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miR-122 is a liver-specific microRNA (miRNA) that binds to two sites (S1 and S2) on the 5= untranslated region (UTR) of the hepatitis C virus (HCV) genome and promotes the viral life cycle. It positively affects viral RNA stability, translation, and replication, but the mechanism is not well understood. To unravel the roles of miR-122 binding at each site alone or in combination, we employed miR-122 binding site mutant viral RNAs, Hep3B cells (which lack detectable miR-122), and complementation with wild-type miR-122, an miR-122 with the matching mutation, or both. We found that miR-122 binding at either site alone increased replication equally, while binding at both sites had a cooperative effect. Xrn1 depletion rescued miR-122-unbound fulllength RNA replication to detectable levels but not to miR-122-bound levels, confirming that miR-122 protects HCV RNA from Xrn1, a cytoplasmic 5=-to-3= exoribonuclease, but also has additional functions. In cells depleted of Xrn1, replication levels of S1-bound HCV RNA were slightly higher than S2-bound RNA levels, suggesting that both sites contribute, but their contributions may be unequal when the need for protection from Xrn1 is reduced. miR-122 binding at S1 or S2 also increased translation equally, but the effect was abolished by Xrn1 knockdown, suggesting that the influence of miR-122 on HCV translation reflects protection from Xrn1 degradation. Our results show that occupation of each miR-122 binding site contributes equally and cooperatively to HCV replication but suggest somewhat unequal contributions of each site to Xrn1 protection and additional functions of miR-122. [ABSTRACT FROM AUTHOR]

Abstract: The study of Hepatitis C Virus (HCV) has benefitted from the use of the Huh7 cell culture system, but until recently there were no other widely used alternatives to this cell line. Here we render another human hepatoma cell line, Hep3B, permissive to the complete virus life cycle by supplementation with the liver-specific microRNA miR-122, known to aid HCV RNA accumulation. When supplemented, Hep3B cells produce J6/JFH-1 virus titres indistinguishable from those produced by Huh7.5 cells. Interestingly, we were able to detect and characterize miR-122-independent replication of di-cistronic replicons in Hep3B cells. Further, we show that Argonaute-2 (Ago2) is required for miR-122-dependent replication, but dispensable for miR-122-independent replication, confirming Ago2''s role in mediating the activity of miR-122. Thus Hep3B cells are a model system for the study of HCV, and miR-122 independent replication is a model to identify proteins involved in the function of miR-122. [Copyright &y& Elsevier]

Abstract: MicroRNA-122 (miR-122) promotes Hepatitis C Virus (HCV) RNA stability, accumulation, and translation through hybridization with the 5′ untranslated region (5′ UTR) of the HCV genome. Depletion of Dicer and TRBP, proteins involved in miRNA biogenesis, reduced HCV RNA accumulation, mature duplex miR-122 abundance, and miR-122 directed mRNA translation suppression, suggesting roles in miR-122 processing. HCV RNA accumulation independent of endogenous mature duplex miR-122 was not affected by Dicer knockdown, suggesting that Dicer is required solely for miR-122 biogenesis, but TRBP knockdown reduced HCV RNA accumulation in this system, suggesting an additional role in supporting HCV RNA accumulation. Mature duplex miR-122 and pre-miR-122 hairpin, but not single-stranded miR-122 (guide or ⁎ strand), augmented HCV RNA accumulation and translation, and Dicer and TRBP were essential for the activity of pre-miR-122 in mouse fibroblasts. Thus, canonical miRNA processing and strand selection is essential for the activity of miR-122 on HCV translation and RNA accumulation. [Copyright &y& Elsevier]

Hepatitis C virus (HCV) infection causes significant morbidity, and efficient mouse models would greatly facilitate virus studies and the development of effective vaccines and new therapeutic agents. Entry factors, innate immunity, and host factors needed for viral replication represent the initial barriers that restrict HCV infection of mouse cells. Experiments in this paper consider early postentry steps of viral infection and investigate the roles of interferon regulatory factors (IRF-3 and IRF-9) and microRNA (miR-122) in promoting HCV replication in mouse embryo fibroblasts (MEFs) that contain viral subgenomic replicons. While wild-type murine fibroblasts are restricted for HCV RNA replication, deletion of IRF-3 alone can facilitate replicon activity in these cells. This effect is thought to be related to the inactivation of the type I interferon synthesis mediated by IRF-3. Additional deletion of IRF-9 to yield IRF-3-/- IRF-9-/-/ MEFs, which have blocked type I interferon signaling, did not increase HCV replication. Expression of liver-specific miR-122 in MEFs further stimulated the synthesis of HCV replicons in the rodent fibroblasts. The combined effects of miR-122 expression and deletion of IRF-3 produced a cooperative stimulation of HCV subgenome replication. miR-122 and IRF-3 are independent host factors that are capable of influencing HCV replication, and our findings could help to establish mouse models and other cell systems that support HCV growth and particle formation. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) replication requires annealing of a liver specific microRNA, miR-122 to 2 sites on 5′ untranslated region (UTR). While, microRNAs downregulate gene expression by binding to the 3′ untranslated region of the target mRNA, in this case, the microRNA anneals to the 5′UTR of the viral genomes and upregulates the viral lifecycle. In this review, we explore the current understandings of the mechanisms by which miR-122 promotes the HCV lifecycle, and its contributions to pathogenesis. Annealing of miR-122 has been reported to (a) stimulate virus translation by promoting the formation of translationally active internal ribosome entry site (IRES) RNA structure, (b) stabilize the genome, and (c) induce viral genomic RNA replication. MiR-122 modulates lipid metabolism and suppresses tumor formation, and sequestration by HCV may influence virus pathogenesis. We also discuss the possible use of miR-122 as a biomarker for chronic hepatitis and as a therapeutic target. Finally, we discuss roles for miR-122 and other microRNAs in promoting other viruses. [ABSTRACT FROM AUTHOR]

Reverse genetic methods to manipulate viral genomes are key tools in modern virological experimentation. They allow for the generation of reporter virus genomes to simplify the assessment of virus growth and for the analysis of the impact of specific mutations in the genome on virus phenotypes. For SARS-CoV-2, reverse genetic systems are complicated by the large size of the viral genome and the instability of certain genomic sections in bacteria requiring the use of low-copy number bacterial artificial chromosome plasmids (bacmids). However, even with the use of bacmids, faithfully amplifying SARS-CoV-2 bacmids is often challenging. In this chapter, we describe a detailed protocol to grow SARS-CoV-2 bacmids and highlight the challenges and optimal techniques to produce large quantities of SARS-CoV-2 bacmids that are free of deletions and mutations. Overall, this chapter has recapitulated an overview of the maxi-preparation procedure for large unstable bacmids like SARS-CoV-2 to facilitate downstream applications., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The 5' untranslated region (UTR) of the hepatitis C virus (HCV) genome forms RNA structures that regulate virus replication and translation. The region contains an internal ribosomal entry site (IRES) and a 5'-terminal region. Binding of the liver-specific microRNA (miRNA) miR-122 to two 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 action is still unresolved. 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 replication of wild-type HCV genomes 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 display an enhanced translation phenotype that correlates with their ability to replicate independently of miR-122. Further, 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. Finally, 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. Thus, we provide a model suggesting that translation stimulation and genome stabilization are the primary roles for miR-122 in promoting HCV. IMPORTANCE The unusual and essential role of miR-122 in promoting HCV propagation is incompletely understood. To better understand its role, we have analyzed HCV mutants capable of replicating independently of miR-122. Our data show that the ability of viruses to replicate independently of miR-122 correlates with enhanced virus translation but that genome stabilization is required to restore efficient HCV replication. This suggests that viruses must gain both abilities to escape the need for miR-122 and impacts the possibility that HCV can evolve to replicate outside the liver., Competing Interests: The authors declare no conflict of interest.

The recent emergence of SARS-CoV-2 in the human population has caused a global pandemic. The virus encodes two proteases, M pro and PL pro , that are thought to play key roles in the suppression of host protein synthesis and immune response evasion during infection. To identify the specific host cell substrates of these proteases, active recombinant SARS-CoV-2 M pro and PL pro were added to A549 and Jurkat human cell lysates, and subtiligase-mediated N-terminomics was used to capture and enrich protease substrate fragments. The precise location of each cleavage site was identified using mass spectrometry. Here, we report the identification of over 200 human host proteins that are potential substrates for SARS-CoV-2 M pro and PL pro and provide a global mapping of proteolysis for these two viral proteases in vitro. Modulating proteolysis of these substrates will increase our understanding of SARS-CoV-2 pathobiology and COVID-19.

Despite the advancement in antiviral therapy, Hepatitis C remains a global health challenge and one of the leading causes of hepatitis related deaths worldwide. Hepatitis C virus, the causative agent, is a positive strand RNA virus that requires a liver specific microRNA called miR-122 for its replication. Unconventional to the canonical role of miRNAs in translation suppression by binding to 3'Untranslated Region (UTR) of messenger RNAs, miR-122 binds to two sites on the 5'UTR of viral genome and promotes viral propagation. In this review, we describe the unique relationship between the liver specific microRNA and HCV, the current knowledge on the mechanisms by which the virus uses miR-122 to promote the virus life cycle, and how miR-122 impacts viral tropism and pathogenesis. We will also discuss the use of anti-miR-122 therapy and its impact on viral evolution of miR-122-independent replication. This review further provides insight into how viruses manipulate host factors at the initial stage of infection to establish a successful infection.

A liver-specific microRNA, miR-122, anneals to the hepatitis C virus (HCV) genomic 5' terminus and is essential for virus replication in cell culture. However, bicistronic HCV replicons and full-length RNAs with specific mutations in the 5' untranslated region (UTR) can replicate, albeit to low levels, without miR-122. In this study, we have identified that HCV RNAs lacking the structural gene region or having encephalomyocarditis virus internal ribosomal entry site (EMCV IRES)-regulated translation had reduced requirements for miR-122. In addition, we found that a smaller proportion of cells supported miR-122-independent replication compared a population of cells supporting miR-122-dependent replication, while viral protein levels per positive cell were similar. Further, the proportion of cells supporting miR-122-independent replication increased with the amount of viral RNA delivered, suggesting that establishment of miR-122-independent replication in a cell is affected by the amount of viral RNA delivered. HCV RNAs replicating independently of miR-122 were not affected by supplementation with miR-122, suggesting that miR-122 is not essential for maintenance of an miR-122-independent HCV infection. However, miR-122 supplementation had a small positive impact on miR-122-dependent replication, suggesting a minor role in enhancing ongoing virus RNA accumulation. We suggest that miR-122 functions primarily to initiate an HCV infection but has a minor influence on its maintenance, and we present a model in which miR-122 is required for replication complex formation at the beginning of an infection and also supports new replication complex formation during ongoing infection and after infected cell division. IMPORTANCE The mechanism by which miR-122 promotes the HCV life cycle is not well understood, and a role in directly promoting genome amplification is still debated. In this study, we have shown that miR-122 increases the rate of viral RNA accumulation and promotes the establishment of an HCV infection in a greater number of cells than in the absence of miR-122. However, we also confirm a minor role in promoting ongoing virus replication and propose a role in the initiation of new replication complexes throughout a virus infection. This study has implications for the use of anti-miR-122 as a potential HCV therapy.

(1) Background: There is a strong need for prevention and treatment strategies for COVID-19 that are not impacted by SARS-CoV-2 mutations emerging in variants of concern. After virus infection, host ER resident sigma receptors form direct interactions with non-structural SARS-CoV-2 proteins present in the replication complex. (2) Methods: In this work, highly specific sigma receptor ligands were investigated for their ability to inhibit both SARS-CoV-2 genome replication and virus induced cellular toxicity. This study found antiviral activity associated with agonism of the sigma-1 receptor (e.g., SA4503), ligation of the sigma-2 receptor (e.g., CM398), and a combination of the two pathways (e.g., AZ66). (3) Results: Intermolecular contacts between these ligands and sigma receptors were identified by structural modeling. (4) Conclusions: Sigma receptor ligands and drugs with off-target sigma receptor binding characteristics were effective at inhibiting SARS-CoV-2 infection in primate and human cells, representing a potential therapeutic avenue for COVID-19 prevention and treatment.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Annealing of the liver-specific microRNA, miR-122, to the Hepatitis C virus (HCV) 5' UTR is required for efficient virus replication. By using siRNAs to pressure escape mutations, 30 replication-competent HCV genomes having nucleotide changes in the conserved 5' untranslated region (UTR) were identified. In silico analysis predicted that miR-122 annealing induces canonical HCV genomic 5' UTR RNA folding, and mutant 5' UTR sequences that promoted miR-122-independent HCV replication favored the formation of the canonical RNA structure, even in the absence of miR-122. Additionally, some mutant viruses adapted to use the siRNA as a miR-122-mimic. We further demonstrate that small RNAs that anneal with perfect complementarity to the 5' UTR stabilize and promote HCV genome accumulation. Thus, HCV genome stabilization and life-cycle promotion does not require the specific annealing pattern demonstrated for miR-122 nor 5' end annealing or 3' overhanging nucleotides. Replication promotion by perfect-match siRNAs was observed in Ago2 knockout cells revealing that other Ago isoforms can support HCV replication. At last, we present a model for miR-122 promotion of the HCV life cycle in which miRNA annealing to the 5' UTR, in conjunction with any Ago isoform, modifies the 5' UTR structure to stabilize the viral genome and promote HCV RNA accumulation.

Hepatitis C virus (HCV) recruits two molecules of the liver-specific microRNA-122 (miR-122) to the 5' end of its genome. This interaction promotes viral RNA accumulation, but the precise mechanism(s) remain incompletely understood. Previous studies suggest that miR-122 is able to protect the HCV genome from 5' exonucleases (Xrn1/2), but this protection is not sufficient to account for the effect of miR-122 on HCV RNA accumulation. Thus, we investigated whether miR-122 was also able to protect the viral genome from innate sensors of RNA or cellular pyrophosphatases. We found that miR-122 does not play a protective role against recognition by PKR, RIG-I-like receptors, or IFITs 1 and 5. However, we found that knockdown of both the cellular pyrophosphatases, DOM3Z and DUSP11, was able to rescue viral RNA accumulation of subgenomic replicons in the absence of miR-122. Nevertheless, pyrophosphatase knockdown increased but did not restore viral RNA accumulation of full-length HCV RNA in miR-122 knockout cells, suggesting that miR-122 likely plays an additional role(s) in the HCV life cycle, beyond 5' end protection. Overall, our results support a model in which miR-122 stabilizes the HCV genome by shielding its 5' terminus from cellular pyrophosphatase activity and subsequent turnover by exonucleases (Xrn1/2).

MicroRNAs (miRNAs) are small non-coding RNAs that function as part of RNA-induced silencing complexes that repress the expression of target genes. Over the past few years, miRNAs have been found to mediate complex regulation of a wide variety of mammalian viral infections, including Hepatitis C virus (HCV) infection. Here, we focus on a highly abundant, liver-specific miRNA, miR-122. In a unique and unusual interaction, miR-122 binds to two sites in the 5' untranslated region (UTR) of the HCV genome and promotes viral RNA accumulation. We will discuss what has been learned about this important interaction to date, provide insights into how miR-122 is able to modulate HCV RNA accumulation, and how miR-122 might be exploited for antiviral intervention., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Hepatitis C Virus (HCV) is a serious global health problem, infecting almost 3% of the world's population. The lack of model systems for studying this virus limit research options in vaccine and therapeutic development, as well as for studying the pathogenesis of chronic HCV infection. Herein we make use of the liver-specific microRNA miR-122 to render mouse cell lines permissive to HCV replication in an attempt to develop additional model systems for the identification of new features of the virus and its life cycle. We have determined that some wild-type and knockout mouse cell lines--NCoA6 and PKR knockout embryonic fibroblasts--can be rendered permissive to transient HCV sub-genomic RNA replication upon addition of miR-122, but we did not observe replication of full-length HCV RNA in these cells. However, other wild-type and knockout cell lines cannot be rendered permissive to HCV replication by addition of miR-122, and in fact, different NCoA6 and PKR knockout cell line passages and isolates from the same mice demonstrated varying permissiveness phenotypes and eventually complete loss of permissiveness. When we tested knockdown of NCoA6 and PKR in Huh7.5 cells, we saw no substantial impact in sub-genomic HCV replication, which we would expect if these genes were inhibitory to the virus' life cycle. This leads us to conclude that along with the influence of specific gene knockouts there are additional factors within the cell lines that affect their permissiveness for HCV replication; we suggest that these may be epigenetically regulated, or modulated by cell line immortalization and transformation.

The unusual role for miR-122 in promoting the hepatitis C virus (HCV) life cycle was first identified in 2005, but its mechanism of action remains uncharacterized. The virus appears to use the microRNA (miRNA) in a way that is opposed to that of normal miRNAs. Instead of interacting with sequences in the 3'-untranslated region (UTR), miR-122 binds to two sites in the 5'-UTR, and instead of silencing gene expression or promoting the degradation of the viral RNA, it stabilizes the genome and potently augments the efficiency by which HCV RNA accumulates in infected cells. This review discusses the current knowledge and models for the mechanism by which miR-122 promotes the HCV life cycle. Annealing of miR-122 to the HCV genome requires particular base pairing, stimulates translation, and stabilizes the viral genome by blocking degradation by host exonucleases, but these functions are unlikely to be the whole story. We will discuss other possible functions for miR-122, the stages of the HCV life cycle at which miR-122 may influence HCV, and other related viruses that may be similarly regulated by miR-122. Despite our lack of detailed mechanistic information, antagonism of miR-122 is emerging as a powerful method to inhibit HCV infections, and unique to other HCV treatment strategies does not, thus far, appear to induce emergence of escape mutants. Used alone or in combination with other antiviral drugs, miR-122 antagonists could be useful to both inhibit the virus and provide selective pressure to inhibit the development of resistance., (Copyright © 2013 John Wiley & Sons, Ltd.)

Hepatitis C Virus (HCV) infection-induced liver disease is a growing problem worldwide, and is the primary cause of liver failure requiring liver transplantation in North America. Improved therapeutic strategies are required to control and possibly eradicate HCV infections, and to modulate HCV-induced liver disease. Cellular microRNAs anneal to and regulate mRNA translation and stability and form a regulatory network that modulates virtually every cellular process. Thus, miRNAs are promising cellular targets for therapeutic intervention for an array of diseases including cancer, metabolic diseases, and virus infections. In this review we outline the features of miRNA regulation and how miRNAs may be targeted in strategies to modulate HCV replication and pathogenesis. In particular, we highlight miR-122, a miRNA that directly modulates the HCV life cycle using an unusual mechanism. This miRNA is very important since miR-122 antagonists dramatically reduced HCV titres in HCV-infected chimpanzees and humans and currently represents the most likely candidate to be the first miRNA-based therapy licensed for use. However, we also discuss other miRNAs that directly or indirectly alter HCV replication efficiency, liver cirrhosis, fibrosis and the development of hepatocellular carcinoma (HCC). We also discuss a few miRNAs that might be targets to treat HCV in cases of HCV/HIV co-infection. Finally, we review methods to deliver miRNA antagonists and mimics to the liver. In the future, it may be possible to design and deliver specific combinations of miRNA antagonists and mimics to cure HCV infection or to limit liver pathogenesis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Hepatitis C virus (HCV) infection causes significant morbidity, and efficient mouse models would greatly facilitate virus studies and the development of effective vaccines and new therapeutic agents. Entry factors, innate immunity, and host factors needed for viral replication represent the initial barriers that restrict HCV infection of mouse cells. Experiments in this paper consider early postentry steps of viral infection and investigate the roles of interferon regulatory factors (IRF-3 and IRF-9) and microRNA (miR-122) in promoting HCV replication in mouse embryo fibroblasts (MEFs) that contain viral subgenomic replicons. While wild-type murine fibroblasts are restricted for HCV RNA replication, deletion of IRF-3 alone can facilitate replicon activity in these cells. This effect is thought to be related to the inactivation of the type I interferon synthesis mediated by IRF-3. Additional deletion of IRF-9 to yield IRF-3(-/-) IRF-9(-/-) MEFs, which have blocked type I interferon signaling, did not increase HCV replication. Expression of liver-specific miR-122 in MEFs further stimulated the synthesis of HCV replicons in the rodent fibroblasts. The combined effects of miR-122 expression and deletion of IRF-3 produced a cooperative stimulation of HCV subgenome replication. miR-122 and IRF-3 are independent host factors that are capable of influencing HCV replication, and our findings could help to establish mouse models and other cell systems that support HCV growth and particle formation.

The host cell-specific factor 1 gene (hcf-1) of the baculovirus Autographa californica multiple nucleopolyhedrovirus is required for efficient virus growth in TN368 cells but is dispensable for virus replication in SF21 cells. However, the mechanism of action of hcf-1 is unknown. To begin to understand its function in virus replication we have investigated the expression and localization pattern of HCF-1 in infected cells. Analysis of virus-infected TN368 cells showed that hcf-1 is expressed at an early time in the virus life cycle, between 2 and 12 h postinfection, and localized the protein to punctate nuclear foci. Through coprecipitation experiments we have confirmed that HCF-1 self-associates into dimers or higher-order structures. We also found that overexpression of HCF-1 repressed expression from the hcf-1 promoter in transient reporter assays. Mutagenesis of cysteine residues within a putative RING finger domain in the amino acid sequence of HCF-1 abolished self-association activity and suggests that the RING domain may be involved in this protein-protein interaction. A different but overlapping set of cysteine residues were required for efficient gene repression activity. Functional analysis of HCF-1 mutants showed that the cysteine amino acids required for both self-association and gene repression activities of HCF-1 were also required for efficient late-gene expression and occlusion body formation in TN368 cells. Mutational analysis also identified essential charged and hydrophobic amino acids located between two of the essential cysteine residues. We propose that HCF-1 is a RING finger-containing protein whose activity requires HCF-1 self-association and gene repression activity.

RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects over 270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both an mRNA and a replication template, making it an attractive target for therapeutic approaches using short interfering RNA (siRNA). We have shown previously that double-stranded siRNA molecules designed to target the HCV genome block gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. However, we now show that this block is not complete. After several treatments with a highly effective siRNA, we have shown growth of replicon RNAs that are resistant to subsequent treatment with the same siRNA. However, these replicon RNAs were not resistant to siRNA targeting another part of the genome. Sequence analysis of the siRNA-resistant replicons showed the generation of point mutations within the siRNA target sequence. In addition, the use of a combination of two siRNAs together severely limited escape mutant evolution. This suggests that RNA interference activity could be used as a treatment to reduce the devastating effects of HCV replication on the liver and the use of multiple siRNAs could prevent the emergence of resistant viruses.

Objective: To compare 3 pressure-reduction devices for effectiveness in prevention of heel ulcers in moderate-risk to high-risk patients., Design: A prospective quasi-experimental 3-group design was used., Setting and Subjects: A sample of 338 "moderate-risk to high-risk" adult inpatients, ages 18 to 97, at 2 medical centers in South Texas were studied., Instruments: The Braden Scale for Pressure Ulcer Risk and investigator-developed history and skin assessment tools were used., Methods: Subjects were randomly assigned to the High-Cushion Kodel Heel Protector (bunny boot), Egg Crate Heel Lift Positioner (egg crate), or EHOB Foot Waffle Air Cushion (foot waffle). Data are demographics, Braden scores, comorbidities, skin assessments, lengths of stay, and costs of devices. Analyses were Chi-square, analysis of variance, and regression., Results: Of 240 subjects with complete data, 77 (32%) were assigned to the bunny boot group, 87 (36.3%) to the egg crate, and 76 (31.7%) to the foot waffle. Twelve ulcers developed in 240 subjects (5% incidence). Six subjects had only 1 foot. Eleven ulcers were Stage I (nonblanchable erythema), and 1 was Stage II (partial thickness). Overall incidence was 3.9% for the bunny boot, 4.6% for the egg crate, and 6.6% for the foot waffle (not significantly different among groups). The bunny boot with pillows was most cost effective (F[3], N = 240) = 1.342, p

Purpose: To provide physicians and nurses with an overview of the impact of obesity on postoperative wound healing and how preplanning protocols can minimize skin and wound care problems in this patient population., Target Audience: This continuing education activity is intended for physicians and nurses with an interest in reducing skin and wound care problems in their patients who are obese., Objectives: After reading the article and taking the test, the participant will be able to: 1. Identify obesity-related changes in body systems and how these impede wound healing. 2. Identify complications of postoperative wound healing in obese patients and the assessments and intervention strategies that can reduce these complications. 3. Identify skin and wound care considerations for obese patients and the role of preplanning protocols in avoiding problems.

RNA interference, (RNAi) is a phenomenon by which the introduction of double stranded RNA (dsRNA) into cells induces targeted degradation of RNA molecules with homologous sequences. In the laboratory, RNAi has become a valuable tool for analysis of gene function through suppression of specific gene products. In addition, RNAi has shown great promise for use in therapeutic strategies designed to suppress the expression of pathogenic genes. In this review, viral expression and animal model systems that indicate the potential benefits of RNAi will be discussed. The major obstacle for the use of short interfering RNA in a laboratory or clinical setting is the need for efficient and sustained delivery of dsRNA to mammalian cells. Here, current methods that are being used to induce RNAi, both in cultured cells and in animal models, will be described with a focus on some of the most promising applications.

Intrinsic and extrinsic factors affect wound healing. High risk factors for the obese patients include infection, seromas, anastomatic leaks, and incision dehiscence. Tissue perfusion is an issue of great concern and is a key factor in most assessments. Obesity adds another dimension to the needs of the patient and presents challenges to nurses. From routine evaluations to specialized assessments with attention to bariatric equipment needs, a thorough understanding of wound healing and potential problems of obese patients, and knowledge of interventions is needed. Nonjudgmental attitudes are imperative in planning care for the obese patients. Following a review of physiological needs and nursing interventions, a case study details one woman's surgical complications.

RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects >270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both a messenger RNA and replication template, making it an attractive target for the study of RNA interference. Double-stranded small interfering RNA (siRNA) molecules designed to target the HCV genome were introduced through electroporation into a human hepatoma cell line (Huh-7) that contained an HCV subgenomic replicon. Two siRNAs dramatically reduced virus-specific protein expression and RNA synthesis to levels that were 90% less than those seen in cells treated with negative control siRNAs. These same siRNAs protected naive Huh-7 cells from challenge with HCV replicon RNA. Treatment of cells with synthetic siRNA was effective >72 h, but the duration of RNA interference could be extended beyond 3 weeks through stable expression of complementary strands of the interfering RNA by using a bicistronic expression vector. These results suggest that a gene-therapeutic approach with siRNA could ultimately be used to treat HCV.