scholarly journals Location specific annealing of miR-122 and other small RNAs defines an Hepatitis C Virus 5′ UTR regulatory element with distinct impacts on virus translation and genome stability

2020 ◽  
Vol 48 (16) ◽  
pp. 9235-9249 ◽  
Author(s):  
Rasika D Kunden ◽  
Sarah Ghezelbash ◽  
Juveriya Q Khan ◽  
Joyce A Wilson
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Small Rna ◽  
Small Rnas ◽  
Rna Structure ◽  
Genome Stability ◽  
Regulatory Element ◽  
Entry Site ◽  
Genome Stabilization

Abstract Hepatitis C virus (HCV) replication requires annealing of a liver specific small-RNA, miR-122 to 2 sites on 5′ untranslated region (UTR). Annealing has been reported to (a) stabilize the genome, (b) stimulate translation and (c) promote the formation of translationally active Internal Ribosome Entry Site (IRES) RNA structure. In this report, we map the RNA element to which small RNA annealing promotes HCV to nucleotides 1–44 and identify the relative impact of small RNA annealing on virus translation promotion and genome stabilization. We mapped the optimal region on the HCV genome to which small RNA annealing promotes virus replication to nucleotides 19–37 and found the efficiency of viral RNA accumulation decreased as annealing moved away from this region. Then, by using a panel of small RNAs that promote replication with varying efficiencies we link the efficiency of lifecycle promotion with translation stimulation. By contrast small RNA annealing stabilized the viral genome even if they did not promote virus replication. Thus, we propose that miR-122 annealing promotes HCV replication by annealing to an RNA element that activates the HCV IRES and stimulates translation, and that miR-122 induced HCV genome stabilization is insufficient alone but enhances virus replication.

scholarly journals Location specific small RNA annealing to the HCV 5’ UTR promotes Hepatitis C Virus replication by favoring IRES formation and stimulating virus translation

2020 ◽  
Author(s):  
Rasika D. Kunden ◽  
Sarah Ghezelbash ◽  
Juveriya Q. Khan ◽  
Joyce A. Wilson
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Small Rna ◽  
Small Rnas ◽  
Rna Structure ◽  
Mechanistic Model ◽  
Genome Replication ◽  
Entry Site ◽  
Genome Stabilization

ABSTRACTHepatitis C virus (HCV) genome replication requires annealing of a liver specific small-RNA, miR-122 to 2 sites on 5’ untranslated region (UTR). Annealing has been reported to a) stabilize the genome, b) promote translation, and c) induce the canonical HCV 5’ UTR Internal Ribosome Entry Site (IRES) structure. In this report we identify the relative impact of small RNA annealing on the three functions ascribed to miR-122 and generate a mechanistic model for miR-122 promotion of HCV. First, we identified that perfectly complementary small RNAs that anneal to different locations on the HCV 5’ UTR stimulate replication with varying efficiencies and mapped the region on the HCV genome to which small RNA annealing promotes virus replication. Second, by using a panel of small RNAs that promote with varying efficiencies we link HCV replication induction with translation stimulation and 5’ UTR RNA structure modifications. However, replication promotion was not linked to genome stabilization since all small RNAs tested could stabilize the viral genome regardless of their ability to promote replication. Thus, we propose that miR-122 annealing promotes HCV replication primarily by activating the HCV IRES and stimulating translation, and that miR-122-induced HCV genome stabilization is insufficient alone but enhances virus replication.Graphical Abstract

scholarly journals Enhanced virus translation enables miR-122-independent Hepatitis C Virus propagation

2021 ◽  
Author(s):  
Mamata Panigrahi ◽  
Michael Palmer ◽  
Joyce A Wilson
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Rna Structure ◽  
Genome Stability ◽  
Internal Ribosomal Entry Site ◽  
Terminal Region ◽  
Translation Regulation ◽  
Virus Propagation ◽  
Entry Site

The 5’UTR of the Hepatitis C Virus genome forms RNA structures that regulate virus replication and translation. The region contains a viral internal ribosomal entry site and a 5’ terminal region. Binding of the liver specific miRNA, miR-122, to two conserved binding sites in the 5’ terminal region regulates viral replication, translation, and genome stability, and is essential for efficient virus replication, but its precise mechanism of its action is still under debate. A current hypothesis is that miR-122 binding stimulates viral translation by facilitating the viral 5’ UTR to form the translationally active HCV IRES RNA structure. While miR-122 is essential for detectable virus replication in cell culture, several viral variants with 5’ UTR mutations exhibit low level replication in the absence of miR-122. We show that HCV mutants capable of replicating independently of miR-122 also replicate independently of other microRNAs generated by the canonical miRNA synthesis pathway. Further, we also show that the mutant genomes display an enhanced translation phenotype that correlates with their ability to replicate independently of miR-122. Finally, we provide evidence that translation regulation is the major role for miR-122, and show that miR-122-independent HCV replication can be rescued to miR-122-dependent levels by the combined impacts of 5’ UTR mutations that stimulate translation, and by stabilizing the viral genome by knockdown of host exonucleases and phosphatases that degrade the genome. Thus, we provide a model suggesting that translation stimulation and genome stabilization are the primary roles for miR-122 in the virus life cycle.

scholarly journals Secondary Structure and Hybridization Accessibility of Hepatitis C Virus 3′-Terminal Sequences

2002 ◽  
Vol 76 (19) ◽  
pp. 9563-9574 ◽  
Author(s):  
Robert M. Smith ◽  
Cherie M. Walton ◽  
Catherine H. Wu ◽  
George Y. Wu
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Secondary Structure ◽  
Rna Structure ◽  
Genome Stability ◽  
Rnase H ◽  
Hcv Rna ◽  
Positive Strand ◽  
Negative Strand

ABSTRACT The 3′-terminal sequences of hepatitis C virus (HCV) positive- and negative-strand RNAs contribute cis-acting functions essential for viral replication. The secondary structure and protein-binding properties of these highly conserved regions are of interest not only for the further elucidation of HCV molecular biology, but also for the design of antisense therapeutic constructs. The RNA structure of the positive-strand 3′ untranslated region has been shown previously to influence binding by various host and viral proteins and is thus thought to promote HCV RNA synthesis and genome stability. Recent studies have attributed analogous functions to the negative-strand 3′ terminus. We evaluated the HCV negative-strand secondary structure by enzymatic probing with single-strand-specific RNases and thermodynamic modeling of RNA folding. The accessibility of both 3′-terminal sequences to hybridization by antisense constructs was evaluated by RNase H cleavage mapping in the presence of combinatorial oligodeoxynucleotide libraries. The mapping results facilitated identification of antisense oligodeoxynucleotides and a 10-23 deoxyribozyme active against the positive-strand 3′-X region RNA in vitro.

Interferons alpha, beta, gamma each inhibit hepatitis C virus replication at the level of internal ribosome entry site-mediated translation.

2005 ◽  
Vol 25 (3) ◽  
pp. 580-594 ◽  
Author(s):  
Srikanta Dash ◽  
Ramesh Prabhu ◽  
Sidhartha Hazari ◽  
Frank Bastian ◽  
Robert Garry ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Internal Ribosome Entry Site ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Alpha Beta

scholarly journals Quasispecies Changes with Distinctive Point Mutations in the Hepatitis C Virus Internal Ribosome Entry Site (IRES) Derived from PBMCs and Plasma

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Luca Mercuri ◽  
Emma C. Thomson ◽  
Joseph Hughes ◽  
Peter Karayiannis
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Rna Structure ◽  
Mononuclear Cells ◽  
Point Mutations ◽  
Pcr Cloning ◽  
Entry Site ◽  
Peripheral Blood Mononuclear ◽  
Internal Ribosome Entry

The 5’ untranslated region (UTR) of the hepatitis C virus (HCV) genome contains the internal ribosome entry site (IRES), a highly conserved RNA structure essential for cap-independent translation of the viral polyprotein. HCV, apart from the liver, is thought to be associated with lymphocyte subpopulations of peripheral blood mononuclear cells (PBMCs), in lymph nodes and brain tissue. In this study, RT-PCR, cloning, and sequence analysis were employed to investigate the quasispecies nature of the 5’UTR following extraction of viral RNA from PBMCs and plasma of HCV infected individuals. The nucleotide variation between IRES-derived sequences from PBMCs and plasma indicated the existence of polymorphic sites within the IRES. HCV isolates had divergent variants with unique mutations particularly at positions 107, 204, and 243 of the IRES. Most of the PBMC-derived sequences contained an A-A-A variant at these positions. The mutations associated with the IRESes suggested the presence of unique quasispecies populations in PBMCs compared with plasma.

scholarly journals Small Interfering RNA Targeted to Hepatitis C Virus 5′ NontranslatedRegion Exerts Potent Antiviral Effect

2006 ◽  
Vol 81 (2) ◽  
pp. 669-676 ◽  
Author(s):  
Tatsuo Kanda ◽  
Robert Steele ◽  
Ranjit Ray ◽  
Ratna B. Ray
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Hcv Infection ◽  
Therapeutic Modality ◽  
Genome Replication ◽  
Entry Site ◽  
Hcv Genotype ◽  
Conserved Sequence

ABSTRACT Hepatitis C virus (HCV) is a major cause of cirrhosis and hepatocellular carcinoma. Interferon alone or together with ribavirin is the only therapy for HCV infection; however, a significant number of HCV-infected individuals do not respond to this treatment. Therefore, the development of new therapeutic options against HCV is a matter of urgency. In the present study, we have examined vectors carrying short hairpin RNA (shRNA) targeting the 5′ nontranslated conserved region of the HCV genome for inhibition of virus replication. Initially, three sequences were selected, and all three shRNAs (psh-53, psh-274, and psh-375) suppressed HCV internal ribosome entry site (IRES)-mediated translation to different degrees in Huh-7 cells. Next, we introduced siRNA into Huh-7.5 cells persistently infected with HCV genotype 2a (JFH1). The most efficient inhibition of JFH1 replication was observed with psh-274, targeted to the portion from subdomain IIId to IIIe of the IRES. Subsequently, Huh-7.5 cells stably expressing psh-274 further displayed a significant reduction in HCV JFH1 replication. The effect of psh-274 on cell-culture-grown HCV genotype 1a (H77) was also evaluated, and inhibition of virus replication and infectivity titers was observed. In the absence of a cell-culture-grown HCV genotype 1b, the effects of psh-274 on subgenomic and full-length replicons were examined, and efficient inhibition of genome replication was observed. Therefore, we have identified a conserved sequence targeted to the HCV genome that can inhibit replication of different genotypes, suggesting the potential of siRNA as an additional therapeutic modality against HCV infection.

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