Hepatitis C virus promotes virion secretion through cleavage of the Rab7 adaptor protein RILP Ann L. Wozniaka, Abby Longa, Kellyann N. Jones-Jamtgaarda, and Steven A. Weinmana,1 a
Department of Internal Medicine and Liver Center, University of Kansas Medical Center, Kansas City, KS 66160
Edited by Francis V. Chisari, The Scripps Research Institute, La Jolla, CA, and approved September 16, 2016 (received for review May 6, 2016)
Hepatitis C virus (HCV) is an enveloped RNA virus that modifies intracellular trafficking processes. The mechanisms that HCV and other viruses use to modify these events are poorly understood. In this study, we observed that two different RNA viruses, HCV and Sendai, cause inhibition of ras-related protein Rab-7 (Rab7)-dependent endosome–lysosome fusion. In both cases, viral infection causes cleavage of the Rab7 adaptor protein RILP (Rab interacting lysosomal protein), which is responsible for linking Rab7 vesicles to dynein motor complexes. RILP cleavage results in the generation of a cleaved RILP fragment (cRILP) missing the N terminus of the molecule. Although RILP localizes in a perinuclear fashion, cRILP moves to the cell periphery. Both knockdown of RILP and expression of cRILP reproduced the HCV-induced trafficking defect, and restoring full-length RILP reversed the trafficking effects of virus. For the first 3 d after electroporation of HCV RNA, intracellular virus predominates over secreted virus, but the quantity of intracellular virus then rapidly declines as secreted virus dominates. The transition from the intracellular-predominant to the secretion-predominant phenotype corresponds to the time course of cRILP generation. Expressing cRILP directly prevents intracellular virus accumulation at early times without affecting net virus production. The ability of cRILP to promote virus secretion could be prevented by a kinesin inhibitor. HCV thus modifies cellular trafficking by cleaving RILP, which serves to redirect Rab7-containing vesicles to a kinesindependent trafficking mode promoting virion secretion. Cleavage of a Rab adaptor protein is thus a mechanism by which viruses modify trafficking patterns of infected cells. trafficking
| kinesin | endocytosis | Sendai virus | hepatitis C virus
T
he hepatitis C virus (HCV) is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is a positive-stranded RNA virus. It replicates on modified ER membranes (1), and virions are subsequently assembled on the surface of ER-associated lipid droplets (2). The processes necessary for virion trafficking to the plasma membrane and exocytosis have not been well defined. There is considerable evidence that HCV alters several membrane trafficking steps. It rearranges the ER to form the membranous web replication complex (3, 4), and it suppresses the fusion of autophagosomes and lysosomes, resulting in the accumulation of autophagic vacuoles (5). Although the mechanisms responsible for HCVinduced membrane rearrangements are poorly understood, it is logical to assume that redirection of vesicle trafficking pathways by HCV serve the viral lifecycle by initiating genome replication, preventing virion degradation, and optimizing virion secretion. A key molecule in the regulation of membrane trafficking is rasrelated protein Rab-7 (Rab7), a member of the small GTPase family that acts as a switch triggering the assembly of vesicle microtubule motor protein linkage complexes (6). Rab7 facilitates the maturation of endosomes and autophagosomes and also promotes the microtubule-directed trafficking of cargos from the late endosome or autophagosome to the lysosome. Rab proteins link to the dynein and kinesin motor complexes through a series of adaptor proteins. These adaptor/effector proteins confer specificity for specific Rabs and ensure that the Rab proteins do not overlap in the pathways that they regulate. Rabs and their effectors have been shown to be targets for infectious agents. Chlamydia releases proteins to recruit specific
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Rabs, redirecting them to the Golgi (7), and Salmonella interacts with the GTPase effector SKIP to assure its localization in the perinuclear region (8). Prior studies have shown the involvement of several Rab proteins in the HCV lifecycle as well (9, 10). RILP, Rab interacting lysosomal protein, is a Rab7 adaptor protein responsible for linking Rab7-containing vesicles to the dynein motor complexes to drive microtubule minus-end–directed transport toward the microtubule organizing center (MTOC) (11, 12). RILP interacts with Rab7 through its C-terminal domain, whereas its N-terminal domain recruits the dynein–dynactin motor. The Rab7– RILP complex acts on many Rab7-containing compartments including phagosomes, melanosomes, and major histocompatibility complex class II-containing compartments (13–15). These dyneinlinked motor complexes act in opposition to kinesin-linked motor complexes, resulting in a “tug of war” for vesicular trafficking (16). In the course of studying viral effects on cellular trafficking, we observed that both HCV and Sendai virus (SeV) infection induced the cleavage of RILP to generate a C-terminal fragment of the protein that no longer mediated inward trafficking and enhanced outward trafficking of Rab7-containing vesicles. The accumulation of this modified adaptor protein increased over the first several days of HCV infection and caused several trafficking changes in the host cell that promoted efficient secretion and minimal intracellular accumulation of infectious virions. These results suggest that viral-induced modification of Rab adaptor proteins is another mechanism by which viruses modify host cells to promote their lifecycle. Results HCV Alters Endosome–Lysosome Trafficking. To examine the effect of
HCV infection on endocytic trafficking, we measured EGF-induced Significance Viruses frequently alter cellular processes to promote their own lifecycle, whereas cells adapt by initiating antiviral responses. Therefore, the outcome of viral disease depends on the balance of these factors. Hepatitis C virus (HCV) induces drastic modifications in cellular vesicle trafficking that appear to be required for the formation of viral replication complexes and the assembly/secretion of viral particles. We show that HCV promotes the production and secretion of infectious virus particles by disrupting Rab7 complexes, shifting microtubuleassociated vesicles from the inwardly directed dynein motor to the outwardly directed kinesin motor complex. This shift targets them toward the cell surface. This finding that viruses cleave the Rab adaptor proteins provides insight into how viruses alter trafficking to promote virus propagation. Author contributions: A.L.W. and S.A.W. designed research; A.L.W., A.L., and K.N.J.-J. performed research; A.L.W. and S.A.W. analyzed data; and A.L.W. and S.A.W. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
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Fig. 1. HCV alters endosome–lysosome trafficking. (A) JFH-1 inhibits EGFinduced EGFR degradation. Cells were serum-starved and EGF was added for the indicted times. Cells were then harvested and analyzed for EGFR by immunoblotting. (B) The average amount of EGFR remaining in response to exogenous EGF (n ≥ 5). Data are presented as mean ± SE. *P ≤ 0.05 compared with control at time 0 min. (C) Rab7 immunofluorescence is increased after JFH-1 infection. Huh-7.5 cells were infected with JFH-1 and assayed after 3 d. Cells were subsequently immunostained for Rab7 (red) as described in Methods. Viral replication was detected by immunostaining for core protein (green). Representative images showing both infected and noninfected (*) cells are shown. (D) JFH-1 infection did not alter Rab7 protein levels in total cell lysate. (E) The amount of active Rab7 is unchanged after JFH-1 infection. GTP-bound Rab7 was pulled down from control and JFH-1– infected Huh-7.5 cell lysates. The presence of Rab7 represents the amount of active Rab7. (F) The intracellular distribution of Rab7 is not affected by JFH-1 infection. Cells were homogenized in a hypotonic buffer and subjected to differential centrifugation, yielding a TH, a 17,000 × g heavy membrane pellet (P), and a 17,000 × g supernatant. (G) The average amount of Rab7 present in the TH of uninfected and JFH-1–infected Huh-7.5 cells as measured by densitometry analysis (n = 5). Data are presented as mean ± SE.
HCV Enhances RILP Degradation. The increase in Rab7 immunofluorescence without a corresponding increase in either total Rab7 or active Rab7 protein levels suggested that HCV either altered the conformation of Rab7 or increased the accessibility of the Rab7 antibody to its epitope. Therefore, we next determined whether HCV infection changes Rab7 binding partners. RILP is a Rab7 adaptor protein that bridges Rab7-containing vesicles to dynein motor complexes. Strikingly, when HCV infection was allowed to proceed to a point where 95–100% of the cells were infected, the level of RILP protein was reduced to
