scholarly journals Revealing in real-time a multistep assembly mechanism for SV40 virus-like particles

2020 ◽  
Vol 6 (16) ◽  
pp. eaaz1639 ◽  
Author(s):  
Mariska G. M. van Rosmalen ◽  
Douwe Kamsma ◽  
Andreas S. Biebricher ◽  
Chenglei Li ◽  
Adam Zlotnick ◽  
...  
Keyword(s):  
Real Time ◽  
Optical Tweezers ◽  
Self Assembly ◽  
Persistence Length ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Contour Length ◽  
Sv40 Virus ◽  
Virus Like Particles ◽  
Assembly Mechanism

Many viruses use their genome as template for self-assembly into an infectious particle. However, this reaction remains elusive because of the transient nature of intermediate structures. To elucidate this process, optical tweezers and acoustic force spectroscopy are used to follow viral assembly in real time. Using Simian virus 40 (SV40) virus-like particles as model system, we reveal a multistep assembly mechanism. Initially, binding of VP1 pentamers to DNA leads to a significantly decreased persistence length. Moreover, the pentamers seem able to stabilize DNA loops. Next, formation of interpentamer interactions results in intermediate structures with reduced contour length. These structures stabilize into objects that permanently decrease the contour length to a degree consistent with DNA compaction in wild-type SV40. These data indicate that a multistep mechanism leads to fully assembled cross-linked SV40 particles. SV40 is studied as drug delivery system. Our insights can help optimize packaging of therapeutic agents in these particles.

Switch from Polymorphic to Homogenous Self‐Assembly of Virus‐Like Particles of Simian Virus 40 through Double‐Cysteine Substitution

Small ◽  
2020 ◽  
Vol 16 (45) ◽  
pp. 2004484
Author(s):  
Chengchen Xu ◽  
Weiwei Zhu ◽  
Hanjing Mao ◽  
Wenjing Zhang ◽  
Gen‐Quan Yin ◽  
...  
Keyword(s):  
Self Assembly ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Virus Like Particles ◽  
Cysteine Substitution

scholarly journals Virus‐Like Particles: Switch from Polymorphic to Homogenous Self‐Assembly of Virus‐Like Particles of Simian Virus 40 through Double‐Cysteine Substitution (Small 45/2020)

Small ◽  
2020 ◽  
Vol 16 (45) ◽  
pp. 2070248
Author(s):  
Chengchen Xu ◽  
Weiwei Zhu ◽  
Hanjing Mao ◽  
Wenjing Zhang ◽  
Gen‐Quan Yin ◽  
...  
Keyword(s):  
Self Assembly ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Virus Like Particles ◽  
Cysteine Substitution

scholarly journals Real-time assembly of an artificial virus elucidated at the single-particle level

2019 ◽  
Author(s):  
Margherita Marchetti ◽  
Douwe Kamsma ◽  
Ernesto Cazares Vargas ◽  
Armando Hernandez García ◽  
Paul van der Schoot ◽  
...  
Keyword(s):  
Real Time ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Single Particle ◽  
Self Assembly ◽  
De Novo ◽  
Particle Growth ◽  
Single Particle Tracking ◽  
Contour Length ◽  
Assembly Pathways

AbstractWhile the structure of a variety of viruses has been resolved at atomistic detail, their assembly pathways remain largely elusive. Key unresolved issues in assembly are the nature of the critical nucleus starting particle growth, the subsequent self-assembly reaction and the manner in which the viral genome is compacted. These issues are difficult to address in bulk approaches and are effectively only accessible by tracking the dynamics of assembly of individual particles in real time, as we show here. With a combination of single-molecule techniques we study the assembly into rod-shaped virus-like particles (VLPs) of artificial capsid polypeptides, de-novo designed previously. Using fluorescence optical tweezers we establish that oligomers that have pre-assembled in solution bind to our DNA template. If the oligomer is smaller than a pentamer, it performs one-dimensional diffusion along the DNA, but pentamers and larger oligomers are essentially immobile and nucleate VLP growth. Next, using real-time multiplexed acoustic force spectroscopy, we show that DNA is compacted in regular steps during VLP growth. These steps, of ∼30 nm of DNA contour length, fit with a DNA packaging mechanism based on helical wrapping of the DNA around the central protein core of the VLP. By revealing how real-time, single particle tracking of VLP assembly lays bare nucleation and growth principles, our work opens the doors to a new fundamental understanding of the complex assembly pathways of natural virus particles.

scholarly journals Enhancement of DNA-mediated gene transfer by high-Mr carrier DNA in synchronized CV-1 cells

1985 ◽  
Vol 225 (2) ◽  
pp. 529-533 ◽  
Author(s):  
A J Strain ◽  
W A H Wallace ◽  
A H Wyllie
Keyword(s):  
Cell Cycle ◽  
Gene Transfer ◽  
Transfection Efficiency ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
S Phase ◽  
Sv40 Virus ◽  
G2 Phase ◽  
Cycle Dependent ◽  
Sv40 Dna

Synchronized CV-1 cells were transfected with SV40 (simian virus 40) DNA-calcium phosphate co-precipitates. In the presence of carrier DNA, the transfection efficiency of SV40 DNA was decreased 5-fold in S-phase cells and was increased 4-fold in preparations of mitotically enriched cells as compared with asynchronous controls. No difference was observed when carrier DNA was omitted, when cells had progressed through S-phase and into G2-phase, or when the infectivity of cells to intact SV40 virus was tested. These results highlight the importance of cell-cycle-dependent factors on DNA-mediated gene transfer.

Photoluminescent organic polymer nanofilms formed in water through a self-assembly formation mechanism

2019 ◽  
Vol 7 (11) ◽  
pp. 3286-3293 ◽  
Author(s):  
Baoxi Feng ◽  
Zhen Xu ◽  
Jiayu Wang ◽  
Fei Feng ◽  
Lin Wang ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Real Time ◽  
Formation Mechanism ◽  
Self Assembly ◽  
Organic Polymer ◽  
Assembly Mechanism ◽  
Real Time Visualization ◽  
Dynamics Simulations

A self-assembly mechanism is demonstrated for the formation of polymer nanofilms based on real-time visualization and molecular dynamics simulations.

scholarly journals Enhanced transformation by a simian virus 40 recombinant virus containing a Harvey murine sarcoma virus long terminal repeat.

1983 ◽  
Vol 3 (3) ◽  
pp. 325-339 ◽  
Author(s):  
M Kriegler ◽  
M Botchan
Keyword(s):  
Long Terminal Repeat ◽  
Recombinant Virus ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Viral Dna ◽  
Sv40 Virus ◽  
Transformed Cell ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Murine Sarcoma

We have constructed a recombinant simian virus 40 (SV40) DNA containing a copy of the Harvey murine sarcoma virus long terminal repeat (LTR). This recombinant viral DNA was converted into an infectious SV40 virus particle and subsequently infected into NIH 3T3 cells (either uninfected or previously infected with Moloney leukemia virus). We found that this hybrid virus, SVLTR1, transforms cells with 10 to 20 times the efficiency of SV40 wild type. Southern blot analysis of these transformed cell genomic DNAs revealed that simple integration of the viral DNA within the retrovirus LTR cannot account for the enhanced transformation of the recombinant virus. A restriction fragment derived from the SVLTR-1 virus which contains an intact LTR was readily identified in a majority of the transformed cell DNAs. These results suggest that the LTR fragment which contains the attachment sites and flanking sequences for the proviral DNA duplex may be insufficient by itself to facilitate correct retrovirus integration and that some other functional element of the LTR is responsible for the increased transformation potential of this virus. We have found that a complete copy of the Harvey murine sarcoma virus LTR linked to well-defined structural genes lacking their own promoters (SV40 early region, thymidine kinase, and G418 resistance) can be effectively used to promote marker gene expression. To determine which element of the LTR served to enhance the biological activity of the recombinant virus described above, we deleted DNA sequences essential for promoter activity within the LTR. SV40 virus stocks reconstructed with this mutated copy of the Harvey murine sarcoma virus LTR still transform mouse cells at an enhanced frequency. We speculate that when the LTR is placed more than 1.5 kilobases from the SV40 early promoter, the cis-acting enhancer element within the LTR can increase the ability of the SV40 promoter to effectively operate when integrated in a murine chromosome. These data are discussed in terms of the apparent cell specificity of viral enhancer elements.

Influence of Nanochannel Inlet Structure Upon DNA Capture Ratio

2011 ◽  
Author(s):  
Jiahao Wu ◽  
Hong Wang ◽  
Jinsoo Kim ◽  
Freddy Murphy ◽  
Steven A. Soper ◽  
...  
Keyword(s):  
Real Time ◽  
Persistence Length ◽  
Contour Length ◽  
Dna Molecules ◽  
Channel Diameter ◽  
Dna Molecule ◽  
Capture Ratio ◽  
Sequence Variations ◽  
Dna Capture ◽  
Dna Chain

DNA molecule will be stretched to its near full contour length inside a nanochannel when the channel diameter is less than the DNA persistence length.1–3 It provides the possibility of real time lab-free-analysis of analysis, such as screening of sequence variations of DNA molecules.3 The key process for this nanochannel-based analysis is to drive DNA molecule electrophoretically through the nanochannel and read out the information of the DNA chain while it is passing the channel.2, 3

Electron Microscopic Observations on the Rescue of SV40 Virus From Transformed Cells

1972 ◽  
Vol 30 ◽  
pp. 278-279
Author(s):  
Ronald Glaser ◽  
Ross Farrugia
Keyword(s):  
Electron Microscopy ◽  
Cell Fusion ◽  
Sendai Virus ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Transformed Cells ◽  
Density Gradients ◽  
Electron Microscopic ◽  
Sv40 Virus ◽  
Virus Particles

Several laboratories have reported that simian virus 40 (SV40) was rescued from transformed cells when the nonproducing cells were cocultivated or fused in the presence of ultraviolet inactivated Sendai virus (UV-SV), to potentially susceptible cells. Evidence obtained from studies in which nuclei from heterokaryons were isolated and separated on density gradients, indicated that rescued virus was first detected in the transformed nuclei of the heterokaryons formed during cell fusion. The present study was performed to determine how long after fusion SV40 virus particles could be found in the nuclei of the heterokaryons and to investigate the site of rescue by electron microscopy.

Cryo-Electron Microscopy and Image Analysis of SV40

1985 ◽  
Vol 43 ◽  
pp. 316-317
Author(s):  
T. S. Baker ◽  
J. Drak ◽  
M. Bina
Keyword(s):  
Electron Microscopy ◽  
Simian Virus 40 ◽  
Carbon Support ◽  
Simian Virus ◽  
Thin Layers ◽  
Sv40 Virus ◽  
Virus Particles ◽  
Frozen Solution ◽  
Cryo Electron Microscopy ◽  
Icosahedral Capsid

The discovery that the T=7 icosahedral capsid of polyoma virus is composed of 72 pentameric capsomers rather than 12 pentamers and 60 hexamers as predicted by constraints of quasi-equivalence has prompted an examination of SV40 virus by electron microscopy to determine whether the capsids of other members of the papovavirus family are similarly constructed.Thin layers of buffered aqueous solutions (∼4 mg/ml) of Simian virus 40 (strain WT776) were prepared for cryo-microscopy using recently developed procedures. Images of virus particles suspended in thin layers of vitreous ice over holes in the carbon support film and maintained at -170°C were recorded using minimal irradiation conditions. Figure 1 shows a typical field in which the frozen solution is similar in thickness to the virus particles (∼49 nm diameter). Particles appear to be excluded or squeezed away from the thinnest regions of solution (e.g., the clear region bordered by particles at the top of Fig. 1).

Enhanced transformation by a simian virus 40 recombinant virus containing a Harvey murine sarcoma virus long terminal repeat

1983 ◽  
Vol 3 (3) ◽  
pp. 325-339
Author(s):  
M Kriegler ◽  
M Botchan
Keyword(s):  
Long Terminal Repeat ◽  
Recombinant Virus ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Viral Dna ◽  
Sv40 Virus ◽  
Transformed Cell ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Murine Sarcoma

We have constructed a recombinant simian virus 40 (SV40) DNA containing a copy of the Harvey murine sarcoma virus long terminal repeat (LTR). This recombinant viral DNA was converted into an infectious SV40 virus particle and subsequently infected into NIH 3T3 cells (either uninfected or previously infected with Moloney leukemia virus). We found that this hybrid virus, SVLTR1, transforms cells with 10 to 20 times the efficiency of SV40 wild type. Southern blot analysis of these transformed cell genomic DNAs revealed that simple integration of the viral DNA within the retrovirus LTR cannot account for the enhanced transformation of the recombinant virus. A restriction fragment derived from the SVLTR-1 virus which contains an intact LTR was readily identified in a majority of the transformed cell DNAs. These results suggest that the LTR fragment which contains the attachment sites and flanking sequences for the proviral DNA duplex may be insufficient by itself to facilitate correct retrovirus integration and that some other functional element of the LTR is responsible for the increased transformation potential of this virus. We have found that a complete copy of the Harvey murine sarcoma virus LTR linked to well-defined structural genes lacking their own promoters (SV40 early region, thymidine kinase, and G418 resistance) can be effectively used to promote marker gene expression. To determine which element of the LTR served to enhance the biological activity of the recombinant virus described above, we deleted DNA sequences essential for promoter activity within the LTR. SV40 virus stocks reconstructed with this mutated copy of the Harvey murine sarcoma virus LTR still transform mouse cells at an enhanced frequency. We speculate that when the LTR is placed more than 1.5 kilobases from the SV40 early promoter, the cis-acting enhancer element within the LTR can increase the ability of the SV40 promoter to effectively operate when integrated in a murine chromosome. These data are discussed in terms of the apparent cell specificity of viral enhancer elements.

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