Critical Role of Cyclophilin A and Its Prolyl-Peptidyl Isomerase Activity in the Structure and Function of the Hepatitis C Virus Replication Complex

Replication of hepatitis C virus (HCV) RNA occurs on intracellular membranes, and the replication complex (RC) contains viral RNA, nonstructural proteins, and cellular cofactors. We previously demonstrated that cyclophilin A (CyPA) is an essential cofactor for HCV infection and the intracellular target of cyclosporine''s anti-HCV effect. Here we investigate the mechanism by which CyPA facilitates HCV replication. Cyclosporine treatment specifically blocked the incorporation of NS5B into the RC without affecting either the total protein level or the membrane association of the protein. Other nonstructural proteins or viral RNAs in the RC were not affected. NS5B from the cyclosporine-resistant replicon was resistant to this disruption of RC incorporation. We also isolated membrane fractions from both naïve and HCV-positive cells and found that CyPA is recruited into membrane fractions in HCV-replicating cells via an interaction with RC-associated NS5B, which is sensitive to cyclosporine treatment. Finally, we introduced point mutations in the prolyl-peptidyl isomerase (PPIase) motif of CyPA and demonstrated a critical role of this motif in HCV replication in cDNA rescue experiments. We propose a model in which the incorporation of the HCV polymerase into the RC depends on its interaction with a cellular chaperone protein and in which cyclosporine inhibits HCV replication by blocking this critical interaction and the PPIase activity of CyPA. Our results provide a mechanism of action for the cyclosporine-mediated inhibition of HCV and identify a critical role of CyPA''s PPIase activity in the proper assembly and function of the HCV RC.Hepatitis C virus (HCV), of the family Flaviviridae, is an enveloped, positive-stranded RNA virus. Spread mostly by blood-borne transmission, HCV infects more than 170 million people worldwide. The viral genome is composed of a single open reading frame (ORF) plus 5′- and 3′-nontranslated regions. The ORF encodes a large polyprotein that is cleaved by cellular and viral proteases into 10 viral proteins. The structural proteins, including the capsid protein (core), two glycoproteins (E1 and E2), and a small ion channel protein (p7), reside in the N-terminal half of the polyprotein. The rest of the ORF encodes six nonstructural (NS) proteins: NS2, NS3, NS4A, NS4B, NS5A, and NS5B. NS3 through NS5B assemble into a replication complex (RC) and are necessary and sufficient for HCV RNA replication in cell culture (8, 42). NS3 is a multifunctional protein with both a serine protease and an RNA helicase activity. The protease activity is responsible for cleavage at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions (5), and the helicase activity is probably required to unwind the double-stranded RNA intermediates formed during replication (38). NS4A serves as an essential cofactor for the NS3 protease and anchors the NS3 protein to intracellular membranes (25, 36, 39). NS4B induces the formation of a “membranous web” that is probably the site of HCV replication (16). It also contains a GTP-binding motif that is required for replication (17). The web is derived from the endoplasmic reticulum (ER) compartment, although proteins of early-endosome origin have also been found to locate to the web (62). NS5A is a phosphoprotein and an integral component of the viral RC. The precise function of NS5A in replication is still unknown but appears to be regulated by phosphorylation and its interaction with several cellular proteins (19, 22, 24, 51, 52, 59, 63, 67). In addition, it may be involved in the transition from replication and particle formation (4, 45, 64). NS5B is the RNA-dependent RNA polymerase that is responsible for copying the RNA genome of the virus during replication. Several cellular cofactors interact with NS5B and modulate its activity in the context of the viral RC (22, 24, 35, 69, 71).Positive-stranded RNA viruses alter the intracellular membranes of host cells to form an RC in which RNA replication occurs. Modifications include the proliferation and reorganization of certain cellular membranes (1). HCV forms an RC associated with altered cellular membranes (16, 23), and crude RCs (CRCs) that maintain the replicase activity in vitro can be isolated by membrane sedimentation or flotation techniques (2, 3, 18, 27, 37).Cyclosporine is a widely used immunosuppressive and anti-inflammatory drug for organ transplant patients. It functions by forming an inhibitory complex with cyclophilins (CyPs) that inhibits the phosphatase activity of calcineurin, which is important for T-cell activation. In recent years, cyclosporine and its derivatives have been shown to be highly effective in suppressing HCV replication in vitro (44, 49, 53, 68) and in vivo (30). The mechanism of this inhibition is independent of its immunosuppressive function and distinct from that of interferon (IFN) (44, 53, 56, 68).We recently showed that HCV infection in vitro is inhibited when CyPA, a major intracellular target of cyclosporine, is downregulated by RNA interference, and mutations in NS5B that confer cyclosporine-resistant binding to CyPA contribute to the cyclosporine resistance of the replicons harboring these mutations (56, 71). Here we report that CyPA is recruited into the HCV RC together with NS5B in HCV replicon or in HCV-infected cells. Cyclosporine disrupts the association between RC-incorporated NS5B and CyPA and results in an exclusion of the polymerase from the viral RC. We also show that the prolyl-peptidyl isomerase (PPIase) motif of CyPA is essential for HCV replication.     

The Hepatitis C Virus NS4B Protein Can trans-Complement Viral RNA Replication and Modulates Production of Infectious Virus

Studies of the hepatitis C virus (HCV) life cycle have been aided by development of in vitro systems that enable replication of viral RNA and production of infectious virus. However, the functions of the individual proteins, especially those engaged in RNA replication, remain poorly understood. It is considered that NS4B, one of the replicase components, creates sites for genome synthesis, which appear as punctate foci at the endoplasmic reticulum (ER) membrane. In this study, a panel of mutations in NS4B was generated to gain deeper insight into its functions. Our analysis identified five mutants that were incapable of supporting RNA replication, three of which had defects in production of foci at the ER membrane. These mutants also influenced posttranslational modification and intracellular mobility of another replicase protein, NS5A, suggesting that such characteristics are linked to focus formation by NS4B. From previous studies, NS4B could not be trans-complemented in replication assays. Using the mutants that blocked RNA synthesis, defective NS4B expressed from two mutants could be rescued in trans-complementation replication assays by wild-type protein produced by a functional HCV replicon. Moreover, active replication could be reconstituted by combining replicons that were defective in NS4B and NS5A. The ability to restore replication from inactive replicons has implications for our understanding of the mechanisms that direct viral RNA synthesis. Finally, one of the NS4B mutations increased the yield of infectious virus by five- to sixfold. Hence, NS4B not only functions in RNA replication but also contributes to the processes engaged in virus assembly and release.Recent estimates predict that the prevalence of hepatitis C virus (HCV) infection is approximately 2.2% worldwide, equivalent to about 130 million persons (22). The virus typically establishes a chronic infection that frequently leads to serious liver disease (1), and current models indicate that both morbidity and mortality as a consequence of HCV infection will continue to rise for about the next 20 years (10, 11, 29).HCV is the only assigned species of the Hepacivirus genus within the family Flaviviridae. The virus can be classified into six genetic groups or clades (numbered 1 to 6) and then further separated into subtypes (e.g., 1a, 1b, 2a, 2b, etc.) (53, 55). HCV has a single-stranded, positive-sense RNA genome that is approximately 9.6 kb in length (reviewed in reference 46). Genomic RNA carries a single open reading frame flanked by 5′ and 3′ nontranslated regions, which are important for both replication and translation (19, 20, 34, 47, 56). Viral RNA is translated by the host ribosomal machinery, and the resultant polyprotein is co- and posttranslationally cleaved to generate the mature viral proteins. The structural proteins (core, E1, and E2) and a small hydrophobic polypeptide called p7 are produced by the cellular proteases signal peptidase and signal peptide peptidase (28, 45, 54). Two virus-encoded proteases, the NS2-3 autoprotease and the NS3 serine protease (5, 13, 26), are responsible for maturation of the nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). With the exception of NS2, the NS proteins are necessary for genome replication (8, 40) and form replication complexes (RCs), which are located at the endoplasmic reticulum (ER) membrane (14, 24, 52, 57, 59). The functions of all viral constituents of RCs have not been characterized in detail. It is known that NS5B is the RNA-dependent RNA polymerase (6), while NS3 possesses helicase and nucleoside triphosphatase activities in addition to acting as a protease (32, 58). However, the precise roles of the other proteins remain to be firmly established.Expression of NS4B, one of the replicase proteins, generates rearrangements at the ER membrane that have been termed the “membranous web” (14, 24) and “membrane-associated foci” (MAFs) (25). Detection of viral RNA at such foci suggests that NS4B is involved in creating the sites where genome synthesis occurs (18, 24, 59). It is predicted that NS4B has an amphipathic α-helix within its N-terminal region, which is followed by four transmembrane domains (TMDs) in the central portion of the protein (17, 42). As a result, the majority of NS4B is likely to be tightly anchored to membranes, and experimental evidence indicates that it has characteristics consistent with an integral membrane protein (27). It is thought that after membrane association, NS4B rearranges membranes into a network, thereby generating foci which act as a “scaffold” to facilitate RNA replication. The mechanisms engaged in formation of foci are not known but include the notion that the NS4B N terminus can translocate into the ER lumen, resulting in rearrangement of cellular membranes (41, 42). Alternatively, palmitoylation, a lipid modification, might facilitate polymerization of NS4B, in turn promoting formation of RCs on the ER membrane (68).Apart from inducing membranous changes required for replication, NS4B may perform other tasks in HCV RNA synthesis. For example, studies of cell culture adaptive mutations in subgenomic replicons (SGRs) have identified amino acid changes that can stimulate RNA production (39), suggesting that NS4B may exert a regulatory role in determining replication efficiency. In support of a regulatory function, replacement of NS4B sequences in an SGR from strain H77 (a genotype 1a strain) with those from strain Con-1 (a genotype 1b strain) gave higher levels of replication than for a wild-type (wt) strain H77 SGR (7). The corresponding replacement of strain Con-1 NS4B sequences with those from strain H77 reduced the replication efficiency of a Con-1 SGR (7). Moreover, interactions of NS4B with the RC can affect the behavior of other replicase proteins. For example, NS4B is needed for hyperphosphorylation of NS5A (35, 48) and restricts its intracellular movement (30).To try to gain greater insight into the functional organization of the components that constitute RCs, trans-complementation assays using defective and helper SGRs have been established (2, 64). Such studies reveal that the only protein capable of trans-complementation is NS5A, while active replication cannot be restored for replicons harboring deleterious mutations in NS3, NS4B, and NS5B. These data led to the conclusion that functional NS5A may be able to exchange between RCs (2), whereas, by inference, such exchange would not be possible for other HCV replicase proteins. In transient-replication assays, complementation by NS5A also relied on its expression as part of a polyprotein (minimally NS3-NS5A), and production of the protein alone failed to restore replication for an inactive SGR (2). However, in a separate study, stable expression of wt NS5A was capable of complementing a defective replicon (64). Thus, different assay systems can give dissimilar results for complementation by NS5A.In this study, we have created a series of mutations in the NS4B gene of HCV strain JFH1 (31) to explore the function of the protein in the HCV life cycle. We focused our attention on the C-terminal portion of NS4B, downstream from the predicted TMD regions, since it is relatively well conserved and is predicted to lie on the cytosolic side of the ER membrane (15, 42). Our analysis examines the impact of mutations on replication efficiency and the intracellular characteristics of the mutants compared to the behavior of the wt protein. In addition, we have utilized this series of mutants to reassess trans-complementation of NS4B in replication assays. Finally, we also analyze the impact of mutations which do not affect replication on the production of infectious virus to determine whether NS4B plays a role in virus assembly and release.     

A Single Point Mutation in Nonstructural Protein NS2 of Bovine Viral Diarrhea Virus Results in Temperature-Sensitive Attenuation of Viral Cytopathogenicity

For Bovine viral diarrhea virus (BVDV), the type species of the genus Pestivirus in the family Flaviviridae, cytopathogenic (cp) and noncytopathogenic (ncp) viruses are distinguished according to their effect on cultured cells. It has been established that cytopathogenicity of BVDV correlates with efficient production of viral nonstructural protein NS3 and with enhanced viral RNA synthesis. Here, we describe generation and characterization of a temperature-sensitive (ts) mutant of cp BVDV strain CP7, termed TS2.7. Infection of bovine cells with TS2.7 and the parent CP7 at 33°C resulted in efficient viral replication and a cytopathic effect. In contrast, the ability of TS2.7 to cause cytopathogenicity at 39.5°C was drastically reduced despite production of high titers of infectious virus. Further experiments, including nucleotide sequencing of the TS2.7 genome and reverse genetics, showed that a Y1338H substitution at residue 193 of NS2 resulted in the temperature-dependent attenuation of cytopathogenicity despite high levels of infectious virus production. Interestingly, TS2.7 and the reconstructed mutant CP7-Y1338H produced NS3 in addition to NS2-3 throughout infection. Compared to the parent CP7, NS2-3 processing was slightly decreased at both temperatures. Quantification of viral RNAs that were accumulated at 10 h postinfection demonstrated that attenuation of the cytopathogenicity of the ts mutants at 39.5°C correlated with reduced amounts of viral RNA, while the efficiency of viral RNA synthesis at 33°C was not affected. Taken together, the results of this study show that a mutation in BVDV NS2 attenuates viral RNA replication and suppresses viral cytopathogenicity at high temperature without altering NS3 expression and infectious virus production in a temperature-dependent manner.The pestiviruses Bovine viral diarrhea virus-1 (BVDV-1), BVDV-2, Classical swine fever virus (CSFV), and Border disease virus (BDV) are causative agents of economically important livestock diseases. Together with the genera Flavivirus, including several important human pathogens like Dengue fever virus, West Nile virus, Yellow fever virus, and Tick-borne encephalitis virus, and Hepacivirus (human Hepatitis C virus [HCV]), the genus Pestivirus constitutes the family Flaviviridae (8, 20). All members of this family are enveloped viruses with a single-stranded positive-sense RNA genome encompassing one large open reading frame (ORF) flanked by 5′ and 3′ nontranslated regions (NTR) (see references 8 and 28 for reviews). The ORF encodes a polyprotein which is co- and posttranslationally processed into the mature viral proteins by viral and cellular proteases. For BVDV, the RNA genome is about 12.3 kb in length and encodes a polyprotein of about 3,900 amino acids. The first third of the ORF encodes a nonstructural (NS) autoprotease and four structural proteins, while the remaining part of the genome encodes NS proteins which share many common characteristics and functions with the corresponding NS proteins encoded by the HCV genome (8, 28). NS2 of BVDV represents a cysteine autoprotease which is distantly related to the HCV NS2-3 protease (26). NS3, NS4A, NS4B, NS5A, and NS5B are essential components of the pestivirus replicase (7, 10, 49). NS3 possesses multiple enzymatic activities, namely serine protease (48, 52, 53), NTPase (46), and helicase activity (51). NS4A acts as an essential cofactor for the NS3 proteinase. NS5B represents the RNA-dependent RNA polymerase (RdRp) (22, 56). The functions of NS4B and NS5A remain to be determined. NS5A has been shown to be a phosphorylated protein that is associated with cellular serine/threonine kinases (44).According to their effects in tissue culture, two biotypes of pestiviruses are distinguished: cytopathogenic (cp) and noncytopathogenic (ncp) viruses (17, 27). The occurrence of cp BVDV in cattle persistently infected with ncp BVDV is directly linked to the induction of lethal mucosal disease in cattle (12, 13). Previous studies have shown that cp BVDV strains evolved from ncp BVDV strains by different kinds of mutations. These include RNA recombination with various cellular mRNAs, resulting in insertions of cellular protein-coding sequences into the viral genome, as well as insertions, duplications, and deletions of viral sequences, and point mutations (1, 2, 9, 24, 33, 36, 37, 42). A common consequence of all these genetic changes in cp BVDV genomes is the efficient production of NS3 at early and late phases of infection. In contrast, NS3 cannot be detected in cells at late time points after infection with ncp BVDV. An additional major difference is that the cp viruses produce amounts of viral RNA significantly larger than those of their ncp counterparts (7, 32, 50). While there is clear evidence that cell death induced by cp BVDV is mediated by apoptosis, the molecular mechanisms involved in pestiviral cytopathogenicity are poorly understood. In particular, the role of NS3 in triggering apoptosis remains unclear. It has been hypothesized that the NS3 serine proteinase might be involved in activation of the apoptotic proteolytic cascade (21, 55). Furthermore, it has been suggested that the NS3-mediated, enhanced viral RNA synthesis of cp BVDV and subsequently larger amounts of viral double-stranded RNAs may play a crucial role in triggering apoptosis (31, 54).In this study, we describe generation and characterization of a temperature-sensitive (ts) cp BVDV mutant whose ability to cause viral cytopathogenicity at high temperature is strongly attenuated. Our results demonstrate that a single amino acid substitution in NS2 attenuates BVDV cytopathogenicity at high temperature without affecting production of infectious viruses and expression of NS3 in a temperature-dependent manner.     

A Short N-Terminal Peptide Motif on Flavivirus Nonstructural Protein NS1 Modulates Cellular Targeting and Immune Recognition

Flavivirus NS1 is a versatile nonstructural glycoprotein, with intracellular NS1 functioning as an essential cofactor for viral replication and cell surface and secreted NS1 antagonizing complement activation. Even though NS1 has multiple functions that contribute to virulence, the genetic determinants that regulate the spatial distribution of NS1 in cells among different flaviviruses remain uncharacterized. Here, by creating a panel of West Nile virus-dengue virus (WNV-DENV) NS1 chimeras and site-specific mutants, we identified a novel, short peptide motif immediately C-terminal to the signal sequence cleavage position that regulates its transit time through the endoplasmic reticulum and differentially directs NS1 for secretion or plasma membrane expression. Exchange of two amino acids within this motif reciprocally changed the cellular targeting pattern of DENV or WNV NS1. For WNV, this substitution also modulated infectivity and antibody-induced phagocytosis of infected cells. Analysis of a mutant lacking all three conserved N-linked glycosylation sites revealed an independent requirement of N-linked glycans for secretion but not for plasma membrane expression of WNV NS1. Collectively, our experiments define the requirements for cellular targeting of NS1, with implications for the protective host responses, immune antagonism, and association with the host cell sorting machinery. These studies also suggest a link between the effects of NS1 on viral replication and the levels of secreted or cell surface NS1.West Nile virus (WNV) is a single-stranded, positive-sense enveloped RNA Flavivirus that cycles in nature between birds and Culex mosquitoes. It is endemic in parts of Africa, Europe, the Middle East, and Asia, and outbreaks occur annually in North America. More than 29,000 human cases of severe WNV infection have been diagnosed in the United States since its entry in 1999, and millions have been infected and remain undiagnosed (9). Humans can develop a febrile illness that progresses to a flaccid paralysis, meningitis, or encephalitis syndrome (59). Dengue virus (DENV) is a genetically related flavivirus that is transmitted by Aedes aegypti and Aedes albopictus mosquitoes and causes clinical syndromes in humans, ranging from an acute self-limited febrile illness (dengue fever [DF]) to a severe and life-threatening vascular leakage and bleeding diathesis (dengue hemorrhagic fever/dengue shock syndrome [DHF/DSS]). Globally, DENV causes an estimated 50 million infections annually, resulting in 500,000 hospitalizations and ∼22,000 deaths (45).The ∼10.7-kb Flavivirus RNA genome is translated as a single polyprotein, which is then cleaved into three structural proteins (C, prM/M, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by virus- and host-encoded proteases (39). The multifunctional NS proteins include an RNA-dependent RNA polymerase and methyltransferase (NS5), a helicase and protease (NS3), accessory proteins that form part of the viral replication complex, and immune evasion molecules (33, 34). Flavivirus NS1 is a 48-kDa nonstructural glycoprotein with two or three N-linked glycans, depending on the flavivirus, and is absent from the virion. The Japanese encephalitis virus (JEV) serogroup (West Nile, Japanese, Murray Valley, and St. Louis encephalitis viruses) generate NS1 and NS1′ proteins, the latter of which is a product of a ribosomal frameshift event that occurs at a heptanucleotide motif located at the beginning of the NS2A gene (25, 47).NS1 is an essential gene as it is required for efficient viral RNA replication (34, 41, 44). In infected mammalian cells, NS1 is synthesized as a soluble monomer, dimerizes after posttranslational modification in the lumen of the endoplasmic reticulum (ER), and accumulates extracellularly as higher-order oligomers, including hexamers (16, 26, 64, 65). Soluble NS1 binds back to the plasma membrane of uninfected cells through interactions with sulfated glycosaminoglycans (5). In infected cells, NS1 is also directly transported to and expressed on the plasma membrane although it lacks a transmembrane domain or canonical targeting motif. The mechanism of cell surface expression of flavivirus NS1 in infected cells remains uncertain although some fraction may be linked through an atypical glycosyl-phosphatidylinositol anchor (30, 50) or lipid rafts (49).NS1 has been implicated in having pathogenic consequences in flavivirus infection. The high levels of NS1 in the serum of DENV-infected patients correlate with severe disease (4, 37). NS1 has been proposed to facilitate immune complex formation (4), elicit auto-antibodies that react with host matrix proteins (21), damage endothelial cells via antibody-dependent complement-mediated cytolysis (38), or directly enhance infection (1). Flavivirus NS1 also has direct immune evasion functions and antagonizes complement activation on cell surfaces and in solution. WNV NS1 attenuates the alternative pathway of complement activation by binding the complement-regulatory protein factor H (11, 36), and DENV, WNV, and YFV NS1 proteins bind C1s and C4 in a complex to promote efficient degradation of C4 to C4b (3).Although NS1 is absent from the virion, antibodies against it can protect against infection in vivo. Immunization with purified NS1 or passive administration of some anti-WNV, anti-yellow fever virus (YFV), and anti-DENV NS1 monoclonal antibodies (MAbs) protect mice against lethal virus challenge (12, 13, 17, 22, 27, 29, 31, 32, 56-58). Initial studies with isotype switch variants and F(ab′)₂ fragments of anti-YFV NS1 MAbs suggested that the Fc region of anti-NS1 MAbs was required for protection (58). Subsequent mechanistic studies with WNV NS1 indicated that only MAbs recognizing cell surface-associated NS1 trigger Fc-γ receptor I- and/or IV-mediated phagocytosis and clearance of infected cells (13).In this study, we identify a reciprocal relationship between the secretion and cell surface expression patterns of WNV and DENV NS1s. Using WNV-DENV NS1 chimeras and point mutants, we identified a novel short peptide motif immediately C-terminal to the signal sequence cleavage position that directs NS1 for secretion or to the plasma membrane. These studies begin to explain how NS1 regulates its localization to several cellular compartments (ER, cell surface, and extracellular space) and have implications for viral infectivity, association with the host cell sorting machinery, and protective immune responses.     

Hepatitis C Virus NS2 Protein Contributes to Virus Particle Assembly via Opposing Epistatic Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes

The hepatitis C virus NS2 protein has been recently implicated in virus particle assembly. To further understand the role of NS2 in this process, we conducted a reverse genetic analysis of NS2 in the context of a chimeric genotype 2a infectious cell culture system. Of 32 mutants tested, all were capable of RNA replication and 25 had moderate-to-severe defects in virus assembly. Through forward genetic selection for variants capable of virus spread, we identified second-site mutations in E1, E2, NS2, NS3, and NS4A that suppressed NS2 defects in assembly. Two suppressor mutations, E1 A78T and NS3 Q221L, were further characterized by additional genetic and biochemical experiments. Both mutations were shown to suppress other NS2 defects, often with mutual exclusivity. Thus, several NS2 mutants were enhanced by NS3 Q221L and inhibited by E1 A78T, while others were enhanced by E1 A78T and inhibited by NS3 Q221L. Furthermore, we show that the NS3 Q221L mutation lowers the affinity of native, full-length NS3-NS4A for functional RNA binding. These data reveal a complex network of interactions involving NS2 and other viral structural and nonstructural proteins during virus assembly.Hepatitis C virus (HCV) is a major cause of acute and chronic liver disease and contributes to the development of hepatocellular carcinoma. HCV is an enveloped, positive-strand RNA virus, the type member of the Hepacivirus genus in the family Flaviviridae (43). HCV exhibits high levels of sequence diversity that cluster into seven major genotypes and numerous subtypes (21).HCV genomes are 9.6 kb and encode a single long open reading frame of ∼3,011 codons (43). Translation of this genome produces a large polyprotein that is co- and posttranslationally processed by viral and host proteases into 10 distinct products. The N-terminal one-third of the polyprotein encodes the structural proteins, which are thought to compose the virus particle. These include an RNA-binding nucleocapsid protein, core (C), and two viral envelope glycoproteins, E1 and E2. E1 and E2 are type I membrane proteins that coordinately fold into a heterodimer complex (36). The remainder of the genome encodes the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B, which mediate the intracellular aspects of the viral life cycle. In addition, a small viroporin-like protein, p7, resides between the structural and NS genes.HCV encodes two proteases, the NS2-NS3 cysteine autoprotease and the NS3-NS4A serine protease. The only known substrate of the NS2-NS3 autoprotease is the NS2/3 junction. This enzyme is encoded by the C-terminal 121 amino acids (aa) of NS2, which forms a homodimer with twin composite active sites composed of two residues from one chain and one residue from the other (45). In addition, the serine protease domain of NS3 plays a noncatalytic role in stimulating NS2/3 cleavage (69). Upstream of the cysteine protease domain, the N-terminal hydrophobic region of NS2 mediates interaction with cellular membranes. While the membrane topology of NS2 is not yet fully known (67, 80), N-terminal cleavage by endoplasmic reticulum-resident signal peptidase and C-terminal cleavage by the cytosolic NS2-NS3 cysteine protease indicate that NS2 likely contains one or three transmembrane (TM) domains.The NS3-NS4A serine protease is encoded by the N-terminal domain of NS3 and is responsible for downstream cleavages at the NS3/4A, NS4A/B, NS4B/5A, and NS5A/B junctions. NS4A, a small (54-aa), membrane-anchored protein, acts as a cofactor for the serine protease activity by helping to complete the chymotrypsin-like fold of NS3 (14, 46). In addition to polyprotein processing, NS3-NS4A serine protease helps to dampen the innate antiviral response by cleaving cellular proteins involved in signal transduction (65).The C-terminal region of NS3 encodes an RNA helicase/NTPase activity that is essential for viral replication, although it is not yet clear which specific step(s) of the replication cycle requires this activity (29, 33). Interestingly, the NS3 serine protease and RNA helicase domains enhance each other''s activities, suggesting that proteolysis and RNA replication may be functionally coordinated (5, 6). In addition, NS4A helps to promote RNA-stimulated ATP hydrolysis by the NS3 helicase domain (4).In addition to their role in polyprotein processing, emerging evidence indicates that NS2 and NS3-NS4A participate in virus particle assembly (52). Prior work showed that NS2 is not essential for RNA replication of subgenomic replicons engineered to express NS3 through NS5B (44). The first evidence for an additional function of NS2 came from the construction of improved chimeric genotype 2a cDNA clones that replicated to high titers in cell culture (HCVcc). Pietschmann and colleagues showed that the Jc1 chimera containing a J6-JFH1 junction between the first and second putative TM domains of NS2 yielded higher-titer viruses than the original infectious J6/JFH chimera (41, 58). Furthermore, a number of adaptive mutations that improve virus production have been mapped to NS2 and NS3 (22, 23, 27, 53, 64, 68, 82). By using bicistronic constructs to express NS2 and NS3 independently of NS2/3 cleavage, two groups showed that full-length NS2, but not uncleaved NS2-NS3 or the NS2 cysteine protease active sites, was required for virus production (24, 25). Moreover, a limited number of mutations in NS2 were shown to inhibit virus assembly (24, 79, 83).Despite these observations, the role of NS2 in virus assembly remains unclear. We have therefore undertaken a genetic analysis to target conserved residues in NS2 for site-directed mutagenesis and identified a number of key residues that are important for virus assembly. Further analysis revealed that a network of genetic interactions among NS2, E1-E2, and NS3-NS4A helps to direct virus assembly. Finally, a suppressor mutation in NS3 was shown to influence functional RNA binding by the RNA helicase/ATPase.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Mutations within a Conserved Region of the Hepatitis C Virus E2 Glycoprotein That Influence Virus-Receptor Interactions and Sensitivity to Neutralizing Antibodies

Cell culture-adaptive mutations within the hepatitis C virus (HCV) E2 glycoprotein have been widely reported. We identify here a single mutation (N415D) in E2 that arose during long-term passaging of HCV strain JFH1-infected cells. This mutation was located within E2 residues 412 to 423, a highly conserved region that is recognized by several broadly neutralizing antibodies, including the mouse monoclonal antibody (MAb) AP33. Introduction of N415D into the wild-type (WT) JFH1 genome increased the affinity of E2 to the CD81 receptor and made the virus less sensitive to neutralization by an antiserum to another essential entry factor, SR-BI. Unlike JFH1_WT, the JFH1_N415D was not neutralized by AP33. In contrast, it was highly sensitive to neutralization by patient-derived antibodies, suggesting an increased availability of other neutralizing epitopes on the virus particle. We included in this analysis viruses carrying four other single mutations located within this conserved E2 region: T416A, N417S, and I422L were cell culture-adaptive mutations reported previously, while G418D was generated here by growing JFH1_WT under MAb AP33 selective pressure. MAb AP33 neutralized JFH1_T416A and JFH1_I422L more efficiently than the WT virus, while neutralization of JFH1_N417S and JFH1_G418D was abrogated. The properties of all of these viruses in terms of receptor reactivity and neutralization by human antibodies were similar to JFH1_N415D, highlighting the importance of the E2 412-423 region in virus entry.Hepatitis C virus (HCV), which belongs to the Flaviviridae family, has a positive-sense single-stranded RNA genome encoding a polyprotein that is cleaved by cellular and viral proteases to yield mature structural and nonstructural proteins. The structural proteins consist of core, E1 and E2, while the nonstructural proteins are p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (42). The hepatitis C virion comprises the RNA genome surrounded by the structural proteins core (nucleocapsid) and E1 and E2 (envelope glycoproteins). The HCV glycoproteins lie within a lipid envelope surrounding the nucleocapsid and play a major role in HCV entry into host cells (21). The development of retrovirus-based HCV pseudoparticles (HCVpp) (3) and the cell culture infectious clone JFH1 (HCVcc) (61) has provided powerful tools to study HCV entry.HCV entry is initiated by the binding of virus particles to attachment factors which are believed to be glycosaminoglycans (2), low-density lipoprotein receptor (41), and C-type lectins such as DC-SIGN and L-SIGN (12, 37, 38). Upon attachment at least four entry factors are important for particle internalization. These include CD81 (50), SR-BI (53) and the tight junction proteins claudin-1 (15) and occludin (6, 36, 51).CD81, a member of the tetraspanin family, is a cell surface protein with various functions including tissue differentiation, cell-cell adhesion and immune cell maturation (34). It consists of a small and a large extracellular loop (LEL) with four transmembrane domains. Viral entry is dependent on HCV E2 binding to the LEL of CD81 (3, 50). The importance of HCV glycoprotein interaction with CD81 is underlined by the fact that many neutralizing antibodies compete with CD81 and act in a CD81-blocking manner (1, 5, 20, 45).SR-BI is a multiligand receptor expressed on liver cells and on steroidogenic tissue. It binds to high-density lipoproteins (HDL), low-density lipoproteins (LDL), and very low-density lipoproteins (VLDL) (31). The SR-BI binding site is mapped to the hypervariable region 1 (HVR-1) of HCV E2 (53). SR-BI ligands, such as HDL and oxidized LDL have been found to affect HCV infectivity (4, 14, 58-60). Indeed, HDL has been shown to enhance HCV infection in an SR-BI-dependent manner (4, 14, 58, 59). Antibodies against SR-BI and knockdown of SR-BI in cells result in a significant inhibition of viral infection in both the HCVpp and the HCVcc systems (5, 25, 32).Although clearly involved in entry and immune recognition, the more downstream function(s) of HCV glycoproteins are poorly understood, as their structure has not yet been solved. Nonetheless, mutational analysis and mapping of neutralizing antibody epitopes have delineated several discontinuous regions of E2 that are essential for HCV particle binding and entry (24, 33, 45, 47). One of these is a highly conserved sequence spanning E2 residues 412 to 423 (QLINTNGSWHIN). Several broadly neutralizing monoclonal antibodies (MAbs) bind to this epitope. These include mouse monoclonal antibody (MAb) AP33, rat MAb 3/11, and the human MAbs e137, HCV1, and 95-2 (8, 16, 44, 45, 49). Of these, MAbs AP33, 3/11, and e137 are known to block the binding of E2 to CD81.Cell culture-adaptive mutations within the HCV glycoproteins are valuable for investigating the virus interaction(s) with cellular receptors (18). In the present study, we characterize an asparagine-to-aspartic acid mutation at residue 415 (N415D) in HCV strain JFH1 E2 that arose during the long-term passaging of infected human hepatoma Huh-7 cells. Alongside N415D, we also characterize three adjacent cell culture adaptive mutations reported previously and a novel substitution generated in the present study by propagating virus under MAb AP33 selective pressure to gain further insight into the function of this region of E2 in viral infection.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Lipid Phosphate Phosphatase 3 Stabilization of β-Catenin Induces Endothelial Cell Migration and Formation of Branching Point Structures

Endothelial cell (EC) migration, cell-cell adhesion, and the formation of branching point structures are considered hallmarks of angiogenesis; however, the underlying mechanisms of these processes are not well understood. Lipid phosphate phosphatase 3 (LPP3) is a recently described p120-catenin-associated integrin ligand localized in adherens junctions (AJs) of ECs. Here, we tested the hypothesis that LPP3 stimulates β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) to induce EC migration and formation of branching point structures. In subconfluent ECs, LPP3 induced expression of fibronectin via β-catenin/LEF-1 signaling in a phosphatase and tensin homologue (PTEN)-dependent manner. In confluent ECs, depletion of p120-catenin restored LPP3-mediated β-catenin/LEF-1 signaling. Depletion of LPP3 resulted in destabilization of β-catenin, which in turn reduced fibronectin synthesis and deposition, which resulted in inhibition of EC migration. Accordingly, reexpression of β-catenin but not p120-catenin in LPP3-depleted ECs restored de novo synthesis of fibronectin, which mediated EC migration and formation of branching point structures. In confluent ECs, however, a fraction of p120-catenin associated and colocalized with LPP3 at the plasma membrane, via the C-terminal cytoplasmic domain, thereby limiting the ability of LPP3 to stimulate β-catenin/LEF-1 signaling. Thus, our study identified a key role for LPP3 in orchestrating PTEN-mediated β-catenin/LEF-1 signaling in EC migration, cell-cell adhesion, and formation of branching point structures.Angiogenesis, the formation of new blood vessels, involves several well-coordinated cellular processes, including endothelial cell (EC) migration, synthesis and deposition of extracellular matrix proteins, such as fibronectin, cell-cell adhesion, and formation of branching point structures (1-3, 19, 33); however, less is known about the underlying mechanisms of these processes (6, 8, 12, 14, 16, 17). For example, adherens junctions (AJs), which mediate cell-cell adhesion between ECs, may be involved in limiting the extent of cell migration (2, 14, 38, 40). VE-cadherin, a protein found in AJs, is a single-pass transmembrane polypeptide responsible for calcium-dependent homophilic interactions through its extracellular domains (2, 38, 40). The VE-cadherin cytoplasmic domain interacts with the Armadillo domain-containing proteins, β-catenin, γ-catenin (plakoglobin), and p120-catenin (p120ctn) (2, 15, 38, 40, 43). Genetic and biochemical evidence documents a crucial role of β-catenin in regulating cell adhesion as well as proliferation secondary to the central position of β-catenin in the Wnt signaling pathway (13, 16, 25, 31, 44). In addition, the juxtamembrane protein p120ctn regulates AJ stability via binding to VE-cadherin (2, 7, 9, 15, 21, 28, 32, 43). The absence of regulation or inappropriate regulation of β-catenin and VE-cadherin functions is linked to cardiovascular disease and tumor progression (2, 6).We previously identified lipid phosphate phosphatase 3 (LPP3), also known as phosphatidic acid phosphatase 2b (PAP2b), in a functional assay of angiogenesis (18, 19, 41, 42). LPP3 not only exhibits lipid phosphatase activity but also functions as a cell-associated integrin ligand (18, 19, 35, 41, 42). The known LPPs (LPP1, LPP2, and LPP3) (20-23) are six transmembrane domain-containing plasma membrane-bound enzymes that dephosphorylate sphingosine-1-phosphate (S1P) and its structural homologues, and thus, these phosphatases generate lipid mediators (4, 5, 23, 35, 39). All LPPs, which contain a single N-glycosylation site and a putative lipid phosphatase motif, are situated such that their N and C termini are within the cell (4, 5, 22, 23, 35, 39). Only the LPP3 isoform contains an Arg-Gly-Asp (RGD) sequence in the second extracellular loop, and this RGD sequence enables LPP3 to bind integrins (18, 19, 22). Transfection experiments with green fluorescent protein (GFP)-tagged LPP1 and LPP3 showed that LPP1 is apically sorted, whereas LPP3 colocalized with E-cadherin at cell-cell contact sites with other Madin-Darby canine kidney (MDCK) cells (22). Mutagenesis and domain swapping experiments established that LPP1 contains an apical targeting signal sequence (FDKTRL) in its N-terminal segment. In contrast, LPP3 contains a dityrosine (109Y/110Y) basolateral sorting motif (22). Interestingly, conventional deletion of Lpp3 is embryonic lethal, since the Lpp3 gene plays a critical role in extraembryonic vasculogenesis independent of its lipid phosphatase activity (11). In addition, an LPP3-neutralizing antibody was shown to prevent cell-cell interactions (19, 42) and angiogenesis (42). Here, we addressed the hypothesis that LPP3 plays a key role in EC migration, cell-cell adhesion, and formation of branching point structures by stimulating β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) signaling.     

Bocavirus Infection Induces Mitochondrion-Mediated Apoptosis and Cell Cycle Arrest at G2/M Phase

Bocavirus is a newly classified genus of the family Parvovirinae. Infection with Bocavirus minute virus of canines (MVC) produces a strong cytopathic effect in permissive Walter Reed/3873D (WRD) canine cells. We have systematically characterized the MVC infection-produced cytopathic effect in WRD cells, namely, the cell death and cell cycle arrest, and carefully examined how MVC infection induces the cytopathic effect. We found that MVC infection induces an apoptotic cell death characterized by Bax translocalization to the mitochondrial outer membrane, disruption of the mitochondrial outer membrane potential, and caspase activation. Moreover, we observed that the activation of caspases occurred only when the MVC genome was replicating, suggesting that replication of the MVC genome induces apoptosis. MVC infection also induced a gradual cell cycle arrest from the S phase in early infection to the G₂/M phase at a later stage, which was confirmed by the upregulation of cyclin B1 and phosphorylation of cdc2. Cell cycle arrest at the G₂/M phase was reproduced by transfection of a nonreplicative NS1 knockout mutant of the MVC infectious clone, as well as by inoculation of UV-irradiated MVC. In contrast with other parvoviruses, only expression of the MVC proteins by transfection did not induce apoptosis or cell cycle arrest. Taken together, our results demonstrate that MVC infection induces a mitochondrion-mediated apoptosis that is dependent on the replication of the viral genome, and the MVC genome per se is able to arrest the cell cycle at the G₂/M phase. Our results may shed light on the molecular pathogenesis of Bocavirus infection in general.The Bocavirus genus is newly classified within the subfamily Parvovirinae of the family Parvoviridae (21). The currently known members of the Bocavirus genus include bovine parvovirus type 1 (BPV1) (17), minute virus of canines (MVC) (57), and the recently identified human bocaviruses (HBoV, HBoV2, and HBoV3) (4, 7, 36).MVC was first recovered from canine fecal samples in 1970 (10). The virus causes respiratory disease with breathing difficulty (14, 32, 49) and enteritis with severe diarrhea (11, 39), which often occurs with coinfection with other viruses (39), spontaneous abortion of fetuses, and death of newborn puppies (14, 29). Pathological lesions in fetuses in experimental infections were found in the lymphoid tissue of the lung and small intestine (14). MVC was isolated and grown in the Walter Reed/3873D (WRD) canine cell line (10), which is derived from a subdermoid cyst of an irradiated male dog (10). The full-length 5.4-kb genome of MVC was recently mapped with palindromic termini (60). Under the control of a single P6 promoter, through the mechanism of alternative splicing and alternative polyadenylation, MVC expresses two nonstructural proteins (NS1 and NP1) and two capsid proteins (VP1 and VP2). Like the NS1 proteins of other parvoviruses, the NS1 of MVC is indispensable for genome replication. The NP1 protein, which is unique to the Bocavirus genus, appears to be critical for optimal viral replication, as the NP1 knockout mutant of MVC suffers from severe impairment of replication (60). A severe cytopathic effect during MVC infection of WRD cells has been documented (10, 60).The HBoV genome has been frequently detected worldwide in respiratory specimens from children under 2 years old with acute respiratory illnesses (2, 34, 55). HBoV is associated with acute expiratory wheezing and pneumonia (3, 34, 55) and is commonly detected in association with other respiratory viruses (34, 55). Further studies are necessary, however, to identify potential associations of HBoV infection with clinical symptoms or disease of acute gastroenteritis (7, 36). The full-length sequence of infectious MVC DNA (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ214110","term_id":"219665308","term_text":"FJ214110"}}FJ214110) that we have reported shows 52.6% identity to HBoV, while the NS1, NP1, and VP1 proteins are 38.5%, 39.9%, and 43.7% identical to those of HBoV, respectively (60).The cytopathic effect induced during parvovirus infection has been widely documented, e.g., in infections with minute virus of mice (MVM) (13), human parvovirus B19 (B19V) (58), parvovirus H-1 (25, 52), and BPV1 (1). In Bocavirus, cell death during BPV1 infection of embryonic bovine tracheal cells has been shown to be achieved through necrosis, independent of apoptosis (1). B19V-induced cell death of primary erythroid progenitor cells has been shown to be mainly mediated by an apoptotic pathway (58) in which the nonstructural protein 11kDa plays a key role (16). In contrast, the MVM-induced cytopathic effect has been revealed to be mediated by NS1 interference with intracellular casein kinase II (CKII) signaling (22, 44, 45), a nonapoptotic cell death. Oncolytic parvovirus H-1 infections can induce either apoptosis or nonapoptotic cell death, depending on the cell type (25, 40). Therefore, the mechanisms underlying parvovirus infection-induced cell death vary, although NS1 has been widely shown to be involved in both apoptotic and nonapoptotic cell death. The nature of the cytopathic effect during Bocavirus MVC infection has not been studied.Parvovirus replication requires infected cells at the S phase. Infection with parvovirus has been revealed to accompany a cell cycle perturbation that mostly leads to an arrest in the S/G₂ phase or the G₂/M phase during infection (30, 33, 42, 47, 65). MVM NS1 expression induces an accumulation of sensitive cells in the S/G₂ phase (6, 46, 47). Whether MVC infection-induced cell death is accompanied by an alternation of cell cycle progression and whether the viral nonstructural protein is involved in these processes have not been addressed.In this study, we found, in contrast with other members of the family Parvoviridae, expression of both the nonstructural and structural proteins of MVC by transfection did not induce cell death or cell cycle arrest. However, the cytopathic effect induced during MVC infection is a replication-coupled, mitochondrion-mediated and caspase-dependent apoptosis, accompanied with a gradual cell cycle arrest from the S phase to the G₂/M phase, which is facilitated by the MVC genome.     

Involvement of Creatine Kinase B in Hepatitis C Virus Genome Replication through Interaction with the Viral NS4A Protein

Persistent infection with hepatitis C virus (HCV) is a major cause of chronic liver diseases. The aim of this study was to identify host cell factor(s) participating in the HCV replication complex (RC) and to clarify the regulatory mechanisms of viral genome replication dependent on the host-derived factor(s) identified. By comparative proteome analysis of RC-rich membrane fractions and subsequent gene silencing mediated by RNA interference, we identified several candidates for RC components involved in HCV replication. We found that one of these candidates, creatine kinase B (CKB), a key ATP-generating enzyme that regulates ATP in subcellular compartments of nonmuscle cells, is important for efficient replication of the HCV genome and propagation of infectious virus. CKB interacts with HCV NS4A protein and forms a complex with NS3-4A, which possesses multiple enzyme activities. CKB upregulates both NS3-4A-mediated unwinding of RNA and DNA in vitro and replicase activity in permeabilized HCV replicating cells. Our results support a model in which recruitment of CKB to the HCV RC compartment, which has high and fluctuating energy demands, through its interaction with NS4A is important for efficient replication of the viral genome. The CKB-NS4A association is a potential target for the development of a new type of antiviral therapeutic strategy.Hepatitis C virus (HCV) infection represents a significant global healthcare burden, and current estimates suggest that a minimum of 3% of the world''s population is chronically infected (4, 19). The virus is responsible for many cases of severe chronic liver diseases, including cirrhosis and hepatocellular carcinoma (4, 16, 19). HCV is a positive-stranded RNA virus belonging to the family Flaviviridae. Its ∼9.6-kb genome is translated into a single polypeptide of about 3,000 amino acids (aa), in which the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B reside in the C-terminal half region (6, 34, 44). NS4A, a small 7-kDa protein, functions as a cofactor for NS3 to enhance NS3 enzyme activities such as serine protease and helicase activities. The hydrophobic N-terminal region of NS4A, which is predicted to form a transmembrane α-helix, is responsible for membrane anchorage of the NS3-4A complex (8, 44, 50), and the central region of NS4A is important for the interaction with NS3 (10, 44). A recent study demonstrated the involvement of the C terminus of NS4A in the regulation of NS5A hyperphosphorylation and viral replication (28).The development of HCV replicon technology several years ago accelerated research on viral RNA replication (7, 44). Furthermore, a robust cell culture system for propagation of infectious HCV particles was developed using a viral genome of HCV genotype 2a, JFH-1 strain, enabling us to study every process in the viral life cycle (27, 47, 54). RNA derived from genotype 1a, HCV H77, containing cell-culture adaptive mutations, also produces infectious viruses (52). Using these systems, it has been reported that the HCV genome replicates in a distinct, subcellular replication complex (RC) compartment, which includes NS3-5B and the viral RNA (2, 14, 33). The RC forms in a distinct compartment with high concentrations of viral and cellular components located on detergent-resistant membrane (DRM) structures, possibly a lipid-raft structure (2, 41), which may protect the RC from external proteases and nucleases. Almost all processes in viral replication are dependent on the host cell''s machinery and involve intimate interaction between viral and host proteins. However, the functional roles of host factors interacting with the HCV RC in viral genome replication remain ambiguous.To gain a better understanding of cellular factors that are components of the HCV RC and that function as regulators of viral replication, a comparative proteomic analysis of DRM fractions from HCV replicon and parental cells and subsequent RNA interference (RNAi) silencing of selected genes were performed. We identified creatine kinase B (CKB) as a key factor for the HCV genome replication. CKB catalyzes the reversible transfer of the phosphate group of phosphocreatine (pCr) to ADP to yield ATP and creatine and is known to play important roles in local delivery and cellular compartmentalization of ATP (48, 51). The findings obtained here suggest that recruitment of CKB to the HCV RC, through CKB interaction with NS4A, is essential for maintenance or enhancement of viral replicase activity.