Understanding the role of paraspeckle components NEAT1 and PSPC1 in HCV life cycle and pathogenesis

Abstract

Hepatitis C virus (HCV) is an enveloped, small, icosahedral, positive-sense single-stranded RNA virus that belongs to the Hepacivirus genus and the Flaviviridae family. HCV causes acute and chronic hepatitis which can range in severity from a moderate condition to a serious life-long condition involving liver cirrhosis and cancer. Around 1.5 million new cases of the hepatitis C virus are reported each year, with an estimated 58 million people worldwide carrying the infection. According to the WHO report, 2,90,000 people died of hepatitis C infection in 2019, primarily due to cirrhosis and hepatocellular carcinoma (HCC). HCV infection causes dysregulation of various host factors and signaling pathways, which might result in HCC. Several cellular entities, including paraspeckle components, are involved in the viral life cycle, pathogenesis, and disease progression. Paraspeckles are subnuclear bodies, composed of proteins like Paraspeckle Component 1 (PSPC1), Splicing Factor Proline and Glutamine Rich (PSF), and Non-POU Domain Containing Octamer Binding (NONO), etc. that bind to lncRNA Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) and form a paraspeckle. They trap various proteins and mRNAs, hindering their function and thereby affecting the cell’s continued response to stress. To understand the molecular basis of HCC, we have studied some of the key components of paraspeckle, the lncRNA NEAT1 and PSPC1 protein in HCV life cycle and pathogenesis. The specific aims of this study are: 1a. Understanding the role of NEAT1 in HCV-induced Hepatocellular Carcinoma 1b. Elucidation of the role of NEAT1 on the HCV life cycle 2. Possible role of paraspeckle protein PSPC1 in the HCV life cycle Part 1a: Understanding the role of NEAT1 in HCV-induced Hepatocellular Carcinoma NEAT1 lncRNA is a major component of paraspeckles that has been linked to several malignancies. Analysis of ‘The Cancer Genome Atlas (TCGA)’ database and validation in HCV-induced HCC tissue and serum samples showed significant high expression of NEAT1 in patients with liver cancer. Moreover, it was observed that NEAT1 levels increased with HCV infection, along with the size and number of the paraspeckles. To further understand the mechanism of NEAT1-induced HCC progression, we selected one of its targets, miR-9-5p, which regulates Beta Ig-H3 (BGH3) mRNA levels. Interestingly, miR-9-5p levels were found to be downregulated upon HCV infection, whereas BGH3 levels were upregulated showing a reverse correlation. Partial knockdown of NEAT1 increased miR-9-5p levels and decreased BGH3 levels, corroborating our initial findings. BGH3 levels were also found to be upregulated in HCV-induced HCC which is consistent with TCGA data and could be directly correlated with NEAT1 levels. As a known oncogene, BGH3 is directly linked to HCC progression mediated by NEAT1. Direct-acting antiviral (DAA)-based treatment is likely to minimize the progression of liver disease and liver-related mortality linked to HCV infection. However, in cirrhotic patients, the risk of HCC persists even after HCV treatment. Elevated levels of NEAT1 were found in serum samples of patients even after treatment with direct-acting antivirals (DAA), indicating that NEAT1 might be a molecular trigger that promotes pathogenesis. Collectively, these findings provide molecular insights into HCV-induced HCC progression via the NEAT1-miR-9-BGH3 axis. Part 1b: Elucidation of the role of NEAT1 on the HCV life cycle The expression of NEAT1 gets elevated in response to viral infection as a stress-induced lncRNA upregulation strategy. However, there are no reports on how NEAT1 or paraspeckles are involved in HCV infection. Interestingly, siRNA-mediated partial silencing of NEAT1 in Huh 7.5 cells increased both positive and negative-strand viral RNA, suggesting that NEAT1 negatively affects the HCV life cycle. NEAT1 resides in the paraspeckles, where it is associated with the proteins, PSF, NONO, and PSPC1. Several reports have indicated that in normal cells, PSF protein is bound to the promoter of several immune-responsive genes and represses their transcription. Upon viral infection, increased NEAT1 level sequesters PSF from the promoter of immune-related genes, thereby increasing their expression. PSF can bind to the DDX60 promoter and inhibit its transcription. We observed that DDX60 level decreases upon silencing of NEAT1 in the background of HCV infection. It appears, upon HCV infection, NEAT1 sequesters PSF from the DDX60 promoter, and hence DDX60 transcription increases. DDX60 is known to inhibit HCV RNA replication, which is regulated by NEAT1, and PSF. Results suggest that NEAT1 regulates HCV RNA by regulating DDX60 levels.

Part 2: Possible role of paraspeckle protein PSPC1 in the HCV life cycle Paraspeckle protein PSPC1 (Paraspeckle Component 1) binds to poly(A), poly(G), and poly(U) RNA homopolymers, transports lipid-related genes from nucleus to cytoplasm and increases cell proliferation. PSPC1 is also known for regulating the life cycle of different viruses, but no reports are available on its role during HCV infection. We investigated the role of PSPC1 in HCV life cycle. Earlier, from our lab, we reported several RNA binding proteins of the host, such as HuR, La, and PTB, which relocalizes from the nucleus to the cytoplasm, bind to HCV RNA, and affect its life cycle. Similarly, PSPC1 protein was also found to be relocalized to cytoplasm upon HCV infection. Partial silencing of PSPC1 increased HCV RNA levels, suggesting negative regulation by PSPC1. Immuno-pulldown using anti-PSPC1 antibody followed by RT-qPCR suggested the binding of PSPC1 protein to HCV RNA. Bioinformatics analysis and UV-crosslinking experiments validated the binding of PSPC1 in the HCV 5’ UTR in the SLIV region. Competition UV-crosslinking experiment using cytoplasmic extract (S10) of Huh7.5 cells suggested that PSPC1 can replace host factors from the HCV 5’ UTR. We further observed that PSPC1 protein levels gradually decreased upon HCV infection. It appears, that as a host response, PSPC1 reduces HCV RNA level, and to prevent that as a viral response, HCV decreases PSPC1 protein level. Taken together, in this study the NEAT1/miR-9-5p/BGH3 axis has been linked to the disease progression. Results put forward the idea of using the NEAT1 level as a potential biological marker for disease prognosis to help better strategize the follow-up treatment approaches. Also, the study unravels the dual control of the HCV life cycle by the Paraspeckle components, NEAT1, and PSPC1, which in turn contributes to viral pathogenesis.