Functional Characterization of Nuclear Localization and Export Signals in Hepatitis C Virus Proteins and Their Role in the Membranous Web

Abstract

The hepatitis C virus (HCV) is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS) and nuclear export signals (NES) have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC) proteins (termed nucleoporins or Nups) are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin α5 (IPOA5/kapα1), importin β3 (IPO5/kap β3), and exportin 1 (XPO1/CRM1) both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication.

Proteomics of HCV virions reveals an essential role for the nucleoporin Nup98 in virus morphogenesis

“…We propose that HCV hijacks Nup98 physiological functions to transport HCV structural components from different cellular compartments, thus coordinating virion morphogenesis. Accordingly, several HCV proteins, including Core, contain functional nuclear localization signal and nuclear export signal sequences that are required to bind cargo proteins and cross the NPC (17). This hypothesis would be in agreement with the model proposed by Neufeldt et al (16), which suggests the insertion of NPC in the cytoplasm of infected cells to compartmentalize virus replication/assembly.…”

The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites

“…Previously, we have shown that components of the nuclear pore complex (NPC) redistribute to regions of HCV replication and assembly within the MW, and we provided evidence supporting a role for the nuclear transport machinery in the formation and function of the MW [37,38]. Comprised of~30 distinct proteins, termed nucleoporins (Nups), the NPC is a large macromolecular structure that facilitates bidirectional transport of macromolecules across the nuclear envelope between the cytoplasm and nucleoplasm.…”

Importin β1 targeting by hepatitis C virus NS3/4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response

“…Indeed, many proteomic and functional RNAi genetic screens revealed that modulation of nuclear pore complexes (NPCs), nuclear transport receptors and the RAN‐GTPase system have profound effects on viral replication . For instance, interactions between viral proteins, nucleoporins (Nups), RAN, importin‐α (IMP‐α), importin‐β (IMP‐β) and exportin (EXP) have been observed with hepatitis C virus (HCV), picornavirus, and human immuno‐deficiency virus (HIV)‐1 . In HCV infection, viral proteins (core, NS2, NS3 and NS5A) harbor a nuclear localization signal (NLS) and/or nuclear export signal (NES) suggesting that nuclear‐cytoplasmic shuttling is important for it virus life cycle that is mostly within the cytoplasm .…”

“…For instance, interactions between viral proteins, nucleoporins (Nups), RAN, importin‐α (IMP‐α), importin‐β (IMP‐β) and exportin (EXP) have been observed with hepatitis C virus (HCV), picornavirus, and human immuno‐deficiency virus (HIV)‐1 . In HCV infection, viral proteins (core, NS2, NS3 and NS5A) harbor a nuclear localization signal (NLS) and/or nuclear export signal (NES) suggesting that nuclear‐cytoplasmic shuttling is important for it virus life cycle that is mostly within the cytoplasm . Indeed, Nups were reported to accumulate in virus‐induced endoplasmic reticulum (ER)‐derived membranous structures where HCV replication occurs (so‐called membranous webs), indicating that Nups can gate these cytoplasmic compartments to promote viral RNA replication and to prevent cytosolic RIG‐I‐like receptor (RLR) sensing of viral RNA .…”

“…In HCV infection, viral proteins (core, NS2, NS3 and NS5A) harbor a nuclear localization signal (NLS) and/or nuclear export signal (NES) suggesting that nuclear‐cytoplasmic shuttling is important for it virus life cycle that is mostly within the cytoplasm . Indeed, Nups were reported to accumulate in virus‐induced endoplasmic reticulum (ER)‐derived membranous structures where HCV replication occurs (so‐called membranous webs), indicating that Nups can gate these cytoplasmic compartments to promote viral RNA replication and to prevent cytosolic RIG‐I‐like receptor (RLR) sensing of viral RNA . This is further supported by the findings that silencing IMPβ1, TNPO1 (KPNB2) and RAN, all involved with nucleocytoplasmic transport and elucidated to interact with NS3/4A protein, decreases HCV replication .…”