Amino Acids Enhance Polyubiquitination of Rheb and Its Binding to mTORC1 by Blocking Lysosomal ATXN3 Deubiquitinase Activity.

Amino-acid-induced lysosomal mechanistic target of rapamycin complex 1 (mTORC1) localization through the Rag GTPases is a critical step for its activation by Rheb GTPase. However, how the mTORC1 interacts with Rheb on the lysosome remains elusive. We report that amino acids enhance the polyubiquitination of Rheb (Ub-Rheb), which shows a strong binding preference for mTORC1 and supports its activation, while the Ub-Rheb is subjected to subsequent degradation. Mechanistically, we identified ATXN3 as a Ub-Rheb deubiquitinase whose lysosomal localization is blocked by active Rag heterodimer in response to amino acid stimulation. Consistently, cells lacking functional Rag heterodimer on the lysosome accumulate Ub-Rheb, and blockade of its degradation instigates robust lysosomal mTORC1 localization and its activation without the Ragulator-Rag system. Thus, polyubiquitination of Rheb is an important post-translational modification, which facilitates the binding of mTORC1 to Rheb on the lysosome and is another crosstalk between the amino acid and growth factor signaling for mTORC1 activation.

Translation arrest cancellation of VemP, a secretion monitor in Vibrio, is regulated by multiple cis and trans factors, including SecY.

VemP is a secretory protein in the Vibrio species that monitors cellular protein-transport activity through its translation arrest, allowing expression of the downstream secD2-secF2 genes in the same operon, which encode components of the protein translocation machinery. When cellular protein-transport function is fully active, secD2/F2 expression remains repressed as VemP translation arrest is canceled immediately. The VemP arrest cancellation occurs on the SecY/E/G translocon in a late stage in the translocation process and requires both trans factors, SecD/F and PpiD/YfgM, and a cis element, Arg-85 in VemP; however, the detailed molecular mechanism remains elusive. This study aimed to elucidate how VemP passing through SecY specifically monitors SecD/F function. Genetic and biochemical studies showed that SecY is involved in the VemP arrest cancellation and that the arrested VemP is stably associated with a specific site in the protein-conducting pore of SecY. VemP-Bla reporter analyses revealed that a short hydrophobic segment adjacent to Arg-85 plays a critical role in the regulated arrest cancellation with its hydrophobicity correlating with the stability of the VemP arrest. We identified Gln-65 and Pro-67 in VemP as novel elements important for the regulation. We propose a model for the regulation of the VemP arrest cancellation by multiple cis elements and trans factors with different roles.

Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection.

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs that adopt non-canonical orientations, as compared to other proline-rich motifs. An affinity tag-purification mass spectrometry approach identified additional PPxPxY-containing host proteins hnRNP L, hnRNPUL1, and PEG10, as VP30 interactors. hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV infection, whereas hnRNPUL1 enhances. RBBP6 and hnRNP L modulate VP30 phosphorylation, increase viral transcription, and exert additive effects on viral RNA synthesis. PEG10 has more modest inhibitory effects on EBOV replication. hnRNPUL1 positively affects viral RNA synthesis but in a VP30-independent manner. Binding studies demonstrate variable capacity of the PPxPxY motifs from these proteins to bind VP30, define PxPPPPxY as an optimal binding motif, and identify the fifth proline and the tyrosine as most critical for interaction. Competition binding and hydrogen-deuterium exchange mass spectrometry studies demonstrate that each protein binds a similar interface on VP30. VP30 therefore presents a novel proline recognition domain that is targeted by multiple host proteins to modulate viral transcription.

Insulin granule morphology and crinosome formation in mice lacking the pancreatic ß cell-specific phogrin (PTPRN2) gene.

We recently reported that phogrin, also known as IA-2ß or PTPRN2, forms a complex with the insulin receptor in pancreatic ß cells upon glucose stimulation and stabilizes insulin receptor substrate 2. In ß cells of systemic phogrin gene knockout (IA-2ß-/-) mice, impaired glucose-induced insulin secretion, decreased insulin granule density, and an increase in the number and size of lysosomes have been reported. Since phogrin is expressed not only in ß cells but also in various neuroendocrine cells, the precise impact of phogrin expressed in ß cells on these cells remains unclear. In this study, we performed a comprehensive analysis of morphological changes in RIP-Cre+/-Phogrinflox/flox (ßKO) mice with ß cell-specific phogrin gene knockout. Compared to control RIP-Cre+/- Phogrin+/+ (Ctrl) mice, aged ßKO mice exhibited a decreased density of insulin granules, which can be categorized into three subtypes. While no differences were observed in the density and size of lysosomes and crinosomes, organelles involved in insulin granule reduction, significant alterations in the regions of lysosomes responding positively to carbohydrate labeling were evident in young ßKO mice. These alterations differed from those in Ctrl mice and continued to change with age. These electron microscopic findings suggest that phogrin expression in pancreatic ß cells plays a role in insulin granule homeostasis and crinophagy during aging, potentially through insulin autocrine signaling and other mechanisms.

The molecular and metabolic program by which white adipocytes adapt to cool physiologic temperatures.

Although visceral adipocytes located within the body's central core are maintained at approximately 37°C, adipocytes within bone marrow, subcutaneous, and dermal depots are found primarily within the peripheral shell and generally exist at cooler temperatures. Responses of brown and beige/brite adipocytes to cold stress are well studied; however, comparatively little is known about mechanisms by which white adipocytes adapt to temperatures below 37°C. Here, we report that adaptation of cultured adipocytes to 31°C, the temperature at which distal marrow adipose tissues and subcutaneous adipose tissues often reside, increases anabolic and catabolic lipid metabolism, and elevates oxygen consumption. Cool adipocytes rely less on glucose and more on pyruvate, glutamine, and, especially, fatty acids as energy sources. Exposure of cultured adipocytes and gluteal white adipose tissue (WAT) to cool temperatures activates a shared program of gene expression. Cool temperatures induce stearoyl-CoA desaturase-1 (SCD1) expression and monounsaturated lipid levels in cultured adipocytes and distal bone marrow adipose tissues (BMATs), and SCD1 activity is required for acquisition of maximal oxygen consumption at 31°C.

Inner membrane YfgM-PpiD heterodimer acts as a functional unit that associates with the SecY/E/G translocon and promotes protein translocation.

PpiD and YfgM are inner membrane proteins that are both composed of an N-terminal transmembrane segment and a C-terminal periplasmic domain. Escherichia coli YfgM and PpiD form a stable complex that interacts with the SecY/E/G (Sec) translocon, a channel that allows protein translocation across the cytoplasmic membrane. Although PpiD is known to function in protein translocation, the functional significance of PpiD-YfgM complex formation as well as the molecular mechanisms of PpiD-YfgM and PpiD/YfgM-Sec translocon interactions remain unclear. Here, we conducted genetic and biochemical studies using yfgM and ppiD mutants and demonstrated that a lack of YfgM caused partial PpiD degradation at its C-terminal region and hindered the membrane translocation of Vibrio protein export monitoring polypeptide (VemP), a Vibrio secretory protein, in both E. coli and Vibrio alginolyticus. While ppiD disruption also impaired VemP translocation, we found that the yfgM and ppiD double deletion exhibited no additive or synergistic effects. Together, these results strongly suggest that both PpiD and YfgM are required for efficient VemP translocation. Furthermore, our site-directed in vivo photocrosslinking analysis revealed that the tetratricopeptide repeat domain of YfgM and a conserved structural domain (NC domain) in PpiD interact with each other and that YfgM, like PpiD, directly interacts with the SecG translocon subunit. Crosslinking analysis also suggested that PpiD-YfgM complex formation is required for these proteins to interact with SecG. In summary, we propose that PpiD and YfgM form a functional unit that stimulates protein translocation by facilitating their proper interactions with the Sec translocon.

Reversible autoinhibitory regulation of Escherichia coli metallopeptidase BepA for selective ß-barrel protein degradation.

Escherichia coli periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a ß-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246-mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice.

CRISPR/Cas9 has enabled inducible gene knockout in numerous tissues; however, its use has not been reported in brown adipose tissue (BAT). Here, we developed the brown adipocyte CRISPR (BAd-CRISPR) methodology to rapidly interrogate the function of one or multiple genes. With BAd-CRISPR, an adeno-associated virus (AAV8) expressing a single guide RNA (sgRNA) is administered directly to BAT of mice expressing Cas9 in brown adipocytes. We show that the local administration of AAV8-sgRNA to interscapular BAT of adult mice robustly transduced brown adipocytes and ablated expression of adiponectin, adipose triglyceride lipase, fatty acid synthase, perilipin 1, or stearoyl-CoA desaturase 1 by >90%. Administration of multiple AAV8 sgRNAs led to simultaneous knockout of up to three genes. BAd-CRISPR induced frameshift mutations and suppressed target gene mRNA expression but did not lead to substantial accumulation of off-target mutations in BAT. We used BAd-CRISPR to create an inducible uncoupling protein 1 (Ucp1) knockout mouse to assess the effects of UCP1 loss on adaptive thermogenesis in adult mice. Inducible Ucp1 knockout did not alter core body temperature; however, BAd-CRISPR Ucp1 mice had elevated circulating concentrations of fibroblast growth factor 21 and changes in BAT gene expression consistent with heat production through increased peroxisomal lipid oxidation. Other molecular adaptations predict additional cellular inefficiencies with an increase in both protein synthesis and turnover, and mitochondria with reduced reliance on mitochondrial-encoded gene expression and increased expression of nuclear-encoded mitochondrial genes. These data suggest that BAd-CRISPR is an efficient tool to speed discoveries in adipose tissue biology.

Impact of salt intake on urinary albumin excretion in patients with type 2 diabetic nephropathy: a retrospective cohort study based on a generalized additive model.

Diabetic kidney disease is an important and common cause of end-stage renal disease. Measurement of urinary albumin excretion (UAE) requires the diagnosis of the stage of diabetic nephropathy and the prognosis of renal function. We aimed to analyze the impact of lifestyle modification on UAE in patients with stage 2 and 3 type 2 diabetic nephropathy who received comprehensive medical care, using a generalized additive model (GAM), an explanatory machine learning model. In this retrospective observational study, we used changes in HbA1c, systolic blood pressure (SBP), and diastolic blood pressure (DBP) levels; body mass index (BMI); and daily salt intake as factors contributing to changes in UAE. In total, 269 patients with type 2 diabetic nephropathy were enrolled (stage 2, 217 patients; stage 3, 52 patients). The rankings that contributed to changes in UAE over 6 months by permutation importance were the changes in daily salt intake, HbA1c, SBP, DBP, and BMI. GAM, which predicts the change in UAE, showed that with increase in the changes in salt intake, SBP, and HbA1c, the delta UAE tended to increase. Salt intake was the most contributory factor for the changes in UAE, and daily salt intake was the best lifestyle factor to explain the changes in UAE. Strict control of salt intake may have beneficial effects on improving UAE in patients with stage 2 and 3 diabetic nephropathy.

Longitudinal association between smartphone ownership and depression among schoolchildren under COVID-19 pandemic.

Under the COVID-19 pandemic, concerns regarding prolonged screen time and mental health effects in children have increased. We examined the association of depression with smartphone ownership in school children at four time points: September 2019, July 2020, December 2020, and March 2021. The analysis revealed an interaction between group and time, indicating that depressive symptoms among smartphone owners were significantly more severe than in the other group. These results were clearer for fourth-year students, pointing that smartphone possession at younger ages may be a risk factor for mental health in the new lifestyle caused by the COVID-19 pandemic.

Tree of motility - A proposed history of motility systems in the tree of life.

Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility.

Non-Contact Heart-Rate Measurement Method Using Both Transmitted Wave Extraction and Wavelet Transform.

Continuous monitoring of heart-rate is expected to lead to early detection of physical discomfort. In this study, we propose a non-contact heart-rate measurement method which can be used in an environment such as driver heart-rate monitoring with body movement. The method is based on the electric field strength transmitted through the human body that changes with the diastole and systole of the heart. Unlike conventional displacement detection of the skin surface, we attempted to capture changes in the internal structure of the human body by irradiating the human body with microwaves and acquiring microwaves that pass through the heart. We first estimated the electric field strength transmitted through the heart using three receiving sensors to reduce the body movement effect. Then we decomposed the estimated transmitted electric field using stationary wavelet transform to eliminate significant distortion due to body movement. As a result, we achieved an estimation accuracy of heart-rate as high as 98% in a verification experiment with normal body movement.

In Vivo Dynamics of Reporter Flaviviridae Viruses.

Recombinant viruses possessing reporter proteins have been generated for virus research. In the case of the family Flaviviridae, we recently generated recombinant viruses, including the hepatitis C virus of the genus Hepacivirus, Japanese encephalitis virus (JEV) of the genus Flavivirus, and bovine viral diarrhea virus of the genus Pestivirus; all three viruses possess an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). Here, we further developed the recombinant viruses and investigated their utility in vivo Recombinant viruses harboring HiBiT in the E, NS1, or NS3 protein constructed based on the predicted secondary structure, solvent-accessible surface area, and root mean square fluctuation of the proteins exhibited comparable replication to that of the wild-type virus in vitro The recombinant JEV carrying HiBiT in the NS1 protein exhibited propagation in mice comparable to that of the parental virus, and propagation of the recombinant was monitored by the luciferase activity. In addition, the recombinants of classical swine fever virus (CSFV) possessing HiBiT in the Erns or E2 protein also showed propagation comparable to that of the wild-type virus. The recombinant CSFV carrying HiBiT in Erns exhibited similar replication to the parental CSFV in pigs, and detection of viral propagation of this recombinant by luciferase activity was higher than that by quantitative PCR (qPCR). Taken together, these results demonstrated that the reporter Flaviviridae viruses generated herein are powerful tools for elucidating the viral life cycle and pathogeneses and provide a robust platform for the development of novel antivirals.IMPORTANCEIn vivo applications of reporter viruses are necessary to understand viral pathogenesis and provide a robust platform for antiviral development. In developing such applications, determination of an ideal locus to accommodate foreign genes is important, because insertion of foreign genes into irrelevant loci can disrupt the protein functions required for viral replication. Here, we investigated the criteria to determine ideal insertion sites of foreign genes from the protein structure of viral proteins. The recombinant viruses generated by our criteria exhibited propagation comparable to that of parental viruses in vivo Our proteomic approach based on the flexibility profile of viral proteins may provide a useful tool for constructing reporter viruses, including Flaviviridae viruses.

Rimonabant suppresses RNA transcription of hepatitis B virus by inhibiting hepatocyte nuclear factor 4α.

Chronic infection with hepatitis B virus (HBV) sometime induces lethal cirrhosis and hepatocellular carcinoma. Although nucleot(s)ide analogs are used as main treatment for HBV infection, the emergence of the drug-resistant viruses has become a problem. To discover novel antivirals with low side effects and low risk of emergence of resistant viruses, screening for anti-HBV compounds was performed with compound libraries of inhibitors targeting G-protein-coupled receptors (GPCRs). HepG2-hNTCP C4 cells infected with HBV were treated with various GPCR inhibitors and harvested at 14 day postinfection for quantification of core protein in the first screening or relaxed circular DNA in the second screening. Finally, we identified a cannabinoid receptor 1 inhibitor, rimonabant, as a candidate showing anti-HBV effect. In HepG2-hNTCP C4 cells, treatment with rimonabant suppressed HBV propagation at the viral RNA transcription step but had no effect on entry or covalently closed circular DNA level. The values of half maximal inhibitory concentration, half maximal effective concentration, and selectivity index of rimonabant in primary human hepatocyte (PHH) are 2.77 µm, 40.4 µm, and 14.6, respectively. Transcriptome analysis of rimonabant-treated primary hepatocytes by RNA sequencing revealed that the transcriptional activity of hepatocyte nuclear factor 4α (HNF4α), which is known to stimulate viral RNA synthesis, was depressed. By treatment of PHH with rimonabant, the expression level of HNF4α protein and the production of the messenger RNAs (mRNAs) of downstream factors promoted by HNF4α were reduced while the amount of HNF4α mRNA was not altered. These results suggest that treatment with rimonabant suppresses HBV propagation through the inhibition of HNF4α activity.

Structural basis of Sec-independent membrane protein insertion by YidC.

Newly synthesized membrane proteins must be accurately inserted into the membrane, folded and assembled for proper functioning. The protein YidC inserts its substrates into the membrane, thereby facilitating membrane protein assembly in bacteria; the homologous proteins Oxa1 and Alb3 have the same function in mitochondria and chloroplasts, respectively. In the bacterial cytoplasmic membrane, YidC functions as an independent insertase and a membrane chaperone in cooperation with the translocon SecYEG. Here we present the crystal structure of YidC from Bacillus halodurans, at 2.4 Å resolution. The structure reveals a novel fold, in which five conserved transmembrane helices form a positively charged hydrophilic groove that is open towards both the lipid bilayer and the cytoplasm but closed on the extracellular side. Structure-based in vivo analyses reveal that a conserved arginine residue in the groove is important for the insertion of membrane proteins by YidC. We propose an insertion mechanism for single-spanning membrane proteins, in which the hydrophilic environment generated by the groove recruits the extracellular regions of substrates into the low-dielectric environment of the membrane.

A photo-cross-linking approach to monitor folding and assembly of newly synthesized proteins in a living cell.

Many proteins form multimeric complexes that play crucial roles in various cellular processes. Studying how proteins are correctly folded and assembled into such complexes in a living cell is important for understanding the physiological roles and the qualitative and quantitative regulation of the complex. However, few methods are suitable for analyzing these rapidly occurring processes. Site-directed in vivo photo-cross-linking is an elegant technique that enables analysis of protein-protein interactions in living cells with high spatial resolution. However, the conventional site-directed in vivo photo-cross-linking method is unsuitable for analyzing dynamic processes. Here, by combining an improved site-directed in vivo photo-cross-linking technique with a pulse-chase approach, we developed a new method that can analyze the folding and assembly of a newly synthesized protein with high spatiotemporal resolution. We demonstrate that this method, named the pulse-chase and in vivo photo-cross-linking experiment (PiXie), enables the kinetic analysis of the formation of an Escherichia coli periplasmic (soluble) protein complex (PhoA). We also used our new technique to investigate assembly/folding processes of two membrane complexes (SecD-SecF in the inner membrane and LptD-LptE in the outer membrane), which provided new insights into the biogenesis of these complexes. Our PiXie method permits analysis of the dynamic behavior of various proteins and enables examination of protein-protein interactions at the level of individual amino acid residues. We anticipate that our new technique will have valuable utility for studies of protein dynamics in many organisms.

Identification and characterization of a translation arrest motif in VemP by systematic mutational analysis.

VemP ( Vibrio protein export monitoring polypeptide) is a secretory protein comprising 159 amino acid residues, which functions as a secretion monitor in Vibrio and regulates expression of the downstream V.secDF2 genes. When VemP export is compromised, its translation specifically undergoes elongation arrest at the position where the Gln156 codon of vemP encounters the P-site in the translating ribosome, resulting in up-regulation of V.SecDF2 production. Although our previous study suggests that many residues in a highly conserved C-terminal 20-residue region of VemP contribute to its elongation arrest, the exact role of each residue remains unclear. Here, we constructed a reporter system to easily and exactly monitor the in vivo arrest efficiency of VemP. Using this reporter system, we systematically performed a mutational analysis of the 20 residues (His138-Phe157) to identify and characterize the arrest motif. Our results show that 15 residues in the conserved region participate in elongation arrest and that multiple interactions between important residues in VemP and in the interior of the exit tunnel contribute to the elongation arrest of VemP. The arrangement of these important residues induced by specific secondary structures in the ribosomal tunnel is critical for the arrest. Pro scanning analysis of the preceding segment (Met120-Phe137) revealed a minor role of this region in the arrest. Considering these results, we conclude that the arrest motif in VemP is mainly composed of the highly conserved multiple residues in the C-terminal region.

Characterization of Recombinant Flaviviridae Viruses Possessing a Small Reporter Tag.

The family Flaviviridae consists of four genera, Flavivirus, Pestivirus, Pegivirus, and Hepacivirus, and comprises important pathogens of human and animals. Although the construction of recombinant viruses carrying reporter genes encoding fluorescent and bioluminescent proteins has been reported, the stable insertion of foreign genes into viral genomes retaining infectivity remains difficult. Here, we applied the 11-amino-acid subunit derived from NanoLuc luciferase to the engineering of the Flaviviridae viruses and then examined the biological characteristics of the viruses. We successfully generated recombinant viruses carrying the split-luciferase gene, including dengue virus, Japanese encephalitis virus, hepatitis C virus (HCV), and bovine viral diarrhea virus. The stability of the viruses was confirmed by five rounds of serial passages in the respective susceptible cell lines. The propagation of the recombinant luciferase viruses in each cell line was comparable to that of the parental viruses. By using a purified counterpart luciferase protein, this split-luciferase assay can be applicable in various cell lines, even when it is difficult to transduce the counterpart gene. The efficacy of antiviral reagents against the recombinant viruses could be monitored by the reduction of luciferase expression, which was correlated with that of viral RNA, and the recombinant HCV was also useful to examine viral dynamics in vivo Taken together, our findings indicate that the recombinant Flaviviridae viruses possessing the split NanoLuc luciferase gene generated here provide powerful tools to understand viral life cycle and pathogenesis and a robust platform to develop novel antivirals against Flaviviridae viruses.IMPORTANCE The construction of reporter viruses possessing a stable transgene capable of expressing specific signals is crucial to investigations of viral life cycle and pathogenesis and the development of antivirals. However, it is difficult to maintain the stability of a large foreign gene, such as those for fluorescence and bioluminescence, after insertion into a viral genome. Here, we successfully generated recombinant Flaviviridae viruses carrying the 11-amino-acid subunit derived from NanoLuc luciferase and demonstrated that these viruses are applicable to in vitro and in vivo experiments, suggesting that these recombinant Flaviviridae viruses are powerful tools for increasing our understanding of viral life cycle and pathogenesis and that these recombinant viruses will provide a robust platform to develop antivirals against Flaviviridae viruses.

Host-derived apolipoproteins play comparable roles with viral secretory proteins Erns and NS1 in the infectious particle formation of Flaviviridae.

Amphipathic α-helices of exchangeable apolipoproteins have shown to play crucial roles in the formation of infectious hepatitis C virus (HCV) particles through the interaction with viral particles. Among the Flaviviridae members, pestivirus and flavivirus possess a viral structural protein Erns or a non-structural protein 1 (NS1) as secretory glycoproteins, respectively, while Hepacivirus including HCV has no secretory glycoprotein. In case of pestivirus replication, the C-terminal long amphipathic α-helices of Erns are important for anchoring to viral membrane. Here we show that host-derived apolipoproteins play functional roles similar to those of virally encoded Erns and NS1 in the formation of infectious particles. We examined whether Erns and NS1 could compensate for the role of apolipoproteins in particle formation of HCV in apolipoprotein B (ApoB) and ApoE double-knockout Huh7 (BE-KO), and non-hepatic 293T cells. We found that exogenous expression of either Erns or NS1 rescued infectious particle formation of HCV in the BE-KO and 293T cells. In addition, expression of apolipoproteins or NS1 partially rescued the production of infectious pestivirus particles in cells upon electroporation with an Erns-deleted non-infectious RNA. As with exchangeable apolipoproteins, the C-terminal amphipathic α-helices of Erns play the functional roles in the formation of infectious HCV or pestivirus particles. These results strongly suggest that the host- and virus-derived secretory glycoproteins have overlapping roles in the viral life cycle of Flaviviridae, especially in the maturation of infectious particles, while Erns and NS1 also participate in replication complex formation and viral entry, respectively. Considering the abundant hepatic expression and liver-specific propagation of these apolipoproteins, HCV might have evolved to utilize them in the formation of infectious particles through deletion of a secretory viral glycoprotein gene.

Characterization of miR-122-independent propagation of HCV.

miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV122KO, possessing G28A substitution in the 5'UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocyte-derived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients.