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    Saccharomyces cerevisiae Hrq1 helicase activity is affected by the sequence but not the length of single-stranded DNA
    (Biochemical and Biophysical Research Communications, Elsevier, 2017-05-13) Rogers, Cody M.; Bochman, Matthew L.
    Mutations in the human RecQ4 DNA helicase are associated with three different diseases characterized by genomic instability. To gain insight into how RecQ4 dysfunction leads to these pathologies, several groups have used the Saccharomyces cerevisiae RecQ4 homolog Hrq1 as an experimental model. Hrq1 displays many of the same functions as RecQ4 in vivo and in vitro. However, there is some disagreement in the literature about the effects of single-stranded DNA (ssDNA) length on Hrq1 helicase activity and the ability of Hrq1 to anneal complementary ssDNA oligonucleotides into duplex DNA. Here, we present a side-by-side comparison of Hrq1 and RecQ4 helicase activity, demonstrating that in both cases, long random-sequence 3′ ssDNA tails inhibit DNA unwinding in vitro in a length-dependent manner. This appears to be due to the formation of secondary structures in the random-sequence ssDNA because Hrq1 preferentially unwound poly(dT)-tailed forks independent of ssDNA length. Further, RecQ4 is capable of ssDNA strand annealing and annealing-dependent strand exchange, but Hrq1 lacks these activities. These results establish the importance of DNA sequence in Hrq1 helicase activity, and the absence of Hrq1 strand annealing activity explains the previously identified discrepancies between S. cerevisiae Hrq1 and human RecQ4.
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    Inverted Alu dsRNA structures do not affect localization but can alter translation efficiency of human mRNAs independent of RNA editing
    (Oxford University Press, 2012) Capshew, C.R.; Dusenbury, K.L.; Hundley, H.A.
    With over one million copies, Alu elements are the most abundant repetitive elements in the human genome. When transcribed, interaction between two Alus that are in opposite orientation gives rise to double-stranded RNA (dsRNA). Although the presence of dsRNA in the cell was previously thought to only occur during viral infection, it is now known that cells express many endogenous small dsRNAs, such as short interfering RNA (siRNAs) and microRNA (miRNAs), which regulate gene expression. It is possible that long dsRNA structures formed from Alu elements influence gene expression. Here, we report that human mRNAs containing inverted Alu elements are present in the mammalian cytoplasm. The presence of these long intramolecular dsRNA structures within 3′-UTRs decreases translational efficiency, and although the structures undergo extensive editing in vivo, the effects on translation are independent of the presence of inosine. As inverted Alus are predicted to reside in >5 of human protein-coding genes, these intramolecular dsRNA structures are important regulators of gene expression.
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    The coronavirus endoribonuclease Nsp15 interacts with retinoblastoma tumor suppressor protein
    (American Society for Microbiology, 2012) Bhardwaj, K.; Liu, P.; Leibowitz, J.L.; Kao, C.C.
    Coronaviruses encode an endoribonuclease, Nsp15, which has a poorly defined role in infection. Sequence analysis revealed a retinoblastoma protein-binding motif (LXCXE/D) in the majority of the Nsp15 of the severe acute respiratory syndrome coronavirus (SARS-CoV) and its orthologs in the alpha and beta coronaviruses. The endoribonuclease activity of the SARS-CoV Nsp15 (sNsp15) was stimulated by retinoblastoma protein (pRb) in vitro, and the two proteins can be coimmunoprecipitated from cellular extracts. Mutations in the pRb-binding motif rendered sNsp15 to be differentially modified by ubiquitin in cells, and cytotoxicity was observed upon its expression. Expression of the sNsp15 in cells resulted in an increased abundance of pRb in the cytoplasm, decreased overall levels of pRb, an increased proportion of cells in the S phase of the cell cycle, and an enhanced expression from a promoter normally repressed by pRb. The endoribonuclease activity of the mouse hepatitis virus (MHV) A59 Nsp15 was also increased by pRb in vitro, and an MHV with mutations in the LXCXE/D-motif, named vLC, exhibited a smaller plaque diameter and reduced the virus titer by ~1 log. Overexpression of pRb delayed the viral protein production by wild-type MHV but not by vLC. This study reveals that pRb and its interaction with Nsp15 can affect coronavirus infection and adds coronaviruses to a small but growing family of RNA viruses that encode a protein to interact with pRb.
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    LL37 and Cationic Peptides Enhance TLR3 Signaling by Viral Double-stranded RNAs
    (PLoS, 2011-10) Kao, C. Cheng; San Mateo, Lani; Dragnea, Bogdan; Wu, Linda H.; Jordan, Jarrett L.; Wen, Yahong; Ranjith-Kumar, C. T.; Bhardwaj, Kanchan; Adhikarakunnathu, Sreedevi; Lai, Yvonne
    Background: Toll-like Receptor 3 (TLR3) detects viral dsRNA during viral infection. However, most natural viral dsRNAs are poor activators of TLR3 in cell-based systems, leading us to hypothesize that TLR3 needs additional factors to be activated by viral dsRNAs. The anti-microbial peptide LL37 is the only known human member of the cathelicidin family of anti- microbial peptides. LL37 complexes with bacterial lipopolysaccharide (LPS) to prevent activation of TLR4, binds to ssDNA to modulate TLR9 and ssRNA to modulate TLR7 and 8. It synergizes with TLR2/1, TLR3 and TLR5 agonists to increase IL8 and IL6 production. This work seeks to determine whether LL37 enhances viral dsRNA recognition by TLR3. Methodology/Principal Findings: Using a human bronchial epithelial cell line (BEAS2B) and human embryonic kidney cells (HEK 293T) transiently transfected with TLR3, we found that LL37 enhanced poly(I:C)-induced TLR3 signaling and enabled the recognition of viral dsRNAs by TLR3. The presence of LL37 also increased the cytokine response to rhinovirus infection in BEAS2B cells and in activated human peripheral blood mononuclear cells. Confocal microscopy determined that LL37 could co-localize with TLR3. Electron microscopy showed that LL37 and poly(I:C) individually formed globular structures, but a complex of the two formed filamentous structures. To separate the effects of LL37 on TLR3 and TLR4, other peptides that bind RNA and transport the complex into cells were tested and found to activate TLR3 signaling in response to dsRNAs, but had no effect on TLR4 signaling. This is the first demonstration that LL37 and other RNA-binding peptides with cell penetrating motifs can activate TLR3 signaling and facilitate the recognition of viral ligands. Conclusions/Significance: LL37 and several cell-penetrating peptides can enhance signaling by TLR3 and enable TLR3 to respond to viral dsRNA.
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    Viral Double-Strand RNA-Binding Proteins Can Enhance Innate Immune Signaling by Toll-Like Receptor 3
    (PLoS, 2011-10) Kao, C. Cheng; Ranjith-Kumar, C. T.; Mukhopadhyay, Suchetana; Zlotnick, Adam; Valverde, Rodrigo A.; Tragesser, Brady J.; Bhardwaj, Kanchan; Chen, Alice; Yi, Guanghui; Lai, Yvonne
    Toll-like Receptor 3 (TLR3) detects double-stranded (ds) RNAs to activate innate immune responses. While poly(I:C) is an excellent agonist for TLR3 in several cell lines and in human peripheral blood mononuclear cells, viral dsRNAs tend to be poor agonists, leading to the hypothesis that additional factor(s) are likely required to allow TLR3 to respond to viral dsRNAs. TLR3 signaling was examined in a lung epithelial cell line by quantifying cytokine production and in human embryonic kidney cells by quantifying luciferase reporter levels. Recombinant 1b hepatitis C virus polymerase was found to enhance TLR3 signaling in the lung epithelial BEAS-2B cells when added to the media along with either poly(I:C) or viral dsRNAs. The polymerase from the genotype 2a JFH-1 HCV was a poor enhancer of TLR3 signaling until it was mutated to favor a conformation that could bind better to a partially duplexed RNA. The 1b polymerase also co-localizes with TLR3 in endosomes. RNA-binding capsid proteins (CPs) from two positive-strand RNA viruses and the hepadenavirus hepatitis B virus (HBV) were also potent enhancers of TLR3 signaling by poly(I:C) or viral dsRNAs. A truncated version of the HBV CP that lacked an arginine-rich RNA-binding domain was unable to enhance TLR3 signaling. These results demonstrate that several viral RNA-binding proteins can enhance the dsRNA-dependent innate immune response initiated by TLR3.
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    A Cell-Based Assay for RNA Synthesis by the HCV Polymerase Reveals New Insights on Mechanism of Polymerase Inhibitors and Modulation by NS5A
    (PLoS, 2011-07) Kao, C. Cheng; Bhardwaj, Kanchan; Baxter, Nielson; Wen, Yahong; Ranjith-Kumar, C. T.
    RNA synthesis by the genotype 1b hepatitis C virus (HCV) polymerase (NS5B) transiently expressed in Human embryonic kidney 293T cells or liver hepatocytes was found to robustly stimulate RIG-I-dependent luciferase production from the interferon b promoter in the absence of exogenously provided ligand. This cell-based assay, henceforth named the 5BR assay, could be used to examine HCV polymerase activity in the absence of other HCV proteins. Mutations that decreased de novo initiated RNA synthesis in biochemical assays decreased activation of RIG-I signaling. In addition, NS5B that lacks the C- terminal transmembrane helix but remains competent for RNA synthesis could activate RIG-I signaling. The addition of cyclosporine A to the cells reduced luciferase levels without affecting agonist-induced RIG-I signaling. Furthermore, non- nucleoside inhibitor benzothiadiazines (BTDs) that bind within the template channel of the 1b NS5B were found to inhibit the readout from the 5BR assay. Mutation M414T in NS5B that rendered the HCV replicon resistant to BTD was also resistant to BTDs in the 5BR assay. Co-expression of the HCV NS5A protein along with NS5B and RIG-I was found to inhibit the readout from the 5BR assay. The inhibition by NS5A was decreased with the removal of the transmembrane helix in NS5B. Lastly, NS5B from all six major HCV genotypes showed robust activation of RIG-I in the 5BR assay. In summary, the 5BR assay could be used to validate inhibitors of the HCV polymerase as well as to elucidate requirements for HCV-dependent RNA synthesis.
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    Analysis of Peptides and Proteins in Their Binding to GroEL
    (European Peptide Society and John Wiley & Sons, Ltd., 2010-12) Chen, Lingling; Ramsey, Andrew; Zheng, Zhida; Li, Yali
    The GroEL-GroES is an essential molecular chaperon system that assists protein folding in cell. Binding of various substrate proteins to GroEL is one of the key aspects in GroEL-assisted protein folding. Small peptides may mimic segments of the substrate proteins in contact with GroEL, and allow detailed structural analysis of the interactions. A model peptide SBP has been shown to bind to a region in GroEL that is important for binding of substrate proteins. Here, we investigated whether the observed GroEL-SBP interaction represented those of GroEL-substrate proteins, and whether SBP was able to mimic various aspects of substrate proteins in GroE- assisted protein folding cycle. We found that SBP competed with substrate proteins, including α-lactalbumin, rhodanese, and malate dehydrogenase, in binding to GroEL. SBP stimulated GroEL ATP hydrolysis rate in a manner similar to that of α-lactalbumin. SBP did not prevent GroES from binding to GroEL, and GroES association reduced the ATPase rates of GroEL/SBP and GroEL/α-lactalbumin to a comparable extent. Binding of both SBP and α-lactalbumin to apo GroEL was dominated by hydrophobic interaction. Interestingly, association of α- lactalbumin to GroEL/GroES was thermodynamically distinct from that to GroEL with reduced affinity and decreased contribution from hydrophobic interaction. However, SBP did not display such differential binding behaviors to apo GroEL and GroEL/GroES, likely due to the lack of a contiguous polypeptide chain that links all of the bound peptide fragments. Nevertheless, studies using peptides provide valuable information on the nature of GroEL-substrate protein interaction, which is central to understand the mechanism of GroEL-assisted protein folding.
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    Conformational changes in the Hepatitis B virus core protein are consistent with a role for allostery in virus assembly
    (American Society for Microbiology, 2010-02-01) Zlotnick, Adam; Katen, Sarah P.; Packianathan, Charles
    In infected cells, virus components must be organized at the right place and time to ensure assembly of infectious virions. From a different perspective, assembly must be prevented until all components are available. Hypothetically, this can be achieved by allosterically controlling assembly. Consistent with this hypothesis, here we show that the structure of hepatitis B virus (HBV) core protein dimer, which can spontaneously self-assemble, is incompatible with capsid assembly. Systematic differences between core protein in dimer and capsid conformations demonstrate linkage between the intradimer interface and interdimer contact surface. These structures also provide explanations for the capsid-dimer selectivity of some antibodies and activity of assembly effectors. Solution studies suggest that the assembly-inactive state is more accurately an ensemble of conformations. Simulations show that allostery supports controlled assembly and results in capsids that are resistant to dissociation. We propose that allostery, as demonstrated in HBV, is common to most self-assembling viruses.