns5b ; figure 1 ) . hcv proteins derived from the amino - terminal third of the precursor include core , e1 and e2 structural proteins . a crucial function of core protein is assembly of the viral nucleocapsid . the aa sequence of this protein is well conserved among different hcv strains compared to other hcv proteins . the non - structural ( ns ) proteins ns3-ns5b are thought to assemble into a membrane - associated hcv rna replicase complex . ns4a serves as a cofactor for ns3 and is involved in targeting ns3 to the er membrane ( wolk et al . , 2000 ) . ns4b plays a role in the remodeling of host - cell membranes , probably to generate a site for replicase assembly . ns5a is also thought to play an important but undefined role in viral rna replication . the role of hcv ns proteins in assembly of the infectious virion is described below . the hcv genome and polyprotein . post - translational cleavages by spp ( signal peptide peptidase ) , sp ( signal peptidase ) , ns2 - 3 pro ( ns2ns3 cysteine protease ) , and ns3 pro/4a ( ns3 serine protease and ns4a complex ) lead to the production of functional hcv proteins . functions of each protein in the viral lifecycle are indicated below the open reading frame . the core is 191 aa in length and consists of three distinct predicted domains : an n - terminal domain that is two - thirds hydrophilic , a c - terminal domain that is one - third hydrophobic , and a short signal peptide sequence of the downstream protein e1 . a c - terminal membrane - anchor of the core is further cleaved by a signal peptide peptidase . the mature core is estimated to be 177179 aa ( ogino et al . , 2004 ; okamoto et al . , maturation of the core by a signal peptide peptidase is required for virion production ( okamoto et al . biophysical techniques have been used to demonstrate that the mature core is a dimeric alpha - helical protein ( boulant et al . , 2005 ) . its aa sequence is highly conserved among different hcv strains , compared with other hcv proteins . thus , core is used in most serologic assays since anti - core antibodies are highly prevalent among hcv - infected individuals . core protein is found on er membranes , on the surface of lipid droplets ( lds ) , on the mitochondrial outer membrane , and to some extent , in the nucleus ( moradpour et al . , 1996 ; barba et al . , 1997 ; , 1998 ; hope et al . , 2002 ; suzuki et al . , 2005 ) . following is a proposed mechanism of translocation of core to membranes within the er network such as lds ( mclauchlan et al . after being processed by a signal peptide peptidase , a large part of the core remains within cytoplasmic leaflets of the er membrane due to preservation of the original transmembrane domain . the cytoplasmic leaflets then swell as lipid accumulates between the two membrane leaflets . as a result , the core is translocated along with part of the er membrane to the surface of a nascent ld before the droplet buds off the er . the proteasome - dependent degradation pathway then participates in post - translational modification of the core ( suzuki et al . , 2001 , 2009 ; ubiquitin ligase e6ap and proteasome activator pa28gamma are key regulators in determining the fate of the core , and thereby play a role in virus production ( shirakura et al . the 120 n - terminal residues of the core protein ( domain i ) contain multiple positively charged residues that are implicated in rna binding and homo - oligomerization . it is therefore likely that this domain is a prerequisite for assembly of the hcv nucleocapsid ( kunkel et al . , 2001 ; it has been shown that a region extending from aa 72 to 91 is responsible for auto - oligomerization of core ( nakai et al . , 2006 ) . although conclusive data for direct packaging of the hcv genome into the viral capsid is lacking , the viral rna sequence of the core protein through to the ns2 region appears not to contain a cis - acting packaging signal . a subgenomic replicon rna carrying the ns3ns5b region can be encapsidated in the viral trans - packaging system ( ishii et al . , 2008 ; steinmann et al . , 2008 ; adair et al . , 2009 ; masaki et al . , in addition , the rna sequence encoding the first 62 aa of core contains highly conserved structures including two stem - loops that are important for rna translation / replication ( mcmullan et al . , 2007 ) . domain ii ( aa 120170 ) is predicted to form two alpha - helices that enable core to associate with membrane proteins and lipids . it has been proposed that domain ii folding occurs in a membrane environment and is critical for the folding of domain i ( boulant et al . , 2005 ) . in addition , a cysteine residue at aa 128 of domain ii creates a disulfide bond to produce a core protein dimer that is required for particle formation ( kushima et al . , 2010 ) . domain iii , pertaining to 20 residues at the c - terminal , is highly hydrophobic and serves as a signal sequence for e1 . although little is known about the molecular mechanisms governing assembly of core into nucleocapsids , comprehensive mutagenesis studies have enabled identification of various aa residues which are essential for hcv morphogenesis ( murray et al . two n - glycosylated envelope proteins e1 and e2 are exposed on the surface of the virus as a heterodimer that mediates viral attachment to host - cell receptors and facilitates virus entry ( op de beeck et al . , 2004 ; vieyres et al . , 2010 ) . ectodomains of e1 and e2 are translocated inside the er lumen and their transmembrane domains are inserted in the membrane of this compartment . e1 contains four to five n - linked glycans and e2 has 11 n - glycosylation sites . hcv glycans , which are thought to contain a mixture of complex and high mannose side - chain , play a role in envelope protein folding and formation of the e1/e2 complexes . the importance of incorporating n - linked glycans of the envelope proteins into infectious virions has been demonstrated ( helle et al . , 2010 ) . a recent study has shown that hcv infection activates the er - associated degradation pathway , which in turn controls the fate of e1 and e2 and modulates virus production ( saeed et al . , 2011 ) . a role of viral ns proteins in hcv production was first suggested following the observation that jfh-1-derived ns proteins are required to generate infectious virus from intra- and inter - genotype chimeric constructs ( lindenbach et al . , 2005 ; pietschmann et al . , evidence supporting a role of ns2 , ns3 , and ns5a in the assembly or release of infectious hcv has come from mutational analyses ( jones et al . , 2007 ; miyanari et al . , 2007 ; appel et al . , 2008 ; jirasko et al . , 2008 ; ns5a is a hydrophilic phosphoprotein which plays a key role in viral rna replication and is involved in modulation of cell signaling pathways and the interferon response ( huang et al . , 2007 ) . ns5a is associated with membrane mediated by a unique amphipathic alpha helix which is located at its n - terminus ( moradpour et al . , 2005 ) , and part of ns5a localizes to lds when expressed alone or as the viral polyprotein ( shi et al . , 2002 ) . experiments based on hcv genomes containing mutated ns5a have shown that some mutants result in failure of association of ns5a with lds and failure of production of infectious particles ( miyanari et al . , 2007 ) . further studies have revealed that the c - terminal region of ns5a plays a key role in hcv production ( appel et al . , 2008 ; masaki et al . , 2008 ; tellinghuisen et al . , substitutions at a serine cluster of the ns5a c - terminus ( aa 2428 , 2430 , and 2433 ) which have no impact on viral rna replication , have been observed to inhibit the interaction between ns5a and core , thereby suggesting that an association between ns5a and core might be involved in virus production ( masaki et al . , 2008 ) . structural analyses have shown that the n - terminal region of ns5a forms a  claw - like  dimer , which might accommodate rna and interact with viral and cellular proteins and membranes ( huang et al . , 2005 ; tellinghuisen et al . , it appears that recruitment of ns5a to lds , thereby enabling interaction with the core , is crucial for virion assembly . one can imagine that newly synthesized hcv rna bound to ns5a is released from the replication complex - containing membrane compartment and can then be captured by core through interaction with the c - terminal region of ns5a at the surface of lds or ld - associated membranes . consequently , viral rna becomes encapsidated and virion assembly proceeds ( figure 2 ) . in this regard , a study has revealed an interaction between ns5a and apoe , suggesting that recruitment of apoe by ns5a is important for the assembly and release of hcv particles ( benga et al . , 2010 ) . after accumulation of synthesized genome rna and the viral proteins , the hcv particles are assembled in an er - related compartment in close connection with the vldl pathway . the viral replication complex , which is composed of ns3ns5b and host factors , is a specialized structure protected by cellular membrane . newly synthesized viral rnas are recruited to surfaces of lipid droplet ( ld ) , where er membrane is associated , possibly via interaction between ns5a and core . thus , the rnas enable to associate with core , thereby proceeding encapsidation and nucleocapsid formation . the nucleocapsid is presumably inserted into the lipid core of the luminal ld and buds into the er lumen with incorporation of e1 and e2 . ns3ns4a is another component of the viral replication complex that exhibits serine protease , as well as rna helicase and rna - stimulated ntpase activity , necessary for viral rna replication . it is now apparent that ns3ns4a also contributes to viral assembly ( yi et al . , 2007 ; ma et al . , 2008 ; han et al . , 2009 ; a previous study has provided genetic evidence that two major subdomains of the ns3 helicase , one demonstrating ntpase activity and the other associated with rna binding , are involved in the early stages of virion assembly , independent of their roles as enzymes ( ma et al . a separate investigation has revealed a contribution of the acidic domain of ns4a to both rna replication and virus assembly ( phan et al . , 2011 ) . ns2 is a cysteine protease composed of a highly hydrophobic n - terminal membrane binding domain which forms either three or four transmembrane helices that insert into the er membrane . ns2 also contains a c - terminal globular and cytosolic protease subdomain , that produce zinc - stimulated ns2/3 autoprotease activity together with the n - terminal one - third of ns3 . mutagenesis of ns2 has identified regions or residues that are important for infectious virus production ( jones et al . for example , mutations involving the dimer interface of the protease region or the c - terminus of ns2 impair the production of infectious hcv , while the catalytic activity of ns2 is not required for viral assembly . genetic and biochemical data demonstrate that ns2 is possibly involved in multiple interactions with both hcv structural and ns proteins including e1e2 , p7 , ns3 , and ns5a , suggesting that ns2 has a role in recruiting these viral proteins to sites in close proximity with lds . alternatively , it may act as a scaffold promoting virus assembly ( jirasko et al . , 2010 ; ma et al . , 2011 ; p7 is a 63-aa polypeptide located between e2 and ns2 , and is a membrane - spanning protein localized within the er . although p7 is not required for viral rna replication in cell culture , the protein is essential for hcv infectivity in chimpanzees ( sakai et al . , 2003 ) . subsequent analyses in hcvcc systems have demonstrated that p7 is important for virion production since the introduction of p7 mutations , such as mutations in the basic residues required for its ion channel activity , impair virus production ( jones et al . although it is not yet clear whether the ion channel function of the protein is needed for virus assembly , a recent study has demonstrated that p7 functions as an h channel in native intracellular membranes and links p7-induced ph changes to the production of infectious intracellular virions ( wozniak et al . , 2010 ) . hcvcc contained within low - density fractions from the culture supernatant of virus - producing cells displays greater specific infectivity than virus in high - density fractions ( lindenbach et al . looking also at the behavior of hcv circulating within the sera of infected hosts , it may be that low - density virus associates with lipid and very - low - density lipoprotein ( vldl ) and/or low - density lipoprotein ( ldl ) . furthermore , hcv particles obtained from virus - infected animals , such as chimpanzees and chimeric mice transplanted with human hepatocytes , demonstrate greater infectivity than virus produced in cell cultures . the virus populations derived from these infected animals have been observed to fractionate into lower density fractions than a major population of hcvcc ( lindenbach et al . , 2006 ) . interestingly , the association of cholesterol and sphingolipid with hcv virions has been shown to play a critical role in viral infectivity ( kapadia et al . depletion of cholesterol and sphingomyelin from hcv virions inhibits the infectivity of hcvcc ( aizaki et al . , 2008 ) . the structural requirement of virion - associated cholesterol for infectivity , as well as its influence on buoyant density , and the association of apolipoprotein with hcv , has been further demonstrated by yamamoto et al . ( there is accumulating evidence that assembly and secretion of hcv particles are associated with the vldl assembly pathway . lipoproteins can be differentiated on the basis of their density , which is affected by lipid content and the types of apolipoprotein they contain . vldls are large triglyceride - rich lipoproteins ( 3080  nm in diameter ) containing cholesterol , cholesteryl esters , apob , and other minor apolipoprotein(s ) . vldls carry triglyceride from the liver to peripheral tissue for storage and to provide a source of energy . triglyceride availability and the size of intracellular triglyceride pools are important regulatory factors in the regulation of vldl production . in addition , the microsomal triglyceride transfer protein ( mtp ) , which is responsible for the transport of triglyceride and cholesteryl esters across er membranes , is required for vldl assembly . purified membrane vesicles containing the hcv replication complex are enriched with apob , mtp , and apoe ( huang et al . , 2007 ) . moreover , agents that inhibit vldl assembly also inhibit hcv secretion from cells producing infectious virus ( chang et al . , 2007 ; apob , apoc1 , and apoe have been observed to associate with hcv particles during viral morphogenesis in the hcvcc system ( chang et al . , 2007 ; meunier et al . , 2008 ; jiang and luo , 2009 ; owen et al . , 2009 ; benga et al . , , 2007 ; jiang and luo , 2009 ; owen et al . , 2009 ; benga et al . , 2010 ; hishiki et al . , these findings demonstrate that apoe is important for hcv infectivity , suggesting that hcv virions are assembled as apoe - enriched lipoprotein particles . a study in which virion - associated cholesterol was depleted and replenished with exogenous sterol analogs has provided evidence that virion - associated cholesterol contributes to the interaction between hcv and apoe ( yamamoto et al . , 2011 ) . apoe is a polymorphic protein with three major isoforms : apoe2 , apoe3 , and apoe4 . differential roles of apoe isoforms on infectious hcv production have been revealed : ectopic expression of apoe3 or apoe4 enables recovery of infectious hcv , while apoe2 has little influence on virus production ( hishiki et al . , 2010 ) . not surprisingly , apoe2 demonstrates significantly less ldl receptor binding activity than apoe3 and apoe4 ( davignon et al . , 1988 ) . in fact , secretion of e1 and e2 within the culture supernatant is reduced by treatment with mtp inhibitors ( icard et al . , 2009 ) . vldl maturation occurs by acquisition of lipids from lds either in the er lumen or in the golgi apparatus . it is likely that hcv envelopment takes place in a lipid - enriched microdomain at the er membrane where luminal lds or vldl precursors are generated ( figure 2 ) , in keeping with evidence of increased cholesterol content among hcv particles compared to host - cell membranes ( aizaki et al . , 2008 ) . neutral lipids such as triglycerides and cholesterol esters are stored within cytosolic ld in cells . ld is thought to be a source of neutral lipid for metabolism and membrane synthesis . neutral lipids form the ld core and are surrounded by an outer layer of amphipathic lipids such as phospholipids and cholesterol . prior to the availability of a tissue culture system for virion production , hcv core was shown to associate with er membranes and on the surface of lds in heterologous expression systems in mammalian cells ( moradpour et al . , 1996 ; barba et al . , 1997 ) . early studies of cells infected with hcv jfh-1 indicate that core is detectable at the er and on the surface of lds in association with the er ( rouille et al . , 2006 ) . core associates with lds in a time - dependent manner and disruption of this process coincides with a loss of virion production ( boulant et al . , 2007 ) . it has subsequently been demonstrated that lds are directly involved in the production of infectious hcv and that core recruits viral non - structural proteins as well as the replication complex to the ld - associated membranes , suggesting that the association between core and lds is required at a certain stage of hcv morphogenesis ( miyanari et al . , 2007 ) . fluorescent labeling and functional imaging of core in living cells has recently been used to visualize core during hcv assembly ( counihan et al . , 2011 ) . core has been observed to move to the surface of large , immobile lds soon after protein translation . diacylglycerol acyltransferases ( dgats ) catalyze the final step of triglyceride synthesis and are crucial for ld biogenesis . a study has revealed that dgat1 interacts with core and is required for trafficking of the core to ld . disrupting translocation of the core to ld by inhibiting dgat1 activity or through knockdown of the dgat1 gene impairs virus production ( herker et al . , 2010 ) . thus , there is now increasing evidence that lds play a central role in the production of infectious hcv and participate in virus assembly . however , one study has demonstrated that , while core derived from hcv jfh-1 is strongly associated with cytoplasmic lds , minimal core from a hcv clone with higher assembly efficiency is detectable on lds ( shavinskaya et al . , 2007 ) . thus , it remains debatable whether hcv assembly is initiated on the surface of lds or at sites where er cisternae are in contact with lds . based on the current evidence , a model for nucleocapsid formation following the initial phase of assembly is demonstrated in figure 2 . two potential models to explain hcv nucleocapsid formation , including the model shown in figure 2 , have been proposed by bartenschlager et al . evidence regarding the assembly of infectious hcv particles has accumulated over the past several years since the availability of hcvcc systems . a variety of key factors in hcv morphogenesis have been identified , including the requirement for components of the vldl biosynthetic machinery and viral ns proteins . however , there are still essential questions to be answered . detailed mechanisms pertaining to nucleotide formation , genome packaging , and the way in which hcv interacts with the lipoprotein / vldl pathway , as well as the precise role of various ns proteins and p7 in hcv assembly , remain unclear . structural studies will be important to clarify the exact composition of the hcv virion , as well as similarities and differences between hcvcc generated in huh-7-derived cells and the  lipoviroparticles  produced by circulating virus within infected individuals . the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .