A Peptide Derived from RNA Recognition Motif 2 of Human La Protein Binds to Hepatitis C Virus Internal Ribosome Entry Site, Prevents Ribosomal Assembly, and Inhibits Internal Initiation of Translation

Abstract

Human La protein is known to interact with hepatitis C virus (HCV) internal ribosome entry site (IRES) and stimulate translation. Previously, we demonstrated that mutations within HCV SL IV lead to reduced binding to La-RNA recognition motif 2 (RRM2) and drastically affect HCV IRES-mediated translation. Also, the binding of La protein to SL IV of HCV IRES was shown to impart conformational alterations within the RNA so as to facilitate the formation of functional initiation complex. Here, we report that a synthetic peptide, LaR2C, derived from the C terminus of La-RRM2 competes with the binding of cellular La protein to the HCV IRES and acts as a dominant negative inhibitor of internal initiation of translation of HCV RNA. The peptide binds to the HCV IRES and inhibits the functional initiation complex formation. An Huh7 cell line constitutively expressing a bicistronic RNA in which both cap-dependent and HCV IRES-mediated translation can be easily assayed has been developed. The addition of purified TAT-LaR2C recombinant polypeptide that allows direct delivery of the peptide into the cells showed reduced expression of HCV IRES activity in this cell line. The study reveals valuable insights into the role of La protein in ribosome assembly at the HCV IRES and also provides the basis for targeting ribosome-HCV IRES interaction to design potent antiviral therapy.Hepatitis C virus (HCV), a member of the Flaviviridae family, is an enveloped positive-sense, single-stranded RNA virus (10). The 9.6-kb-long genome encodes a single polyprotein of about 3,000 amino acids. The polyprotein is processed by host cell and viral proteases into three major structural proteins and several nonstructural proteins necessary for viral replication (4, 18). HCV causes a variety of liver diseases in humans, including liver cirrhosis and hepatocellular carcinoma (20,32). It is estimated that about 3% of the world population is infected with HCV, and about 85% of infected individuals develop chronic infection. Current options for treating HCV involve alpha interferon alone or in combination with ribavirin (6, 13). However, these treatments fail to achieve sustained virological response in the majority of patients, thus emphasizing the need for novel therapeutic approaches to combat HCV infection.Translation initiation of HCV occurs in a cap-independent manner wherein the ribosomes are recruited onto an internal ribosome entry site (IRES) located mostly within the 5Ј untranslated region (UTR) and extending a few nucleotides into the coding region (31, 37, 39). HCV IRES has been shown to form three complex stem-loops and a pseudoknot, which encompasses the initiator AUG codon (7). Although the HCV IRES binds to the 40S ribosomal subunit specifically and stably even in absence of any initiation factors, efficient translation requires canonical initiation factors like eIF2 and eIF3 (21,27,33) and other noncanonical trans-acting cellular proteins including polypyrimidine tract-binding protein (1), La autoantigen (2), poly(rC) binding protein (1...

Virological effects and safety of combined double filtration plasmapheresis (DFPP) and interferon therapy in patients with chronic hepatitis C: A preliminary study

“…An NS3 protease inhibitor has been developed as a novel antiviral agent, and clinical studies have been performed using this agent; however, the clinical application of this drug will take time because of the occurrence of certain adverse events [8]. The development of an NS5 polymerase inhibitor and an internal ribosome entry site (IRES)-targeting agent is in progress [9][10][11].…”

Structural Determinant of Human La Protein Critical for Internal Initiation of Translation of Hepatitis C Virus RNA

“…32 P]UTP to generate the labeled HCV IRES RNA as described previously (25). The HCV IRES-containing monocistronic construct (HCV luciferase) was linearized with XhoI to prepare HCV Luc RNA, and the HCV bicistronic construct was linearized with PmeI to generate capped bicistronic RNA (Rluc-HCV IRES-Fluc) to use in the in vitro translation studies.…”

“…Recently, we have demonstrated that a 24-mer peptide (LaR2C) derived from the C terminus of RRM (230-300) of La protein competes with the cellular La protein binding to the HCV IRES and interferes with the functional initiation complex formation (25). It appears that LaR2C interferes with 48S ribosome complexes, rendering it incompetent for 60S joining during internal initiation of translation of HCV RNA (25).…”

Targeted delivery of hepatitis C virus-specific short hairpin RNA in mouse liver using Sendai virosomes

“…Since the mechanism of ribosome assembly is unique and fundamentally different from the cap-dependent translation of the host cell mRNA, it can be targeted to selectively inhibit viral protein synthesis and consequently its replication (Dasgupta et al, 2004;Hellen & Sarnow, 2001;Trowbridge & Gowans, 1998). Previously, we have demonstrated the importance of the GCAC sequence near the iAUG for ribosome assembly onto HCV IRES RNA (Pudi et al, 2003(Pudi et al, , 2005. In fact, human La protein (an important host factor) has been shown to bind to the above sites and a peptide derived from the RNA-binding region of La protein has been shown to interfere with the ribosome assembly and inhibit viral RNA translation (Ali et al, 2000;Mondal et al, 2008;Pudi et al, 2004Pudi et al, , 2005.…”

“…Previously, we have demonstrated the importance of the GCAC sequence near the iAUG for ribosome assembly onto HCV IRES RNA (Pudi et al, 2003(Pudi et al, , 2005. In fact, human La protein (an important host factor) has been shown to bind to the above sites and a peptide derived from the RNA-binding region of La protein has been shown to interfere with the ribosome assembly and inhibit viral RNA translation (Ali et al, 2000;Mondal et al, 2008;Pudi et al, 2004Pudi et al, , 2005. Here, we have investigated the ability of an shRNA targeting this region of HCV IRES to inhibit translation and replication of viral RNA.…”