TIM-1 Promotes Hepatitis C Virus Cell Attachment and Infection

Abstract

Human TIM and TAM family proteins were recently found to serve as phosphatidylserine (PS) receptors which promote infections by many different viruses, including dengue virus, West Nile virus, Ebola virus, Marburg virus, and Zika virus. In the present study, we provide substantial evidence demonstrating that TIM-1 is important for efficient infection by hepatitis C virus (HCV). The knockdown of TIM-1 expression significantly reduced HCV infection but not HCV RNA replication. Likewise, TIM-1 knockout in Huh-7.5 cells remarkably lowered HCV cell attachment and subsequent HCV infection. More significantly, the impairment of HCV infection in the TIM-1 knockout cells could be restored completely by ectopic expression of TIM-1 but not TIM-3 or TIM-4. Additionally, HCV infection and cell attachment were inhibited by PS but not by phosphatidylcholine (PC), demonstrating that TIM-1-mediated enhancement of HCV infection is PS dependent. The exposure of PS on the HCV envelope was confirmed by immunoprecipitation of HCV particles with a PS-specific monoclonal antibody. Collectively, these findings demonstrate that TIM-1 promotes HCV infection by serving as an attachment receptor for binding to PS exposed on the HCV envelope.IMPORTANCE TIM family proteins were recently found to enhance infections by many different viruses, including several members of the Flaviviridae family. However, their importance in HCV infection has not previously been examined experimentally. The TIM family proteins include three members in humans: TIM-1, TIM-3, and TIM-4. The findings derived from our studies demonstrate that TIM-1, but not TIM-3 or TIM-4, promotes HCV infection by functioning as an HCV attachment factor. Knockout of the TIM-1 gene resulted in a remarkable reduction of HCV cell attachment and infection. PS-containing liposomes blocked HCV cell attachment and subsequent HCV infection. HCV particles could also be precipitated with a PS-specific monoclonal antibody. These findings suggest that TIM-1 and its binding ligand, PS, may serve as novel targets for antiviral intervention. KEYWORDS hepatitis C virus, TIM-1, attachment, infection, receptor, TIM-3, TIM-4 H epatitis C virus (HCV) is an enveloped RNA virus containing a 9.6-kb singlestranded RNA genome of positive polarity (1). It is the prototype member of the Hepacivirus genus in the Flaviviridae family (2, 3). The viral RNA genome consists of a long open reading frame (ORF), encoding a single polyprotein, and untranslated regions (UTRs) at both the 5= and 3= ends. Upon translation, the viral polyprotein precursor is cleaved by cellular peptidases and the viral NS2/NS3 metalloprotease and NS3/4A serine protease into 10 individual structural and nonstructural (NS) proteins, designated core (C), envelope proteins 1 and 2 (E1 and E2), p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (4). The structural proteins C, E1, and E2 are essential for the formation of HCV particles (5). The NS3 to NS5B proteins are the minimal set of viral proteins required for HCV RNA replication, although a...

Determinants in the Ig Variable Domain of Human HAVCR1 (TIM-1) Are Required To Enhance Hepatitis C Virus Entry

“…However, binding of PS to a PS receptor alone is not sufficient to mediate the HAVCR1-HCV interaction, since mHavcr1 did not enhance HCV infection, suggesting that the HAVCR1 IgV may also interact with other molecules at the virion surface, such as E1E2. Our results are consistent with previous studies showing that the human HAVCR1 (TIM-1) homologs HAVCR2 (also known as TIM-3) and TIM-4, which are also PS receptors, did not enhance HCV infection (26) and indicate that apoptotic mimicry may not be the only mechanism by which HAVCR1 enhances HCV infection.…”

“…3E). This requirement for relatively high concentrations of PS-liposomes to achieve inhibition of HCVcc entry is consistent with previously published data on the role of HAVCR-1 (TIM-1) in HCV entry (26). Similarly, treatment of viral particles with a micromolar concentration of annexin V, a cellular protein that binds to PS, partially inhibited HCVcc entry ( Fig.…”

“…HAVCR1 (TIM-1) has been implicated in cell entry of enveloped viruses by apoptotic mimicry via interaction with envelope phospholipids (23). Because HAVCR1 polymorphisms have been associated with the outcome of HCV infection (24,25) and HAVCR1 (TIM-1) enhances HCV infection in cell culture (26), we further examined the interaction of HCV with HAVCR1 to understand the mechanism(s) involved in the infection enhancement. To do so, we studied entry into human hepatoma cells using cell culturederived (HCVcc) and HCV envelope-pseudotyped (HCVpp) viruses.…”

Hepatitis C Virus Entry: An Intriguingly Complex and Highly Regulated Process

“…Interestingly, in addition to their role in viral attachment, these host factors have been shown to also contribute to later steps of the viral life cycle, such as post-binding steps [52,53], internalization [54] or replication [55]. Recently, TIM-1/human hepatitis A virus cellular receptor 1 (HAVCR1)/CD365, a phosphatidylserine receptor that serves as a host factor for various Flaviviridae viruses has been identified as an additional factor contributing to HCV attachment via interaction with phosphatidylserine exposed on the HCV envelope [56]. It has been suggested that HCV-TIM-1 interaction may stabilize/enhance viral attachment and promote subsequent interaction with the main entry factors [57].…”

Title: Mechanisms of Host Cell Binding and Neurotropism of Zika Virus

“…Given that MDCK cells are still permissive for ZIKV replication 40 , we hypothesized that a more generalized receptor may be contributing to viral adsorption when ZVBM is NAGylated. The association of human AXL with ZIKV adsorption 31–33 suggests that viral phosphatidyl serine (PS) may facilitate entry into certain host cells by binding Gas6, which in turn binds AXL, as is seen with multiple viruses 41–45 . We pretreated ZIKV strains MR_766 and PRVABC59 with the PS-binding protein Annexin V prior to infection of Vero cell monolayers.…”