Single Particle Observation of SV40 VP1 Polyanion-Induced Assembly Shows That Substrate Size and Structure Modulate Capsid Geometry

Li, Chenglei; Kneller, Andrew R.; Jacobson, Stephen C.; Zlotnick, Adam

doi:10.1021/acschembio.6b01066

Cited by 16 publications

(21 citation statements)

References 51 publications

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“…SV40 favors stepwise assembly of the capsid that occur concomitantly with nucleic acid binding [56]. Consistent with our results, SV40 assembly has shown tertiary structure of the cargo and length of cargo influence the assembly of the virion [57]. Computational models show that weak CP-CP interactions favor initial formation of a disordered CP-nucleic acid oligomer that then assembles, while strong interactions favor co-assembly [19,20,21].…”

Section: Discussionsupporting

confidence: 86%

Length of encapsidated cargo impacts stability and structure ofin vitroassembled alphavirus core-like particles

Rayaprolu¹,

Moore²,

Wang³

et al. 2017

J. Phys.: Condens. Matter

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In vitro assembly of alphavirus nucleocapsid cores, called core-like particles (CLPs), requires a polyanionic cargo. There are no sequence or structure requirements to encapsidate single-stranded nucleic acid cargo. In this work, we wanted to determine how the length of the cargo impacts the stability and structure of the assembled CLPs. We hypothesized that cargo neutralizes the basic region of the alphavirus capsid protein and if the cargo is long enough, it will also act to scaffold the CP monomers together. Experimentally we found that CLPs encapsidating short 27mer oligonucleotides were less stable than CLPs encapsidating 48mer or 90mer oligonucleotides under different chemical and thermal conditions. Furthermore, cryo-EM studies showed there were structural differences between CLPs assembled with 27mer and 48mer cargo. To mimic the role of the cargo in CLP assembly we made a mutant (4D) where we substituted a cluster of four Lys residues in the CP with four Asp residues. We found that these few amino acid substitutions were enough to initiate CLP assembly in the absence of cargo. The cargo-free 4D CLPs show higher resistance to ionic strength and increased temperature compared to wild-type cargo containing CLPs suggesting their CLP assembly mechanism might also be different.

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Section: Discussionsupporting

confidence: 86%

Length of encapsidated cargo impacts stability and structure ofin vitroassembled alphavirus core-like particles

Rayaprolu¹,

Moore²,

Wang³

et al. 2017

J. Phys.: Condens. Matter

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“…CCMV has, for instance, a preference for packaging of polystyrene sulfonate (PSS), which favors a collapsed state over singlestranded RNA, which acts like a many-branched polymer (43). For SV40, it is shown that tertiary structure of PSS or RNA and the size of the substrate affect the assembly (19). All these experiments show that the template around which the capsid forms decisively influences assembly.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2020

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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.

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“…We used two protocols to obtain VP1 pentamers. Initially, we followed the procedure described by Li et al (41). Briefly, DNA-filled VLPs were dissociated by dialysis against 20 mM Tris-Cl (pH 8.9), 50 mM NaCl, 2 mM dithiothreitol (DTT), and 5 mM EDTA at 4 C. The dialysate was centrifuged to remove aggregated protein and then loaded on a Superose 6 column (GE Lifesciences, Chicago, IL) equilibrated in 100 mM 3-(N-morpholino)propanesulfonic acid (MOPS; pH 7.2), 250 mM NaCl.…”

Section: Methodsmentioning

confidence: 99%

Effect of dsDNA on the Assembly Pathway and Mechanical Strength of SV40 VP1 Virus-like Particles

Rosmalen

Zlotnick

et al. 2018

Biophysical Journal

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Simian virus 40 (SV40) is a possible vehicle for targeted drug delivery systems because of its low immunogenicity, high infectivity, and high transfection efficiency. To use SV40 for biotechnology applications, more information is needed on its assembly process to efficiently incorporate foreign materials and to tune the mechanical properties of the structure. We use atomic force microscopy to determine the effect of double-stranded DNA packaging, buffer conditions, and incubation time on the morphology and strength of virus-like particles (VLPs) composed of SV40 VP1 pentamers. DNA-induced assembly results in a homogeneous population of native-like, $45 nm VLPs. In contrast, under high-ionic-strength conditions, the VP1 pentamers do not seem to interact consistently, resulting in a heterogeneous population of empty VLPs. The stiffness of both in-vitro-assembled empty and DNA-filled VLPs is comparable. Yet, the DNA increases the VLPs' resistance to large deformation forces by acting as a scaffold, holding the VP1 pentamers together. Both disulfide bridges and Ca 2þ , important in-vitro-assembly factors, affect the mechanical stability of the VLPs: the reducing agent DTT makes the VLPs less resistant to mechanical stress and prone to damage, whereas Ca 2þ-chelating EDTA induces a marked softening of the VLP. These results show that negatively charged polymers such as DNA can be used to generate homogeneous particles, thereby optimizing VLPs as vessels for drug delivery. Moreover, the storage buffer should be chosen such that VP1 interpentamer interactions are preserved.

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Single Particle Observation of SV40 VP1 Polyanion-Induced Assembly Shows That Substrate Size and Structure Modulate Capsid Geometry

Cited by 16 publications

References 51 publications

Length of encapsidated cargo impacts stability and structure ofin vitroassembled alphavirus core-like particles

Length of encapsidated cargo impacts stability and structure ofin vitroassembled alphavirus core-like particles

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

Effect of dsDNA on the Assembly Pathway and Mechanical Strength of SV40 VP1 Virus-like Particles

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