Abstract
Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to distinct characteristics of these apolipoproteins that influence HCV assembly and cell entry. This will guide future research to precisely pinpoint how apolipoproteins function during virus assembly and cell entry.
Highlights
Recent estimates indicate that 80 million people worldwide are chronically infected with Hepatitis C virus (HCV), which is causing chronic liver disease leading to life-threatening conditions like cirrhosis and hepatocellular carcinoma [1]
Indicate that amphipathic alpha-helices within ApoC1 are the key determinant for its role in production of infectious HCV particles. It is well-known that during its replication cycle HCV closely interacts with lipoprotein metabolism, with the very low density lipoproteins (VLDL) pathway, as several components of this pathway like ApoB, Apolipoprotein E (ApoE) and microsomal triglyceride transfer protein (MTTP) have been implicated in HCV morphogenesis [9,10,11,12,13]
Previous studies could show that expression of ApoE alone is sufficient to restore production of infectious HCV progeny in non-liver cells, indicating that ApoB and MTTP are not essential [14, 15]
Summary
Recent estimates indicate that 80 million people worldwide are chronically infected with Hepatitis C virus (HCV), which is causing chronic liver disease leading to life-threatening conditions like cirrhosis and hepatocellular carcinoma [1]. Several liver cell expressed apolipoproteins, including ApoB and the exchangeable apolipoproteins E, A1, and C1 have been identified on HCV particles suggesting that they could act during virus assembly and may influence cell entry [4, 18,19,20,21]. The ApoCs are believed to modulate receptor interactions and influence enzymes involved in lipid metabolism[27] The role of these other exchangeable apolipoproteins in the HCV replication cycle has not been explored. We show that all ApoErelated exchangeable apolipoproteins can redundantly take over the role of ApoE in HCV assembly in non-liver cells and that amphipathic alpha-helical repeats within these proteins are the key determinant for their function. Distinct differences in the assembly efficacy, specific infectivity of released viruses and the ratio between extracellular and intracellular infectious virus production indicate that specific properties of these apolipoproteins influence these steps of the HCV replication cycle
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