Infectivity of Hepatitis C Virus Is Influenced by Association with Apolipoprotein E Isoforms

Hepatitis C virus (HCV) is a causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV in circulating blood associates with lipoproteins such as very low density lipoprotein (VLDL) and low-density lipoprotein (LDL). Although these associations suggest that lipoproteins are important for HCV infectivity, the roles of lipoproteins in HCV production and infectivity are not fully understood. To clarify the roles of lipoprotein in the HCV life cycle, we analyzed the effect of apolipoprotein E (ApoE), a component of lipoprotein, on virus production and infectivity. The production of infectious HCV was significantly reduced by the knockdown of ApoE. When an ApoE mutant that fails to be secreted into the culture medium was used, the amount of infectious HCV in the culture medium was dramatically reduced; the infectious HCV accumulated inside these cells, suggesting that infectious HCV must associate with ApoE prior to virus release. We performed rescue experiments in which ApoE isoforms were ectopically expressed in cells depleted of endogenous ApoE. The ectopic expression of the ApoE2 isoform, which has low affinity for the LDL receptor (LDLR), resulted in poor recovery of infectious HCV, whereas the expression of other isoforms, ApoE3 and ApoE4, rescued the production of infectious virus, raising it to an almost normal level. Furthermore, we found that the infectivity of HCV required both the LDLR and scavenger receptor class B, member I (SR-BI), ligands for ApoE. These findings indicate that ApoE is an essential apolipoprotein for HCV infectivity.Hepatitis C virus (HCV) infection is a major global health problem. More than 170 million people worldwide are infected with HCV. HCV causes chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (18). A member of the family Flaviviridae, HCV has a positive-sense, single-stranded RNA genome that is packaged into an enveloped viral particle. The genome encodes a large precursor polyprotein, which is cleaved by host and viral proteases to generate at least 10 functional viral proteins: core, envelope protein 1 (E1), E2, p7, nonstructural protein 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B (12, 13). Core associates with the lipid droplet (LD). The role of this association remained elusive until robust HCV replication systems became available (32). We previously showed that the LD is an important organelle for HCV production (23). In hepatocytes, the LD is physiologically important as a lipid source for the production of lipoproteins such as very low density lipoprotein (VLDL) (11). VLDL is synthesized in the liver as a triglyceride/cholesterol ester-rich particle (diameter, 30 to 100 nm) surrounded by apolipoproteins such as apolipoprotein B100 (abbreviated as ApoB throughout), ApoC''s, and ApoE. VLDL is released into blood vessels to be delivered as a lipid source to peripheral cells, and it is also readsorbed by liver cells after processing (5).HCV particles circulating in the blood of HCV carriers associate with lipoproteins, such as low-density lipoprotein (LDL), VLDL, and chylomicrons; thus, these are termed lipo-viro particles (LVPs) (1, 26). Purified LVPs from circulating blood contain triglyceride, ApoB, ApoB48, ApoCII, ApoCIII, ApoE, and virus components such as HCV RNA and core (8), indicating that the LVP has dual viral and lipoprotein characteristics. The HCVcc strain, which contains a chimeric HCV-2a genome with a structural region from HCV-J6 and nonstructural/noncoding regions from an infectious JFH1 virus, can establish long-term infection in chimpanzees. Viruses recovered from the chimpanzee contain infectious virus particles with a slightly low density, suggesting that an in vivo association with low-density factors influences infectivity (19). However, the role of a lipoprotein-like component of LVPs in virus replication is not clear. Moreover, the mechanism by which LVPs are generated during HCV production is unknown.When HCV-producing cells are treated with an inhibitor of microsomal triglyceride transfer protein (MTP) or with ApoB-specific small interfering RNA (siRNA), the production of HCV particles is suppressed (10, 14, 25). Therefore, lipoprotein biosynthesis appears to play an important role in the production of infectious HCV and its egress from infected cells. ApoB, ApoC1, and ApoE associate with infectious virus particles in the HCVcc infection/replication system (4, 6, 15, 22, 27). Furthermore, ApoE depletion suppresses the production of infectious HCV (4, 6, 15, 27). These reports strongly suggest the importance of lipoprotein function to the HCV life cycle. However, the precise roles of lipoproteins and apolipoproteins in virus production and infectivity are not fully understood.We analyzed the production of HCV from cells in which apolipoprotein production was knocked down with siRNA. We found that ApoE is required for the infectivity of HCV, a finding consistent with other reports (4, 6, 15). ApoE is a polymorphic protein with three major isoforms: ApoE2, ApoE3, and ApoE4. The three isoforms differ by amino acid substitutions at one or two sites (residues 130 and 176) on the 317-amino-acid chain of the ApoE molecule. The polymorphism of ApoE influences its multiple functions due to isoform-dependent differences in receptor-binding activity and lipoprotein association preference. For example, ApoE2 has drastically lower LDL receptor (LDLR) binding activity than ApoE3 and ApoE4 (7). In the present study, we investigated the role of ApoE isoforms in virus production and infectivity.(Part of this study was presented at the 16th International Symposium on Hepatitis C Virus and Related Viruses, Nice, France, 3 to 7 October 2009.)     

Proteasomal and autophagic pathways converge on lipid droplets

Apolipoprotein B (apoB) is the primary protein of very low-density lipoproteins (VLDL). We found that apoB accumulated on the surface of cytoplasmic lipid droplets (LDs) of hepatocytes when the proteasomal or autophagic processes were suppressed. ApoB associated with LDs was poly-ubiquitinated and surrounded by autophagic vacuoles. Moreover, proteasomal subunits were concentrated around LDs. Our data suggest that apoB that is destined to be degraded remains adhered to LDs until it is broken down by the proteasomal and autophagic pathways. We speculate that the LD surface serves as a platform to prevent hydrophobic apoB from forming aggregates, and that LDs may play a similar role for other aggregation-prone hydrophobic proteins.     

Inhibition of ADP-ribosylation suppresses aberrant accumulation of lipidated apolipoprotein B in the endoplasmic reticulum

ApoB-crescent, an endoplasmic reticulum (ER)-lipid droplet amalgamation structure, is a useful marker to indicate aberrant lipidated apolipoprotein B accumulation in the hepatocyte ER. Blockade of the ER-to-Golgi transport by either vesicle transport inhibitors or dominant-negative Arf1 caused a significant increase in ApoB-crescents. However, a low concentration of Brefeldin A induced the same result without affecting protein secretion, suggesting ADP-ribosylation as an additional mechanism. ADP-ribosylation inhibitors not only suppressed the increase of ApoB-crescents, but also rapidly dissolved existing ApoB-crescents. These results implicate the involvement of ADP-ribosylation in the ApoB-crescent formation and maintenance process at the ER.     

Nuclear lipid droplets form in the inner nuclear membrane in a seipin-independent manner

Nuclear lipid droplets (LDs) in hepatocytes are derived from precursors of very-low-density lipoprotein in the ER lumen, but it is not known how cells lacking the lipoprotein secretory function form nuclear LDs. Here, we show that the inner nuclear membrane (INM) of U2OS cells harbors triglyceride synthesis enzymes, including ACSL3, AGPAT2, GPAT3/GPAT4, and DGAT1/DGAT2, and generates nuclear LDs in situ. mTOR inhibition increases nuclear LDs by inducing the nuclear translocation of lipin-1 phosphatidic acid (PA) phosphatase. Seipin, a protein essential for normal cytoplasmic LD formation in the ER, is absent in the INM. Knockdown of seipin increases nuclear LDs and PA in the nucleus, whereas seipin overexpression decreases these. Seipin knockdown also up-regulates lipin-1β expression, and lipin-1 knockdown decreases the effect of seipin knockdown on nuclear LDs without affecting PA redistribution. These results indicate that seipin is not directly involved in nuclear LD formation but instead restrains it by affecting lipin-1 expression and intracellular PA distribution.     

Warning coloration in sawflyAthalia rosae larva and concealing coloration in butterflyPieris rapae larva feeding on similar plants evolved through individual selection

Green larvae of the butterfly Pieris rapae and black larvae of the sawfly Athalia rosae feed on green leaves of the same cruciferous plants. To demonstrate that P. rapae has concealing coloration and that A. rosae has warning coloration, the larvae of the two species were supplied to naive chicks Gallus gallus on white, green or black backgrounds. P. rapae larvae were palatable and their green body color acted as a concealing coloration. On the other hand, A. rosae larvae were unpalatable and their black body color acted as a warning coloration. There is a general consensus that warning coloration is an altruistic character which needs victims, and thus can evolve through kin selection or green beard selection. However, black A. rosae larvae were seldom injured by chicks' attack, in particular, on the green background. Therefore, the warning coloration of A. rosae larvae can be a selfish character and hence can evolve through individual selection as well as concealing coloration of P. rapae.     

Comparative population studies of threePieris butterflies,P. rapae,P. melete andP. napi,living in the same area

Utilization of patchy habitats by adult populations of three Pieris butterflies, P. rapae, P. melete and P. napi was studied throughout the flight season in an area of their coexistence, about 3×1.5 km, in a farm village in the mountains in Inabu, Aichi Prefecture. Field study was by the mark-recapture method. Results were analyzed by dispersal distances and recapture duration decay curves for adults of different age-classes estimated on the basis of physical condition of their wings, together with supplementary information of daliy egg-laying rate of females, obtained in field cages. Sexually immature, mated femals of P. rapae after teneral stage showed a migratory flight. On the other hand, reproductive females and all males of P. rapae were strongly resident within suitable habitats, and reproductive females begun to lay eggs abundantly at sunny places of newly suitable areas within a short period. P. melete seemed to disperse gradually from emerged stites and females of this species continued to lay some constant numbers of eggs for more than ten days over a wider area. P. napi appeared more like P. melete than P. rapae. The habitats of the three species can be characterized as follows:P. rapae, temporary, continued for pre-reproductive females but localized for reproductive females and all males, and unstable;P. melete, permanent, widespread, and stable;P. napi, permanent, localized, and stable. The numbers of generations of P. rapae, P. melete and P. napi were estimated to be about six, three and three, respectively. Seasonal fluctuations in the number of adults were influenced by the stability of their habitats, i. e., the population size fluctuated sharply in P. rapae, but it was much more stable in P. melete and P. napi. In view of these results, it can be said that P. rapae fits the general characteristics of a r-strategist whereas P. melete and P. napi are more K-strategic than P. rapae.     

Role of ORPs in sterol transport from plasma membrane to ER and lipid droplets in mammalian cells

In this study, we investigated the mechanisms of sterol transport from the plasma membrane (PM) to the endoplasmic reticulum (ER) and lipid droplets (LDs) in HeLa cells. By overexpressing all mammalian oxysterol-binding protein-related proteins (ORPs), we found that especially ORP1S and ORP2 enhanced PM-to-LD sterol transport. This reflected the stimulation of transport from the PM to the ER, rather than from the ER to LDs. Double knockdown of ORP1S and ORP2 inhibited sterol transport from the PM to the ER and LDs, suggesting a physiological role for these ORPs in the process. A two phenylalanines in an acidic tract (FFAT) motif in ORPs that mediates interaction with VAMP-associated proteins (VAPs) in the ER was not necessary for the enhancement of sterol transport by ORPs. However, VAP-A and VAP-B silencing slowed down PM-to-LD sterol transport. This was accompanied by enhanced degradation of ORP2 and decreased levels of several FFAT motif-containing ORPs, suggesting a role for VAPs in sterol transport by stabilization of ORPs.