Quantitative Lipid Droplet Proteome Analysis Identifies Annexin A3 as a Cofactor for HCV Particle Production

Abstract

Lipid droplets are vital to hepatitis C virus (HCV) infection as the putative sites of virion assembly, but morphogenesis and egress of virions remain ill defined. We performed quantitative lipid droplet proteome analysis of HCV-infected cells to identify co-factors of that process. Our results demonstrate that HCV disconnects lipid droplets from their metabolic function. Annexin A3 (ANXA3), a protein enriched in lipid droplet fractions, strongly impacted HCV replication and was characterized further: ANXA3 is recruited to lipid-rich fractions in HCV-infected cells by the viral core and NS5A proteins. ANXA3 knockdown does not affect HCV RNA replication but severely impairs virion production with lower specific infectivity and higher density of secreted virions. ANXA3 is essential for the interaction of viral envelope E2 with apolipoprotein E (ApoE) and for trafficking, but not lipidation, of ApoE in HCV-infected cells. Thus, we identified ANXA3 as a regulator of HCV maturation and egress.

Functional innate immunity restricts Hepatitis C Virus infection in induced pluripotent stem cell–derived hepatocytes

“…The HCV reporter Jc1 p7-GLuc-2A-NS2 was cloned as described 34 , but with a modified F2A site. For shRNA constructs, the primers listed below were cloned into a modified pSicoR-MS1-Puro vector 61 using the following target sequences: IRF3, GGAGGCAGTACTTCTGATA; RIG-I, GAAACTTGCCAGTTATATA, STAT1, GGTGGTATTTAGTCTATTA; STAT2, GAACAGTCCTGTTCAGAAA; TLR3, GGAGATTCCAGATTATAAA, and NT-control, GTCAAGTCTCACTTGCGTC described in 62 . Complete primer sequences are available on request.…”

Protein Interactions during the Flavivirus and Hepacivirus Life Cycle

“…They showed dramatic changes in lipid droplet protein association. In particular, HCV infection causes recruitment of annexin A3 to these organelles and this relocalization is necessary for a virus particle maturation step after capsid envelopment (172) (Fig. 3D).…”

Control of Drosophila Growth and Survival by the Lipid Droplet-Associated Protein CG9186/Sturkopf

“…LD-associated proteins are key to their diverse functions. On top of a core set of a few dozen of LD-associated proteins (e.g., reviewed in Hodges and Wu, 2010), proteomic studies with LDs isolated from diverse cell lines, tissues, and organisms revealed the presence of likely hundreds of additional proteins (Beller et al, 2006;Cermelli et al, 2006;Ding et al, 2012;Fei et al, 2011;Li et al, 2016;Rö sch et al, 2016;Schmidt et al, 2013;Vrablik et al, 2015;Zhang et al, 2011). While the role of selected LD-associated proteins, such as the perilipins, in energy metabolism regulation is well investigated (e.g., reviewed in Kimmel and Sztalryd, 2016), many proteins still lack a detailed analysis.…”