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Title: HCV Induces the Expression of Rubicon and UVRAG to Temporally Regulate the Maturation of Autophagosomes and Viral Replication}
Article:
Abstract:
Hepatitis C virus( HCV) induces autophagy to enhance its replication. However, how HCV regulates the autophagic pathway remains largely unclear. In this report, we demonstrated that HCV infection could induce the expression of Rubicon and UVRAG, which inhibited and stimulated the maturation of autophagosomes, respectively. The induction of Rubicon by HCV was prompt whereas the induction of UVRAG was delayed, resulting in the accumulation of autophagosomes in the early time points of viral infection. The role of Rubicon in inhibiting the maturation of autophagosomes in HCV-infected cells was confirmed by siRNA knockdown and the over-expression of Rubicon, which enhanced and suppressed the maturation of autophagosomes, respectively. Rubicon played a positive role in HCV replication, as the suppression of its expression reduced HCV replication and its over-expression enhanced HCV replication. In contrast, the over-expression of UVRAG facilitated the maturation of autophagosomes and suppressed HCV replication. The HCV subgenomic RNA replicon, which expressed only the nonstructural proteins, could also induce the expression of Rubicon and the accumulation of autophagosomes. Further analysis indicated that the HCV NS4B protein was sufficient to induce Rubicon and autophagosomes. Our results thus indicated that HCV, by differentially inducing the expression of Rubicon and UVRAG, temporally regulated the autophagic flux to enhance its replication.
Introduction:
Hepatitis C virus( HCV) is an important human pathogen that can cause severe liver diseases including cirrhosis and hepatocellular carcinoma. It belongs to the flavivirus family and has a 9. 6 Kb positive-stranded RNA genome. This genome encodes a polyprotein with a length of slightly more than 3, 000 amino acids. The translation of the HCV polyprotein is mediated by an internal ribosomal entry site( IRES) that comprises most of the 5’-untranslated region and the first few codons of the polyprotein coding sequence. After its synthesis, the HCV polyprotein is processed into structural and nonstructural proteins by cellular and viral proteases[1]. The nonstructural proteins NS3, NS4A, NS4B, NS5A and NS5B are required and sufficient for viral RNA replication[2]. HCV is a hepatotropic virus. It can induce autophagy in its host cells to enhance its replication[3–12]. Autophagy is a catabolic process by which cells remove protein aggregates and damaged organelles for recycling. This process begins by the formation of membrane crescents termed phagophores in the cytosol. The edges of these phagophores will subsequently extend to sequester part of the cytoplasm, leading to the formation of enclosed double-membrane vesicles, known as autophagosomes. Autophagosomes mature by fusing with lysosomes to form autolysosomes, in which the cargos of autophagosomes are digested by lysosomal enzymes for recycling[13]. Autophagy occurs at a basal level in cells during normal conditions and is important for maintaining cellular homeostasis. It can also be induced by stress, such as nutrient starvation. Many protein factors that are important for autophagy have been identified. Class III phosphatidylinositol-3-kinase( PI3KC3) is one of these factors. It catalyzes the formation of phosphatidylinositol-3-phosphate( PI3P) and is important for the initiation of autophagy[14]. PI3KC3 consists of three core components, hVps34, p150 and Beclin-1[14, 15]. This core complex is associated with two mutually exclusive factors Atg14L and UVRAG[16–18]. Atg14L is important for autophagosomal membrane nucleation and expansion, whereas UVRAG plays a more important role in the maturation of autophagosomes[19]. UVRAG can bind to the class C Vps/HOPS complex, which is a guanine nucleotide exchange factor for the small GTPase Rab7, to activate Rab7 and stimulate the fusion between autophagosomes and lysosomes[20]. The UVRAG activity is negatively regulated by Rubicon( RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein), which binds to the UVRAG-PI3KC3 complex to inhibit the maturation of autophagosomes[21]. Rubicon can also sequester UVRAG from C-Vps/HOPS and inhibit the activation of Rab7[22]. Another factor that is important for autophagy is the microtubule-associated protein light-chain 3( LC3). LC3 is a cytosolic protein. During autophagy, it is covalently linked by Atg3, Atg4 and Atg7 to phosphatidylethanolamine( PE), a phospholipid. This lipidation allows LC3 to localize to autophagosomal membranes. LC3 is de-lipidated by Atg4 after the maturation of autophagosomes and released back into the cytosol. It can also be degraded by lysosomal enzymes, if it is localized to the inner membrane of autophagosomes[13]. The lipidation of LC3 and its localization to autophagosomes are often used as markers for autophagy. HCV can induce the accumulation of autophagosomes and use autophagosomal membranes as the site for its RNA replication[5, 12]. However, there are controversies regarding whether HCV can efficiently induce the fusion between autophagosomes and lysosomes[5, 7, 9, 11, 23–25]. In this report, we investigated the mechanism of HCV-induced autophagy. Our results indicated that HCV could induce the expression of both Rubicon and UVRAG. The induction of Rubicon was prompt whereas the induction of UVRAG was delayed, leading to the delayed maturation and the accumulation of autophagosomes in the early time points of HCV infection. Our results thus indicated that HCV, by differentially regulating the expression of Rubicon and UVRAG, temporally regulated the autophagic flux to enhance its replication.
Discussion:
It has previously been shown that HCV infection can induce autophagy. In this report, we demonstrated that the induction of the autophagic response by HCV was rapid and could be detected as early as six hours post-infection( Fig. 1). This induction of autophagic response began with the accumulation of autophagosomes, which matured inefficiently at the early time points of HCV infection( Fig. 1C). This inefficient maturation of autophagosomes was due to the induction of Rubicon by HCV, as the suppression of Rubicon expression facilitated the maturation of autophagosomes( Fig. 2), and the over-expression of Rubicon had the opposite effect( Fig. 3). These results indicated that the accumulation of autophagosomes in the early stage of HCV infection was at least partially due to the inhibition of the maturation of autophagosomes( i. e., the reduction of the autophagic “off-rate”). In contrast to the early time points, the maturation of autophagosomes was efficient at 48 hours post-infection( Fig. 1). The increased maturation efficiency of autophagosomes at this time point was apparently due to the induction of UVRAG by HCV, which overcame the suppressive effect of Rubicon, as the over-expression of UVRAG enhanced the maturation of autophagosomes in HCV-infected cells( Fig. 4). The model of how HCV regulates the maturation of autophagosome at different stage of infection is illustrated in Fig. 8. Note that in spite of the increase of the maturation efficiency of autophagosomes at the latter time point, the overall number of autophagic vacuoles remained high at that time point( Fig. 1B and 1C). It is conceivable that this was due to the higher autophagic initiation rate( i. e., the autophagic “on-rate”) caused by the higher expression level of UVRAG, which is known to enhance the initiation of autophagy[18]. Our studies also demonstrated that Rubicon enhanced HCV replication whereas UVRAG inhibited HCV replication( Fig. 5). By using cells that harbored the HCV subgenomic RNA replicon, we demonstrated that the effect of Rubicon on HCV was most likely on HCV RNA replication( Fig. 6 and S6 Fig.). As our previous studies indicated that autophagosomal membranes could serve as the sites for HCV RNA replication, it is likely that Rubicon enhanced HCV RNA replication by increasing the pool of autophagosomes in infected cells. This could also explain why the over-expression of UVRAG, which reduced the pool of autophagosomes by facilitating their maturation, would reduce HCV RNA replication. Our results thus are consistent with the model that HCV induces the expression of Rubicon to enhance the accumulation of autophagosomes, which serve as sites for HCV RNA replication in the early stage of HCV replication. However, due to the extensive reorganization of membrane structures in the later stage of infection, autophagosomes may be replaced by other membrane structures such as the smaller double membrane vesicles( DMVs)[29, 30], which may also be derivatives of autophagosomes due to their structural similarities, and become dispensable for HCV RNA replication. This model will also explain why previous studies indicated that autophagy is only important for HCV replication in the early stage, but not in the later stage, of infection[4]. We had also studied the mechanism of Rubicon induction by HCV. Our results indicated that HCV NS4B was sufficient to induce its expression and the accumulation of autophagosomes( Fig. 7). HCV NS4B has previously been shown to interact with Rab5 and Vps34 to regulate autophagy[8, 31]. It is unclear whether these activities of NS4B contributed to its effect on Rubicon. Previous studies indicated that HCV induced the autophagic response via the UPR[5, 7, 11]. It is interesting that NS4B by itself was sufficient to induce the cleavage of ATF6, an important event of the UPR( Fig. 7A). This result is consistent with a previous report, which indicated that HCV NS4B by itself could induce the UPR[32]. It is conceivable that NS4B might induce Rubicon via the UPR, which could activate a number of transcription factors including ATF6. Some of these transcription factors might be involved in the up-regulation of the Rubicon gene, as our results indicated that the induction of Rubicon by HCV was likely a transcriptional event( S3 Fig.). Many viruses can perturb the autophagic pathway to enhance their replications and, similar to HCV, some viruses can inhibit the fusion between autophagosomes and lysosomes[33–38]. For example, poliovirus and coxsakievirus B3 induce autophagosomes to support its replication[39, 40]. Rotavirus also blocks the maturation of autophagosomes and use the autophagic vacuoles for the transport of viral proteins from ER to viroplasms where the viral genome replication and particle assembly take place[36]. Dengue virus-2( DENV-2), however, uses amphisomes, an intermediate autophagic vacuole prior to the formation of autolysosomes, for its protein translation and RNA replication[41]. In contrast to DENV-2, DENV-3, a different DENV serotype, uses both amphisomes and autolysosomes for its replication[42]. Kaposi's sarcoma-associated herpesvirus( KSHV) can also inhibit the maturation of autophagosomes to enhance its replication[43]. Interestingly, KSHV inhibits the maturation of autophagosomes by using its K7 protein to bind to Rubicon. This previous observation on KSHV K7 indicates that the effect of HCV on Rubicon and the regulation of autophagosomal maturation is not unique. It is likely that this may be a mechanism frequently exploited by viruses to control the autophagic pathway in their host cells for their replication.
HCV induces autophagy to benefit its replication . In this report , we demonstrated that HCV infection could induce the expression of both Rubicon and UVRAG , which inhibited and stimulated the maturation of autophagosomes , respectively . The induction of Rubicon by HCV was prompt whereas the induction of UVRAG was delayed , resulting in the accumulation of autophagosomes in the early time points of viral infection . Rubicon positively regulated HCV replication , apparently by increasing the pool of autophagosomes , which could serve as the sites for HCV RNA replication . On the contrary , UVRAG , which reduced the pool of autophagosomes , inhibited HCV replication if it was over-expressed prior to HCV infection . The analysis of HCV genes indicated that the HCV NS4B protein was sufficient to induce the expression of Rubicon and the accumulation of autophagosomes . Our results thus revealed a novel mechanism used by a virus to temporally regulate the autophagic flux for its replication .