Hepatitis C Virus NS5A Targets Nucleosome Assembly Protein NAP1L1 To Control the Innate Cellular Response

ABSTRACT Hepatitis C virus (HCV) is a single-stranded positive-sense RNA hepatotropic virus. Despite cellular defenses, HCV is able to replicate in hepatocytes and to establish a chronic infection that could lead to severe complications and hepatocellular carcinoma. An important player in subverting the host response to HCV infection is the viral nonstructural protein NS5A, which, in addition to its role in replication and assembly, targets several pathways involved in the cellular response to viral infection. Several unbiased screens identified nucleosome assembly protein 1-like 1 (NAP1L1) as an interaction partner of HCV NS5A. Here we confirmed this interaction and mapped it to the C terminus of NS5A of both genotype 1 and 2. NS5A sequesters NAP1L1 in the cytoplasm, blocking its nuclear translocation. However, only NS5A from genotype 2 HCV, and not that from genotype 1, targets NAP1L1 for proteosome-mediated degradation. NAP1L1 is a nuclear chaperone involved in chromatin remodeling, and we demonstrated the NAP1L1-dependent regulation of specific pathways involved in cellular responses to viral infection and cell survival. Among those, we showed that lack of NAP1L1 leads to a decrease of RELA protein levels and a strong defect of IRF3 TBK1/IKKε-mediated phosphorylation, leading to inefficient RIG-I and Toll-like receptor 3 (TLR3) responses. Hence, HCV is able to modulate the host cell environment by targeting NAP1L1 through NS5A. IMPORTANCE Viruses have evolved to replicate and to overcome antiviral countermeasures of the infected cell. Hepatitis C virus is capable of establishing a lifelong chronic infection in the liver, which could develop into cirrhosis and cancer. Chronic viruses are particularly able to interfere with the cellular antiviral pathways by several different mechanisms. In this study, we identified a novel cellular target of the viral nonstructural protein NS5A and demonstrated its role in antiviral signaling. This factor, called nucleosome assembly protein 1-like 1 (NAP1L1), is a nuclear chaperone involved in the remodeling of chromatin during transcription. When it is depleted, specific signaling pathways leading to antiviral effectors are affected. Therefore, we provide evidence for both a novel strategy of virus evasion from cellular immunity and a novel role for a cellular protein, which has not been described to date.

Histone Chaperone Nap1 Is a Major Regulator of Histone H2A-H2B Dynamics at the InducibleGALLocus

Histone chaperones, like nucleosome assembly protein 1 (Nap1), play a critical role in the maintenance of chromatin architecture. Here, we use theGALlocus inSaccharomyces cerevisiaeto investigate the influence of Nap1 on chromatin structure and histone dynamics during distinct transcriptional states. When theGALlocus is not expressed, cells lacking Nap1 show an accumulation of histone H2A-H2B but not histone H3-H4 at this locus. Excess H2A-H2B interacts with the linker DNA between nucleosomes, and the interaction is independent of the inherent DNA-binding affinity of H2A-H2B for these particular sequences as measuredin vitro. When theGALlocus is transcribed, excess H2A-H2B is reversed, and levels of all chromatin-bound histones are depleted in cells lacking Nap1. We developed anin vivosystem to measure histone exchange at theGALlocus and observed considerable variability in the rate of exchange across the locus in wild-type cells. We recapitulate this variability within vitronucleosome reconstitutions, which suggests a contribution of DNA sequence to histone dynamics. We also find that Nap1 is required for transcription-dependent H2A-H2B exchange. Altogether, these results indicate that Nap1 is essential for maintaining proper chromatin composition and modulating the exchange of H2A-H2Bin vivo.

Nucleosome Assembly Protein 1-Like 1 (Nap1l1) Regulates the Proliferation of Murine Induced Pluripotent Stem Cells

Background/Aims: To investigate whether nucleosome assembly protein 1-like 1 (Nap1l1) regulates the proliferation of induced pluripotent stem cells (iPSC) and the potential mechanisms. Methods: Nap1l1-knockdown-iPSC and Nap1l1-overexpression-iPSC were constructed by transfection of lentiviral particles. The proliferation of iPSC was detected by MTT analysis, and cell cycle was analyzed by flow cytometry. Results: Nap1l1 overexpression promoted iPSC proliferation and induced G2/M transition compared to their control iPSC while Nap1l1-knockdown-iPSC dramatically displayed the reduced proliferation and accumulated G2/M phase cells. Further analysis showed that Nap1l1 overexpression in iPSC increased the expression of cyclin B1, downregulated the expression of p21 and p27, while knockdown of Nap1l1 showed the opposite effects. In addition, overexpression of Nap1l1 promoted the phosphorylation of AKT and ERK in iPSC, while knockdown of Nap1l1 inhibited the effects. However, these effects displayed in Nap1l1-overexpression-iPSC were greatly suppressed by the inhibition of AKT or ERK signaling. Conclusions: The results indicate that Nap1l1 promotes the proliferation of iPSC attributable to G2/M transition caused by downregulation of p27 and p21, and upregulation of cyclin B1, the activation of AKT or ERK is involved in the process. The present study has revealed a novel molecular mechanism involved in the proliferation of iPSC.