A Charged Performance by gp17 in Viral Packaging

Gene therapy and transgenic research have advanced quickly in recent years due to the development of CRISPR technology. The rapid development of CRISPR technology has been largely benefited by chemical engineering. Firstly, chemical or synthetic substance enables spatiotemporal and conditional control of Cas9 or dCas9 activities. It prevents the leaky expression of CRISPR components, as well as minimizes toxicity and off-target effects. Multi-input logic operations and complex genetic circuits can also be implemented via multiplexed and orthogonal regulation of target genes. Secondly, rational chemical modifications to the sgRNA enhance gene editing efficiency and specificity by improving sgRNA stability and binding affinity to on-target genomic loci, and hence reducing off-target mismatches and systemic immunogenicity. Chemically-modified Cas9 mRNA is also more active and less immunogenic than the native mRNA. Thirdly, nonviral vehicles can circumvent the challenges associated with viral packaging and production through the delivery of Cas9-sgRNA ribonucleoprotein complex or large Cas9 expression plasmids. Multi-functional nanovectors enhance genome editing in vivo by overcoming multiple physiological barriers, enabling ligand-targeted cellular uptake, and blood-brain barrier crossing. Chemical engineering can also facilitate viral-based delivery by improving vector internalization, allowing tissue-specific transgene expression, and preventing inactivation of the viral vectors in vivo. This review aims to discuss how chemical engineering has helped improve existing CRISPR applications and enable new technologies for biomedical research. The usefulness, advantages, and molecular action for each chemical engineering approach are also highlighted.

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Nucleotide-dependent DNA gripping and an end-clamp mechanism regulate the bacteriophage T4 viral packaging motor

Nature Communications ◽

10.1038/s41467-018-07834-2 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 10

Author(s):

Mariam Ordyan ◽

Istiaq Alam ◽

Marthandan Mahalingam ◽

Venigalla B. Rao ◽

Douglas E. Smith

Keyword(s):

Bacteriophage T4 ◽

Viral Packaging

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Vertical silicon nanowires as a universal platform for delivering biomolecules into living cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0909350107 ◽

2010 ◽

Vol 107 (5) ◽

pp. 1870-1875 ◽

Cited By ~ 399

Author(s):

Alex K. Shalek ◽

Jacob T. Robinson ◽

Ethan S. Karp ◽

Jin Seok Lee ◽

Dae-Ro Ahn ◽

...

Keyword(s):

Small Molecules ◽

High Throughput ◽

Silicon Nanowires ◽

Mammalian Cells ◽

Living Cells ◽

Diverse Range ◽

Viral Packaging ◽

Neuronal Progenitor ◽

Efficient Delivery ◽

Surface Modified

A generalized platform for introducing a diverse range of biomolecules into living cells in high-throughput could transform how complex cellular processes are probed and analyzed. Here, we demonstrate spatially localized, efficient, and universal delivery of biomolecules into immortalized and primary mammalian cells using surface-modified vertical silicon nanowires. The method relies on the ability of the silicon nanowires to penetrate a cell’s membrane and subsequently release surface-bound molecules directly into the cell’s cytosol, thus allowing highly efficient delivery of biomolecules without chemical modification or viral packaging. This modality enables one to assess the phenotypic consequences of introducing a broad range of biological effectors (DNAs, RNAs, peptides, proteins, and small molecules) into almost any cell type. We show that this platform can be used to guide neuronal progenitor growth with small molecules, knock down transcript levels by delivering siRNAs, inhibit apoptosis using peptides, and introduce targeted proteins to specific organelles. We further demonstrate codelivery of siRNAs and proteins on a single substrate in a microarray format, highlighting this technology’s potential as a robust, monolithic platform for high-throughput, miniaturized bioassays.

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Influenza A and B Virus Intertypic Reassortment through Compatible Viral Packaging Signals

Journal of Virology ◽

10.1128/jvi.01440-14 ◽

2014 ◽

Vol 88 (18) ◽

pp. 10778-10791 ◽

Cited By ~ 56

Author(s):

S. F. Baker ◽

A. Nogales ◽

C. Finch ◽

K. M. Tuffy ◽

W. Domm ◽

...

Keyword(s):

Influenza A ◽

Viral Packaging ◽

B Virus ◽

Packaging Signals

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Construction of Retroviral Vectors with Improved Safety, Gene Expression, and Versatility

Journal of Virology ◽

10.1128/jvi.72.2.994-1004.1998 ◽

1998 ◽

Vol 72 (2) ◽

pp. 994-1004 ◽

Cited By ~ 44

Author(s):

Seon Hee Kim ◽

Seung Shin Yu ◽

Jong Sang Park ◽

Paul D. Robbins ◽

Chung Sun An ◽

...

Keyword(s):

Gene Expression ◽

Mutational Analysis ◽

Leukemia Virus ◽

Retroviral Vectors ◽

Retroviral Vector ◽

Upstream Region ◽

Target Cells ◽

Internal Ribosome Entry ◽

Viral Packaging ◽

Or Gene

ABSTRACT Murine leukemia virus (MLV)-based retroviral vectors are the most frequently used gene delivery vehicles. However, the current vectors are still not fully optimized for gene expression and viral titer, and many genetic and biochemical features of MLV-based vectors are poorly understood. We have previously reported that the retroviral vector MFG, where the gene of interest is expressed as a spliced mRNA, is superior in the level of gene expression with respect to other vectors compared in the study. As one approach to developing improved retroviral vectors, we have systematically performed mutational analysis of the MFG retroviral vector. We demonstrated that the entiregag coding sequence, together with the immediate upstream region, could be deleted without significantly affecting viral packaging or gene expression. To our knowledge, this region is included in all currently available retroviral vectors. In addition, almost the entire U3 region could be replaced with the heterologous human cytomegalovirus immediately-early promoter without deleterious effects. We could also insert internal ribosome entry sites (IRES) and multicloning sites into MFG without adverse effects. Based on these observations, we have constructed a series of new, improved retroviral constructs. These vectors produced viral titers comparable to MFG, expressed high levels of gene expression, and stably transferred genes to the target cells. Our vectors are more convenient to use because of the presence of multicloning sites and IRESs, and they are also more versatile because they can be readily converted to various applications. Our results have general implications regarding the design and development of improved retroviral vectors for gene therapy.

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Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection

10.1101/453068 ◽

2018 ◽

Author(s):

Autumn T. LaPointe ◽

Joaquín Moreno-Contreras ◽

Kevin J. Sokoloski

Keyword(s):

Viral Infection ◽

Growth Kinetics ◽

Sindbis Virus ◽

Nonstructural Protein ◽

Severe Disease ◽

Protein Accumulation ◽

Genomic Rnas ◽

Viral Growth ◽

Viral Packaging ◽

The Impact

AbstractAlphaviruses are arthropod-borne RNA viruses that are capable of causing severe disease and are a significant burden to public health. Alphaviral replication results in the production of both capped and noncapped viral genomic RNAs, which are packaged into virions during the infections of vertebrate and invertebrate cells. However, the roles that the noncapped genomic RNAs (ncgRNAs) play during alphaviral infection have yet to be exhaustively characterized. Here, the importance of the ncgRNAs to alphaviral infection was assessed by using mutants of the nsP1 protein of Sindbis virus (SINV), which altered the synthesis of the ncgRNAs during infection by modulating the protein’s capping efficiency. Specifically, point mutants at residues Y286A and N376A decreased capping efficiency, while a point mutant at D355A increased the capping efficiency of the SINV genomic RNA during genuine viral infection. Viral growth kinetics were significantly reduced for the D355A mutant relative to wild type infection, whereas the Y286A and N376A mutants showed modest decreases in growth kinetics. Overall genomic translation and nonstructural protein accumulation was found to correlate with increases and decreases in capping efficiency. However, genomic, minus strand, and subgenomic viral RNA synthesis was largely unaffected by the modulation of alphaviral capping activity. In addition, translation of the subgenomic vRNA was found to be unimpacted by changes in capping efficiency. The mechanism by which decreased presence of ncgRNAs reduced viral growth kinetics was through the impaired production of viral particles. Collectively, these data illustrate the importance of ncgRNAs to viral infection and suggests that they play in integral role in the production of viral progeny.ImportanceAlphaviruses have been the cause of both localized outbreaks and large epidemics of severe disease. Currently, there are no strategies or vaccines which are either safe or effective for preventing alphaviral infection or treating alphaviral disease. This deficit of viable therapeutics highlights the need to better understand the mechanisms behind alphaviral infection in order to develop novel antiviral strategies for alphaviral disease. In particular, this study details a previously uncharacterized aspect of the alphaviral life cycle, the importance of noncapped genomic viral RNAs to alphaviral infection. This offers new insights into the mechanisms of alphaviral replication and the impact of the noncapped genomic RNAs on viral packaging.

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Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate

10.1101/2020.06.11.147199 ◽

2020 ◽

Cited By ~ 16

Author(s):

Christiane Iserman ◽

Christine Roden ◽

Mark Boerneke ◽

Rachel Sealfon ◽

Grace McLaughlin ◽

...

Keyword(s):

Small Molecules ◽

Mammalian Cells ◽

Upper Airway ◽

Dependent Manner ◽

Rna Sequences ◽

Concentration Dependent Manner ◽

Health Crisis ◽

Viral Packaging ◽

N Protein ◽

Human Body Temperature

AbstractA mechanistic understanding of the SARS-CoV-2 viral replication cycle is essential to develop new therapies for the COVID-19 global health crisis. In this study, we show that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with the viral genome, and propose a model of viral packaging through LLPS. N-protein condenses with specific RNA sequences in the first 1000 nts (5’-End) under physiological conditions and is enhanced at human upper airway temperatures. N-protein condensates exclude non-packaged RNA sequences. We comprehensively map sites bound by N-protein in the 5’-End and find preferences for single-stranded RNA flanked by stable structured elements. Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules thus presenting screenable processes for identifying antiviral compounds effective against SARS-CoV-2.

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Interaction of the Adenovirus IVa2 Protein with Viral Packaging Sequences

Journal of Virology ◽

10.1128/jvi.74.6.2687-2693.2000 ◽

2000 ◽

Vol 74 (6) ◽

pp. 2687-2693 ◽

Cited By ~ 65

Author(s):

Wei Zhang ◽

Michael J. Imperiale

Keyword(s):

Dna Sequences ◽

Dna Packaging ◽

Late Promoter ◽

Apparent Effect ◽

Packaging Signal ◽

Viral Dna ◽

Viral Packaging ◽

Infected Cells ◽

Major Late Promoter ◽

Viral Dna Packaging

ABSTRACT We have demonstrated previously that the adenovirus L1 52/55-kDa protein binds to the viral IVa2 protein in infected cells. The significance of this interaction was unclear, however, based on the known functions of these two proteins: the 52/55-kDa protein is required for viral DNA packaging, while the IVa2 protein is a transactivator of the major late promoter (MLP). In this report, we have attempted to elucidate a role for each of the two proteins in the other's known function. There is no apparent effect of the 52/55-kDa protein on the interaction of the IVa2 protein with the MLP. Surprisingly, however, we found that the IVa2 protein can interact with the adenoviral packaging signal and that this interaction involves DNA sequences that have previously been demonstrated to be required for packaging.

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Complete nucleotide sequence of a mouse VL30 retro-element.

Molecular and Cellular Biology ◽

10.1128/mcb.8.8.2989 ◽

1988 ◽

Vol 8 (8) ◽

pp. 2989-2998 ◽

Cited By ~ 35

Author(s):

S E Adams ◽

P D Rathjen ◽

C A Stanway ◽

S M Fulton ◽

M H Malim ◽

...

Keyword(s):

Nucleotide Sequence ◽

Complete Nucleotide Sequence ◽

Leukemia Virus ◽

Open Reading Frames ◽

Long Terminal Repeats ◽

Base Pairs ◽

Viral Packaging ◽

Protein Functions ◽

Active Promoter ◽

Reading Frames

The complete nucleotide sequence of a mouse retro-element is presented. The cloned element is composed of 4,834 base pairs (bp) with long terminal repeats of 568 bp separated by an internal region of 3,698 bp. The element did not appear to have any open reading frames that would be capable of encoding the functional proteins that are normally produced by retro-elements. However, some regions of the genome showed some homology to retroviral gag and pol open reading frames. There was no region in VL30 corresponding to a retroviral env gene. This implies that VL30 is related to retrotransposons rather than to retroviruses. The sequence also contained regions that were homologous to known reverse transcriptase priming sites and viral packaging sites. These observations, combined with the known transcriptional capacity of the VL30 promoter, suggest that VL30 relies on protein functions of other retro-elements, such as murine leukemia virus, while maintaining highly conserved cis-active promoter, packaging, and priming sites necessary for its replication and cell-to-cell transmission.

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DNA crunching by a viral packaging motor: Compression of a procapsid-portal stalled Y-DNA substrate

Virology ◽

10.1016/j.virol.2009.11.047 ◽

2010 ◽

Vol 398 (2) ◽

pp. 224-232 ◽

Cited By ~ 42

Author(s):

Krishanu Ray ◽

Chandran R. Sabanayagam ◽

Joseph R. Lakowicz ◽

Lindsay W. Black

Keyword(s):

Viral Packaging ◽

Dna Substrate

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