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

Abstract

Human La protein has been implicated in facilitating internal ribosome entry site (IRES)-mediated translation of hepatitis C virus (HCV).The mechanism of internal initiation of translation of hepatitis C virus (HCV) RNA is unique and fundamentally different from the cap-dependent translation of host cell mRNAs, and thus, the HCV internal ribosome entry site (IRES) offers a potential target for developing novel antiviral therapeutics (6).The ribosome assembly at the HCV IRES has been shown to be prokaryotic-like and requires a minimal number of initiation factors (23). The HCV IRES has been shown to be capable of binding directly to 40S ribosomal subunit with the help of the ribosomal protein S5 (10, 18). Although, HCV IRES binds to the 40S ribosomal subunit specifically and stably even in the absence of any initiation factors, efficient translation requires some of the canonical initiation factors and noncanonical trans-acting factors, possibly to facilitate ribosome binding and to ensure proper positioning of the initiator AUG (iAUG) codon in the P site (13). Several cellular trans-acting factors have been reported to be critically required for HCV IRES-mediated translation, which includes human La autoantigen (2).Human La protein was originally identified in the sera from patients with systemic lupus erythematosus and Sjögren's syndrome (28). Earlier, it was reported in the literature that La protein contains three RNA recognition motifs (RRMs), which were putatively located between residues 1 and 100 (previously named RRM1), 101 and 208 (previously, RRM2), and 209 and 300 (previously, RRM3) (12, 24). However, according to the recent nomenclature, the first structured domain in human La is termed "La motif" located between residues 16 and 75 ], followed by two RRMs, RRM (112-184) and RRM (230-300) (22,29). Although, based on the structure determinations, the precise boundaries of the structured cores were found to be slightly different , RRM (229-327)], they largely encompass the above regions (1, 17). La becomes associated with the 3Ј termini of many newly synthesized small RNAs made by RNA polymerase III, as well as certain small RNAs synthesized by other RNA polymerases (20,31). The N terminal 80 amino acid (aa) residues termed the La motif and the adjacent RRM part have been shown to be required for high-affinity binding with polymerase III transcripts, where the La motif helps in specific recognition for the polyuridylate (UUU OH ) sequence at the 3Ј end of the RNA. The C-terminal RRM (230-300) has been shown to have a beta sheet comprising five strands and a long C-terminal helix that binds to the putative RNA binding site (17). The central RRM (112-184) has been shown to possess a classical RRMtype fold containing four stranded beta sheets backed by two alpha helices. It also has an additional beta strand inserted between alpha 2 and beta 4. The helix alpha 3 sheet of RRM (112-184) is predominantly hydrophilic and protrudes away

MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR

“…Less likely, HCV RNA synthesis could have a notyet-identified nuclear phase. The 3′-terminal U stretch of U6 could associate with La protein, a protein required for IRESmediated translation of HCV RNA (23,24). Thus, in future studies it would be of great interest to determine whether U3, U6, and ATAD1, as well as associated proteins, are involved in the HCV replication process.…”

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

“…Transfection of shDNA constructs into cells harbouring HCV replicons was performed as described previously (Roy et al, 2008). Briefly, Huh7 cells harbouring the monocistronic HCV (genotype 2a) replicon, were transfected with either pSUPER vector (a generous gift from Dr Rene Bernards, Netherlands) alone (as control) or with 0.5 mM of the plasmid constructs encoding sh-SLIV or Nsp-sh RNA.…”

“…Semiquantitative RT-PCR was performed for HCV IRES positive and negative strand RNAs using standard protocols, as described previously (Roy et al, 2008). The PCR products were analysed on 1.2 % agarose gels.…”

“…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.…”

“…peptides etc. (Dasgupta et al, 2004;Pawlotsky et al, 2007;Roy et al, 2008). However, to date, a suitable system for selective and efficient delivery of small nucleic acid molecules into liver cells is lacking.…”