Hepatitis C virus subverts liver-specific microRNA, miR-122, to upregulate viral RNA abundance in both infected cultured cells and in the liver of infected chimpanzees. These findings have identified miR-122 as an attractive antiviral target. Thus, it is imperative to know whether a distinct functional complex exists between miR-122 and the viral RNA versus its normal cellular target mRNAs. Toward this goal, effects on viral RNA abundance of mutated miR-122 duplex molecules, bound at each of the two target sites in the viral genome, were compared to effects on microRNA-or siRNA-mediated regulation of reporter target mRNAs. It was found that miR-122 formed an unusual microRNA complex with the viral RNA that is distinct from miR-122 complexes with reporter mRNAs. Notably, miR-122 forms an oligomeric complex in which one miR-122 molecule binds to the 5′ terminus of the hepatitis C virus (HCV) RNA with 3′ overhanging nucleotides, masking the 5′ terminal sequences of the HCV genome. Furthermore, specific internal nucleotides as well as the 3′ terminal nucleotides in miR-122 were absolutely required for maintaining HCV RNA abundance but not for microRNA function. Both miR-122 molecules utilize similar internal nucleotides to interact with the viral genome, creating a bulge and tail in the miR-122 molecules, revealing tandemly oriented oligomeric RNA complexes. These findings suggest that miR-122 protects the 5′ terminal viral sequences from nucleolytic degradation or from inducing innate immune responses to the RNA terminus. Finally, this remarkable microRNA-mRNA complex could be targeted with compounds that inactivate miR-122 or interfere with this unique RNA structure. H epatitis C virus (HCV) infection is a global health problem with an estimated 170 million people infected worldwide (reviewed in ref. 1). HCV-infected individuals typically develop persistent infections that can lead to chronic hepatitis, cirrhosis, and hepatocellular carcinoma (2). Currently, there are no vaccines available, and the clinical efficacies of modern therapeutics are limited. Thus, understanding fundamental aspects of hostvirus interactions in HCV infection is important for the discovery of unique anti-HCV treatments. HCV is a hepatotrophic, positive-sense RNA virus that belongs to the family Flaviviridae. The HCV genome contains a single open reading frame encoding the viral polyprotein, which is subsequently cleaved into at least 10 viral proteins by host and viral proteases. The HCV open reading frame is flanked by 5′ and 3′ noncoding regions (NCRs) that contain RNA elements that are important for viral replication and translation (reviewed in ref.3). Recently, interactions between the liver-specific microRNA, miR-122, with two sites in the HCV 5′NCR (Fig. 1A) have been shown to be essential to maintain HCV RNA abundance during virus infection in cultured cells (4, 5) and in infected chimpanzees (6).MicroRNAs (miRNAs) are small, noncoding RNAs that are predicted to regulate at least one-third of all human mRNAs (7,8). As a general ru...

SUMMARY The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory disease coronavirus 2 (SARS-CoV-2), has led to millions of confirmed cases and deaths worldwide. Efficient diagnostic tools are in high demand, as rapid and large-scale testing plays a pivotal role in patient management and decelerating disease spread. This paper reviews current technologies used to detect SARS-CoV-2 in clinical laboratories as well as advances made for molecular, antigen-based, and immunological point-of-care testing, including recent developments in sensor and biosensor devices. The importance of the timing and type of specimen collection is discussed, along with factors such as disease prevalence, setting, and methods. Details of the mechanisms of action of the various methodologies are presented, along with their application span and known performance characteristics. Diagnostic imaging techniques and biomarkers are also covered, with an emphasis on their use for assessing COVID-19 or monitoring disease severity or complications. While the SARS-CoV-2 literature is rapidly evolving, this review highlights topics of interest that have occurred during the pandemic and the lessons learned throughout. Exploring a broad armamentarium of techniques for detecting SARS-CoV-2 will ensure continued diagnostic support for clinicians, public health, and infection prevention and control for this pandemic and provide advice for future pandemic preparedness.

Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family. Although ZIKV infection is typically mild and self-limiting in healthy adults, infection has been associated with neurological symptoms such as Guillain-Barré syndrome, and a causal link has been established between fetal microcephaly and ZIKV infection during pregnancy. These risks, and the magnitude of the ongoing ZIKV pandemic, have created an urgent need for the development of animal models to study the immune response to ZIKV infection. Previous animal models have primarily focused on pathogenesis in immunocompromised mice. In this study, we provide a model of ZIKV infection in wild-type immunocompetent C57BL/6 mice, and have provided an analysis of the immune response to infection. We evaluated the activation of several innate immune cell types, and studied the kinetics, phenotype, and functionality of T cell responses to ZIKV infection. Our results demonstrate that ZIKV infection is mild in wild-type immunocompetent C57BL/6 mice, resulting in minimal morbidity. Our data establish that at the peak of the adaptive response, antigen-experienced CD4+ T cells polarize to a Th1 phenotype, and antigen-experienced CD8+ T cells exhibit an activated effector phenotype, producing both effector cytokines and cytolytic molecules. Furthermore, we have identified a novel ZIKV CD8+ T cell epitope in the envelope protein that is recognized by the majority of responding cells. Our model provides an important reference point that will help dissect the impact of polymorphisms in the circulating ZIKV strains on the immune response and ZIKV pathogenesis. In addition, the identification of a ZIKV epitope will allow for the design of tetramers to study epitope-specific T cell responses, and will have important implications for the design and development of ZIKV vaccine strategies.

). Genome-scale ordered RNA structure (GORS) was widely distributed in many animal and plant viruses, much greater in extent than RNA structures required for viral translation or replication, but in mammalian viruses was associated with host persistence. To substantiate the existence of large-scale RNA structure differences between viruses, a large set of alignments of mammalian RNA viruses and rRNA sequences as controls were examined by thermodynamic methods (to calculate minimum free energy differences) and by algorithmically independent RNAz and Pfold methods. These methods produced generally concordant results and identified substantial differences in the degrees of evolutionarily conserved, sequence order-dependent RNA secondary structure between virus genera and groups. A probe hybridization accessibility assay was used to investigate the physical nature of GORS. Transcripts of hepatitis C virus (HCV), hepatitis G virus/GB virus-C (HGV/GBV-C), and murine norovirus, which are predicted to be structured, were largely inaccessible to hybridization in solution, in contrast to the almost universal binding of probes to a range of unstructured virus transcripts irrespective of G؉C content. Using atomic force microscopy, HCV and HGV/GBV-C RNA was visualized as tightly compacted prolate spheroids, while under the same experimental conditions the predicted unstructured poliovirus and rubella virus RNA were pleomorphic and had extensively single-stranded RNA on deposition. Bioinformatic and physical characterization methods both identified fundamental differences in the configurations of viral genomic RNA that may modify their interactions with host cell defenses and their ability to persist.The genetic material of the three domains of life is doublestranded DNA. In contrast, many of the viruses that parasitize these organisms have RNA-based genomes. In addition to their capacity to directly encode proteins, RNA genomes have the ability to form secondary and higher-order RNA structures that contribute to their stability and can form sequence motifs in a structural context that may function during the virus life cycle. For example, several virus groups have evolved internal ribosome entry sites that function in ribosome recruitment and the initiation of translation. A range of different types of internal ribosome entry site elements are recognized, all characterized by extensive (200-to 500-nucleotide [nt]) internal RNA-RNA base pairing (5). Similarly, genome replication per se or the packaging of the genome into capsid precursors can involve the interaction of the virus polymerase and/or other viral proteins with defined RNA structural elements (9,16,18,28,36,46). Typically, RNA structures involved in these replication functions take the form of discrete stem loops of various sizes, frequently with evolutionarily highly conserved sequences in terminal loops that are involved in RNA-protein or RNA-RNA interactions.Of the many methods devised to predict RNA secondary structures, the algorithm used in MFOLD and RNAFold ...

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression at the post-transcriptional level. Through this activity, they are implicated in almost every cellular process investigated to date. Hence, it is not surprising that miRNAs play diverse roles in regulation of viral infections and antiviral responses. Diverse families of DNA and RNA viruses have been shown to take advantage of cellular miRNAs or produce virally encoded miRNAs that alter host or viral gene expression. MiRNA-mediated changes in gene expression have been demonstrated to modulate viral replication, antiviral immune responses, viral latency, and pathogenesis. Interestingly, viruses mediate both canonical and non-canonical interactions with miRNAs to downregulate specific targets or to promote viral genome stability, translation, and/or RNA accumulation. In this review, we focus on recent findings elucidating several key mechanisms employed by diverse virus families, with a focus on miRNAs at the host–virus interface during herpesvirus, polyomavirus, retroviruses, pestivirus, and hepacivirus infections.

RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs.

Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with the liver-specific microRNA, miR-122. miR-122 binds to two sites in the 5′ untranslated region (UTR) and this interaction promotes HCV RNA accumulation, although the precise role of miR-122 in the HCV life cycle remains unclear. Using biophysical analyses and Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) we investigated miR-122 interactions with the 5′ UTR. Our data suggests that miR-122 binding results in alteration of nucleotides 1–117 to suppress an alternative secondary structure and promote functional internal ribosomal entry site (IRES) formation. Furthermore, we demonstrate that two hAgo2:miR-122 complexes are able to bind to the HCV 5′ terminus simultaneously and SHAPE analyses revealed further alterations to the structure of the 5′ UTR to accommodate these complexes. Finally, we present a computational model of the hAgo2:miR-122:HCV RNA complex at the 5′ terminus of the viral genome as well as hAgo2:miR-122 interactions with the IRES–40S complex that suggest hAgo2 is likely to form additional interactions with SLII which may further stabilize the HCV IRES. Taken together, our results support a model whereby hAgo2:miR-122 complexes alter the structure of the viral 5′ terminus and promote formation of the HCV IRES.

The hepatitis C virus (HCV) replicates on a membrane protein complex composed of viral proteins, replicating RNA, and altered cellular membranes. Small-molecule inhibitors of cellular lipid-cholesterol metabolism such as 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 all show a negative effect on HCV replication. Perturbation of host cell lipid and cholesterol metabolism can disrupt replication complexes by altering membranous structures where replication occurs. Changes in cholesterol and (or) lipid composition can have a general effect on membrane structure. Alternatively, metabolic changes can exert a more subtle influence over replication complexes by altering localization of host proteins through alterations in lipid anchoring. Here, we use Huh-7 cells harboring subgenomic HCV replicons to demonstrate that 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 do not disrupt the membranous web where replication occurs, whereas cholesterol-depleting agents such as beta-cyclodextrin do. Cellular imaging suggests that the HCV RNA can remain associated with subcellular compartments connected with replication complexes in the presence of metabolic inhibitors. Therefore, at least 2 different molecular mechanisms are possible for the inhibition of HCV replication through the modulation of cellular lipid and cholesterol metabolism.