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1
Bansho, Yohsuke, Taro Furubayashi, Norikazu Ichihashi, and Tetsuya Yomo. "Host–parasite oscillation dynamics and evolution in a compartmentalized RNA replication system." Proceedings of the National Academy of Sciences 113, no. 15 (March 28, 2016): 4045–50. http://dx.doi.org/10.1073/pnas.1524404113.
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To date, various cellular functions have been reconstituted in vitro such as self-replication systems using DNA, RNA, and proteins. The next important challenges include the reconstitution of the interactive networks of self-replicating species and investigating how such interactions generate complex ecological behaviors observed in nature. Here, we synthesized a simple replication system composed of two self-replicating host and parasitic RNA species. We found that the parasitic RNA eradicates the host RNA under bulk conditions; however, when the system is compartmentalized, a continuous oscillation pattern in the population dynamics of the two RNAs emerges. The oscillation pattern changed as replication proceeded mainly owing to the evolution of the host RNA. These results demonstrate that a cell-like compartment plays an important role in host–parasite ecological dynamics and suggest that the origin of the host–parasite coevolution might date back to the very early stages of the evolution of life.
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Dramé-Maigné, Adèle, Anton S. Zadorin, Iaroslava Golovkova, and Yannick Rondelez. "Quantifying the Performance of Micro-Compartmentalized Directed Evolution Protocols." Life 10, no. 2 (February 13, 2020): 17. http://dx.doi.org/10.3390/life10020017.
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High-throughput, in vitro approaches for the evolution of enzymes rely on a random micro-encapsulation to link phenotypes to genotypes, followed by screening or selection steps. In order to optimise these approaches, or compare one to another, one needs a measure of their performance at extracting the best variants of a library. Here, we introduce a new metric, the Selection Quality Index (SQI), which can be computed from a simple mock experiment, performed with a known initial fraction of active variants. In contrast to previous approaches, our index integrates the effect of random co-encapsulation, and comes with a straightforward experimental interpretation. We further show how this new metric can be used to extract general protocol efficiency trends or reveal hidden selection mechanisms such as a counterintuitive form of beneficial poisoning in the compartmentalized self-replication protocol.
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Mulaj, Mentor, Tatiana Miti, and Martin Muschol. "Self-Replication of Transthyretin Amyloid Aggregates from Native Tetramers in vitro." Biophysical Journal 108, no. 2 (January 2015): 45a. http://dx.doi.org/10.1016/j.bpj.2014.11.280.
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Wang, Li-juan, Hou-xiu Wang, Longhe Jiang, and Chun-yang Zhang. "Development of an in Vitro Autocatalytic Self-Replication System for Biosensing Application." ACS Sensors 3, no. 12 (November 21, 2018): 2675–83. http://dx.doi.org/10.1021/acssensors.8b01171.
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Rupp, Brigitte, Zsolt Ruzsics, Torsten Sacher, and Ulrich H. Koszinowski. "Conditional Cytomegalovirus Replication In Vitro and In Vivo." Journal of Virology 79, no. 1 (January 1, 2005): 486–94. http://dx.doi.org/10.1128/jvi.79.1.486-494.2005.
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ABSTRACT We have established a conditional gene expression system for cytomegalovirus which allows regulation of genes independently from the viral replication program. Due to the combination of all elements required for regulated expression in the same viral genome, conditional viruses can be studied in different cell lines in vitro and in the natural host in vivo. The combination of a self-sufficient tetracycline-regulated expression cassette and Flp recombinase-mediated insertion into the viral genome allowed fast construction of recombinant murine cytomegaloviruses carrying different conditional genes. The regulation of two reporter genes, the essential viral M50 gene and a dominant-negative mutant gene (m48.2) encoding the small capsid protein, was analyzed in more detail. In vitro, viral growth was regulated by the conditional expression of M50 by 3 orders of magnitude and up to a millionfold when the dominant-negative small capsid protein mutant was used. In vivo, viral growth of the dominant-negative mutant was reduced to detection limits in response to the presence of doxycycline in the organs of mice. We believe that this conditional expression system is applicable to genetic studies of large DNA viruses in general.
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Hwang, Yung, Melinda Futran, Daniel Hidalgo, Divya Ramalingam Iyer, Nicholas Rhind, and Merav Socolovsky. "Global Increase in Replication Fork Speed during a p57KIP2-Regulated Erythroid Cell Fate Switch." Blood 128, no. 22 (December 2, 2016): 698. http://dx.doi.org/10.1182/blood.v128.22.698.698.
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Abstract Cell cycle regulators are increasingly implicated in cell fate decisions such as the acquisition or loss of pluripotency and self-renewal potential. The cell cycle mechanisms that regulate these cell fate decisions are largely unknown. Here we studied an S phase- dependent cell fate switch in the erythroid fetal liver, in which murine early erythroid progenitors transition in vivo from a self-renewal state into a phase of active erythroid gene transcription and concurrent maturational cell divisions. In the fetal liver, this transition corresponds to the transition from subset S0 (CD71-low, Ter119-negative) to subset S1 (CD71-high, Ter119-negative). We found that the S0 to S1 transition takes place during an S phase that is abruptly shorter (decreasing from 7 hours to 4 hours). Further, self-renewing S0 cells uniquely express the cyclin-dependent kinase (CDK) inhibitor p57KIP2 during S phase. To investigate its potential role, we studied DNA replication in vitro and in vivo in p57KIP2 -deficient fetal liver progenitors, employing a variety of techniques, including DNA combing. We found that S0 erythroid progenitors are dependent on p57KIP2-mediated slowing of replication forks for self-renewal, either in vivo, or in dexamethasone-dependent expansion cultures in vitro. The switch from self-renewal in S0 to differentiation in wild-type S1 progenitors entails rapid downregulation of p57KIP2 with a consequent global increase in replication fork speed and an abruptly shorter S phase. In the absence of p57KIP2, replication fork processivity increases prematurely in self-renewing S0 cells, prior to the activation of the erythroid transcriptional program (Figure 1), resulting in replicative stress and cell death. It is well established that differentiation leads to reprogramming of DNA replication, reflected by changes to origin usage and to the timing of replication of chromatin domains. Here we find that the replication program is fundamentally altered in additional key respects: the global processivity of replication forks, regulated by CDK activity, increases abruptly with the switch from self-renewal to differentiation, affecting DNA synthesis rates and S phase duration. Our results are also of interest since the regulation of replication kinetics was thought to be primarily via the regulation of origin firing efficiency, rather than via fork processivity. Here we found no difference in the former (there was no significant change in inter-origin distances, Figure 1). While the full significance of faster forks to the activation of the erythroid transcriptional program is yet to be understood, a recent report found that T cell help leads to faster forks and a shorter S phase in B cells (Gitlin et al., Science 349, 643-646 2015). Regulation of global fork speed may therefore be an intrinsic part of physiological developmental programs. Disclosures No relevant conflicts of interest to declare.
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Sugiyama, Kazuo, Kenji Suzuki, Takahide Nakazawa, Kenji Funami, Takayuki Hishiki, Kazuya Ogawa, Satoru Saito, et al. "Genetic Analysis of Hepatitis C Virus with Defective Genome and Its Infectivity in Vitro." Journal of Virology 83, no. 13 (April 15, 2009): 6922–28. http://dx.doi.org/10.1128/jvi.02674-08.
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ABSTRACT Replication and infectivity of hepatitis C virus (HCV) with a defective genome is ambiguous. We molecularly cloned 38 HCV isolates with defective genomes from 18 patient sera. The structural regions were widely deleted, with the 5′ untranslated, core, and NS3-NS5B regions preserved. All of the deletions were in frame, indicating that they are translatable to the authentic terminus. Phylogenetic analyses showed self-replication of the defective genomes independent of full genomes. We generated a defective genome of chimeric HCV to mimic the defective isolate in the serum. By using this, we demonstrated for the first time that the defective genome, as it is circulating in the blood, can be encapsidated as an infectious particle by trans complementation of the structural proteins.
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Zitzmann, Carolin, Christopher Dächert, Bianca Schmid, Hilde van der Schaar, Martijn van Hemert, Alan S. Perelson, Frank J. M. van Kuppeveld, Ralf Bartenschlager, Marco Binder, and Lars Kaderali. "Mathematical modeling of plus-strand RNA virus replication to identify broad-spectrum antiviral treatment strategies." PLOS Computational Biology 19, no. 4 (April 4, 2023): e1010423. http://dx.doi.org/10.1371/journal.pcbi.1010423.
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Plus-strand RNA viruses are the largest group of viruses. Many are human pathogens that inflict a socio-economic burden. Interestingly, plus-strand RNA viruses share remarkable similarities in their replication. A hallmark of plus-strand RNA viruses is the remodeling of intracellular membranes to establish replication organelles (so-called “replication factories”), which provide a protected environment for the replicase complex, consisting of the viral genome and proteins necessary for viral RNA synthesis. In the current study, we investigate pan-viral similarities and virus-specific differences in the life cycle of this highly relevant group of viruses. We first measured the kinetics of viral RNA, viral protein, and infectious virus particle production of hepatitis C virus (HCV), dengue virus (DENV), and coxsackievirus B3 (CVB3) in the immuno-compromised Huh7 cell line and thus without perturbations by an intrinsic immune response. Based on these measurements, we developed a detailed mathematical model of the replication of HCV, DENV, and CVB3 and showed that only small virus-specific changes in the model were necessary to describe the in vitro dynamics of the different viruses. Our model correctly predicted virus-specific mechanisms such as host cell translation shut off and different kinetics of replication organelles. Further, our model suggests that the ability to suppress or shut down host cell mRNA translation may be a key factor for in vitro replication efficiency, which may determine acute self-limited or chronic infection. We further analyzed potential broad-spectrum antiviral treatment options in silico and found that targeting viral RNA translation, such as polyprotein cleavage and viral RNA synthesis, may be the most promising drug targets for all plus-strand RNA viruses. Moreover, we found that targeting only the formation of replicase complexes did not stop the in vitro viral replication early in infection, while inhibiting intracellular trafficking processes may even lead to amplified viral growth.
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Jain, Bhawana, Amita Jain, Om Prakash, Ajay K. Singh, Tanushree Dangi, Mastan Singh, and K. P. Singh. "In-vitro validation of self designed siRNA targeting non-structural 1 gene of Influenza A virus." South Asian Journal of Experimental Biology 4, no. 6 (February 4, 2015): 315–22. http://dx.doi.org/10.38150/sajeb.4(6).p315-322.
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The genomic variability makes Influenza A virus (IAV) difficult to be con-trolled by existing vaccines or anti-influenza drugs. Viral gene targeting siRNA induces the RNAi mechanism in the host and silents the gene by cleaving mRNA. Objective was to develop siRNA targeting non-structural 1 gene and to validate its efficiency in vitro. siRNA was designed rationally, targeting the most conserved region (delineated with the help of multiple sequence align-ment) of NS1 gene of IAV strains. To choose the most efficient siRNA, three levels screening method was developed. Ultimately one siRNA duplex was selected on the basis of its unique position in conserved region. siRNA effica-cy was confirmed in vitro on commonly used Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e. Influenza A/H3N2 [A/India/LKO864/2011(H3N2)] and Influenza A/pdmH1N1 [A/India/LKO2151/2012(H1N1)]. Of total 173 strains worldwide and 30 strains from India, 32 bp long (position 561 - 592) conserved region was identified. The longest ORF of NS1 gene was targeted by the selected siRNA, which showed 65.5% inhibition in replication of Influenza A/pdmH1N1 and 67.2% inhibition in replication of Influenza A/H3N2 at 48 hpi on MDCK cell line. This study shows that siRNA targeting NS1 may be quite effective in controlling IAV rep-lication so can be used as anti-IAV therapeutic agent.
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Bourne, Christina, Sejin Lee, Bollu Venkataiah, Angela Lee, Brent Korba, M. G. Finn, and Adam Zlotnick. "Small-Molecule Effectors of Hepatitis B Virus Capsid Assembly Give Insight into Virus Life Cycle." Journal of Virology 82, no. 20 (August 6, 2008): 10262–70. http://dx.doi.org/10.1128/jvi.01360-08.
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ABSTRACT The relationship between the physical chemistry and biology of self-assembly is poorly understood, but it will be critical to quantitatively understand infection and for the design of antivirals that target virus genesis. Here we take advantage of heteroaryldihydropyrimidines (HAPs), which affect hepatitis B virus (HBV) assembly, to gain insight and correlate in vitro assembly with HBV replication in culture. Based on a low-resolution crystal structure of a capsid-HAP complex, a closely related series of HAPs were designed and synthesized. These differentially strengthen the association between neighboring capsid proteins, alter the kinetics of assembly, and give rise to aberrant structures incompatible with a functional capsid. The chemical nature of the HAP variants correlated well with the structure of the HAP binding pocket. The thermodynamics and kinetics of in vitro assembly had strong and predictable effects on product morphology. However, only the kinetics of in vitro assembly had a strong correlation with inhibition of HBV replication in HepG2.2.15 cells; there was at best a weak correlation between assembly thermodynamics and replication. The correlation between assembly kinetics and virus suppression implies a competition between successful assembly and misassembly, small molecule induced or otherwise. This is a predictive and testable model for the mechanism of action of assembly effectors.
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Titolo, Steve, Alex Pelletier, Anne-Marie Pulichino, Karine Brault, Elizabeth Wardrop, Peter W. White, Michael G. Cordingley, and Jacques Archambault. "Identification of Domains of the Human Papillomavirus Type 11 E1 Helicase Involved in Oligomerization and Binding to the Viral Origin." Journal of Virology 74, no. 16 (August 15, 2000): 7349–61. http://dx.doi.org/10.1128/jvi.74.16.7349-7361.2000.
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ABSTRACT The E1 helicase of papillomavirus is required, in addition to host cell DNA replication factors, during the initiation and elongation phases of viral episome replication. During initiation, the viral E2 protein promotes the assembly of enzymatically active multimeric E1 complexes at the viral origin of DNA replication. In this study we used the two-hybrid system and chemical cross-linking to demonstrate that human papillomavirus type 11 (HPV11) E1 can self-associate in yeast and form hexamers in vitro in a reaction stimulated by single-stranded DNA. Self-association in yeast was most readily detected using constructs spanning the E1 C-terminal domain (amino acids 353 to 649) and was dependent on a minimal E1-E1 interaction region located between amino acids 353 and 431. The E1 C-terminal domain was also able to oligomerize in vitro but, in contrast to wild-type E1, did so efficiently in the absence of single-stranded DNA. Sequences located between amino acids 191 and 353 were necessary for single-stranded DNA to modulate oligomerization of E1 and were also required, together with the rest of the C terminus, for binding of E1 to the origin. Two regions within the C-terminal domain were identified as important for oligomerization: the ATP-binding domain and region A, which is located within the minimal E1-E1 interaction domain and is one of four regions of E1 that is highly conserved with the large T antigens of simian virus 40 and polyomavirus. Amino acid substitutions of highly conserved residues within the ATP-binding domain and region A were identified that reduced the ability of E1 to oligomerize and bind to the origin in vitro and to support transient DNA replication in vivo. These results support the notion that oligomerization of E1 occurs primarily through the C-terminal domain of the protein and is allosterically regulated by DNA and ATP. The bipartite organization of the E1 C-terminal domain is reminiscent of that found in other hexameric proteins and suggests that these proteins may oligomerize by a similar mechanism.
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Woerman, Amanda L., Sabeen A. Kazmi, Smita Patel, Atsushi Aoyagi, Abby Oehler, Kartika Widjaja, Daniel A. Mordes, Steven H. Olson, and Stanley B. Prusiner. "Familial Parkinson’s point mutation abolishes multiple system atrophy prion replication." Proceedings of the National Academy of Sciences 115, no. 2 (December 26, 2017): 409–14. http://dx.doi.org/10.1073/pnas.1719369115.
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In the neurodegenerative disease multiple system atrophy (MSA), α-synuclein misfolds into a self-templating conformation to become a prion. To compare the biological activity of α-synuclein prions in MSA and Parkinson’s disease (PD), we developed nine α-synuclein−YFP cell lines expressing point mutations responsible for inherited PD. MSA prions robustly infected wild-type, A30P, and A53T α-synuclein–YFP cells, but they were unable to replicate in cells expressing the E46K mutation. Coexpression of the A53T and E46K mutations was unable to rescue MSA prion infection in vitro, establishing that MSA α-synuclein prions are conformationally distinct from the misfolded α-synuclein in PD patients. This observation may have profound implications for developing treatments for neurodegenerative diseases.
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Borghesi, Lisa, Qi Yang, and Brandt Esplin. "Cell intrinsic E47 is required for stem cell self-renewal and differentiation but is dispensable for short-term myeloid development (36.2)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 36.2. http://dx.doi.org/10.4049/jimmunol.184.supp.36.2.
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Abstract The immune system is constantly replenished by a rare population of hematopoietic stem cells (HSC) residing in the bone marrow of adult organisms. E-proteins, the widely expressed basic helix-loop-helix (bHLH) transcription factors, appear to contribute to HSC activity, but their specific cell intrinsic functions remain undefined. In contrast to a recent report, we show that E47 is dispensable for the short-term myeloid differentiation of HSCs. In our quantitative assays, E47 deficient progenitors show competent myeloid production in vitro and in vivo as well as under burden of a pathogen mimic. We also show that long-term myeloid and lymphoid differentiation is compromised due to the progressive loss of the self-renewal potential of HSCs in vivo. Compromised self-renewal of E47 null HSCs is associated with over-proliferation and premature exhaustion under replication stress. These observations suggest that cell-intrinsic E47 is dispensable for the short-term myeloid repopulation activity of HSCs and, by contrast, that E47 plays an essential cell-autonomous role in the self-renewal of HSCs by preventing hyperproliferation-associated exhaustion following replication stress.
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Risager, Peter Christian, Ulrik Fahnøe, Maria Gullberg, Thomas Bruun Rasmussen, and Graham J. Belsham. "Analysis of classical swine fever virus RNA replication determinants using replicons." Journal of General Virology 94, no. 8 (August 1, 2013): 1739–48. http://dx.doi.org/10.1099/vir.0.052688-0.
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Self-replicating RNAs (replicons), with or without reporter gene sequences, derived from the genome of the Paderborn strain of classical swine fever virus (CSFV) have been produced. The full-length viral cDNA, propagated within a bacterial artificial chromosome, was modified by targeted recombination within Escherichia coli. RNA transcripts were produced in vitro and introduced into cells by electroporation. The translation and replication of the replicon RNAs could be followed by the accumulation of luciferase (from Renilla reniformis or Gaussia princeps) protein expression (where appropriate), as well as by detection of CSFV NS3 protein production within the cells. Inclusion of the viral E2 coding region within the replicon was advantageous for replication efficiency. Production of chimeric RNAs, substituting the NS2 and NS3 coding regions (as a unit) from the Paderborn strain with the equivalent sequences from the highly virulent Koslov strain or the vaccine strain Riems, blocked replication. However, replacing the Paderborn NS5B coding sequence with the RNA polymerase coding sequence from the Koslov strain greatly enhanced expression of the reporter protein from the replicon. In contrast, replacement with the Riems NS5B sequence significantly impaired replication efficiency. Thus, these replicons provide a system for determining specific regions of the CSFV genome required for genome replication without the constraints of maintaining infectivity.
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Sekulovic, Sanja, Vala Gylfadottir, Irma Vulto, Maura Gasparetto, Yasmine Even, Christy Brookes, Clayton Smith, et al. "Prolonged self-renewal activity unmasks telomerase control of telomere homeostasis and function of mouse hematopoietic stem cells." Blood 118, no. 7 (August 18, 2011): 1766–73. http://dx.doi.org/10.1182/blood-2010-11-319632.
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Abstract Strategies for expanding hematopoietic stem cells (HSCs) could have significant utility for transplantation-based therapies. However, deleterious consequences of such manipulations remain unknown. Here we examined the impact of HSC self-renewal divisions in vitro and in vivo on their subsequent regenerative and continuing ability to sustain blood cell production in the absence of telomerase. HSC expansion in vitro was obtained using a NUP98-HOXA10hd transduction strategy and, in vivo, using a serial transplant protocol. We observed ∼ 10kb telomere loss in leukocytes produced in secondary mice transplanted with HSCs regenerated in primary recipients of NUP98-HOXA10hd-transduced and in vitro-expanded Tert−/− HSCs 6 months before. The second generation leukocytes also showed elevated expression of γH2AX (relative to control) indicative of greater accumulating DNA damage. In contrast, significant telomere shortening was not detected in leukocytes produced from freshly isolated, serially transplanted wild-type (WT) or Tert−/− HSCs, suggesting that HSC replication posttransplant is not limited by telomere shortening in the mouse. These findings document a role of telomerase in telomere homeostasis, and in preserving HSC functional integrity on prolonged self-renewal stimulation.
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Khromykh, Alexander A., Natasha Kondratieva, Jean-Yves Sgro, Ann Palmenberg, and Edwin G. Westaway. "Significance in Replication of the Terminal Nucleotides of the Flavivirus Genome." Journal of Virology 77, no. 19 (October 1, 2003): 10623–29. http://dx.doi.org/10.1128/jvi.77.19.10623-10629.2003.
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ABSTRACT Point mutations that resulted in a substitution of the conserved 3′-penultimate cytidine in genomic RNA or the RNA negative strand of the self-amplifying replicon of the Flavivirus Kunjin virus completely blocked in vivo replication. Similarly, substitutions of the conserved 3′-terminal uridine in the RNA negative or positive strand completely blocked replication or caused much-reduced replication, respectively. The same preference for cytidine in the 3′-terminal dinucleotide was noted in reports of the in vitro activity of the RNA-dependent RNA polymerase (RdRp) for the other genera of Flaviviridae that also employ a double-stranded RNA (dsRNA) template to initiate asymmetric semiconservative RNA positive-strand synthesis. The Kunjin virus replicon results were interpreted in the context of a proposed model for initiation of RNA synthesis based on the solved crystal structure of the RdRp of φ6 bacteriophage, which also replicates efficiently using a dsRNA template with conserved 3′-penultimate cytidines and a 3′-terminal pyrimidine. A previously untested substitution of the conserved pentanucleotide at the top of the 3′-terminal stem-loop of all Flavivirus species also blocked detectable in vivo replication of the Kunjin virus replicon RNA.
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Park, Mi-Young, Young-Eui Kim, Myong-Rang Seo, Jae-Rin Lee, Chan Hee Lee, and Jin-Hyun Ahn. "Interactions among Four Proteins Encoded by the Human Cytomegalovirus UL112-113 Region Regulate Their Intranuclear Targeting and the Recruitment of UL44 to Prereplication Foci." Journal of Virology 80, no. 6 (March 15, 2006): 2718–27. http://dx.doi.org/10.1128/jvi.80.6.2718-2727.2006.
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ABSTRACT Four phosphoproteins, of 34, 43, 50, and 84 kDa, with common amino termini are synthesized via alternative splicing from the UL112-113 region of the human cytomegalovirus genome. Although genetic studies provided evidence that both the UL112 and UL113 loci in the viral genome are required for efficient viral replication, whether the four proteins play specific roles or cooperate in replication is not understood. Here we present evidence, using in vitro and in vivo coimmunoprecipitation assays, that the four UL112-113 proteins both self-interact and interact with each other. A mapping study of the 84-kDa protein showed that the N-terminal region encompassing amino acids 1 to 125, which is shared in all UL112-113 proteins and highly conserved among betaherpesviruses, is required for both self-interaction and nuclear localization as foci. Further localization studies revealed that, unlike the 43-, 50-, and 84-kDa proteins, which were distributed as nuclear punctate forms, the 34-kDa form was located predominantly in the cytoplasm. However, when all four proteins were coexpressed simultaneously, all of the UL112-113 proteins were efficiently localized to the promyelocytic leukemia oncogenic domains. We also found that the ability of the UL112-113 proteins to relocate UL44 (the viral polymerase processivity factor) to prereplication foci relied on self-interaction and reached maximal levels when the four proteins were coexpressed. Therefore, our data suggest that interactions occurring among UL112-113 proteins via their shared N-terminal regions are important to both their intranuclear targeting and the recruitment of UL44 to subnuclear sites for viral replication.
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Serio, Tricia R., Anil G. Cashikar, Anthony S. Kowal, George J. Sawicki, and Susan L. Lindquist. "Self-perpetuating changes in Sup35 protein conformation as a mechanism of heredity in yeast." Biochemical Society Symposia 68 (August 1, 2001): 35–43. http://dx.doi.org/10.1042/bss0680035.
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Recently, a novel mode of inheritance has been described in the yeast Saccharomyces cerevisiae. The mechanism is based on the prion hypothesis, which posits that self-perpetuating changes in the conformation of single protein, PrP, underlie the severe neurodegeneration associated with the transmissible spongiform enchephalopathies in mammals. In yeast, two prions, [URE3] and [PSI+], have been identified, but these factors confer unique phenotypes rather than disease to the organism. In each case, the prion-associated phenotype has been linked to alternative conformations of the Ure2 and Sup35 proteins. Remarkably, Ure2 and Sup35 proteins existing in the alternative conformations have the unique capacity to transmit this physical state to the newly synthesized protein in vivo. Thus, a mechanism exists to ensure replication of the conformational information that underlies protein-only inheritance. We have characterized the mechanism by which Sup35 conformational information is replicated in vitro. The assembly of amyloid fibres by a region of Sup35 encompassing the N-terminal 254 amino acids faithfully recapitulates the in vivo propagation of [PSI+]. Mutations that alter [PSI+] inheritance in vivo change the kinetics of amyloid assembly in vitro in a complementary fashion, and lysates from [PSI+] cells, but not [psi-] cells, accelerate assembly in vitro. Using this system we propose a mechanism by which the alternative conformation of Sup35 is adopted by an unstructured oilgomeric intermediate at the time of assembly.
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Han, Dongran, Xiaodong Qi, Cameron Myhrvold, Bei Wang, Mingjie Dai, Shuoxing Jiang, Maxwell Bates, et al. "Single-stranded DNA and RNA origami." Science 358, no. 6369 (December 14, 2017): eaao2648. http://dx.doi.org/10.1126/science.aao2648.
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Self-folding of an information-carrying polymer into a defined structure is foundational to biology and offers attractive potential as a synthetic strategy. Although multicomponent self-assembly has produced complex synthetic nanostructures, unimolecular folding has seen limited progress. We describe a framework to design and synthesize a single DNA or RNA strand to self-fold into a complex yet unknotted structure that approximates an arbitrary user-prescribed shape. We experimentally construct diverse multikilobase single-stranded structures, including a ~10,000-nucleotide (nt) DNA structure and a ~6000-nt RNA structure. We demonstrate facile replication of the strand in vitro and in living cells. The work here thus establishes unimolecular folding as a general strategy for constructing complex and replicable nucleic acid nanostructures, and expands the design space and material scalability for bottom-up nanotechnology.
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Gao, Hong-Qiang, Stefan G. Sarafianos, Edward Arnold, and Stephen H. Hughes. "RNase H Cleavage of the 5′ End of the Human Immunodeficiency Virus Type 1 Genome." Journal of Virology 75, no. 23 (December 1, 2001): 11874–80. http://dx.doi.org/10.1128/jvi.75.23.11874-11880.2001.
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ABSTRACT The synthesis of retroviral DNA is initiated near the 5′ end of the RNA. DNA synthesis is transferred from the 5′ end to the 3′ end of viral RNA in an RNase H-dependent step. In the case of human immunodeficiency virus type 1 (HIV-1) (and certain other retroviruses that have complex secondary structures at the ends of the viral RNA), there is the possibility that DNA synthesis can lead to a self-priming event that would block viral replication. The extent of RNase H cleavage must be sufficient to allow the strand transfer reaction to occur, but not so extensive that self-priming occurs. We have used a series of model RNA substrates, with and without a 5′ cap, to investigate the rules governing RNase H cleavage at the 5′ end of the HIV-1 genome. These in vitro RNase H cleavage reactions produce an RNA fragment of the size needed to block self-priming but still allow strand transfer. The cleavages seen in vitro can be understood in light of the structure of HIV-1 reverse transcriptase in a complex with an RNA/DNA substrate.
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Kato, Takanobu, Takuya Matsumura, Theo Heller, Satoru Saito, Ronda K. Sapp, Krishna Murthy, Takaji Wakita, and T. Jake Liang. "Production of Infectious Hepatitis C Virus of Various Genotypes in Cell Cultures." Journal of Virology 81, no. 9 (February 14, 2007): 4405–11. http://dx.doi.org/10.1128/jvi.02334-06.
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ABSTRACT A unique hepatitis C virus (HCV) strain JFH-1 has been shown to replicate efficiently in cell culture with production of infectious HCV. We previously developed a DNA expression system containing HCV cDNA flanked by two self-cleaving ribozymes to generate HCV particles in cell culture. In this study, we produced HCV particles of various genotypes, including 1a (H77), 1b (CG1b), and 2a (J6 and JFH-1), in the HCV-ribozyme system. The constructs also contain the secreted alkaline phosphatase gene to control for transfection efficiency and the effects of culture conditions. After transfection into the Huh7-derived cell line Huh7.5.1, continuous HCV replication and secretion were confirmed by the detection of HCV RNA and core antigen in the culture medium. HCV replication levels of strains H77, CG1b, and J6 were comparable, whereas the JFH-1 strain replicates at a substantially higher level than the other strains. To evaluate the infectivity in vitro, the culture medium of JFH-1-transfected cells was inoculated into naive Huh7.5.1 cells. HCV proteins were detected by immunofluorescence 3 days after inoculation. To evaluate the infectivity in vivo, the culture medium from HCV genotype 1b-transfected cells was inoculated into a chimpanzee and caused a typical course of HCV infection. The HCV 1b propagated in vitro and in vivo had sequences identical to those of the HCV genomic cDNA used for cell culture transfection. The development of culture systems for production of various HCV genotypes provides a valuable tool not only to study the replication and pathogenesis of HCV but also to screen for antivirals.
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Zhang, Chaozheng, Yueyong Liu, Liyue Liu, Zhiyong Lou, Hongyan Zhang, Hongqin Miao, Xuebo Hu, Yanping Pang, and Bingsheng Qiu. "Rice black streaked dwarf virus P9-1, an α-helical protein, self-interacts and forms viroplasms in vivo." Journal of General Virology 89, no. 7 (July 1, 2008): 1770–76. http://dx.doi.org/10.1099/vir.0.2008/000109-0.
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Replication and assembly of viruses from the family Reoviridae are thought to take place in discrete cytoplasmic inclusion bodies, commonly called viral factories or viroplasms. Rice black streaked dwarf virus (RBSDV) P9-1, a non-structural protein, has been confirmed to accumulate in these intracellular viroplasms in infected plants and insects. However, little is known about its exact function. In this study, P9-1 of RBSDV-Baoding was expressed in Escherichia coli as a His-tagged fusion protein and analysed using biochemical and biophysical techniques. Mass spectrometry and circular dichroism spectroscopy studies showed that P9-1 was a thermostable, α-helical protein with a molecular mass of 41.804 kDa. A combination of gel-filtration chromatography, chemical cross-linking and a yeast two-hybrid assay was used to demonstrate that P9-1 had the intrinsic ability to self-interact and form homodimers in vitro and in vivo. Furthermore, when transiently expressed in Arabidopsis protoplasts, P9-1 formed large, discrete viroplasm-like structures in the absence of infection or other RBSDV proteins. Taken together, these results suggest that P9-1 is the minimal viral component required for viroplasm formation and that it plays an important role in the early stages of the virus life cycle by forming intracellular viroplasms that serve as the sites of virus replication and assembly.
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Hricik, Todd, Anna Sophia McKenney, Ross L. Levine, Giulia Federici, Ann Zeuner, Emanuel F. Petricoin, Agostino Tafuri, et al. "Transcriptosome and Phospho-Proteomic Analyses of Erythroblasts Expanded in Vitro From Normal Donors (ND) and From Patients with Polycythemia Vera (PV)." Blood 120, no. 21 (November 16, 2012): 2860. http://dx.doi.org/10.1182/blood.v120.21.2860.2860.
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Abstract Abstract 2860 Expansion of EBs is observed when cells from ND are cultured with the glucocorticoid receptor α(GRα) agonist dexamethasone (DXM). In addition, EBs can be cultured from patients with PV, a disease characterized by erythrocytosis, in the presence (+) or absence (−) of DXM (Varricchio et al. Blood. 2011;118:425). In cultures of ND, DXM induces expansion by inducing EBs into a reversible self-replication state and attenuating STAT5 activation mediated by the erythropoietin receptor (EPO-R). In cultures of PV, EB expansion is due to acquisition of a constitutive self-replication state characterized by expression of a dominant negative GRβ isoform which can attenuate EPO-R signalling in a DXM-independent manner. The homogeneity in morphology and biological properties of EBs generated by ND in cultures + DXM and by PV patients in cultures +/− DXM suggests that comparisons between the transcriptosome and phosphoproteosome of these different isolates may allow identification of mechanisms which induce PV EBs constitutively into self-replication or that mediate effects of GRα not inhibited by GRβ. Gene expression studies. We performed microarray analyses of EBs obtained in vitro from 5 ND + DXM and from 3 PV (all JAK2 V617F positive) + and - DXM. ANOVA analyses between ND + DXM vs. PV - DXM identified 55 differentially expressed genes (FDR<0.05). Notably, the majority of these genes is linked to lipid metabolism and likely represents GRβ-indipendent effects of GRα. ANOVA analyses of ND + DXM vs. PV + DXM did not reveal significant differences in gene expression, confirming previous studies of specific targets by quantitative RT-PCR which indicated the similar expression profile of the 2 populations. Supervised analysis of a subset of genes found to be differentially expressed in human EBs (Nandi et al, Blood 2011:117:96) revealed no significantly differentially expressed genes between PV + DXM and PV – DXM (paired t-test) and between PV + DXM and ND + DXM (ANOVA). We identified only 3 genes (CA1, CA2 and UBE3C) in this signature which were differentially expressed between PV – DXM and ND. Phosphoproteomic studies. We obtained phosphoproteomic profile of EBs obtained in vitro from 3 ND + DXM and from 3 PV (all JAK2 V617F positive) +/− DXM using a Reverse-phase Protein MicroArray including 170 validated antibodies against signaling proteins representative of pathways controlling proliferation/survival/migration/adhesion. Unsupervised hierarchical clustering and non-parametric statistical analysis were used to compare the activation status of a first set of 120 signaling proteins. In conclusion, the gene expression profile of EBs from PV and ND largely overlap, with the exception of a subset of genes important in metabolic and signaling pathways while phosphoproteomic analysis reveals important differences in cell signaling between PV and ND EBs. We suggest that integrated gene expression and proteomic analyses may reveal differential networks which govern normal and malignant erythroid expansion. Disclosures: Tafuri: Sigma Tau Pharmaceuticals: Research Funding.
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van Leeuwen, Hans C., Chantal B. E. M. Reusken, Marko Roeten, Tim J. Dalebout, Jose Ignacio Riezu-Boj, Juan Ruiz, and Willy J. M. Spaan. "Evolution of naturally occurring 5′ non-translated region variants of hepatitis C virus genotype 1b in selectable replicons." Journal of General Virology 85, no. 7 (July 1, 2004): 1859–66. http://dx.doi.org/10.1099/vir.0.79924-0.
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Quasispecies shifts are essential for the development of persistent hepatitis C virus (HCV) infection. Naturally occurring sequence variations in the 5′ non-translated region (NTR) of the virus could lead to changes in protein expression levels, reflecting selective forces on the virus. The extreme 5′ end of the virus' genome, containing signals essential for replication, is followed by an internal ribosomal entry site (IRES) essential for protein translation as well as replication. The 5′ NTR is highly conserved and has a complex RNA secondary structure consisting of several stem–loops. This report analyses the quasispecies distribution of the 5′ NTR of an HCV genotype 1b clinical isolate and found a number of sequences differing from the consensus sequence. The consensus sequence, as well as a major variant located in stem–loop IIIa of the IRES, was investigated using self-replicating HCV RNA molecules in human hepatoma cells. The stem–loop IIIa mutation, which is predicted to disrupt the stem structure, showed slightly lower translation efficiency but was severely impaired in the colony formation of selectable HCV replicons. Interestingly, during selection of colonies supporting autonomous replication, mutations emerged that restored the base pairing in the stem–loop. Recloning of these altered IRESs confirmed that these second site revertants were more efficient in colony formation. In conclusion, naturally occurring variants in the HCV 5′ NTR can lead to changes in their replication ability. Furthermore, IRES quasispecies evolution was observed in vitro under the selective pressure of the replicon system.
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Driscoll, Mark D., and Stephen H. Hughes. "Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Can Prevent Self-Priming of Minus-Strand Strong Stop DNA by Promoting the Annealing of Short Oligonucleotides to Hairpin Sequences." Journal of Virology 74, no. 19 (October 1, 2000): 8785–92. http://dx.doi.org/10.1128/jvi.74.19.8785-8792.2000.
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ABSTRACT Understanding how viral components collaborate to convert the human immunodeficiency virus type 1 genome from single-stranded RNA into double-stranded DNA is critical to the understanding of viral replication. Not only must the correct reactions be carried out, but unwanted side reactions must be avoided. After minus-strand strong stop DNA (−sssDNA) synthesis, degradation of the RNA template by the RNase H domain of reverse transcriptase (RT) produces single-stranded DNA that has the potential to self-prime at the imperfectly base-paired TAR hairpin, making continued DNA synthesis impossible. Although nucleocapsid protein (NC) interferes with −sssDNA self-priming in reverse transcription reactions in vitro, NC alone did not prevent self-priming of a synthetic −sssDNA oligomer. NC did not influence DNA bending and therefore cannot inhibit self-priming at hairpins by directly blocking hairpin formation. Using DNA oligomers as a model for genomic RNA fragments, we found that a 17-base DNA fragment annealed to the 3′ end of the −sssDNA prevented self-priming in the presence of NC. This implies that to avoid self-priming, an RNA-DNA hybrid that is more thermodynamically stable than the hairpin must remain within the hairpin region. This suggests that NC prevents self-priming by generating or stabilizing a thermodynamically favored RNA-DNA heteroduplex instead of the kinetically favored TAR hairpin. In support of this idea, sequence changes that increased base pairing in the DNA TAR hairpin resulted in an increase in self-priming in vitro. We present a model describing the role of NC-dependent inhibition of self-priming in first-strand transfer.
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Zufferey, Romain, Thomas Dull, Ronald J. Mandel, Anatoly Bukovsky, Dulce Quiroz, Luigi Naldini, and Didier Trono. "Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery." Journal of Virology 72, no. 12 (December 1, 1998): 9873–80. http://dx.doi.org/10.1128/jvi.72.12.9873-9880.1998.
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ABSTRACT In vivo transduction of nondividing cells by human immunodeficiency virus type 1 (HIV-1)-based vectors results in transgene expression that is stable over several months. However, the use of HIV-1 vectors raises concerns about their safety. Here we describe a self-inactivating HIV-1 vector with a 400-nucleotide deletion in the 3′ long terminal repeat (LTR). The deletion, which includes the TATA box, abolished the LTR promoter activity but did not affect vector titers or transgene expression in vitro. The self-inactivating vector transduced neurons in vivo as efficiently as a vector with full-length LTRs. The inactivation design achieved in this work improves significantly the biosafety of HIV-derived vectors, as it reduces the likelihood that replication-competent retroviruses will originate in the vector producer and target cells, and hampers recombination with wild-type HIV in an infected host. Moreover, it improves the potential performance of the vector by removing LTR sequences previously associated with transcriptional interference and suppression in vivo and by allowing the construction of more-stringent tissue-specific or regulatable vectors.
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Chen, Mingzhou, Tomoaki Ogino, and Amiya K. Banerjee. "Mapping and Functional Role of the Self-Association Domain of Vesicular Stomatitis Virus Phosphoprotein." Journal of Virology 80, no. 19 (October 1, 2006): 9511–18. http://dx.doi.org/10.1128/jvi.01035-06.
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ABSTRACT The phosphoprotein (P protein) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNA-dependent RNA polymerase complex and plays a central role in viral transcription and replication. Using both the yeast two-hybrid system and coimmunoprecipitation assays, we confirmed the self-association of the P protein of Indiana serotype (Pind) and heterotypic interaction between Pind and the P protein of New Jersey serotype (Pnj). Furthermore, by using various truncation and deletion mutants of Pind, the self-association domain of the Pind protein was mapped to amino acids 161 to 210 within the hinge region. The self-association domain of Pind protein is not required for its binding to nucleocapsid and large proteins. We further demonstrated that the self-association domain of Pind protein is essential for VSV transcription in a minireplicon system and that a synthetic peptide spanning amino acids 191 to 210 in the self-association domain of Pind protein strongly inhibited the transcription of the VSV genome in vitro in a dose-dependent manner. These results indicated that the self-association domain of Pind protein plays a critical role in VSV transcription.
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Schregel, Vera, Sabrina Auerochs, Ramona Jochmann, Katja Maurer, Thomas Stamminger, and Manfred Marschall. "Mapping of a self-interaction domain of the cytomegalovirus protein kinase pUL97." Journal of General Virology 88, no. 2 (February 1, 2007): 395–404. http://dx.doi.org/10.1099/vir.0.82393-0.
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The human cytomegalovirus-encoded protein kinase pUL97 is a determinant of efficient virus replication and fulfils several regulatory functions. In particular, pUL97 interacts with and phosphorylates viral and cellular proteins. Substrate phosphorylation has regulatory consequences on viral replicative stages such as DNA synthesis, transcription and nuclear capsid egress. pUL97, in accordance with related herpesviral protein kinases, possesses strong autophosphorylation activity. Here, we demonstrate that pUL97 shows a pronounced potential to self-interact. Self-interaction of pUL97 is not dependent on its kinase activity, as seen with a catalytically inactive point mutant. The property of self-interaction maps to the amino acid region 231–280 which is separable from the postulated kinase domain. The detection of high-molecular-mass complexes of pUL97 suggests the formation of dimers and oligomers. Importantly, the analysis of pUL97 mutants by in vitro kinase assays demonstrated a correlation between self-interaction and protein kinase activity, i.e. all mutants lacking the ability to self-interact were negative or reduced in their kinase activity. Thus, our findings provide novel insights into the pUL97 structure–activity relationship suggesting an importance of self-interaction for pUL97 functionality.
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Shuchat, Sholom, Gilad Yossifon, and Mahmoud Huleihel. "Perfusion in Organ-on-Chip Models and Its Applicability to the Replication of Spermatogenesis In Vitro." International Journal of Molecular Sciences 23, no. 10 (May 12, 2022): 5402. http://dx.doi.org/10.3390/ijms23105402.
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Organ/organoid-on-a-chip (OoC) technologies aim to replicate aspects of the in vivo environment in vitro, at the scale of microns. Mimicking the spatial in vivo structure is important and can provide a deeper understanding of the cell–cell interactions and the mechanisms that lead to normal/abnormal function of a given organ. It is also important for disease models and drug/toxin testing. Incorporating active fluid flow in chip models enables many more possibilities. Active flow can provide physical cues, improve intercellular communication, and allow for the dynamic control of the environment, by enabling the efficient introduction of biological factors, drugs, or toxins. All of this is in addition to the fundamental role of flow in supplying nutrition and removing waste metabolites. This review presents an overview of the different types of fluid flow and how they are incorporated in various OoC models. The review then describes various methods and techniques of incorporating perfusion networks into OoC models, including self-assembly, bioprinting techniques, and utilizing sacrificial gels. The second part of the review focuses on the replication of spermatogenesis in vitro; the complex process whereby spermatogonial stem cells differentiate into mature sperm. A general overview is given of the various approaches that have been used. The few studies that incorporated microfluidics or vasculature are also described. Finally, a future perspective is given on elements from perfusion-based models that are currently used in models of other organs and can be applied to the field of in vitro spermatogenesis. For example, adopting tubular blood vessel models to mimic the morphology of the seminiferous tubules and incorporating vasculature in testis-on-a-chip models. Improving these models would improve our understanding of the process of spermatogenesis. It may also potentially provide novel therapeutic strategies for pre-pubertal cancer patients who need aggressive chemotherapy that can render them sterile as well asfor a subset of non-obstructive azoospermic patients with maturation arrest, whose testes do not produce sperm but still contain some of the progenitor cells.
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Mears, Harriet V., Edward Emmott, Yasmin Chaudhry, Myra Hosmillo, Ian G. Goodfellow, and Trevor R. Sweeney. "Ifit1 regulates norovirus infection and enhances the interferon response in murine macrophage-like cells." Wellcome Open Research 4 (May 15, 2019): 82. http://dx.doi.org/10.12688/wellcomeopenres.15223.1.
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Background: Norovirus, also known as the winter vomiting bug, is the predominant cause of non-bacterial gastroenteritis worldwide. Disease control is predicated on a robust innate immune response during the early stages of infection. Double-stranded RNA intermediates generated during viral genome replication are recognised by host innate immune sensors in the cytoplasm, activating the strongly antiviral interferon gene programme. Ifit proteins (interferon induced proteins with tetratricopeptide repeats), which are highly expressed during the interferon response, have been shown to directly inhibit viral protein synthesis as well as regulate innate immune signalling pathways. Ifit1 is well-characterised to inhibit viral translation by sequestration of eukaryotic initiation factors or by directly binding to the 5' terminus of foreign RNA, particularly those with non-self cap structures. However, noroviruses have a viral protein, VPg, covalently linked to the 5' end of the genomic RNA, which acts as a cap substitute to recruit the translation initiation machinery. Methods: Ifit1 knockout RAW264.7 murine macrophage-like cells were generated using CRISPR-Cas9 gene editing. These cells were analysed for their ability to support murine norovirus infection, determined by virus yield, and respond to different immune stimuli, assayed by quantitative PCR. The effect of Ifit proteins on norovirus translation was also tested in vitro. Results: Here, we show that VPg-dependent translation is completely refractory to Ifit1-mediated translation inhibition in vitro and Ifit1 cannot bind the 5' end of VPg-linked RNA. Nevertheless, knockout of Ifit1 promoted viral replication in murine norovirus infected cells. We then demonstrate that Ifit1 promoted interferon-beta expression following transfection of synthetic double-stranded RNA but had little effect on toll-like receptor 3 and 4 signalling. Conclusions: Ifit1 is an antiviral factor during norovirus infection but cannot directly inhibit viral translation. Instead, Ifit1 stimulates the antiviral state following cytoplasmic RNA sensing, contributing to restriction of norovirus replication.
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Pellerin, Marie, Edouard Hirchaud, Yannick Blanchard, Nicole Pavio, and Virginie Doceul. "Characterization of a Cell Culture System of Persistent Hepatitis E Virus Infection in the Human HepaRG Hepatic Cell Line." Viruses 13, no. 3 (March 4, 2021): 406. http://dx.doi.org/10.3390/v13030406.
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Hepatitis E virus (HEV) is considered as an emerging global health problem. In most cases, hepatitis E is a self-limiting disease and the virus is cleared spontaneously without the need of antiviral therapy. However, immunocompromised individuals can develop chronic infection and liver fibrosis that can progress rapidly to cirrhosis and liver failure. The lack of efficient and relevant cell culture system and animal models has limited our understanding of the biology of HEV and the development of effective drugs for chronic cases. In the present study, we developed a model of persistent HEV infection in human hepatocytes in which HEV replicates efficiently. This HEV cell culture system is based on differentiated HepaRG cells infected with an isolate of HEV-3 derived from a patient suffering from acute hepatitis E. Efficient replication was maintained for several weeks to several months as well as after seven successive passages on HepaRG naïve cells. Moreover, after six passages onto HepaRG, we found that the virus was still infectious after oral inoculation into pigs. We also showed that ribavirin had an inhibitory effect on HEV replication in HepaRG. In conclusion, this system represents a relevant and efficient in vitro model of HEV replication that could be useful to study HEV biology and identify effective antiviral drugs against chronic HEV infection.
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Kuwasaki, T. "Inhibition of human immunodeficiency virus 1 replication in vitro by a self-stabilized oligonucleotide with 2'-O-methyl-guanosine-uridine quadruplex motifs." Journal of Antimicrobial Chemotherapy 51, no. 4 (March 13, 2003): 813–19. http://dx.doi.org/10.1093/jac/dkg174.
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Holec, Sara A. M., Jisoo Lee, Abby Oehler, Lyn Batia, Aryanna Wiggins-Gamble, Jeffrey Lau, Felicia K. Ooi, et al. "The E46K mutation modulates α-synuclein prion replication in transgenic mice." PLOS Pathogens 18, no. 12 (December 1, 2022): e1010956. http://dx.doi.org/10.1371/journal.ppat.1010956.
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In multiple system atrophy (MSA), the α-synuclein protein misfolds into a self-templating prion conformation that spreads throughout the brain, leading to progressive neurodegeneration. While the E46K mutation in α-synuclein causes familial Parkinson’s disease (PD), we previously discovered that this mutation blocks in vitro propagation of MSA prions. Recent studies by others indicate that α-synuclein adopts a misfolded conformation in MSA in which a Greek key motif is stabilized by an intramolecular salt bridge between residues E46 and K80. Hypothesizing that the E46K mutation impedes salt bridge formation and, therefore, exerts a selective pressure that can modulate α-synuclein strain propagation, we asked whether three distinct α-synuclein prion strains could propagate in TgM47+/- mice, which express human α-synuclein with the E46K mutation. Following intracranial injection of these strains, TgM47+/- mice were resistant to MSA prion transmission, whereas recombinant E46K preformed fibrils (PFFs) transmitted neurological disease to mice and induced the formation of phosphorylated α-synuclein neuropathology. In contrast, heterotypic seeding following wild-type (WT) PFF–inoculation resulted in preclinical α-synuclein prion propagation. Moreover, when we inoculated TgM20+/- mice, which express WT human α-synuclein, with E46K PFFs, we observed delayed transmission kinetics with an incomplete attack rate. These findings suggest that the E46K mutation constrains the number of α-synuclein prion conformations that can propagate in TgM47+/- mice, expanding our understanding of the selective pressures that impact α-synuclein prion replication.
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López, Lissett, Ana Urzainqui, Elvira Domínguez, and Juan Antonio García. "Identification of an N-terminal domain of the plum pox potyvirus CI RNA helicase involved in self-interaction in a yeast two-hybrid system." Journal of General Virology 82, no. 3 (March 1, 2001): 677–86. http://dx.doi.org/10.1099/0022-1317-82-3-677.
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Potyvirus CI RNA helicase is a protein involved in RNA genome replication and virus movement. The protein aggregates in the cytoplasm of infected cells to form typical cylindrical inclusions. A yeast two-hybrid system was used to analyse interactions of the CI RNA helicase from plum pox potyvirus (PPV) with itself and with other viral proteins. No interactions could be detected of full-length CI protein with itself or with PPV P3/6K1, NIa, NIb or CP proteins. However, positive self-interactions were detected for N-terminal fragments of the CI protein, allowing the mapping of a CI–CI binding domain to the N-terminal 177 aa of the protein. Further deletion analysis suggested that several regions of this domain contribute to the interaction. Moreover, pull-down experiments demonstrate that, at least in vitro, full-length PPV CI protein is able to self-interact in the absence of other virus or plant factors.
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Nahhas, Alaa F., and Thomas J. Webster. "Targeting the Conserved Sequence of the Substrate for the Proteinase of Severe-Acute-Respiratory-Syndrome-Coronavirus-2 (SARS-CoV-2) Using Nano-Networks: Efficacy, Stability, and No Cytotoxicity." Journal of Biomedical Nanotechnology 18, no. 4 (April 1, 2022): 1158–63. http://dx.doi.org/10.1166/jbn.2022.3330.
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Herein, we designed a nano peptide that contains three important motifs for targeting the chemotrypsin-like cysteine protease (3CLpro) which is the enzyme responsible for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) replication. The novel nano peptide contains the Nap Phe-Phe motif that is responsible for peptide self-assembly, an octapeptide (Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe) motif where the enzyme recognizes the substrate and induces enzyme sensitivity, and a tetrapeptide motif which is positively charged containing the peptide (Lys)4 that facilitates penetration into a cell. The nano peptide was characterized using Proton Nuclear Magnetic Resonance (H-NMR) and Liquid Chromatography-Mass Spectrometry (LC-MS) to confirm its structure. In vitro results showed that the presently formulated nano peptide was not cytotoxic to fibroblasts for up to 72 hours, bound to 3CLpro, inhibited SARS-CoV-2 Omicron variant virus replication, and was stable for binding for up to one week in culture. In this manner, this timely study demonstrates that this novel nano peptide should be studied for a wide range of Coronavirus Disease (COVID-19) prophylactic or therapeutic applications.
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Petrucelli, Alex, Mahadevaiah Umashankar, Patricia Zagallo, Michael Rak, and Felicia Goodrum. "Interactions between Proteins Encoded within the Human CytomegalovirusUL133-UL138Locus." Journal of Virology 86, no. 16 (June 6, 2012): 8653–62. http://dx.doi.org/10.1128/jvi.00465-12.
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We previously described a novel genetic locus within the ULb′ region of the human cytomegalovirus (HCMV) genome that, while dispensable for replication in fibroblasts, suppresses replication in hematopoietic progenitors and augments replication in endothelial cells. This locus, referred to as theUL133-UL138locus, encodes four proteins, pUL133, pUL135, pUL136, and pUL138. In this work, we have mapped the interactions among these proteins. An analysis of all pairwise interactions during transient expression revealed a robust interaction between pUL133 and pUL138. Potential interactions between pUL136 and both pUL133 and pUL138 were also revealed. In addition, each of theUL133-UL138locus proteins self-associated, suggesting a potential to form higher-order homomeric complexes. As both pUL133 and pUL138 function in promoting viral latency in CD34+hematopoietic progenitor cells (HPCs) infectedin vitro, we further focused on this interaction. pUL133 and pUL138 are the predominant complex detected when all proteins are expressed together and require no other proteins in the locus for their association. During infection, the interaction between pUL133 and pUL138 or pUL136 can be detected. A recombinant virus that fails to express both pUL133 and pUL138 exhibited a latency phenotype similar to that of viruses that fail to express either pUL133 or pUL138, indicating that these proteins function cooperatively in latency and do not have independent functions that additively contribute to HCMV latency. These studies identify protein interactions among proteins encoded by theUL133-UL138locus and demonstrate an important interaction impacting the outcome of HCMV infection.
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Joseph, Aviva, Jian Hua Zheng, Antonia Follenzi, Teresa DiLorenzo, Kaori Sango, Jaime Hyman, Ken Chen, et al. "Lentiviral Vectors Encoding Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T-Cell Receptor Genes Efficiently Convert Peripheral Blood CD8 T Lymphocytes into Cytotoxic T Lymphocytes with Potent In Vitro and In Vivo HIV-1-Specific Inhibitory Activity." Journal of Virology 82, no. 6 (January 9, 2008): 3078–89. http://dx.doi.org/10.1128/jvi.01812-07.
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ABSTRACT The human immunodeficiency virus type 1 (HIV-1)-specific CD8 cytotoxic T-lymphocyte (CTL) response plays a critical role in controlling HIV-1 replication. Augmenting this response should enhance control of HIV-1 replication and stabilize or improve the clinical course of the disease. Although cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection in immunocompromised patients can be treated by adoptive transfer of ex vivo-expanded CMV- or EBV-specific CTLs, adoptive transfer of ex vivo-expanded, autologous HIV-1-specific CTLs had minimal effects on HIV-1 replication, likely a consequence of the inherently compromised qualitative function of HIV-1-specific CTLs derived from HIV-1-infected individuals. We hypothesized that this limitation could be circumvented by using as an alternative source of HIV-1-specific CTLs, autologous peripheral CD8+ T lymphocytes whose antigen specificity is redirected by transduction with lentiviral vectors encoding HIV-1-specific T-cell receptor (TCR) α and β chains, an approach used successfully in cancer therapy. To efficiently convert peripheral CD8 lymphocytes into HIV-1-specific CTLs that potently suppress in vivo HIV-1 replication, we constructed lentiviral vectors encoding the HIV-1-specific TCR α and TCR β chains cloned from a CTL clone specific for an HIV Gag epitope, SL9, as a single transcript linked with a self-cleaving peptide. We demonstrated that transduction with this lentiviral vector efficiently converted primary human CD8 lymphocytes into HIV-1-specific CTLs with potent in vitro and in vivo HIV-1-specific activity. Using lentiviral vectors encoding an HIV-1-specific TCR to transform peripheral CD8 lymphocytes into HIV-1-specific CTLs with defined specificities represents a new immunotherapeutic approach to augment the HIV-1-specific immunity of infected patients.
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Le, Hang Phuong, Xiaoyan Ma, Jorge Vaquero, Megan Brinkmeyer, Fei Guo, Wolf-Dietrich Heyer, and Jie Liu. "DSS1 and ssDNA regulate oligomerization of BRCA2." Nucleic Acids Research 48, no. 14 (July 1, 2020): 7818–33. http://dx.doi.org/10.1093/nar/gkaa555.
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Abstract The tumor suppressor BRCA2 plays a key role in initiating homologous recombination by facilitating RAD51 filament formation on single-stranded DNA. The small acidic protein DSS1 is a crucial partner to BRCA2 in this process. In vitro and in cells (1,2), BRCA2 associates into oligomeric complexes besides also existing as monomers. A dimeric structure was further characterized by electron microscopic analysis (3), but the functional significance of the different BRCA2 assemblies remains to be determined. Here, we used biochemistry and electron microscopic imaging to demonstrate that the multimerization of BRCA2 is counteracted by DSS1 and ssDNA. When validating the findings, we identified three self-interacting regions and two types of self-association, the N-to-C terminal and the N-to-N terminal interactions. The N-to-C terminal self-interaction of BRCA2 is sensitive to DSS1 and ssDNA. The N-to-N terminal self-interaction is modulated by ssDNA. Our results define a novel role of DSS1 to regulate BRCA2 in an RPA-independent fashion. Since DSS1 is required for BRCA2 function in recombination, we speculate that the monomeric and oligomeric forms of BRCA2 might be active for different cellular events in recombinational DNA repair and replication fork stabilization.
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Scalise, Mariangela, Fabiola Marino, Luca Salerno, Eleonora Cianflone, Claudia Molinaro, Nadia Salerno, Antonella De Angelis, Giuseppe Viglietto, Konrad Urbanek, and Daniele Torella. "From Spheroids to Organoids: The Next Generation of Model Systems of Human Cardiac Regeneration in a Dish." International Journal of Molecular Sciences 22, no. 24 (December 7, 2021): 13180. http://dx.doi.org/10.3390/ijms222413180.
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Organoids are tiny, self-organized, three-dimensional tissue cultures that are derived from the differentiation of stem cells. The growing interest in the use of organoids arises from their ability to mimic the biology and physiology of specific tissue structures in vitro. Organoids indeed represent promising systems for the in vitro modeling of tissue morphogenesis and organogenesis, regenerative medicine and tissue engineering, drug therapy testing, toxicology screening, and disease modeling. Although 2D cell cultures have been used for more than 50 years, even for their simplicity and low-cost maintenance, recent years have witnessed a steep rise in the availability of organoid model systems. Exploiting the ability of cells to re-aggregate and reconstruct the original architecture of an organ makes it possible to overcome many limitations of 2D cell culture systems. In vitro replication of the cellular micro-environment of a specific tissue leads to reproducing the molecular, biochemical, and biomechanical mechanisms that directly influence cell behavior and fate within that specific tissue. Lineage-specific self-organizing organoids have now been generated for many organs. Currently, growing cardiac organoid (cardioids) from pluripotent stem cells and cardiac stem/progenitor cells remains an open challenge due to the complexity of the spreading, differentiation, and migration of cardiac muscle and vascular layers. Here, we summarize the evolution of biological model systems from the generation of 2D spheroids to 3D organoids by focusing on the generation of cardioids based on the currently available laboratory technologies and outline their high potential for cardiovascular research.
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Silveira, Guilherme F., Pryscilla F. Wowk, Allan H. D. Cataneo, Paula F. dos Santos, Murilo Delgobo, Marco A. Stimamiglio, Maria Lo Sarzi, et al. "Human T Lymphocytes Are Permissive for Dengue Virus Replication." Journal of Virology 92, no. 10 (March 7, 2018): e02181-17. http://dx.doi.org/10.1128/jvi.02181-17.
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ABSTRACTDengue virus (DV) infection can cause either a self-limiting flu-like disease or a threatening hemorrhage that may evolve to shock and death. A variety of cell types, such as dendritic cells, monocytes, and B cells, can be infected by DV. However, despite the role of T lymphocytes in the control of DV replication, there remains a paucity of information on possible DV-T cell interactions during the disease course. In the present study, we have demonstrated that primary human naive CD4+and CD8+T cells are permissive for DV infection. Importantly, both T cell subtypes support viral replication and secrete viable virus particles. DV infection triggers the activation of both CD4+and CD8+T lymphocytes, but preactivation of T cells reduces the susceptibility of T cells to DV infection. Interestingly, the cytotoxicity-inducing protein granzyme A is highly secreted by human CD4+but not CD8+T cells after exposure to DVin vitro. Additionally, using annexin V and polycaspase assays, we have demonstrated that T lymphocytes, in contrast to monocytes, are resistant to DV-induced apoptosis. Strikingly, both CD4+and CD8+T cells were found to be infected with DV in acutely infected dengue patients. Together, these results show that T cells are permissive for DV infectionin vitroandin vivo, suggesting that this cell population may be a viral reservoir during the acute phase of the disease.IMPORTANCEInfection by dengue virus (DV) causes a flu-like disease that can evolve to severe hemorrhaging and death. T lymphocytes are important cells that regulate antibody secretion by B cells and trigger the death of infected cells. However, little is known about the direct interaction between DV and T lymphocytes. Here, we show that T lymphocytes from healthy donors are susceptible to infection by DV, leading to cell activation. Additionally, T cells seem to be resistant to DV-induced apoptosis, suggesting a potential role as a viral reservoir in humans. Finally, we show that both CD4+and CD8+T lymphocytes from acutely infected DV patients are infected by DV. Our results raise new questions about DV pathogenesis and vaccine development.
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Deszcz, Luiza, Regina Cencic, Carla Sousa, Ernst Kuechler, and Tim Skern. "An Antiviral Peptide Inhibitor That Is Active against Picornavirus 2A Proteinases but Not Cellular Caspases." Journal of Virology 80, no. 19 (October 1, 2006): 9619–27. http://dx.doi.org/10.1128/jvi.00612-06.
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ABSTRACT The replication of many viruses is absolutely dependent on proteolytic cleavage. Infected cells also use this biological mechanism to induce programmed cell death in response to viral infection. Specific inhibitors for both viral and cellular proteases are therefore of vital importance. We have recently shown that the general caspase inhibitor zVAD.fmk inhibits not only caspases, but also the 2A pro of human rhinoviruses (HRVs) (L. Deszcz, J. Seipelt, E. Vassilieva, A. Roetzer, and E. Kuechler, FEBS Lett. 560:51-55, 2004). Here, we describe a derivative of zVAD.fmk that inhibits HRV2 2A pro but that has no effect on caspase 9. This gain in specificity was achieved by replacing the aspartic acid of zVAD.fmk with methionine to generate zVAM.fmk. Methionine was chosen because an oligopeptide with methionine at the P1 position was a much better substrate than an oligopeptide with an alanine residue, which is found at the P1 position of the wild-type HRV2 2A pro cleavage site. zVAM.fmk inhibits the replication of HRV type 2 (HRV2), HRV14, and HRV16. In contrast to zVAD.fmk, however, zVAM.fmk did not inhibit apoptosis induced by puromycin in HeLa cells. zVAM.fmk inhibited in vitro the intermolecular cleavage of eukaryotic initiation factor 4GI (eIF4GI) by HRV2 2A pro at nanomolar concentrations. However, much higher concentrations of zVAM.fmk were required to inhibit HRV14 2A pro cleavage of eIF4GI. In contrast, intramolecular self-processing of HRV14 2A pro was much more susceptible to inhibition by zVAM.fmk than that of HRV2 2A pro , suggesting that zVAM.fmk inhibits HRV2 and HRV14 replication by targeting different reactions of the same proteinase.
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Guo, Hui Shan, Juan José López-Moya, and Juan Antonio García. "Mitotic Stability of Infection-Induced Resistance to Plum Pox Potyvirus Associated with Transgene Silencing and DNA Methylation." Molecular Plant-Microbe Interactions® 12, no. 2 (February 1999): 103–11. http://dx.doi.org/10.1094/mpmi.1999.12.2.103.
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Plum pox potyvirus (PPV) infection of transgenic Nicotiana benthamiana plants that expressed the PPV NIb RNA replicase carrying a Gly to Val mutation at the GDD motif (NIbV lines) induced a phenotype of virus resistance and transgene silencing, which was not transmissible to the progeny after self-fertilization (H. S. Guo and J. A. García, Mol. Plant-Microbe Interact. 10:160-170, 1997). Here, we demonstrate that the induced resistance of NIbV plants is mitotically stable after plant propagation by grafting and by in vitro regeneration. Virus replication or residual virus RNA seem not to be required to maintain transgene silencing and virus resistance. Analysis by PCR (polymerase chain reaction) amplification after treatment with methylation-sensitive restriction nucleases indicates that DNA methylation is associated with establishment and maintenance of transgene silencing and virus resistance. Restoration of transgene activity and susceptibility to PPV in sexual progeny correlated with resetting of transgene DNA methylation. On the basis of these and other published results, we present a general model for post-transcriptional gene silencing in which RNA signals, generated either by a silenced nuclear gene or by virus replication, both activate a specific cytoplasmic RNA degradation pathway and induce changes (in particular, DNA methylation) in homologous nuclear genes that switch them from an active to a silenced status.
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Eriksson, Jesper M., and Elisabeth Haggård-Ljungquist. "The Multifunctional Bacteriophage P2 Cox Protein Requires Oligomerization for Biological Activity." Journal of Bacteriology 182, no. 23 (December 1, 2000): 6714–23. http://dx.doi.org/10.1128/jb.182.23.6714-6723.2000.
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ABSTRACT The Cox protein of bacteriophage P2 is a multifunctional protein of 91 amino acids. It is directly involved in the site-specific recombination event leading to excision of P2 DNA out of the host chromosome. In this context, it functions as an architectural protein in the formation of the excisome. Cox is also a transcriptional repressor of the P2 Pc promoter, thereby ensuring lytic growth. Finally it promotes derepression of prophage P4, a nonrelated defective satellite phage, by activating the P4 PLL promoter that controls P4 DNA replication. In this case it binds upstream of the PLL promoter, which normally is activated by the P4 Delta protein. In this work we have analyzed the native form of the Cox protein in vivo, using a bacteriophage λ cI-based oligomerization assay system, and in vitro, using gel filtration, cross-linking agents, and gel retardation assays. We found that P2 Cox has a strong oligomerization function in vivo as well as in vitro. The in vitro analysis indicates that its native form is a tetramer that can self-associate to octamers. Furthermore we show that oligomerization is necessary for the biological activity by characterizing differentcox mutants and that oligomerization is mediated by the C-terminal region.
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Díaz-Yáñez, Fernando, Ricardo Álvarez, Iván L. Calderón, Juan A. Fuentes, and Fernando Gil. "CdsH Contributes to the Replication of Salmonella Typhimurium inside Epithelial Cells in a Cysteine-Supplemented Medium." Microorganisms 8, no. 12 (December 17, 2020): 2019. http://dx.doi.org/10.3390/microorganisms8122019.
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Salmonella Typhimurium is a facultative, intracellular pathogen whose products range from self-limited gastroenteritis to systemic diseases. Food ingestion increases biomolecules’ concentration in the intestinal lumen, including amino acids such as cysteine, which is toxic in a concentration-dependent manner. When cysteine’s intracellular concentration reaches toxic levels, S. Typhimurium expresses a cysteine-inducible enzyme (CdsH), which converts cysteine into pyruvate, sulfide, and ammonia. Despite this evidence, the biological context of cdsH’s role is not completely clear, especially in the infective cycle. Since inside epithelial cells both cdsH and its positive regulator, ybaO, are overexpressed, we hypothesized a possible role of cdsH in the intestinal phase of the infection. To test this hypothesis, we used an in vitro model of HT-29 cell infection, adding extra cysteine to the culture medium during the infective process. We observed that, at 6 h post-invasion, the wild type S. Typhimurium proliferated 30% more than the ΔcdsH strain in the presence of extra cysteine. This result shows that cdsH contributes to the bacterial replication in the intracellular environment in increased concentrations of extracellular cysteine, strongly suggesting that cdsH participates by increasing the bacterial fitness in the intestinal phase of the S. Typhimurium infection.
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Primadharsini, Putu Prathiwi, Shigeo Nagashima, Masaharu Takahashi, Kazumoto Murata, and Hiroaki Okamoto. "Ritonavir Blocks Hepatitis E Virus Internalization and Clears Hepatitis E Virus In Vitro with Ribavirin." Viruses 14, no. 11 (November 3, 2022): 2440. http://dx.doi.org/10.3390/v14112440.
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Hepatitis E virus (HEV) is increasingly recognized as the leading cause of acute hepatitis. Although HEV infections are mostly self-limiting, a chronic course can develop especially in those with immunocompromised state. Ribavirin is currently used to treat such patients. According to various reports on chronic HEV infections, a sustained virological response (SVR) was achieved in approximately 80% of patients receiving ribavirin monotherapy. To increase the SVR rate, drug combination might be a viable strategy, which we attempted in the current study. Ritonavir was identified in our previous drug screening while searching for candidate novel anti-HEV drugs. It demonstrated potent inhibition of HEV growth in cultured cells. In the present study, ritonavir blocked HEV internalization as shown through time-of-addition and immunofluorescence assays. Its combination with ribavirin significantly increased the efficiency of inhibiting HEV growth compared to that shown by ribavirin monotherapy, even in PLC/PRF/5 cells with robust HEV production, and resulted in viral clearance. Similar efficiency was seen for HEV genotypes 3 and 4, the main causes of chronic infection. The present findings provide insight concerning the advantage of combination therapy using drugs blocking different steps in the HEV life cycle (internalization and RNA replication) as a potential novel treatment strategy for chronic hepatitis E.
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Jain, Mamta K., Mamta K. Jain, Hesham Sadek, James de Lemos, Darren mcGuire, Colby Ayers, Jennifer L. Eiston, et al. "525. Atovaquone for Treatment of COVID-19 (Ataq COVID-19) Trial." Open Forum Infectious Diseases 8, Supplement_1 (November 1, 2021): S363—S364. http://dx.doi.org/10.1093/ofid/ofab466.724.
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Abstract Background Our group performed an in-silico screen to identify FDA approved drugs that inhibit SARS-C0V-2 main protease (Mpro), followed by in vitro viral replication assays, and in vivo pharmacokinetic studies in mice. These studies identified atovaquone as a promising candidate for inhibiting viral replication. Methods Enrolled patients were randomized in a 2:1 fashion to atovaquone 1500 mg twice daily versus matched placebo. Patients received standard of care treatment including remdesivir, dexamethasone, or convalescent plasma as deemed necessary by the treating team. Patients agreed to allow collection of saliva at baseline and twice a day while hospitalized or up to 10 days. Saliva was collected and RNA extracted for viral load (VL) measurement by Real-time PCR. Our primary outcome was to examine the between group differences in log transformed VL(copies/mL) using generalized linear mixed-effect models of repeated measures from all samples. Additional analysis of Atovquone plasma concentrations were examined and correlated with viral load and body mass index (BMI). Results Of the 61 patients enrolled; 41 were received atovaquone and 19 placebo. Overall the population was predominately male Hispanic with a mean age of 51 years. The two groups were balanced (Table 1) with regard to age, gender, race, co-morbidities, days from onset of symptoms, baseline oxygen requirements, and receipt of COVID-19 specific standard of care treatment. A higher proportion with diabetes was noted in the Atovaquone arm. The log10 VL was 5.25 copies/mL vs. 4.79 copies/mL at baseline in the atovaquone vs. placebo group. Although there was a decrease in VL over time, there was no differences between the atovaquone plus standard of care arm versus the standard of care arm (Figure 1). Additional analysis of atovaquone plasma concentration demonstrated a wide variation in atovaquone levels, inverse association between atovaquone levels and BMI (rho -0.44, p=0.03), and Day 5 concentrations and VL (rho -0.54, p=0.005). Figure 1. Mean viral load of COVID-19 over time of atovaquone (blue) vs. placebo (red). Table 1. Baseline characteristics Conclusion Although atovaquone showed promising in vitro antiviral properties for COVID-19, in this pilot study we did not detect a change in VL in patients who received atovaquone compared to placebo, possibly due to failure of patients achieve adequate drug levels. Disclosures Mamta K. Jain, MD, MPH, Gilead Sciences Inc. (Individual(s) Involved: Self): Research Grant or Support, Scientific Research Study Investigator; GlaxoSmithKline (Individual(s) Involved: Self): Scientific Research Study Investigator; Merck (Individual(s) Involved: Self): Scientific Research Study Investigator; Vasgene (Individual(s) Involved: Self): Scientific Research Study Investigator
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Asgari, Samira, Luregn J. Schlapbach, Stéphanie Anchisi, Christian Hammer, Istvan Bartha, Thomas Junier, Geneviève Mottet-Osman, et al. "Severe viral respiratory infections in children with IFIH1 loss-of-function mutations." Proceedings of the National Academy of Sciences 114, no. 31 (July 17, 2017): 8342–47. http://dx.doi.org/10.1073/pnas.1704259114.
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Viral respiratory infections are usually mild and self-limiting; still they exceptionally result in life-threatening infections in previously healthy children. To investigate a potential genetic cause, we recruited 120 previously healthy children requiring support in intensive care because of a severe illness caused by a respiratory virus. Using exome and transcriptome sequencing, we identified and characterized three rare loss-of-function variants in IFIH1, which encodes an RIG-I-like receptor involved in the sensing of viral RNA. Functional testing of the variants IFIH1 alleles demonstrated that the resulting proteins are unable to induce IFN-β, are intrinsically less stable than wild-type IFIH1, and lack ATPase activity. In vitro assays showed that IFIH1 effectively restricts replication of human respiratory syncytial virus and rhinoviruses. We conclude that IFIH1 deficiency causes a primary immunodeficiency manifested in extreme susceptibility to common respiratory RNA viruses.
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Liu, Nanxin, Zeyu Yang, Yuying Liu, Xintao Dang, Qingqing Zhang, Jin Wang, Xueying Liu, Jie Zhang, and Xiaoyan Pan. "Identification of a Putative SARS-CoV-2 Main Protease Inhibitor through In Silico Screening of Self-Designed Molecular Library." International Journal of Molecular Sciences 24, no. 14 (July 13, 2023): 11390. http://dx.doi.org/10.3390/ijms241411390.
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There have been outbreaks of SARS-CoV-2 around the world for over three years, and its variants continue to evolve. This has become a major global health threat. The main protease (Mpro, also called 3CLpro) plays a key role in viral replication and proliferation, making it an attractive drug target. Here, we have identified a novel potential inhibitor of Mpro, by applying the virtual screening of hundreds of nilotinib-structure-like compounds that we designed and synthesized. The screened compounds were assessed using SP docking, XP docking, MM-GBSA analysis, IFD docking, MD simulation, ADME/T prediction, and then an enzymatic assay in vitro. We finally identified the compound V291 as a potential SARS-CoV-2 Mpro inhibitor, with a high docking affinity and enzyme inhibitory activity. Moreover, the docking results indicate that His41 is a favorable amino acid for pi-pi interactions, while Glu166 can participate in salt-bridge formation with the protonated primary or secondary amines in the screened molecules. Thus, the compounds reported here are capable of engaging the key amino acids His41 and Glu166 in ligand-receptor interactions. A pharmacophore analysis further validates this assertion.
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Oo, Adrian, Pouya Hassandarvish, Sek Peng Chin, Vannajan Sanghiran Lee, Sazaly Abu Bakar, and Keivan Zandi. "In silico study on anti-Chikungunya virus activity of hesperetin." PeerJ 4 (October 26, 2016): e2602. http://dx.doi.org/10.7717/peerj.2602.
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BackgroundThe re-emerging,Aedes spp.transmitted Chikungunya virus (CHIKV) has recently caused large outbreaks in a wide geographical distribution of the world including countries in Europe and America. Though fatalities associated with this self-remitting disease were rarely reported, quality of patients’ lives have been severely diminished by polyarthralgia recurrence. Neither effective antiviral treatment nor vaccines are available for CHIKV. Our previous in vitro screening showed that hesperetin, a bioflavonoid exhibits inhibitory effect on the virus intracellular replication. Here, we present a study using the computational approach to identify possible target proteins for future mechanistic studies of hesperetin.Methods3D structures of CHIKV nsP2 (3TRK) and nsP3 (3GPG) were retrieved from Protein Data Bank (PDB), whereas nsP1, nsP4 and cellular factor SPK2 were modeled using Iterative Threading Assembly Refinement (I-TASSER) server based on respective amino acids sequence. We performed molecular docking on hesperetin against all four CHIKV non-structural proteins and SPK2. Proteins preparation and subsequent molecular docking were performed using Discovery Studio 2.5 and AutoDock Vina 1.5.6. The Lipinski’s values of the ligand were computed and compared with the available data from PubChem. Two non-structural proteins with crystal structures 3GPG and 3TRK in complexed with hesperetin, demonstrated favorable free energy of binding from the docking study, were further explored using molecular dynamics (MD) simulations.ResultsWe observed that hesperetin interacts with different types of proteins involving hydrogen bonds, pi-pi effects, pi-cation bonding and pi-sigma interactions with varying binding energies. Among all five tested proteins, our compound has the highest binding affinity with 3GPG at −8.5 kcal/mol. The ligand used in this study also matches the Lipinski’s rule of five in addition to exhibiting closely similar properties with that of in PubChem. The docking simulation was performed to obtain a first guess of the binding structure of hesperetin complex and subsequently analysed by MD simulations to assess the reliability of the docking results. Root mean square deviation (RMSD) of the simulated systems from MD simulations indicated that the hesperetin complex remains stable within the simulation timescale.DiscussionThe ligand’s tendencies of binding to the important proteins for CHIKV replication were consistent with our previous in vitro screening which showed its efficacy in blocking the virus intracellular replication. NsP3 serves as the highest potential target protein for the compound’s inhibitory effect, while it is interesting to highlight the possibility of interrupting CHIKV replication via interaction with host cellular factor. By complying the Lipinski’s rule of five, hesperetin exhibits drug-like properties which projects its potential as a therapeutic option for CHIKV infection.
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Gao, Yong, Michael A. Lobritz, Justin Roth, Measho Abreha, Kenneth N. Nelson, Immaculate Nankya, Dawn M. Moore-Dudley, Awet Abraha, Stanton L. Gerson, and Eric J. Arts. "Targets of Small Interfering RNA Restriction during Human Immunodeficiency Virus Type 1 Replication." Journal of Virology 82, no. 6 (January 16, 2008): 2938–51. http://dx.doi.org/10.1128/jvi.02126-07.
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ABSTRACT Small interfering RNAs (siRNAs) have been shown to effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. The mechanism(s) for this inhibition is poorly understood, as siRNAs may interact with multiple HIV-1 RNA species during different steps of the retroviral life cycle. To define susceptible HIV-1 RNA species, siRNAs were first designed to specifically inhibit two divergent primary HIV-1 isolates via env and gag gene targets. A self-inactivating lentiviral vector harboring these target sequences confirmed that siRNA cannot degrade incoming genomic RNA. Disruption of the incoming core structure by rhesus macaque TRIM5α did, however, provide siRNA-RNA-induced silencing complex access to HIV-1 genomic RNA and promoted degradation. In the absence of accelerated core disruption, only newly transcribed HIV-1 mRNA in the cytoplasm is sensitive to siRNA degradation. Inhibitors of HIV-1 mRNA nuclear export, such as leptomycin B and camptothecin, blocked siRNA restriction. All HIV-1 RNA regions and transcripts found 5′ of the target sequence, including multiply spliced HIV-1 RNA, were degraded by unidirectional 3′-to-5′ siRNA amplification and spreading. In contrast, HIV-1 RNA 3′ of the target sequence was not susceptible to siRNA. Even in the presence of siRNA, full-length HIV-1 RNA is still encapsidated into newly assembled viruses. These findings suggest that siRNA can target only a relatively “naked” cytoplasmic HIV-1 RNA despite the involvement of viral RNA at nearly every step in the retroviral life cycle. Protection of HIV-1 RNA within the core following virus entry, during encapsidation/virus assembly, or within the nucleus may reflect virus evolution in response to siRNA, TRIM5α, or other host restriction factors.